Bioconversion of Pulp and Paper Mills Sludge and Prehydrolysate Stream into  
Ethanol and Cellulase Enzyme 
 
by 
 
Li Kang 
 
 
 
 
A dissertation submitted to the Graduate Faculty of 
Auburn University 
in partial fulfillment of the 
requirements for the Degree of 
Doctor of Philosophy  
 
Auburn, Alabama 
August, 6, 2011 
 
 
 
Copyright 2011 by Li Kang 
 
 
Approved by 
 
Yoon Y. Lee , Chair, Professor of Chemical Engineering 
Christopher B. Roberts, Professor of Chemical Engineering 
Robert P. Chambers, Professor of Chemical Engineering 
Evert C. Duin, Associate Professor of Chemistry and Biochemistry 
 
 
 
 
 
 ii
Abstract 
 
 
In the first part of this dissertation, two different types of paper mill sludges, 
primary sludge and recycle sludge, were evaluated as a feedstock for bioconversion to 
ethanol. The sludges were first subjected to enzymatic conversion to sugars by 
commercial cellulase enzymes. The enzymatic conversion was inefficient because of 
interference by ash present in the sludges with the enzymatic reaction. The main cause 
was that the pH level is dictated by the CaCO
3
 in ash, which was two units higher than 
the pH optimum of cellulase. To alleviate this problem, simultaneous saccharification and 
co-fermentation (SSCF) using cellulase (Spezyme CP) and recombinant Escherichia coli 
(ATCC-55124), and simultaneous saccharification and fermentation (SSF) using 
cellulase and Saccharomyces cerevisiae (ATCC-200062) were applied to the sludges 
without any pretreatment.  Ethanol yields of 75-81% of the theoretical maximum were 
obtained from the SSCF on the basis of total carbohydrates. The yield from the SSF was 
also in the range of 74-80% on the basis of glucan. The SSCF and SSF proceeded under 
stable condition with the pH staying near 5.5, close to the optimum for cellulase. 
Decrease of pH occurred due to carbonic acid and other organic acids formed during 
fermentation. The ash was partially neutralized by the acids produced from the SSCF and 
SSF and acted as a buffer to stabilize the pH during fermentation. When the SSF and 
SSCF were operated in fed-batch mode, the ethanol concentration in the broth increased 
from 25.5 and 32.6 g/L to 42 and 45 g/L, respectively. The ethanol concentration was
 iii
limited by the tolerance of the microorganism in case of SSCF. The ethanol yield in fed-
batch operation dropped to 68% for SSCF and 70% for SSF. 
The second part of this dissertation dealt with the de-ashing process of paper 
sludges. In the first part, it was demonstrated that the paper sludges with the high ash 
content were efficiently converted to ethanol in both SSCF and SSF. However, high ash 
content in the sludge, however, limited the solid loading in the bioreactor causing low 
product concentration. De-ashing of sludges before SSCF and SSF was attempted to 
overcome this difficulty. Bioconversion of the low ash content sludges gave an ethanol 
yield of 80% with cellulase loading of 15 FPU/g-glucan. With lowering the cellulase 
loading to10 FPU/g-glucan, the ethanol yield was reduced to 70%.  High solids loading in 
SSF and SSCF decreased the ethanol yield. High agitation and de-ashing of the sludges 
make up part of the yield loss caused by high solids loading. In addition, substitution of 
the laboratory fermentation medium (peptone and yeast extract) with corn steep liquor 
did not bring about any adverse effects in the fermentation. When the SSCF and SSF 
were operated in fed-batch mode using low-ash content sludges, the ethanol 
concentration in the broth was increased to 48 and 60 g/L, respectively.  
In the third part of the dissertation, production of cellulase from unbleached 
hardwood pulp and kraft paper sludge was investigated. Cellulase on-site production is a 
supplementary unit in a kraft paper mill in parallel with or in place of bioethanol. Using 
cellulase and Saccharomyces cerevisiae on kraft paper sludge, which is waste from the 
kraft pulp making process, it is possible to produce bioethanol. It is feasible and 
convenient to integrate these two processes without any impact on the output of paper 
goods and on the production of bioethanol. To explore this potential, the cellulase 
 iv
enzyme was produced by Trichoderma reesei Rut C-30 and was investigated for its 
characteristics and titers.  Experiments were conducted at different conditions to 
determine the impact of operational factors upon T. reesei Rut C-30 growth and cellulase 
production.  Different concentrations of unbleached hardwood pulp were examined to 
determine the maximum levels of cellulase activity.  The highest titer of 7.5 FPU/mL was 
obtained with use of 4% (w/v) of unbleached kraft processed hardwood pulp. Paper 
sludge was also considered as a potential feedstock for cellulase production. However, it 
has a high ash content which is detrimental to cell growth. It is desirable to remove ash as 
much as possible from the sludge, while retaining carbohydrates. In this study, the de-
ashed sludge pre-processed via physical and chemical treatment was used as substrates 
for cellulase production and ethanol fermentation. The cellulase enzyme produced from 
de-ashed sludge exhibited cellulase activity as high as 8 FPU/mL. The properties of the 
crude cellulase were determined and compared with a commercially available cellulase, 
Spezyme CP, using hardwood pulp and acid-treated corn stover as the substrates.  The 
gross cellulolytic activities and other properties the enzymes produced in this work from 
the unbleached hardwood pulp and kraft paper sludge were comparable with those from 
the commercial enzyme, Spezyme CP.  The in-house cellulase, however, exhibited higher 
xylanase activity than Spezyme CP.  
In the last part of the dissertation, ethanol production from hemicellulose 
prehydrolysate was investigated. Most of the hemicellulose fraction of pulp mill 
feedstock (softwood or hardwood) is released into black liquor during the pulping 
process. The black liquor is combusted to recover chemicals and to generate steam and 
electricity. It is feasible to recover this fraction of carbohydrate and enhance its value by 
 v
converting it into value-added products. Hemicellulose is selectively converted to soluble 
sugars (termed as prehydrolysate) by treating it with hot water. The sugars produced from 
pre-hydrolysis process are mixtures of pentose, hexose, and their oligomers. In this study, 
pectinase and Saccharomyces cerevisiae were used to convert the prehydrolysate into 
ethanol. The prehydrolysate produced from wood also contains toxins, primarily lignin 
and sugar degradation products, which strongly inhibit microbial reaction. De-
toxification of the prehydrolysates was done by overliming (addition of excess CaO).  
When hydrolysate is obtained by treating wood, the total sugar concentration is below 4 
wt. %. Consequently, when the hydrolysate is used as a fermentation substrate, the 
ethanol concentration is less than 2 wt. %, which is far below the level acceptable as the 
distillation feed. Use of the mixture of prehydrolysate and pulp mill sludges as the 
fermentation feed, however, can increase the product concentration. In bioconversion of 
sludge, a certain amount of water is added to attain fluidity required for SSF operation. In 
this study, prehydrolysate, in place of water, was added into the bioreactor along with the 
sludge. Using this procedure, there was a net increase of total sugar concentration in the 
bioreactor above that of the base case, which led to an increase in product concentration.  
The experimental data detailing the proposed bioprocess convertion of the mixed-feed to 
ethanol are presented in this paper. 
 vi
Acknowledgments 
 
 
I would like to thank my advisor, Dr. Yoon Young Lee. He has continually 
inspired me to reach out to new fields, and has shared his excellent knowledge of science 
with me through his creativity and guidance. I would also like to thank Dr. Gopal A. 
Krishnagopalan, Dr. Sunghoon Yoon, Dr. Wei Wang, Dr. Allen J. Smith, Dr. Changshin 
Sunwoo, and Meng Liang for numerous technical discussions and their cooperation 
during the course of this investigation. I would to thank my laboratory colleagues, Dr. 
Rajesh Gupta, Venkata Ramesh Pallapolu, Urvi Kothari, Suan Shi, and Suma Peri, for 
their cooperation during the course of this investigation. I want to thank Dr. Christopher 
Roberts, Dr. Robert Chambers and Dr. Evert C. Duin for their guidance and being in my 
committee.   
Finally, I would like to thank my parents and family members, for their constant 
support and encouragement which have motivated me throughout all of my endeavors. 
The funding for this study was provided by the Masada, LLC, Alabama Center for 
Renewable Resources Engineering, and Laboratory of Biofuels and Biomaterials, Auburn 
University, and their support is gratefully acknowledged. 
 
 
 
 
 
 vii
Table of Contents 
 
 
Abstract............................................................................................................................... ii 
Acknowledgments ............................................................................................................. vi 
List of Tables .................................................................................................................... xii 
List of Figures.................................................................................................................. xiv 
Chapter I. Introduction  .......................................................................................................1 
Chapter II. Literature Review  ............................................................................................5 
 2.1 Background-Bioethanol  ...................................................................................5 
 2.2 Composition and Structure of Biomass  ...........................................................7 
      2.2.1 Cellulose  ..................................................................................................7 
      2.2.2 Hemicellose  ..............................................................................................8 
      2.2.3 Lignin  .....................................................................................................10 
      2.2.4 Extractives and Ash  ...............................................................................12 
      2.2.5 Acetyl Group  ..........................................................................................12 
 2.3 Bioconversion of Lignocellulosic Biomass  ...................................................13 
      2.3.1 Pretreatment  ...........................................................................................14 
              2.3.1.1 Biological Pretreatment  ..............................................................14 
              2.3.1.2 Physical Pretreatment  ..................................................................14 
              2.3.1.3 Chemical Pretreatment  ................................................................15 
      2.3.2 Enzymatic Hydrolysis of Lignocellulosic Biomass  ...............................24
 viii
              2.3.2.1 Cellulase  ......................................................................................24 
             2.3.2.2 Addition of Surfactants  ................................................................27 
              2.3.3.3 Hemicellulase  ..............................................................................29 
              2.3.3.4 Substrates  ....................................................................................31 
      2.3.3 Fermentation  ..........................................................................................34 
 2.4 The Integrated Wood-based Biorefinery  .......................................................40 
      2.4.1 Background of Pulp and Paper-making  .................................................41 
              2.4.1.1 Mechanical Pulp or Thermomechanical Pulp (TMP)  .................41 
              2.4.1.2 Chemithermomechanical Pulp (CTMP)  .....................................42 
              2.4.1.3 Chemical Pulp  .............................................................................42 
              2.4.1.4 Recycled Paper Pulp  ...................................................................44 
              2.4.1.5 Paper Making  ..............................................................................45 
      2.4.2 The Development of an Integrated Wood-based Biorefinery   ...............45 
              2.4.2.1 Hemicellulose Pre-extraction  ......................................................46 
              2.4.2.2 Black Liquor  ...............................................................................48 
              2.4.2.3 Pulp and Paper Waste  .................................................................48 
      2.4.3 Cellulase Production in Kraft Pulp Mills  ...............................................50 
Chapter III. Bioconversion of Kraft Paper Mill Sludges to Ethanol by SSF and SSCF  ..60 
 3.1 Abstract  ..........................................................................................................60 
 3.2 Introduction  ....................................................................................................61 
 3.3 Materials and Methods  ...................................................................................63 
      3.3.1 Materials  ................................................................................................63 
      3.3.2 Contaminant Microorganism Test  .........................................................64 
 ix
      3.3.3 Digestibility Test  ....................................................................................64 
      3.3.4. Simultaneous Saccharification and Fermentation / Co-fermentation  ...66 
      3.3.5. Analytical Methods  ...............................................................................67 
 3.4. Results and Discussion  .................................................................................68 
      3.4.1 Composition  ...........................................................................................68 
      3.4.2 Enzymatic Digestibility  .........................................................................68 
      3.4.3 Microbial Contamination of Sludges  .....................................................70 
      3.4.4 Simultaneous Saccharification and Fermentation  ..................................70 
      3.4.5 Simultaneous Saccharification and Co-Fermentation  ............................74 
      3.4.6 Fed-Batch Operation  ..............................................................................76 
 3.5. Conclusion  ....................................................................................................78 
Chapter IV. De-ashing Treatment of Paper Sludges and Enhancing Ethanol Production by        
                     SSF and SSCF of De-ashing Paper Sludge  .................................................88 
 4.1 Abstract  ..........................................................................................................88 
 4.2 Introduction  ....................................................................................................88 
 4.3 Materials and Methods  ...................................................................................91 
      4.3.1 Materials  ................................................................................................91 
      4.3.2 Colony Forming Unit Test  .....................................................................93 
      4.3.3 Simultaneous Saccharification and Fermentation / Co-fermentation  ....93 
      4.3.4. Analytical Methods  ...............................................................................95 
 4.4. Results and Discussion  .................................................................................96 
      4.4.1 De-ashing of Sludges by Screening  .......................................................96 
      4.4.2 SSF and SSCF of Screened De-ashing of Sludges  ................................97 
      4.4.3 Evaluation of Low-cost Fermentation Medium for Fermentation  .........99 
 x
      4.4.4 Fed-Batch Operation  ............................................................................104 
 4.5. Conclusion  ..................................................................................................105 
Chapter V. Production of Cellulase from Unbleaching Hardwood Pulp and Paper  
                  Sludge  ..........................................................................................................117 
 5.1 Abstract  ........................................................................................................117 
 5.2 Introduction  ..................................................................................................118 
 5.3 Materials and Methods  .................................................................................121 
      5.3.1 Materials  ..............................................................................................121 
      5.3.2 Cellulase Production  ............................................................................122 
      5.3.3 Enzyme Analysis  .................................................................................123 
      5.3.4. Enzymatic Hydrolysis Test  .................................................................125 
      5.3.5. Simultaneous Saccharification and Fermentation (SSF)  ....................125 
      5.3.6. Analytical Methods  .............................................................................127 
 5.4. Results and Discussion  ...............................................................................127 
      5.4.1 Effect of Different pH on Cellulase Production from Hardwood Pulp  127 
      5.4.2 Effect of Different Hardwood Pulp Concentration  ..............................131 
      5.4.3 Enzyme Production from De-ashed and Chemical Treated Sludge.......133 
      5.4.4 Enzymatic Hydrolysis Test  ..................................................................135 
      5.4.5 SSF Experiments  ..................................................................................137 
 5.5. Conclusion  ..................................................................................................138 
Chapter VI. Ethanol Production from the Mixture of Hemicellulose Prehydrolysate and  
                   Paper Sludge, Integrated with Hemicellulose Pre-extraction of Southern  
                   Pine ..............................................................................................................150 
 6.1 Abstract  ........................................................................................................150 
 6.2 Introduction  ..................................................................................................151 
 xi
 6.3 Materials and Methods  .................................................................................154 
      6.3.1 Materials  ..............................................................................................154 
      6.3.2 Pre extraction  .......................................................................................154 
      6.3.3 Composition Analysis of Prehydrolysate  .............................................157 
      6.3.4. Contaminant Microorganism Test  ......................................................158 
      6.3.5. Treatment of Prehydrolysate with CaO  ..............................................158 
      6.3.6. Enzymatic Hydrolysis Test  .................................................................158 
      6.3.7. Separation Hydrolysis and Fermentation and SSF of Prehydrolysate  159 
      6.3.8. SSF of Paper Primary Sludge with or without Prehydrolysate  ...........160 
      6.3.9. Analytical Methods  .............................................................................162 
 6.4. Results and Discussion  ...............................................................................162 
      6.4.1 Pre-extraction of Softwood  ..................................................................162 
      6.4.2 Composition of Softwood Prehydrolysate  ...........................................163 
      6.4.3 Enzymatic Hydrolysis of Softwood Prehydrolysate  ............................165 
      6.4.4 De-toxification and SHF of Softwood Prehydrolysate  ........................167 
      6.4.5 SSF of Softwood Prehydrolysate  .........................................................171 
      6.4.6 SSF of Prehydrolysate with Paper Sludge  ...........................................171 
 6.5. Conclusion  ..................................................................................................172 
Chapter VII. Future Work  ..............................................................................................183 
Bibliography  ..................................................................................................................186 
 
 
 
 xii
List of Tables 
 
 
Table II-1 Composition of sludges and corn stover ..........................................................54 
Table III-1 Composition of sludges and corn stover ........................................................80 
Table III-2 Enzymatic digestibility of paper mill sludges ................................................81 
Table III-3 Ethanol yield from SSF and SSCF of paper mill sludges ..............................82 
Table III-4 Composition analysis of fermentation residues ..............................................83 
Table IV-1 Composition of de-ashed sludges .................................................................107 
Table IV-2 Ethanol yield from SSF and SSCF of treated paper sludges ........................108 
Table IV-3 Ethanol yield from SSF and SSCF of paper sludges at different solid  
                  loading ...........................................................................................................109 
Table IV-4 Ethanol yield from SSF and SSCF of sludges at different agitation  
                    intensity .......................................................................................................110 
Table IV-5 Ethanol yield from SSCF of de-ashed paper sludges on different medium .111 
Table V-1 Enzyme activities and productivity obtained in the cultivation of T. reesei Rut  
                  C-30 on 20 g/L hardwood pulp under different pH control conditions ........139 
Table V-2 Enzyme activity, yield and productivity obtained in the cultivation of T. reesei  
                 Rut C-30 on different concentration of hardwood pulp as substrates ............140 
Table V-3 Enzyme activities obtained in the cultivation of T. reesei Rut C-30 on 40 g/L  
                  hardwood pulp ..............................................................................................141 
Table V-4 Specific activities of cellulolytic and henicellulolytic enzymes of fermentation  
                  broth of alkaline treated sludge .....................................................................142 
Table V-5 Cellulase activity and productivity of T. reesei Rut C-30 in 3-L fermenter  
                  using acid and alkaline treated paper mill sludge as substrates ....................143 
Table VI-1 Enzyme activities detected in commercial enzyme preparations .................174 
 xiii
Table VI-2 Sugar content of first Stage prehydrolysate at different time periods ..........175 
Table VI-3 Sugar content of three stage prehydrolysates ...............................................176 
Table VI-4 Organic compounds content of three stage prehydrolysates ........................177 
Table VI-5 Composition of solid fraction in three stage prehydrolysates ......................178
 
 xiv
List of Figures 
 
 
Figures II-1 Bioethanol production process diagram ........................................................55 
Figures II-2 Pretreatment of biomass ................................................................................56 
Figures II-3 Cellulose hydrolysis by cellulases from Trichoderma reessi .......................57 
Figures II-4 Structure of xylan and the sites of attack by xylanolytic enzymes ...............58 
Figures II-5 Mannanses attack on galactoglucomanna .....................................................59 
Figures III-1 Simultaneous saccharification and fermentation (SSF) of sludges by   
                     Saccharomyces cerevisiae (ATCC-200062).................................................84 
Figures III-2 Buffer capacity titration curves with acetic acid for paper sludge slurries and  
                      deionized water ...........................................................................................85 
Figures III-3 Simultaneous saccharification and cofermentation (SSCF) of sludge by  
                      recombinant E.coli (KO11)..........................................................................87 
Figures III-4 Fed-batch SSF and SSCF of sludges ...........................................................88 
Figures III-5 Simultaneous saccharification and fermentation (SSF) of sludges by  
                      Saccharomyces cerevisiae (ATCC-200062)..............................................112 
Figures IV-1 De-ashing process of the primary sludge ..................................................113 
Figures IV-2 Scanning electron micrograph (SEM) of de-ashed and untreated sludge ..113 
Figures IV-3 Simultaneous saccharification and fermentation (SSF) of de-ashed sludge  
                      by Saccharomyces cerevisiae (ATCC-200062) ........................................114 
Figures IV-4 Simultaneous saccharification and co-fermentation (SSCF) of de-ashed   
                      sludge by Escherichia coli KO11 (ATCC-55124) ....................................115 
Figures IV-5 Fed-Batch SSF and SSCF of sludges and corn stover (S-PS-1) ...............116 
Figures V-1 Cellulase production by T. reesei Rut C-30 under different pH control  
                     conditions ...................................................................................................144 
 xv
Figures V-2 Cellulase production by T. reesei Rut C-30 at different concentration of   
                     hardwood pulp ...........................................................................................145 
Figures V-3 Cellulase production by T. reesei using acid leached sludge .....................146 
Figures V-4 Cellulase production by T. reesei in using alkaline treated sludge .............147 
Figures V-5 Enzymatic hydrolysis of different substrates by HP-enzyme and Spezyme          
                    CP ................................................................................................................148 
Figures V-6 Ethanol production from untreated primary sludge by HP-enzyme and  
                     Spezyme CP ...............................................................................................149 
Figures VI-1 Different commercial enzyme preparation undergo hydrolysis of softwood  
                      prehydrolysate ...........................................................................................179 
Figures VI-2 Composition change of prehydrolysate after de-toxification ....................180 
Figures VI-3 Separation hydrolysis and fermentation (SHF) of prehydrolysate by  
                      Multifect Pectinase FE and Saccharomyces cerevisiae (ATCC-200062) 181 
Figures VI-4 Simultaneous saccharification and fermentation (SSF) of prehydrolysate by  
                      Saccharomyces cerevisiae (ATCC-200062) and Multifect Pectinase FE 182 
 
 1
I. Introduction 
Increasing energy demand, escalating oil prices, concerns about greenhouse gas 
emissions from fossil fuels and energy security have led to great interest in the production 
of fuels from renewable materials. Ethanol has been an economically feasible fuel for 
transportation for several decades. Currently, ethanol is mainly produced from any 
feedstock that contains natural sugars or starch that can be readily converted to sugar, 
such as corn in the United States. However, it is recognized that the corn ethanol cannot 
meet the requirement without causing a major disruption of the food markets (McElroy, 
2006).  
Cellulosic ethanol offers promise as cellulose comes from inexpensive and 
abundant lignocellulosic biomass (Inderwildi and King, 2009). In addition, cellulose does 
not compete with food related agricultural products. It is estimated that 323 million tons 
of cellulosic raw materials are thrown away each year in the U.S. alone that could be used 
to produce ethanol. This includes 36.8 million dry tons of urban wood waste, 90.5 million 
dry tons of primary mill residues, 45 million dry tons of forest residues, and 150.7 million 
dry tons of corn stover & wheat straw (Energy Information Administration,  2007).  
The increasing need of energy and the dire financial performance of the U.S. pulp 
and paper industries bring up the possibility of wood based-biorefinery which would look 
to use all components of wood to make pulp, transportation fuels, power, and chemicals. 
From the pulp and paper making process, there are three potential opportunities, paper 
sludge including kraft paper mill sludge and waste news print, hemicellulose, and black
 2
liquor. The pulp and paper industry produces about 4.1 to 5 million dry tons per year of 
solid waste in U. S., consisting primarily of de-watered sludge, which contains fibers and
other materials produced during paper-making or paper recycling (Fan and Lynd, 2006b). 
Paper sludges are normally disposed by incineration or landfill, which cause substantial 
financial burden and various environmental problems. A better alternative to landfilling 
and incineration is to utilize paper sludge in a biorefinery.  Sludge contains much more 
cellulose and much less hemicellulose and lignin than corn stover. Moreover, the short 
fibers in the sludge are easily digested by cellulase enzymes (Fan and Lynd, 2006a; 
Jeffries et al., 1999; Fan et al., 2003). These features can potentially make bioconversion 
of sludges possible without pretreatment. As one of the major processing steps in 
bioethanol plants, pretreatment is a significant cost factor, second only to feedstock cost 
which accounts for about 30% of the total production cost of ethanol (Galbe and Zacchi, 
2002). Simultaneous saccharification and fermentation (SSF) using cellulase (Spezyme 
CP) and Saccharomyces cerevisiae (ATCC-200062), and simultaneous saccharification 
and co-fermentation (SSCF) using cellulase and recombinant Escherichia coli (ATCC-
55124) were separately applied to the sludges without any pretreatment.  
 Bioconversion of kraft paper sludges with high ash content can be done by both 
fermentation processes. However, the operation of sludges with high ash content is 
difficult. Some degree of de-ashing before SSCF and SSF is therefore necessary. Several 
methods have been applied to recover the fiber and fillers from paper sludge (Leuthold 
and Leuthold, 1996; Maxham, 1994; Simpson and Lam, 1998), which facilitate the reuse 
of fiber or fillers instead of the sludge as a whole. The equipment used to separate fiber or 
 3
fillers from sludge may be as conventional as screens and cleaners (Dorica and Simandl, 
1995). 
The high cost from the usage of enzymes in the hydrolysis step is one of the major 
drawbacks in the production of bioethanol (Steele et al., 2005; Spano et al., 1978). The 
cost of bioethanol and cellulase production could be reduced when immediate processing 
is minimized, without compromising the quality of the final product. Cellulase 
production using substrates directly from the kraft mill could be feasible, convenient, and 
economically viable.  Besides the sulfite wood pulp (Watson et al., 1984), some waste 
items such as pretreated wood (Juhasz et al., 2005; Shin et al., 2000), sawdust (Lo et al., 
2005), waste paper (Royer and Nakas, 1987; Chen and Wayman, 1991; Shin et al., 2000), 
waste cardboard (Szij?rt? et al., 2004) and paper sludge (Royer and Nakas, 1987; 
Maheshwari et al., 1994) could be used as a carbon source for cellulase production. 
In kraft pulping, about 80% of the hemicellulose in the wood is dissolved in the 
black liquor (Biermann, 1996; Sjostrom, 1993). It is technically difficult to separate this 
hemicellulose part from alkaline black liquor before it is burned to generate heat and 
power (Sjostrom, 1993). It is technically feasible to insert a new process in front of the 
digester to extract hemicellulose and convert to ethanol through a fermentation process. 
Hemicelluloses can be removed from lignocellulosic materials by hot water, alkaline, and 
solvent extractive processes (Willfor and Holmbom, 2004; Song et al., 2008; Sears et al., 
1971; Nguyen et al., 1998; Aziz and Sarkanen, 1989). Through additional processes, 
ethanol can be produced from hemicellulose.  
The overall objective of this study is to investigate the technical feasibility of 
establishing a wood-based biorefinery to be integrated with existing kraft paper mills. 
 4
The following specific objectives were set for this study. 
? To investigate the performance of the kraft paper mill sludge using cellulase and 
Saccharomyces cerevisiae (ATCC-200062), or recombinant Escherichia coli 
(ATCC-55124). 
? To develop a process for de-ashing of paper mill sludge. 
? To develop methods for efficient cellulase production from unbleached hardwood 
pulp and paper sludge. 
? To improve the fermentation yield of prehydrolysate by investigating the factors 
affecting (inhibiting) the fermentation step. 
 5
II. Literature Review 
2.1 Background-Bioethanol 
Since 2003, the price of the fossil fuels has been increasing and peaked at $147.30 
July 2008 (http://www.usatoday.com/money/economy/2008-07-11-3815204975_x.htm). 
As countries develop, industry, rapid urbanization, and higher living standards drive up 
energy use. Thriving economies such as China and India are quickly becoming large oil 
consumers. Demand for oil is outstripping the supply of oil and oil depletion is expected 
to cause crude oil prices to go up over the next 50 years. The days of inexpensive fossil 
energy are gone, and we have to turn to new sources of energy, such as, solar, tidal, wind, 
hydro, nuclear and so on.  In addition,   the dependency on oil has two major drawbacks: 
burning fossil fuels may contribute to global warming; and for net-consuming countries 
like the United States, importing oil creates a dependency on oil-producing countries. 
Therefore, shifting society?s dependence away from fossil fuel to alternative renewable 
biomass fuel has attracted great interest. Ethanol is a renewable resource, can be 
produced, unlike petroleum which cannot be produced and in time will be gone. Because 
it is easy to manufacture and process, it is rapidly becoming a common alternative to 
gasoline in some parts of the world, such as in Brazil.  
Ethanol can be mass-produced by fermentation of sugar or by hydration of 
ethylene (CH
2
=CH
2
) from petroleum and other sources. Currently ethanol is being 
produced mainly from starch or sugar in a wide variety of crops, but there has been 
considerable debate about ?Food vs. fuel?. The years 2007?2008 saw a dramatic increase
 6
 in world food prices, creating a global crisis and causing political and economical 
instability and social unrest in both developing and developed nations. Systemic causes 
for the worldwide increases in food prices continue to be a subject of debate. The 
increasing use of biofuels in developed countries is thought to be one of the main causes. 
Recent developments with cellulosic ethanol production and commercialization may 
allay some of these concerns. Cellulosic ethanol is obtained from cellulose, the main 
component of wood, straw and much of the structure of plants. Since cellulose cannot be 
digested by humans, the production of cellulose does not compete with the production of 
food, other than conversion of land from food production to cellulose production. 
Cellulose is present in every plant; in the form of straw, grass, and wood. The whole 
plant can be harvested, unlike corn. It is estimated that 323 million tons of cellulose 
containing raw materials, which could be used to create ethanol, are thrown away each 
year in the US alone. This includes 36.8 million dry tons of urban wood wastes, 90.5 
million dry tons of primary mill residues, 45 million dry tons of forest residues, and 150.7 
million dry tons of corn stover & wheat straw (Energy Information Administration, 
2007). Kim and Dale (Kim, S. and Dale, B.E., 2004) estimated that there is enough 
biomass from wasted crops and crop residues to potentially replace 32% of the global 
gasoline consumption with bioethanol. 
Also, waste paper, paper sludge, and packaging materials comprise a substantial 
part of the solid waste sent to landfills or incineration in the United States each day alone, 
which is definitely a solid waste disposal headache. Finding new landfills is difficult and 
also causes environmental problems. The cost of incineration is not cheap. Reduction of 
the disposal of solid waste through cellulosic ethanol conversion would reduce solid 
 7
waste disposal costs by local and state governments. It is estimated that each person in 
the US throws away 4.4 lb of trash each day, of which 37% contains waste paper, which 
is largely cellulose. That computes to 244 thousand tons per day of discarded waste 
paper. The solid waste raw material that produces cellulosic ethanol is not only free, it 
has a negative cost i.e., ethanol producers can get paid to take it away. Taking paper 
sludge as an example, the current cost of disposal ranges from $0 to $50 per wet ton 
according to Boise Paper Mill (Abston, 2008).  
Besides energy and economic security, environmental benefits are also one of the 
main driving forces behind cellulosic ethanol commercialization. According to the U.S. 
Department of Energy studies conducted by the Argonne Laboratories of the University 
of Chicago, one of the benefits of cellulosic ethanol is that it reduces greenhouse gas 
emissions (GHG) by 85% over reformulated gasoline 
(http://www1.eere.energy.gov/biomass/news.html). However, even though the cellulosic 
biomass is plentiful and low-cost, it is relatively difficult to convert to ethanol (Wyman, 
1996). Unlike starch and sugar, cellulosic biomass is made up of complex structure, 
which has three main biopolymers (cellulose, hemicellulose, and lignin) intertwined 
chemically and physically, rendering it very difficult to producing alcohol.  
2.2 Composition and structure of biomass 
2.2.1 Cellulose 
Cellulose is the main constituent of wood and other biomass, approximately 38-
50% of dry substance and forms the structural framework of the cell walls. Like amylose 
and the main chains of amylopectin, the cellulose molecule is a linear, unbranched 
homopolysaccharide with a repeating unit of D-glucose strung together by ?-glycosidic 
 8
linkages, consisting of 10,000 to 15,000 glucose units (Goldstein, 1981). The ?-
glycosidic linkages in cellulose form linear chains that are highly stable and resistant to 
chemical attack because hydrogen bonding between cellulose chains makes the polymers 
more rigid, inhibiting the flexing of the molecules that must occur in the hydrolytic 
breaking of the glycosidic linkages. The free hydroxyl groups present in the cellulose 
macromolecule are likely to be involved in a number of intra and inter molecular 
hydrogen bonds which may give rise to various ordered crystalline arrangements 
(Lehninger et al., 2004). Thus, bundles of cellulose molecules are aggregated together to 
form microfibrils? diameter of an individual microfibril is around 2-5 nm (Zhang et al. 
2007), via orderly hydrogen bonds and Van der Waal?s forces leading to highly ordered 
(crystalline) regions alternate with less ordered (amorphous) regions resulting in low 
accessibility to enzymes. Microfibrils build up fibrils and, finally, cellulose fibers 
(Sj?str?m, 1993; Zhang et al. 2007). Crystalline cellulose microfibrils are embedded in a 
matrix of hemicellulose, pectin and a variety of proteins (Mansfield and Saddler, 2003). 
Unlike starch, which when ingested in the diet is easily hydrolyzed by ?-
amylases, enzymes in saliva and intestinal secretions that break ?-glycosidic bonds 
between glucose units, cellulose cannot be used as a fuel source by most animals, because 
they lack an enzyme to hydrolyze the ?-linkages. Termites readily digest cellulose (and 
therefore wood), but only because their intestinal tract harbors a symbiotic 
microorganism, Trichonympha, that secretes cellulase, which hydrolyzes the ?-linkages.  
2.2.2 Hemicellulose 
Hemicellulose is a highly complex, branched polymer made up of five different 
monomeric sugars attached through different linkages, approximately 23-32% of dry 
 9
substance. It contains five-carbon sugars (usually D-xylose and L-arabinose), six-carbon 
sugars (D-galactose, D-glucose, and D-mannose), acetic acid, and uronic acid. 
Hemicellulose has a lower degree of polymerization (DP) than cellulose (about 200 vs. 
more than 10,000), and its lower limits have not been clearly defined. Hemicellulose can 
be classified as xylans, mannans, galactans etc. based upon the composition and intra-
structural bonding, and these groups can exist separately as single components or 
collectively (Applegarth and Dutton, 1965; Roelofsen, 1959; Jayme and Tio, 1968). 
Agricultural residues, such as wheat straw and sugarcane bagasse, contain large amounts 
of xylan, some arabinan, and only very small amounts of mannan. Hardwood 
hemicellulose mostly contains xylans while softwood hemicellulose contains 
glucomannan. Xylan is a complex polysaccharide composed of a backbone of ?-1,4-
linked xylopyranosyl residues that, depending on the plant source, can be variably 
substituted by side chains of arabinosyl, glucuronosyl, methylglucuronosyl, acetyl, 
feruloyl and p-coumaroyl residues. Typically, 4-O-methylglucuronic acid linked at C-2 at 
every tenth (on average) 1, 4-linked xylose units (Shimizu, 2001), and every seven out of 
ten xylose units contain an acetyl group attached to them (Bailey and Ollis, 1986). 
The mannan can be classified in four subfamilies: linear mannan, glucomannan, 
galactomannan, and galactoglucomannan (Petkowicz et al., 2001). Each of these 
polysaccharides presents a ?-1, 4-linked backbone containing mannose or a combination 
of glucose and mannose residues (Liepman et al., 2007). In addition, the mannan 
backbone can be substituted with side chains of ?-1, 6-linked galactose residues. The 
major softwood hemicellulose is an O-acetyl-galactoglucomannan (15-25% of wood), 
which is beta-1,4-linked mannose and glucose residues in the ratio 3:1 often with alpha-
 10
1,6-galactose as side group (Timell, 1967; Ethier et al.,1998;Meier, H. 1961; Katz, 1965; 
Lindberg et al., 1973). In hardwood a low amount of glucomannan (2-5% of wood) is 
also present, composed of D-glucose and D-mannose, while softwood also contains 
arabinoglucuronoxylan, which is composed of L-arabinose, 4-O-methyl-glucuronic acid, 
and D-xylose in the molar ratio 1.3:2:10 (Sj?str?m, 1993).Mannan also exists in 
galactomannan forms in the seeds of leguminous plants (Handford et al., 2003; 
Buckeridge et al., 2000). 
2.2.3 Lignin 
Lignin, which is a major non carbohydrate in wood and other native plant material 
and comprises 15-25% of the dry weight of the biomass that encrusts the cell walls and 
cements the cells together, is a relatively inexpensive and structurally appealing similar to 
phenolic polymer (Feldman et al., 2002). It is essentially a random, three-dimensional 
network polymer comprised of variously linked phenyl propane units (Sj?str?m, 1993). It 
is amorphous in nature and plays a critical role in giving structural rigidity to hold plant 
fibers together. The carbohydrate polymers are tightly bound to the lignin by hydrogen 
bond and some covalent bonds (Fan et al., 1982). Lignin cannot be separated from 
biomass without degrading it, and the molecular weight of native lignin still remains 
unclear. It is estimated to have as much as 50 million Daltons (Rowell, 2005).  
Lignin also plays an important role in the transport of water and nutrients and 
decreases the permeation of water through the cell walls. Lignin is proven as resistant to 
microbial degradation, playing an important role in a plant?s natural defense (Basaglia et 
al., 1992; Chandler et al., 1980). The lignin in plant cell walls must be separated from 
carbohydrates during biomass conversion to open the protective lignin structure. 
 11
Therefore, a pretreatment is necessary in order to improve the digestibility of the biomass 
for a subsequent enzymatic treatment. The main target of successful pretreatment is to 
diminish the protective linkage between lignin and polysaccharides (lignin?carbohydrate 
complexes, LCC).  
Most of the current industrial processes have lignin residue which is combusted 
for energy since it has a much higher heating value (HHV): 5062 cal/g on oven dry basis 
(Domalski et al., 1987). A kraft paper mill is one such example; the lignin isolated from 
pulp-making process is burned for energy generation and chemical recycling. However, 
its application is limited since the efficiency of lignin as a fuel is very low, producing less 
than 25% as much energy as fuel oil (Feldman et al., 2002). 
The competitiveness of the lignocellulosic ethanol process can be enhanced by 
creating a market for the lignin. Lignin can be used as a precursor for DMSO, vanilla, 
phenol, and ethylene (Eckert et al., 2007; Lora and Glasser, 2002; Reddy and Yang, 
2005). High-quality lignin has been demonstrated to work as a substitute for polymeric 
materials, such as resins, surfactants, and dispersants. The National Renewable Energy 
Laboratory (NREL) has investigated the potential of converting lignin into a hydrocarbon 
that can be used as a high-octane automobile fuel additive (Montague, 2003). Lignin can 
also be used as a carbon source for carbon fibers (Feldman et al., 2002; Kadla et al., 
2002). The use of lignin-based carbon fiber composite materials could dramatically 
reduce fuel consumption by lowering the overall weight of the vehicle, and a study in 
2002 showed that using 10% lignin can produce enough carbon fiber to replace half of 
the steel used in domestic passenger vehicles (Leitten et al., 2002). 
 
 12
2.3.4 Extractives and Ash 
Extractive components mainly include terpenes, phenolic compounds, inorganic 
material, non-structural sugars and nitrogen containing compounds. Terpenes are a large 
and varied class of hydrocarbons, produced primarily by a wide variety of plants. They 
serve as antiherbivore defense compounds in plants. Phenolic compounds are found in 
heartwood which gives dark color to heartwood and make wood resistant to decay, and are 
also the precursor of lignin. Inorganic material may come from any soluble material that is 
associated with the biomass, such as soil or fertilizer. Nitrogen containing compounds 
include alkaloids, phenazines and protein. Ashes are mainly inorganic components such 
as alkali earth carbonate and oxalates, either in structural or extractable form. Structural ash 
is an inorganic material that is bound into the physical structure of the biomass, while 
extractable ash is inorganic material that can be removed by washing or extracting the 
material.  
2.3.5 Acetyl Group 
Acetyl groups are bound through an ester linkage to hemicellulose chains, 
especially xylans, in wood and other plants. A measure of acetyl content is necessary for 
biomass containing hemicellulose with a xylan backbone, but not for biomass containing 
a mannan backbone. Xylan, in native plant cell walls, is extensively acetylated 
(Holtzapple, 1993). Recent studies indicate that removing acetyl groups from xylan 
greatly enhanced the biomass digestibility through increased swellability (Weimer et al., 
1986), thereby increasing the hydrolysis rate (Grohmann et al., 1989; Kong et al., 1992). 
The natural moisture present in plants hydrolyzes the acetyl groups to acetic acid, 
particularly at elevated temperatures. Deacetylation occurs during the pretreatment 
(Grohmann et al., 1989). 
 13
2.3 Bioconversion of Lignocellulosic Biomass 
There are four stages to produce ethanol using a biological approach (Fig. II-1.): 
1. A pretreatment phase, to make the lignocellulosic biomass amenable to 
hydrolysis; 
2. Enzymatic hydrolysis (cellulose hydrolysis) , to break down the molecules 
into sugars; 
3. Microbial fermentation of the sugar solution; 
4. Distillation to produce 99.5% pure alcohol. 
2.3.1 Pretreatment 
The cellulosic microfibrils are embedded in a matrix of lignin and hemicelluloses 
and this arrangement presents a major accessibility problem for cellulase enzymes. 
Therefore, a pretreatment (Fig.II-2.) is necessary in order to improve the digestibility of 
the biomass for a subsequent enzymatic hydrolysis step (Mosier et al., 2005). Many 
different pretreatment technologies have been developed. Of these many pretreatment 
technologies, some are focused on hemicellulose, some are focused on disrupting the 
highly ordered cellulose, and others are focused on disrupting the lignin-carbohydrate 
complex (Dale et al., 1984). Most pretreatments are done through physical, chemical, and 
biological pretreatment. Physical pretreatment technologies do not use the addition of 
chemicals and usually involve ball-milling, irradiation, steaming, and hydrothermolysis 
(Hsu, 1996). Chemical pretreatment technologies have been proposed and investigated, 
e.g. steam treatment with SO
2
, ammonia fiber explosion (AFEX), ammonia-recycled 
percolation(ARP), lime pretreatment, dilute acid pretreatment , and concentrated acid 
pretreatment (Gong et al.,1999). Biological pretreatment technologies use lignin 
 14
degrading microorganisms to improve substrate accessibility to cellulase enzymes 
(Taniguchi et al., 2005). In order to achieve higher efficiency, some processes are a 
combination of two or more pretreatment methods (McMillan, 1994). The economic and 
environmental concerns, however, limit the applicability of most of known methods. 
2.3.1.1 Biological Pretreatment 
Although lignin is proven to be resistant to microbial degradation and therefore 
plays an important function in a plant?s natural defense (Basaglia et al., 1992; Chandler et 
al., 1980), some microorganisms, particularly white-rot fungi and soft-rot fungi, can 
release the necessary enzymes, such as lignin peroxidases during secondary metabolism, 
to break lignin down (Kirk and Farrell, 1987). White-rot fungi are one of the most 
promising types of organisms for the biological pretreatment (Kirk, 1975). Biological 
pretreatments require low energy input because of the mild conditions of pretreatment, 
but its low rate in comparison to the chemical treatments has prevented its usage in 
commercial scale plants.  
2.3.1.2 Physical Pretreatment 
There are two kinds of physical pretreatment, mechanical pretreatment and non-
mechanical pretreatment. Mechanical pretreatment includes different modes of milling 
(ball, two-roll, compression, dry, wet and vibratory milling), chipping, and grinding to 
reduce the particle size and increase the surface area of the biomass (Caufield et al., 
1974; Matsumura et al., 1977; Puri, 1984). It is believed that these methods not only 
reduces their particle size, but also decrystallizes lignocelluloses. There is some debate 
about whether the benefits derived from the milling generally result from lower 
crystallinity or actually from the smaller particle size (Chang and Holtzapple, 2000). 
 15
Mechanical pretreatment is energy- intensive and capital-intensive, and hence 
unattractive on a commercial scale. The non-mechanical pretreatment options include 
irradiation, pyrolysis, steaming, and microwave treatment (Mandels et al., 1974; 
Beardmore et al., 1980; Mohan et al., 2006). These methods also require considerable 
energy and have a high operating cost.  
2.3.1.3 Chemical Pretreatment 
Most of the chemical pretreatments may need a prior step involving chipping and 
milling. Different chemical treatments have been used over the past few decades for 
removing lignin or hemicellulose, destroying the cellulose crystalline structure, and 
increasing the pore size and surface area, and include steam explosion pretreatment, 
steam explosion with gas or acid, acid pretreatment, alkaline treatment, AFEX 
pretreatment, the organosolv processes, liquid hot water pretreatment (LHW), oxidizing 
reagent pretreatment, cellulose solvent pretreatment, and supercritical fluids etc. 
Steam explosion: Steam explosion has been one of the most thoroughly 
investigated pretreatment technologies involving treating different biomass resources 
(Boussaid et al., 2000; Pan et al., 2004; Yang et al., 2002). In steam explosion the 
feedstock is subjected to high steam pressure (usually at 200?450 psig) and high 
temperatures (usually 150-270?C) for retention times of 20 seconds ?10 minutes, 
followed by suddenly discharging the product to atmospheric pressure. Treated biomass 
with the high pressure and high temperature of the process causes hydrolysis of acetyl 
groups of hemicellulose which release acetic acid, and therefore cause the autohydrolysis 
of hemicellulose. The typical steam explosion pretreatment fractionates feedstock into 
different components. Most of the cellulose and lignin remain in the solid part after 
 16
pretreatment and most of the hemicelluloses, low molecular weight lignin decomposed 
by-product, and extractives are solubilized in the liquid part, thereby leading to much 
higher content lignin in the solid part than that of untreated feedstock. Lignin phenolic 
groups content increases after pretreatment and lignin condensation is observed indicate 
lignin structure is changed (Shevchenko et al., 2001b).  
Depending on different lignocellulosic material, especially for softwood, and 
modifying pretreatment efficiency, by adding SO
2
, CO
2
 and sulfuric acid during steam 
explosion treatment can facilitate the degradation of biomass and improve the enzymatic 
digestibility of biomass (Schell et al., 1998; Zheng et al., 1998; Robinson et al., 2002; 
Shahbazi, et al., 2005; Tucker et al., 2003; Varga et al., 2004). SO
2
 forms sulfuric acid 
and CO
2 
forms carbonic acid, which therefore make the pretreatment environment more 
acidic. Since SO
2
 is very toxic, this can lead to safety and environmental problems. 
Carbon dioxide becomes somewhat attractive because it is a byproduct of ethanol 
fermentation process. Grous et al found the enzymatic digestibility of steam exploded 
poplar to be as high as 90% conversion in 24 hr (Grous et al., 1986). However, in the 
mean time, it also generates different inhibitory compounds for further enzymatic 
hydrolysis and fermentation steps due to autohydrolysis of lignin and hemicellulose 
degradation. Adding acid or SO
2
 improves pretreatment efficiency, but also leads to 
expensive corrosion and high pressure proof equipment, safety issues and environmental 
disposal problems. 
Acid pretreatment: Acid pretreatment has been investigated for a long time 
because of its high pretreatment efficiency with most feedstocks (Grethlein et al., 1984; 
Schell et al., 1992; Nguyen et al., 1998; Schell et al., 2003). Acids used in this 
 17
pretreatment are usually sulfuric acid, hydrochloric acid, and phosphoric acid (Allen et 
al., 2001; Kalman et al., 2002; Saha et al., 2005a; Saha et al., 2005b; Kim and Mazza, 
2008). Usually, sulfuric acid is used because of its low cost. In diluted form (0.1% to 
0.7% sulfuric acid as catalyst), it generally performs at relatively high temperatures (100-
240?C) for a few seconds to 60 minutes depending on temperature. Generally, diluted 
acid pretreatment process, similar to steam explosion with an acid catalyst, solubilizes the 
hemicellulose component of biomass into liquid stream, while leaving most of the 
cellulose fraction intact in the solid part. Both pretreatments remove the lignin-
hemicellulose physical protection and improve the remaining cellulose susceptibility to 
enzymes (Grethlein et al., 1991). Enzymatic digestibility of remaining cellulose is 
improved but total sugar yield from diluted acid pretreatment is low due to hemicellulose 
degradation. To maximize the sugar yields from the hemicellulose and cellulose fractions 
of biomass, a two-stage acid hydrolysis process has been developed (Nguyen et al., 
2000). The first stage is performed under relatively milder conditions to hydrolyze 
hemicellulose, while the second stage is optimized to hydrolyze the more resistant 
cellulose fraction under higher temperatures. The disadvantages of the dilute acid process 
include high investment in equipment associated with acid corrosion, low sugar 
concentration in liquid stream, and high content inhibitors which makes fermentation 
very difficult. Since diluted acid pretreatment at high temperature produces large 
quantities of degradation byproducts and undesirable inhibitory compounds, the 
concentrated acid pretreatment is employed at temperatures which could be as low as 30 
?C at near theoretical yields with little degradation. Arkenol and Masada Resource Group 
plan to use this process in their bioethanol plants (http://www.arkenol.com). The 
 18
hydrogen bonding between cellulose chains is disrupted during the concentrated acid 
pretreatment, and cellulose becomes completely amorphous. The amorphous cellulose is 
extremely susceptible to hydrolysis. Thus, dilution with water at modest temperatures 
provides complete and rapid hydrolysis to glucose, with little degradation. Minimizing 
the use of sulfuric acid and recycling the acid cost-effectively are critical factors in the 
economic feasibility of the process. 
Alkaline treatment: Alkali, such as calcium hydroxide (lime), sodium hydroxide 
and ammonia, has been employed as a pretreatment method for many years (Tarkow and 
Feist, 1969; Chang et al., 2001; Kaar and Holtzapple, 2000; Kim and Holtzapple, 2005; 
Kim and Lee, 2005a and 2005b; Iyer et al., 1996; Kim et al., 2000; Kim and Lee, 1996). 
Alkaline pretreatment causes a swelling effect on the biomass, which is an intra 
crystalline swelling action penetrating both the amorphous and crystalline structure of 
cellulose and resulting in irreversible change in the structure of cellulose. This is 
accompanied by an inter-crystalline swelling action with water, which is a reversible 
change of dehydration of the substrate and has an effect only on the amorphous phase 
(Mishra, 2000). During alkaline pretreatment, the lignin is degraded predominantly by 
cleavage of lignin-hemicellulose bonds, and delignification brings about changes in the 
structure of cellulose, whose DP and crystallinity decrease and accessible surface area 
increases, thus making the biomass more susceptible to enzymatic hydrolysis. A certain 
amount of alkali is required to combine with the acetyl and carbonyl content of biomass 
and around 10-50% of the alkali is consumed during the pretreatment, which is a 
significant economic factor (Wenzl, 1970). 
 19
Among the alkaline reagents, calcium hydroxide seems a good choice considering 
the cost of the chemical, process temperatures of 25-100 
0
C for 1 day to 2 weeks, but it is 
difficult to recover (Hsu, 1996). The cost of sodium hydroxide is relatively high, but the 
recovery has been well documented in the pulp industry for a long time. Aqueous 
ammonia has also been used as a good choice for pretreatment, due to its relatively low 
cost, about one-fourth the cost of sulfuric acid on a molar basis, and its high volatility 
which makes it easy to recover and reuse. Also, ammonia is not severely corrosive, 
unlike sulfuric acid and hydrochloric acid. The ammonolysis cleaves the C-O-C bonds in 
lignin and the ether and ester bonds in the lignin-carbohydrate complex. Two processes, 
based on aqueous ammonia, ammonia recycle percolation (ARP) and soaking in aqueous 
ammonia (SAA), were used by Gupta et al. to improve the enzymatic digestibility of corn 
stover and hybrid poplar wood close to the quantitative maximum. Both processes have 
the high selectivity for lignin reaction and high retention of hemicellulose (Kim, T.H. and 
Lee, Y.Y., 2005a and 2005b; Iyer et al., 1996; Gupta, 2008). In the ARP process, the 
ammonia is continuously fed and withdrawn from a flow-through (percolation) reactor. 
The lignin and other extraneous components are cleanly separated from the biomass 
structure, which prevents recondensation of lignin in the biomass. The SAA is a batch 
process used at a relatively low severity conditions. Because of low severity conditions, 
longer treatment time is required. At a typical condition of 15% NH
3
OH and 60
o 
C, which 
gives a system pressure of about 1 atm (less than 30 psia), a reaction time of several 
hours to one day is required to achieve acceptable level of pretreatment effects. Because 
of low process energy and low equipment cost, the overall processing cost of SAA is 
substantially lower than ARP.  
 20
The Ammonia Fiber Expansion (AFEX) process was developed by Bruce Dale et 
al. in the early 1980?s, where biomass is soaked with liquid anhydrous ammonia at 
temperatures (60 ? 160
o 
C) and at pressures (250-1000 psi) from 5 to 30 min (Dale and 
Moreira, 1982; Wyman et al., 2005; Teymouri et al., 2005). Then, the pressure is released 
suddenly (explosion) resulting in a physical action on the substrate thus facilitating its 
disintegration of the biomass. AFEX decreases the crystallinity of cellulose (Gollapalli et 
al., 2002), retains most of hemicellulose since there is little water present in the process, 
disrupts the hemicellulose-lignin links by ammonolysis, and modifies the structure of 
lignin, therefore reducing non-productive binding between cellulase and lignin (Martinez 
et al., 1991). After the treatment, most of the ammonia can be recovered for reuse and the 
retained ammonia in the biomass could be used as a nitrogen source in the following 
bioconversion (Dale et al., 1985). However, within the reaction conditions of temperature 
(60?C-180?C),  the upper limit of the ammonia pressure is around 450 psia, which is 
relatively higher than a normal pulp mill digester pressure. 
Ammonia works well for feedstocks of low lignin content, especially agricultural 
residues of annual plants such as corn stover, sugarcane bagasse, and wheat straw, but not 
for wood species, especially softwood (Iyer et al., 1996). 
Organosolv Processes:A lot of organic solvents, mainly alcohol, were originally 
used to extract lignin from wood with high purity in order to study lignin and 
carbohydrates (Kleinert, 1974). Organosolv pretreatment breaks down bonds such as ?-
aryl ether and aryl glycerol-?-aryl ether in the lignin-carbohydrate complex (Kleinert, 
1974; Sarkanen et al., 1981), bringing significant changes in the lignin structure and 
decreasing the average molecular weight (Gilarranz et al., 2000). The breakdown 
 21
products, like solubilized lignin, are dissolved in the solvent and can be subsequently 
used to produce valuable coproducts. The organosolv pretreatment is helpful to produce 
substrates highly susceptible to enzyme and therefore increase the enzymatic digestibility 
due to delignification (Pan et al., 2005).  The acid or base catalyst is added into an 
aqueous organic solvent to reduce the reaction time and temperature (Chum et al., 1988; 
Chum et al., 1990; Thring et al., 1990). Based on organosolv processes, Zhang et al 
developed a new cellulose and organic-solvent-based lignocelluloses fractionation 
(COSLIF) technology to separate lignocellulosic biomass into amorphous cellulose, 
hemicellulose, and lignin under modest reaction conditions (atmospheric pressure and 
~50
o 
C) (Zhang et al. 2007).  
However, organosolv processes are still in a developing stage and are not yet 
ready to be commercialized due to the high cost of solvent recovery. Recycling of the 
solvent is important for reduction of cost due to high cost of solvents. Removal of the 
solvent from the pretreated biomass is also important since the solvent could be 
inhibitory to enzymes and microorganisms.  
Liquid hot water pretreatment: Water under high pressure penetrates the biomass 
for up to 15 min at 200?230 
0
C (Mosier et al., 2005). Liquid hot water pretreatment 
(LHW) is also called autohydrolysis, because the hydronium ion from water ionization 
causes the cleavage of the acetyl group under high temperature, which forms the acetic 
acid catalyzing autohydrolysis of hemicellulose (Casebier et al., 1969; Lora and 
Wayman, 1978). Acetic acid catalyzes the hydrolysis of glycosidic linkages in 
hemicellulose and the beta-ether linkages in lignin resulting in the formation of different 
phenolic acids from lignin and some organic acids, such as formic acid and glucuronic 
 22
acid from hemicellulose degradation. Like steam explosion, LHW pretreatment keeps 
most of cellulose and lignin in the solid part after pretreatment and solubilizes most 
hemicelluloses, low molecular weight lignin decomposed by-product and extractives into 
the liquid part. But unlike steam explosion, there is no addition of chemicals in this 
process, and therefore, formation of inhibitory components is much lower and the 
requirement for corrosion-resistant materials is also much lower. In order to minimize 
hydrolysis to monosaccharides, control of pH during pretreatment is employed by adding 
of alkali to maintain the pH constant above 5 and below 7 (Weil et al., 1998). It also 
appears to improve enzymatic digestibility of cellulose, decrease in cellulose crystallinity, 
and lower association of cellulose with lignin (Ladisch et al., 1983; Ladisch et al., 1989; 
Lin et al., 1985). 
Cellulose solvent pretreatment: Cadoxen, an alkali solution of CdO in aqueous 
ethylenediamine was utilized as a cellulose solvent (Ladisch et al., 1978; Tsao, 1978). 
Once cellulose is dissolved, not only is the cellulose structure decrystalized by 
dissolution and subsequent regeneration to a highly active amorphous cellulose form, but 
lignin the protective shell is also disrupted. However, Cadoxen is very aggressive and 
time-consuming to prepare. The cost of these solvents and their toxicity may prohibit 
their commercial application.  
Recently, an ionic liquid for pretreatment of cellulosic biomass was developed as 
a promising green solvent for biomass fractionation. Ionic liquids are nonflammable and 
recyclable cellulose solvents with very low volatility and high thermal stability. 
Carbohydrates and lignin can be simultaneously dissolved in ionic liquids with anion 
activity (e.g. the 1-butyl-3-methylimidazolium cation in the 1-butyl-3-methylimidazolium 
 23
chloride (BMIMCl) and 1-allyl-3-methylimidazolium cation in the 1-allyl-3-
methylimidazolium chloride (AMIMCl)) (Dadi et al., 2006; Reichert et al., 2001). 
However, most data showing the effectiveness of ionic liquids has been developed using 
pure crystalline cellulose, and its applicability to the more complex lignicellulosic 
biomass, toxicity toward enzymes and microorganisms, and recovery cost is unknown.  
Oxidizing reagent: Oxidizing reagents, such as oxygen (McGinnis et al.,1983), 
ozone (Ben-Ghedalia and Miron, 1981;Ben-Ghedalia and Shefet, 1983 Neely, 1984), 
hydrogen peroxide (Wei and Cheng, 1985; Azzam et al., 1989; Bjerre et al., 1996; Kim et 
al., 2000), peracetic acid (Teixeira et al., 2000; Zhao et al.,2007), and chlorine dioxide 
(Sullivan and Hershberger, 1959) can modify the structure of lignin?carbohydrate 
complexes, and therefore enhance the enzymatic digestibility of pretreated biomass. 
Azzam et al. found the enzymatic digestibility of the H
2
O
2
 pretreated cane bagasse to be 
95%
,
 but a significant amount of hemicellulose was lost (Azzam et al., 1989). However, 
in the presence of a reducing agent, high concentrations of H
2
O
2
 will react violently, and 
hydrogen peroxide in high concentrations is an aggressive oxidizer and will corrode 
many materials, including human skin. Chlorine dioxide is very toxic. Ozone was found 
to have high selectivity for lignin removal with slight effect on hemicellulose, low 
treatment temperature/pressure and the absence of fermentation inhibitors after 
pretreatment. However, ozone, like H
2
O
2
 and chloride dioxide, is expensive and cannot 
be recovered (Vidal and Molinier, 1988). In addition, it is usually more difficult to handle 
and store a gas, like oxygen and ozone, than a liquid. 
Supercritical fluids pretreatment: A supercritical fluid is any substance at a 
temperature and pressure above its critical point. It behaves as neither a fluid nor a gas, 
 24
but has some of the properties of each. It can diffuse through solids like a gas, and 
dissolve materials like a liquid. Supercritical fluids are suitable as a substitute for organic 
solvents in a range of industrial and laboratory processes. Carbon dioxide, sulfur dioxide 
and ammonia have been used to treat lignocellulosic biomass. Carbon dioxide becomes 
more attractive because it is a byproduct from ethanol fermentation process. Shah et al 
treated hardwood with supercritical carbon dioxide-sulfur dioxide mixtures to make 
acetone, butanol, and ethanol (ABE) (Shah et al., 1991). Zheng et al. (Zheng et al., 1998) 
used supercritical carbon dioxide to treat Avicel, recycled paper mix, sugarcane bagasse 
and the repulping waste of recycled paper, and then suddenly released the carbon dioxide 
pressure to increase the accessible surface area of the cellulosic substrate to enzymatic 
hydrolysis. However, this technology raises serious questions about equipment costs due 
to high pressure.  
2.3.2 Enzymatic Hydrolysis of Lignocellulosic Biomass 
The enzymatic hydrolysis of cellulose into glucose is a slow and complex process 
because of the physical nature of the substrate. Cellulose in its native form has a highly 
crystalline structure. In addition, the cellulose is embedded in a matrix of lignin and 
hemicellulose, where the number of active enzyme binding sites available is limited. The 
factors that affect the enzymatic hydrolysis of lignocellulosic biomass include cellulase 
property, substrates, and reaction conditions (temperature, pH, etc.). 
2.3.2.1 Cellulase  
Cellulases are an important class of industrial enzymes for various industries, 
such as textile and paper industries, outside the biorefinery area. Cellulase enzymes are 
found in bacteria, some of which are in the digestive tracts of herbivores through 
 25
symbiotic microbes. Fungi and termites also contain the enzymes and are largely 
responsible for the degradation of decaying trees and plants. Both fungi and bacteria can 
produce cellulases, including endo- and exoglucanases, for the hydrolysis of 
lignocellulosics (Tomme et al., 1995).  
Of all fungal genera, including P. chrysosporium and species of Trichoderma, 
Aspergillus, and Penicillium (Lynd et al., 2002), Trichoderma has been most extensively 
studied for cellulase production. Cellulase enzymes from Trichoderma reesei, the most 
frequently studied cellulolytic fungus, are composed of three components - exo-1,4-?-D-
glucanases or cellobiohydrolases or exocellulase, microcrystalline cellulase, avicelase 
(CBHs, EC 3.2.1.91), endo-1,4-?-D-glucanases or endoglucanase, endocellulase, 
CMCase (EGs, EC 3.2.1.4) and ?- glucosidases or cellobiase (?Gs, EC 3.2.1.21).  
In the presence of cellulose, T. reesei secretes CBH I (~60%), CBH II (~15%), a 
number of EGs (~20%), and over 30 other minor proteins (Gritzali et al., 1979). EG I is 
reported to represent 5 to 10 % of the secreted protein, the major endoglucanase (Penttil? 
et al.,1987; Nidetzky and Claeyssens,1994), but EG II is reported to account for most of 
the endoglucanase activity produced by T. reesei (Suominen et al.,1993).Vinzant et al. 
identified five endo-glucanases (EG I/Cel 7B, EG II/Cel 5A, EG III/Cel 12A, EG IV/Cel 
61A,
 
and EG V/Cel 45A), two exo-glucanases (CBH I/Cel 7A, CBH II/Cel 6A), two 
xylanases (XYN I and XYN II), a ?-D-glucosidase, an ?-L-arbinofluranosidase, an acetyl 
xylan esterase, a ?-mannanase, and an ?-glucuronidase by 2-D electrophoresis in the 
cellulase complex of T.reesei (Vinzant et al.,2001).  
The endoglucanases have two distinct structural domains, the catalytic (tadpole 
head) and binding (tail) regions, which are separated by a ?linker? domain that is usually 
 26
glycosylated. Glycosylation is thought to protect the enzyme from proteolytic attack and 
improves the thermal stability of the protein. It also helps in the adsorption of 
endoglucanases into the insoluble cellulose (Langsford et al., 1987; Merivuori et al., 
1985; Olden et al., 1985). Endoglucanases cleave glycosidic bonds within cellulose 
microfibrils, acting preferentially at amorphous cellulose regions. EGs fragment cellulose 
chains to generate reactive ends for CBHs, which act ?processively? to degrade cellulose, 
including crystalline cellulose, from either the reducing (CBH I) or non-reducing (CBH 
II) ends, to generate mainly small oligosaccharides and cellobiose. ?-glucosidase 
hydrolyzes small oligosaccharides and cellobiose to glucose (Fig.II-3.).  
It was found that enzyme preparations containing only endocellulases have little 
effect on native cellulose. On the other hand, those containing both endo and 
exocellulases will cause significant degradation of native cellulose. Thus, the endo and 
exocellulases appear to work in a synergistic or cooperative manner on native high-
molecular-size cellulose. According to a recent study, CBHII of T. reesei had no 
detectable effect on the DP of cotton cellulose and caused only a 3% solubilization; 
however, it rapidly solubilized the lower DP cellulose and acted synergistically with EGI 
to solubilize cotton (Kleman-Leyer et al., 1996). At high concentrations, cellobiose 
inhibits CBH activity. Thus ?-glucosidase, which converts cellobiose into glucose, is 
often required for optimal cellulose performance in conditions where cellobiose 
accumulates, as it relieves end-product inhibition. T. reesei cellulase preparations,
 
supplemented with Aspergillus ?-glucosidase, are used
 
most often for cellulose 
saccharification on an industrial scale
 
(Sternberg et al., 1977). T.
 
reesei produces ?-
glucosidases at low levels compared
 
to other fungi such as Aspergillus species (Reczey et 
 27
al., 1998) and are not sufficient for extensive in
 
vitro saccharification of cellulose, though 
the levels of T. reesei ?-glucosidase are presumably sufficient
 
for growth on cellulose. In 
addition,
 
the ?-glucosidases of T. reesei are subject to product
 
(glucose) inhibition (Chen 
et al., 1992; Gong et al., 1977), while those of Aspergillus
 
species are more glucose 
tolerant (Gunata and Vallier, 1999; Watanabe et al., 1992; Yan and Lin., 1997). 
2.3.2.2 Addition of Surfactants  
Enzymatic hydrolysis of cellulose consists of three steps: adsorption of cellulase 
enzymes onto the surface of the cellulose which takes place at cellulose?liquid interface, 
the biodegradation of cellulose to water soluble sugars, and the desorption of cellulase. 
There are many studies on fungal cellulases showing that, once adsorbed on to the 
surface, desorption of the enzyme is not easy (Otter et al., 1989; Palonen et al., 1999; 
Nidetzky et al., 1994). Many researchers found that the rate of cellulose hydrolysis 
decreases dramatically as the hydrolysis proceeds, called rate retardation (Desai and 
Converse, 1997; Eriksson et al., 2002a). It has been suggested that the rate of hydrolysis 
is proportional to the amount of adsorbed enzymes (Sattler et al., 1989; Sattler et al., 
1988). A major obstacle in the enzymatic conversion of lignocellulose is the adsorption 
of significant amounts of enzyme on exposed lignin surfaces due to unspecific binding of 
cellulases by hydrophobic interactions with lignin. Palonen et al. showed that significant 
amounts of CBH I and EG II could bind to alkali and enzymatic lignin (Palonen et al., 
2004). Surfactant adsorption to lignin prevents unproductive binding of enzymes to lignin 
(Eriksson et al., 2002b). The surfactants used in the enzymatic hydrolysis include 
nonionic surfactant, such as, Tween 20, 80 (Wu and Ju, 1998), polyoxyethylene glycol 
(Park et al.,1992), anionic surfactant, such as, sodium dodecylsulphate (Eriksson et al., 
 28
2002b), cationic Q-86W (Ooshima et al.,1986), sophorolipid, rhamnolipid, and bacitracin 
(Helle et al., 1993). Inhibitory effects have been observed with cationic Q-86W at high 
concentration and anionic surfactant Neopelex F-25 (Ooshima et al., 1986). Nonionic 
surfactants are believed to be more effective for enhancing the cellulose hydrolysis. One 
of the main surfactants that have been investigated for its potential to improve hydrolysis 
is the Tween series (Polysorbate series). The hydrolysis yield of newspaper was increased 
from 41% to 55% by the addition of Tween 80 (Castanon and Wilke, 1981). Ooshima et 
al later showed that Tween 20 enhanced the enzymatic hydrolysis of Avicel from 33% to 
45% within 72 hr (Ooshima et al., 1986).  
Three explanations have been presented the effectiveness of surfactants such as 
Tween in improving hydrolysis, including, 1) decreasing the adsorption of cellulases to 
substrates (Ooshima et al. 1986), 2) increasing the desorption of enzymes from their 
binding sites (Park et al. 1992), and 3) increasing enzyme stability and prevent 
denaturation of enzymes during hydrolysis  (Kaar and Holtzapple 1998).  
Eriksson et al. found that addition of non-ionic and anionic surfactants to a 
hydrolysis mixture of lignocellulose resulted in a decrease in Cel 7A adsorption to the 
residual substrate, and, regardless of the addition of Tween 20, cellulase Cel 7A (CBH I) 
retained 100% of its activity during 96 hr of incubation (Eriksson et al., 2002b). 
Therefore, it was unlikely that surfactants function as stabilizers during hydrolysis.  
Not only surfactants but polymers with various amount of ethylene oxide (EO) 
content, such as poly (ethylene glycol) (PEG), can also bind to lignin by hydrophobic 
interaction and hydrogen bonding and can reduce the unproductive binding of enzymes. 
J. B?rjesson et al found that addition of PEG to enzyme hydrolysis of lignocellulose 
 29
increased the conversion from 42% to 78% in 16 hr, and decreased desorption of Cel 7A 
decreased from 81 to 59% (B?rjesson et al., 2007).  
2.3.2.3 Hemicellulase  
Xylanases and mannanases are currently used in the pulp and paper industry 
where xylanase or mannanase pretreatment facilitates chemical bleaching of pulps 
(Viikari et al., 1994; Bajpai, 2004; Montiel et al., 1999; Bhat et al., 2000). Xylanases do 
not remove lignin-based chromophores directly but instead remove the xylan network 
that interacts with the residual lignin. Similar to this mechanism, xylanases do not work 
in the cellulose but instead remove the covering of xylan from the cellulose microfibrils 
by catalyzing the hydrolysis of the xylan backbone, and therefore increase the access of 
cellulolytic enzymes to cellulose. The effective enzymatic release of D-xylose from xylan 
requires a large variety of hemicellulases (Biely, 1985): endo-1,4-?-xylanase (EC 3.2.1.8) 
(or xylanases), which cleaves bonds within the xylose backbone and produces xylobiose 
and xylooligomers, xylan 1,4-?-xylosidase (EC 3.2.1.37) (or ?-xylosidase) which release 
xylose units from xylobiose and xylooligomers, and side-chain-splitting enzymes such as 
?-L-arabinofuranosidases (EC 3.2.1.55), ??D-glucuronidases (EC 3.2.1), acetyl xylan 
esterases (EC 3.1.1.6), hydroxycinnamic acid esterases (ferulic acid esterases EC 3.1.1.73 
and p-coumaric acid esterases) (Fig.II-4.). 
Like the cellulolytic enzyme, synergistic action was also observed among the 
xylanolytic enzymes, but the synergy is more complex in the cellulolytic enzyme. 
Different components of xylanases involving the main-chain and side-chain hydrolytic 
enzymes work synergistically with each other, and this synergy can be defined as 
homosynergy or heterosynergy (Coughlan et al., 1993). Acetyl groups can hinder the 
 30
approach of enzymes that cleave the xylan backbone; hence the removal of these 
substituents by acetyl xylan esterases facilitates the action of xylanases. McCrae et al 
found that the feruloyl and p-coumaryl esterases aided the release of xylan (McCrae et 
al., 1994). Similarly, xylanases greatly enhance the release of arabinose from 
heteroxylans by the ?-L-arabinofuranosidases (Filho et al., 1993). It was also found that 
the mixture of xylanase and ?-xylosidase was only effective in hydrolyzing the acetyl 
xylan in the presence of acetylxylan esterase (Clarke, 1997; Biely, 1985). Tenkanen et al. 
indicated that unlike cellulases, the endoxylanases of fungi do not bind strongly to xylan, 
mannan, or cellulose (Tenkanen, et al., 1995a). Interestingly, some bacterial 
endoxylanases possess a cellulose-binding domain (CBD), the function of which is 
unknown; the binding domain does not bind to xylan, nor does it enhance xylan 
hydrolysis (Coughlan et al., 1993). 
The enzymatic hydrolysis of galacto(gluco)mannans, present particularly in 
softwoods, is accomplished through the action of endo-?-1,4-mannanase (?-mannanase 
EC 3.2.1.78) which, together with the exo ?-glucosidases (EC 3.2.1.21), randomly cleave 
the beta-mannosidic linkages within the main chain. Additional enzymes, mannan-1, 4-?-
mannosidases (EC 3.2.1.25) and ?-1, 6-galactosidases (EC 3.2.1.22), ?-glucosidases (EC 
3.2.1.21), and acetyl mannan esterases (EC 3.1.1.6) are needed to cleave the side chain, 
which create more sites for subsequent enzymic hydrolysis (Filho, 1998) (Fig.II-5.). 
Similar to the cellulolytic enzyme and xylanolytic enzymes, synergistic action 
also happens in the mannan structure by a variety of main- and side-chain-cleaving 
enzymes. G?bitz et al. found an increased production of monomers from the 
galactomannan by the homeosynergy between ?-mannosidase and two ?-mannanases 
 31
from Sclerotium rolfsii (G?bitz et al., 1996). The galactose release from softwood pulp is 
enhanced by the presence of mannanase in combination with alpha-galactosidase (Clarke 
et al., 2000).  
Since hemicelluloses restrict the access of cellulolytic enzymes by coating 
cellulose fibers, enzyme mixtures supplementation of so-called ??accessory?? enzymes - 
hemicellulase with similar cellulase activity could show differences in performance on 
lignocellulosic biomass if they differ in hemicellulase composition (Berlin et al.,2005a; 
Berlin et al.,2005b; Berlin et al.,2006).  
2.3.2.4 Substrates 
Many researchers have addressed issues concerning how the structural features of 
the substrate including lignin content, hemicellulose content, biomass crystallinity and 
accessible surface area and substrate concentration affect the enzymatic hydrolysis. 
Structural features of the substrate: Chang et al observed that lignin can interfere 
with hydrolysis if it irreversibly binds cellulase or ?-glucosidase (Chang et al., 1981). 
Ooshima et al. observe that cellulase enzymes bind to lignocellulose residue (Ooshima et 
al., 1990). Tarkow et al. observed that the location and nature of carbohydrate-lignin 
bond also strongly influence digestibility (Tarkow and Feist, 1969). Delignification 
causes biomass swelling and modification of the lignin structure, therefore an increase 
the internal surface area and median pore volume (Fan et al., 1982), which leads to a 
reduction of irreversible adsorption of enzyme on lignin (Ooshima et al., 1990) and an 
increase of enzyme accessibility to cellulose (Meunier-Goddik and Penner, 1999; 
Mooney et al., 1998). Only 20-65% of the lignin need be removed to increase the 
susceptibility of enzymatic degradation depending on the sources of cellulose (Fan et al., 
 32
1982). However, because most delignification techniques result in significant 
solubilization of hemicellulose, the delignification may not be the only factor that 
attributed digestibility increase (Wyman, 1996). 
Dilute acid pretreatment studies (Grohmann et al., 1985 and 1986) showed that 
the removal of hemicellulose improves cellulose digestibility despite the high lignin 
content. Even though the results of Burns et al. and Converse et al. suggested that 
hemicellulose removal increases porosity and the specific surface areas accessible to 
cellulase (Converse et al., 1990; Burns et al., 1989), Torget et al reported that lignin 
subjected to high-temperature acid treatments was chemically modified and recondensed 
as an altered lignin polymer, which may play a less detrimental role in digestibility 
(Torget et al., 1991).  
Another factor that may affect enzymatic hydrolysis is biomass crystallinity. 
Dunlap et al discovered an inverse linear relationship between the crystallinity index 
(CrI) and digestibility of natural cellulose (Dunlap et al., 1976). Reese et al. found that 
the cellulose becomes more crystalline with a decrease of the highly accessible 
amorphous portion. They found an increase in crystallinity during hydrolysis of cellulose 
and enzymes (Reese et al.1950). Thus, it is important for complete hydrolysis of cellulose 
to open up the crystalline structure of the cellulose and decrease the resistance to further 
hydrolysis, i.e., the interaction between cellulose chains (Sasaki et al., 1979).  
The CrI is largely influenced by the biomass type and composition. For 
lignocellulosic biomass, the crystallinity index measures the relative amount of 
crystalline cellulose in the total biomass. Many investigations have shown that 
crystallinity correlates inversely with digestibility, but increased CrI after biomass 
 33
pretreatment has been observed in many studies (Chang and Holtzapple, 2000; Kasahara 
et al., 2001; Tanahashi et al., 1983). Caulfield et al found that increased accessible 
surface and decreased particle size rather than crystallinity affect the rate and extent of 
the hydrolysis (Caulfield and Moore, 1974.). It appears likely that the percentage of 
crystallinity is therefore not the main determinant of the enzymatic degradation of 
cellulose (Puri, 1984; and Sawada et al., 1988).  
The accessible surface area of lignocellulose is another key factor that determines 
susceptibility to enzymatic degradation. It is important because contact between enzyme 
molecules and the cellulose surface is required for hydrolysis to proceed. Cellulosic 
particles have both external and internal surfaces, and the external surface is thought to 
be a small fraction of overall surface area for most substrates. Knappert et al. found that 
changes in the internal area are primarily due to changes in the pore size region, and 
hence the increase in accessibility for the enzyme would be significant (Knappert et al., 
1980). But Burns et al. (1989) found that the surface area of pores which are too small to 
be accessible to the enzyme decreases more slowly, presumably because the substrate 
containing these small pores reacts only at the external surface. Thompson et al. conclude 
that the surface area of the substrate in relation to the size of the enzymes is the main 
limiting factor in the enzymatic hydrolysis of lignocellulosic biomass (Thompson et al., 
1992).  
Substrate Concentration: There is a tradeoff between achieving high glucose 
concentrations and achieving high levels and rates of cellulose conversion. This is likely 
due to product inhibition, though non-productive enzyme binding could also play a slight 
 34
role. Varga et al. (Varga et al., 2002) have reported that product inhibition starts to occur 
around 15 g/L glucose. 
It is believed that substrate inhibition is not present at a low substrate 
concentration, and Elshafei et al. (Elshafei et al., 1991) mention that substrate loading 
between 0.5-5% total solids has only a minimal effect on cellulose conversion, though 
there is a substantial effect on hemicellulose conversion for alkaline treated corn stover 
samples. Huang et al. (Huang and Penner, 1991) found that the substrate inhibition 
occurred when the ratio of the microcrystalline substrate Avicel PH 101 to the cellulase 
from Trichoderma reesei (grams of cellulose/FPU of enzyme. FPU is filter paper unit, 
defined as a micromole of reducing sugar as glucose produced by 1 ml of enzyme per 
minute.) was greater than 5. Penner et al. found that the optimum substrate to enzyme 
ratio was 1.25 g of the microcrystalline substrate Avicel per FPU of the cellulase from T. 
reesei (Penner and Liaw, 1994).  
2.3.3 Fermentation 
Fermentation is the phase of the bioconversion process which converts sugars to 
ethanol and carbon dioxide or other valuable products such as numerous organic acids by 
following enzymatic hydrolysis of lignocellulosic biomass. The fermentation reaction is 
caused by microorganism-yeast or bacteria, which feed on the sugars. Three approaches 
have been investigated for ethanol fermentation:  
Consolidated BioProcessing (CBP): This combines all three processes, cellulase 
production, cellulose hydrolysis, and fermentation. They are carried out in one reactor 
with the use of single microorganism or group of microorganisms operating at the same 
conditions. This is also called Direct Microbial Conversion (DMC). The microorganism 
 35
community used in the reactor produces enzymes required for all the steps in the biomass 
to ethanol process (Lynd et al., 1996). Two strategies can be adopted for engineering the 
microorganisms for this purpose. It is estimated that CBP can reduce the cost of 
biological conversion by more than 4 times that of SSCF process, which is an advanced 
process combination with simultaneous saccharification with co-fermentation of hexose 
and pentose sugars, because of the reduced number of vessels required, the reduced cost 
of cellulase production and the reduced time of the process. First, naturally occurring 
cellulolytic microorganism can be engineered for expressing the enzymes required for 
ethanol production. Second, ethanol producing organisms can be improved genetically to 
express the cellulase system (Lynd et al., 2005). However, the ethanol yields are rather 
low, a lot of metabolic by-products are produced, and the organisms usually do not have 
high ethanol tolerance (Ravinder et al., 2000; South et al., 1993). 
Separate Hydrolysis and Fermentation (SHF): In this process, enzymatic 
hydrolysis and fermentation is performed sequentially in two different vessels. In this 
process each step can be conducted at optimal conditions of pH and temperature. 
Cellulolytic enzymes usually perform better at higher temperatures - 50 ?5? C and a pH 
of 4.0?5.0 (Saddler and Gregg,1998), whereas most of the ethanologenic organisms 
proposed for the fermentation of sugars, e.g. Saccharomyces cerevisiae (Olsson and 
Hahn?Ha?gerdal,1993; Tonn et al.,1997) or Zymomonas mobilis (Park et al.,1993; 
Lawford et al.,1997) limit the temperature to below 40?C, usually at 30?C (Philippidis et 
al.,1996). However, glucose and cellobiose accumulation in the hydrolysis step inhibits 
the activity of the cellulases (Stenberg et al., 2000; Xiao et al., 2004; Tengborg et al., 
 36
2001). The build-up of any of these products negatively affects enzymatic hydrolysis in 
the SHF process.  
Simultaneous Saccharification and Fermentation (SSF): SSF combines cellulose 
hydrolysis and fermentation in one step. Because the glucose produced by the hydrolysis 
process is immediately consumed by the microorganism, only very low levels of 
cellobiose and glucose are observed in the reactor. Therefore, this process can greatly 
reduce the product inhibition to cellulase, which in turn increases sugar production rates, 
concentrations, and yields, and decreases enzyme loading requirements and process time. 
There are resulting cost savings due to the reduced number of vessels required compared 
to SHF process. In addition, there is another advantage to reducing requirements for 
sterile conditions because the presence of ethanol in the culture broth helps to avoid 
undesired microbial contamination in SSF (Szczodrak and Targonski, 1989; Wyman et 
al., 1992; Grohmann, 1993). 
The Saccharomyces cerevisiae strains are the most commonly used ethanologens 
for starch and cellulosic biomass sugars and have high efficient fermentation of glucose 
to ethanol and rapid fermentation rates at low pH (Wyman, 1996). In addition, they have 
very high ethanol tolerance. Baker?s yeast?S.cerevisiae?can produce 96.71g/L ethanol 
in the fermentation process (Caylak and Vardar, 1996). This microorganism utilizes six-
carbon sugars, such as glucose, mannose and galactose, as well as the disaccharides 
sucrose and maltose by the Embden-Meyerhof Pathway (EM). 
However, there are some the disadvantages to SSF. The microorganisms used in 
SSF are usually the yeast S. cerevisiae. The optimal temperature for SSF is around 38 ?C, 
which is a compromise between the optimal temperatures for hydrolysis (50 ?C) and 
 37
fermentation (35 ?C) (Philippidis, 1996). This has a negative effect on the productivity 
because the enzymatic hydrolysis is the rate-limiting step in SSF (Philippidis and Smith, 
1995). Thermotolerant yeasts and bacteria have been developed in the SSF to increase the 
temperature close to the optimal temperature of enzymatic hydrolysis. Kadam et al found 
that the thermotolerant yeast, Candida acidothermophilum, produced 80% of the 
theoretical ethanol yield at 40 ?C using dilute-acid pretreated poplar as substrate (Kadam 
and Schmidt, 1997). Ballesteros et al.  found Kluyveromyces marxianus and K. fragilis 
that have the highest ethanol productivity and an ethanol yield of 0.5 g/g cellulose in 78 
hr using Solka Floc 200 as a substrate at 42 ?C from 27 yeast strains (Ballesteros et al., 
1991).  Another disadvantage to SSF is that it is difficult to recover and reuse the yeast in 
an industrial process because of the difficulty in separating the yeast from the solid 
residue after SSF (Galbe and Zacchi, 2002). In addition, ethanol was found to be 
inhibitory to enzymes. Wu et al. found that cellulase lost its original activity dependent 
on ethanol concentrations (Wu and Lee, 1997). 
Simultaneous Saccharification and Co-Fermentation (SSCF): Similar to SSF, 
SSCF combines cellulose hydrolysis and fermentation in one step by using strains with 
the ability to convert both hexose and pentose to ethanol. The main advantages of SSCF 
are that microorganisms can fermentate glucose and xylose into ethanol in one bioreactor, 
and further improve xylan removal which can improve the access of enzymes to cellulose 
(?hgren et al., 2007) and reduce the inhibitory effect of xylose and xylooligomer on the 
cellulose hydrolysis by cellulase (Xiao et al., 2004; Nigam, and Prabhu, 1991; Kim and 
Lee, 2005b; Kumar and Wyman, 2008). 
 38
As stated above, the most commonly used microorganism is Saccharomyces 
cerevisiae; it has very high ethanol tolerance and is very robust. Some carbonyl 
compounds ? furans and phenolics, which are very inhibitory to most of ethanologen 
bacteria, are less inhibitory to yeast (Taherzadeh et al., 2000a; and 2000b), since yeast 
can convert them to the corresponding and less toxic alcohols (Martin and J?nsson, 2003; 
Sarvari Horvath et al., 2003). However, it cannot ferment pentose sugars; as any 
hydrolysis process, the pentose fraction is currently discarded.  In order to improve 
process economics, it is desirable to convert both pentoses and hexoses to ethanol (Lynd 
et al., 1999). Yeasts such as Pichia stipitis, Candida shehatae, and Pachysolen 
tannophilus, which are naturally capable of fermenting both glucose and xylose to 
ethanol, are being used in the fermentation process at laboratory scale (Lynd et al., 1999). 
Pichia stipitis CBS 6054 is one of natural xylose-fermenting yeast, which notably 
ferments xylose to ethanol with reasonable yield and productivity. 
However, these yeast strains are inhibited by byproducts generated during 
pretreatment and enzymatic hydrolysis of the lignocellulose biomass (Hahn-H?gerdal et 
al., 1994). In addition, Chandakant et al. reported that these yeasts produce about one 
fifth the ethanol of S. cerevisiae (Chandakant and Bisaria, 1998).Therefore, a lot of 
research has been focused on metabolic engineering strains of bacteria and yeast able to 
utilize pentose and hexose recently. Anaerobic bacteria Z. mobilis (Lawford et al., 1998), 
the bacteria Escherichia coli (Ingram et al., 1997), and S. cerevisiae (Tonn et al., 1997) 
have been developed to meet the requirements of industrial lignocellulosic biomass 
fermentation. Naturally, Z. mobilis can only use the hexose sugars glucose and fructose to 
produce ethanol with higher yields, due to the production of less biomass, but is less 
 39
robust (Rogers et al., 1979; Lawford et al., 1998). Z. mobilis degrades hexose sugars to 
pyruvate using the Entner-Doudoroff pathway (Sprenger, 1996). The pyruvate is then 
fermented to produce ethanol and carbon dioxide as the only products, similar to yeast. 
But Z. mobilis can not consume pentose. In addition, another big concern in the 
fermentation process is that Z. mobilis is sensitive to various inhibitors. Zhang et al. 
inserted the genes for xylose assimilation and pentose phosphate pathway enzymes, like 
xyloase isomerase, xylulokinase, transketolase, and transladolase from E. coli into the 
bacterium Zymomonas mobilis for effective fermentation of xylose to produce ethanol in 
NREL (Zhang et al., 1995). The researchers at Dupont continued this work. In 2006, 
DuPont's biofuels technology manager William Provine reported that his group has 
recently come up with a Zymomonas strain capable of tolerating up to 10% ethanol 
(Service, 2007).  
E. coli can ferment a number of pentose and hexose sugars into a mixture of acids 
(lactic, acetic, formic, and succinic) and ethanol (Clark, 1989). E. coli KO11 is one of the 
very first ethanologens engineered by Ingram?s group. They transformed pyruvate 
decarboxylase (pdc) and alcohol dehydrogenase II (adh B) from Zymomonas mobilis into 
the parental strain of E. coli KO11, which has the natural ability to utilize several 
pentoses and hexoses, to produce ethanol with high metabolic yield (Ohta et al., 1991). 
However, its growth robustness and ethanol tolerance are relatively low compared to 
yeast (Yomano et al., 1998; Dien et al., 2003; Service, 2007). In order to improve the 
ethanol tolerance of E. coli strain KO11, Ingram?s group isolated mutant strains with 
increased ethanol tolerance by using a novel approach alternating selection for ethanol 
tolerance in liquid and high antibiotic resistance on solid media. The best of these 
 40
mutants, strain E. coli LY01, exhibited more resistant than KO11 to inhibitors generated 
from pretreatment stage (Yomano et al., 1998) and high ethanol tolerance  as high as 100 
g L?1 (Zaldivar et al.,1998).  
Yeasts are still the most promising metabolic-engineering platforms for cellulosic 
ethanol processes as can be demonstrated by their usage in biotechnology for hundreds of 
years, because of their natural tolerance to high ethanol and inhibitors and their low pH 
growing condition which avoids bacterial contaminations. Ho et al. transformed three 
xylose-fermenting genes, xylose reductase and xylitol dehydrogenase genes from Pichia 
stipitis and xylulokinase gene from Saccharomyces cerevisiae, into Saccharomyce spp. 
for the co-fermentation of glucose and xylose with an ethanol yield of 0.30 g/g of initial 
total sugar (Ho et al., 1998). In order to increase the ethanol yield and the tolerance to 
toxic inhibitors, Ho et al further developed stable Saccharomyces yeasts 424A (LNH-
ST), which can ferment a mixture of pure glucose (7%) and xylose (4%) into ethanol in 
30 hours with an ethanol yield of 0.41 g/g of initial total sugar and 80% of the ethanol 
theoretical yield of initial total sugar (Sedlak, 2004). 
2.4 The Integrated Forest Biorefinery 
The pulp and paper industry in United States is facing overseas competitors, 
primarily from Asia, who have installed new technologies and who also have wood and 
labor cost advantages. At the same time, due to the digital revolution, the demand for 
certain grades of papers, particularly newsprint, is decreasing. According to Global Forest 
the decline in newspaper readership and advertising, among many sectors of the 
population, particularly young adults, and along with increasing competition from the 
Internet and other media, has led to consumption falling 10% from 2006 to 2007 (Global 
 41
Annual Forest, Paper & Packaging Industry survey - 2008 edition - Survey of 2007 
Results). The combination of reduced demand in the US along with the increased supply 
of cheap paper is causing a great upset to the pulp and paper industry in the United States, 
which is forcing the pulp and paper industries to find new opportunities in order to 
improve their competitiveness. The integrated ?Biorefinery? process to produce ethanol 
and cellulase as a supplementary unit in kraft paper mills can be developed. It can 
transform the pulp and paper industry into a competitive and innovative one, which allow 
a significant economic growth through new value-added products based on existing 
byproducts or waste at pulp and paper mills (i.e. hemicelluloses, black liquor, and paper 
sludge), while still preserving the main product-paper and paper related products. 
2.4.1 Background of Pulp and Paper-making  
Pulp and paper-making begins with trees as the main raw material - nearly 90%, 
though many lignocellulosic non-woody plants can be used, such as cotton, wheat straw, 
sugar cane waste, flax, bamboo, and linen rags.  The main steps in pulp and paper- 
making are raw material preparation, such as wood debarking and chipping, pulp 
manufacturing, pulp bleaching, paper manufacturing, and fiber recycling. Not all parts of 
wood are useful for making pulp. Bark contains relatively high lignin and ash, and has to 
be removed. Chipping reduces the wood waste to a uniform size. Pulp manufacturing 
converts wood or lignocellulosic non-woody material to pulp fibers for paper 
manufacturing by mechanical or chemical means (Biermann, 1996; Sj?str?m, 1993). 
2.4.1.1 Mechanical Pulp or Thermomechanical Pulp (TMP) 
In the TMP making process, the wood chips are steamed with refiner plate. The 
heat softens the lignin, which allows fiber separation with less damage than in purely 
 42
mechanical pulping. The mechanical pulping process usually has a very high yield, in 
which more than 90% of the wood used in the process is converted into pulp. However, 
the process doesn?t remove much lignin, and therefore most of the lignin remains in the 
pulp. Mechanical pulp can be used without bleaching to make printing papers for 
applications in which low brightness is acceptable, primarily newsprint, and other low-
strength and low brightness applications, such as cardboard. Mechanical pulp is 
sometimes blended with kraft pulp to produce a middle-level product with low cost and 
reasonable strength and color properties, such as magazine. A large amount of energy is 
required to produce mechanical pulp, but it costs less than chemical pulp (Biermann, 
1996; Sj?str?m, 1993). 
2.4.1.2 Chemithermomechanical Pulp (CTMP) 
Chemithermomechanical pulp (CTMP) or chemimechanical making processes 
involve mechanical action and the use of chemicals. The wood chips are softened with 
sodium carbonate, sodium hydroxide, sodium sulfite, and other chemical prior to a drastic 
mechanical action, but not as drastic as a mechanical pulp making. It removes extractives 
and small amounts of hemicellulose but not much lignin. Therefore, the yield of the 
chemimechanical pulping process is also high, 85-95%. The CTMP is stronger than 
TMP, but requires less energy to produce. CTMP can be bleached to produce bleached 
chemithermomechanical pulps (BCTMP) with high yield. (Biermann, 1996; Sj?str?m, 
1993). 
2.4.1.3 Chemical Pulp 
Chemical pulping uses chemicals to dissolve the lignin in the wood under the 
vigorous conditions, freeing the cellulose fibers. 
 43
Sulfite Pulping: The sulfite process uses mixtures of sulfurous acid and/or its 
alkali salts carried out between pH 1.5 and 5 to solubilize lignin through the formation of 
lignosulfonates  and cleavage of lignin bonds. Sulfite pulping has higher yield than kraft 
pulping. The byproduct of sulfite pulping, lignosulfonate has a wide variety of 
applications, such as leather tanning, drilling mud dispersants, ore flotation, and resins. 
However, sulfite pulp is weaker than kraft pulp. Not all species of wood can be pulped 
easily. Cooking cycles are long, and chemical recovery is complicated compare to kraft 
pulping. Therefore, most of sulfite mills have been converted to kraft mills (American 
Forest & Paper Association, 2002).   
Kraft Pulping: In the kraft pulping process, wood chips are cooked with white 
liquor, a mixture of sodium hydroxide and sodium sulfide. It is heated in a digester which 
is capable of holding high pressures and high temperatures. The typical condition of 
delignification is several hours at 130 to 180 ?C. Under these conditions 90~95% lignin, 
all extractive, and some hemicellulose are degraded to give fragments which are soluble 
in the strongly alkaline liquid. The remaining solid is collected and washed to become 
pulp. The combined liquid is called ?black liquor??. The unbleached kraft pulp at this 
point is quite brown, called brown stock.  
To make white paper, the unbleached kraft pulp must undergo additional 
bleaching process to remove the remaining lignin and brighten the pulp. Chlorine and 
hypochlorite were initially used to bleach chemical pulps. Because of environmental 
considerations about the the release of organochlorine compounds into the environment, 
chlorine dioxide, oxygen, ozone, hydrogen peroxide, and other chemicals have been used 
recently  
 44
The chemical recovery process is an important part of the kraft pulping process. 
In this process, the black liquor is concentrated in multiple effect evaporators to 65-70% 
solids and then sent to the recovery boiler to burn. The degraded organic compounds in 
the black liquor have significant energy content and are able to produce enough steam for 
mill operations. The inorganic chemicals (Na
2
S, Na
2
CO
3
, and any impurities) are 
recovered at the base of the recovery boiler. Dissolving these inorganic chemicals from 
the recovery boiler produces green liquor. Converting the Na
2
CO
3
 in the greenliquor to 
NaOH using Ca(OH)
2
 regenerates white liquor used in the pulping process. Calcium 
carbonate precipitates from the white liquor and is heated to become calcium oxide. 
Calcium oxide is reacted with water to regenerate the calcium hydroxide.  
Kraft chemical recovery processes consume an estimated 8.04 million BTU/ ton 
of pulp. However, the recovery boiler in the chemical recovery processes can produce 1 - 
2 times more energy in the form of high pressure steam, which is used to reduce the 
steam pressure for the mill use and generate electricity (Martin et al., 2000). Bark and 
wood residues are often burned in a separate power boiler to generate steam. In addition, 
the water from multiple effect evaporators is usually clean enough to be used in other 
parts of the mill.  
Kraft pulping produces a stronger pulp from a wide variety of tree species, 
compared to mechanical, semichemical, or sulfite pulp and is used in high-strength 
papers, such packaging bag and shipping containers (Biermann, 1996; Sj?str?m, 1993). 
2.4.1.4 Recycled Paper Pulp 
Since paper contains the same fibers as the original plants, these fibers can be 
recycled to make new paper. In the US, approximately 27% of paper is recycled and used 
 45
in applications which require less brightening, such as containerboard and paperboard. 
(American Forest & Paper Association, Paper, Paperboard & Wood Pulp: 2002 Statistics, 
Data through 2001, (2002)).  
Paper recycling processes can use either chemical or mechanical pulp produced 
by mechanical energy with chemicals and water. Paper recycling typically includes 
repulping, screening, and cleaning process. These processes remove contaminants, such 
as plastics staples, and glues. Sometimes, Processes that produce newsprint, tissue or 
other ?bright? grades must undergo a deinking process, which remove printing ink and 
other unwanted elements by adding chemicals (surfactant) during pulping. If white 
recycled paper is needed, the pulp may need to be bleached with hydrogen peroxide, 
chlorine dioxide, or oxygen to increase the brightness of deinked pulp. The recycled pulp 
can be used alone or blended with chemical pulp to give it extra strength or smoothness. 
2.4.1.5 Paper Making 
The pulp is diluted with water to make it 99% water. This slurry enters the 
headbox, and then is sprayed onto a fast-moving, continuous wire screen. Water starts to 
drain from the pulp on the screen, and the fibers quickly begin to bond together to form 
the paper sheet.  
At this point, the paper is uncoated. For printed paper, it may be coated with 
fillers, such as calcium carbonate or clay and titanium dioxide, to increase its strength, 
water resistance, opacity and brightness (Biermann, 1996). 
2.4.2 The Development of an integrated wood- based Biorefinery  
In the emerging cellulosic ethanol market, the opportunity to repurpose existing 
U.S. pulp and paper mills as biorefineries has been brought up. The pulp and paper 
 46
industry in the U.S. is facing global competition and reduced profit. In addition, there has 
been a lack of capital investment in recent years. Therefore, the performance of the U.S. 
pulp and paper sector has been obsolete for the last 10 years. In the last 5 years the 
closures of pulp mills, and the devastation of the local communities where they are based, 
has accelerated. However, the pulp and paper industries have some advantages for 
cellulosic ethanol production. They are large biomass handlers with the know-how and 
highly skilled workforce to operate a complex process industry, and therefore they are a 
natural host for cellulosic ethanol production. A wood-based biorefinery is a plant that 
produces multiple value added chemical products from wood, chemicals and materials. 
From the pulp and paper making process, there are several potential opportunities, 
hemicellulose, black liquor, paper sludge, and waste paper. 
2.4.2.1 Hemicellulose Pre-extraction 
In the United States, 80% of total pulping capacity is chemical pulp. 
Approximately 98% of chemical pulp capacity is kraft pulping, with the remaining 2% 
being the sulfite pulping (American Forest & Paper Association, Paper, Paper board & 
Wood Pulp: 2002 Statistics, Data through 2001, (2002)). 
In kraft pulping, around 80% of the hemicellulose in the wood is dissolved in the 
black liquor (Biermann, 1996; Sj?str?m, 1993). It is difficult to separate this 
hemicellulose part from alkaline black liquor before it is burned to generate heat and 
power and to recover pulping chemicals, since part of the hemicellulose is degraded at 
alkaline conditions (Sjostrom, 1993). 
It is technically feasible to insert a new process before the digester stage to extract 
hemicellulose and convert to ethanol through a fermentation process. Hemicelluloses can 
 47
be removed from lignocellulosic materials by hot water, alkaline, and solvent extractive 
processes (Willfor and Holmbom, 2004; Song et al., 2008; Sears et al., 1971; Nguyen et 
al., 1998; Aziz and Sarkanen, 1989). It has been reported that during alkaline extraction, 
hemicellulose is partially degraded to aliphatic acids (Glasser et al., 2000). Hemicellulose 
exists in an amorphous form and is effectively hydrolyzed into soluble, monomeric 
sugars by dilute-acid pretreatment (Wyman, 1996). Hemicellulose hydrolyzates from 
water or dilute-acid pretreatment can be utilized to produce value-added products, such as 
ethanol, xylitol, (Buhner and Agblevor, 2004; Mussatto et al., 2005; Walther et al., 2001; 
Werpy and Petersen. 2004) and so on. Hemicelluloses have been utilized as plant gum for 
thickeners, adhesives, protective colloids, emulsifiers, and stabilizers as well as a 
biodegradable oxygen barrier films (Hartman et al., 2006; Kamm and Kamm, 2004; 
Kilpel?inen et al., 2007). Oligosaccharides may provide a source of higher value-added 
products, such as animal feed additives (Davis et al., 2002; Fernandez et al., 2002). 
Fufural, a xylose degradation product, can be utilized for production of lubricants, 
coatings, adhesives, and furan resins.  
The optimization of pre-extraction technology for optimal removal of 
hemicellulose should be investigated. Hemicellulose preextraction has to be done without 
reducing pulp yield and strength, since the existing ways of pulp and paper making will 
still remain as heart of wood based biorefinery (Closset et al., 2005; Mensink et al., 
2007). Cellulose fiber is the main component of paper; the minimizing the hydrolysis of 
cellulose should be considered. The fiber strength of the final paper is a key physical 
parameter in the production of paper. Some researchers have noted hemicellulose content 
is related to fiber strength (Sch?nberg et al., 2001). The inhibitors to microorganism, such 
 48
as furans, HMF, furfural, organic acid and phenolic compounds form during the 
preextraction process (Taherzadeh et al.,2000a; Taherzadeh et al., 2000b;Palmqvist et 
al.,2000a; Palmqvist et al.,2000b; Klinke et al.,2004). If the main target of prehydrolysate 
is ethanol production, the minimizing and removal of inhibitors should be done before the 
fermentation step. 
2.4.2.1 Black Liquor  
During the kraft process, 90~95% lignin, all extractives and 80% of the 
hemicellulose in the wood are degraded to give fragments which are soluble in the black 
liquor. The black liquor is concentrated, and then is combusted to produce steam and 
power and to recover the pulping chemicals, NaOH and Na
2
S.(Biermann, 1996). Lignin 
removed from the black liquor by acid precipitation may be used for chemicals. 
Gasification of black liquor is another approach. Gasification of the black liquor produces 
synthesis gas, which may be used as feedstock to produce transportation fuels. Two 
demonstration plants of black liquor gasification are operating in the United States 
(Closset et al., 2004). 
2.4.2.3 Pulp and Paper Waste 
The Pulp and Paper industry generates huge amounts of waste, mainly from the 
papermaking process and waste paper and paperboard. According to the U.S. 
Environmental Protection Agency, waste paper and paperboard made up the largest 
component of all municipal solid waste generated, about 32.7 percent and around 50% of 
them is recovered to make new paper (Municipal Solid Waste Generation, Recycling and 
Disposal in the United States: Facts and Figures, 2007). However, wood fibers can only 
be recovered 5 to 7 times before they become too short and weak to be made into new 
 49
paper. The pulp and paper industry also produces about 4.1 to 5 million dry tons per year 
of solid waste in United States, consisting primarily of de-watered sludges, which contain 
fibers and other materials produced during paper-making or paper recycling (Fan and 
Lynd, 2006b).  The paper sludges are normally disposed by incineration or landfill, which 
causes substantial financial burden and a source of various environmental problems. 
Fiber in the sludge decomposes and produces methane, a greenhouse gas with 23 times 
the heat-trapping power of carbon dioxide. More than one-third of municipal landfill 
solid waste is waste paper and paper sludge, and municipal landfills account for 34 
percent of human related methane emissions to the atmosphere, making landfills the 
single largest source of such emissions, according to EPA?s study (Environmental Paper 
Network, 2007). 
The cost of incineration is high because the burn efficiency of sludge is low, as a 
result of high moisture content (Fan and Lynd, 2006; Scott et al., 1995; Sj?de et al., 
2007). A better alternative to landfilling and incineration is to incorporate paper sludge in 
a biorefinery. Paper sludge has high carbohydrate content. Table II-1 lists the typical 
compositions of primary and recycled sludge. Compared with corn stover, sludge 
contains much higher cellulose, and much less hemicellulose as well as lignin.  The short 
fibers in the sludge are easily digested by cellulase enzymes (Fan and Lynd, 2006a; 
Jeffries et al., 1999; Fan et al., 2003). These features can potentially make bioconversion 
of sludges possible without pretreatment. As one of the major processing units in 
bioethanol plant, pretreatment is a significant cost factor, second only to feedstock cost 
which accounts for about 30% of the total production cost of ethanol (Galbe and Zacchi, 
2002). As a feedstock for bioethanol, paper mill sludges offer two significant economic 
 50
benefits. First, the cost of sludge is essentially zero and may even be a negative number 
considering that the cost of disposal is about $20 per wet ton (Randy Abston, 2008). 
Second, elimination of pretreatment can reduce the production cost of ethanol.  
2.4.3 Cellulase Production in Kraft Pulp Mills 
One of the drawbacks in the production of bioethanol is the high cost from the 
usage of the enzymes utilized in the hydrolysis step (Steele et al., 2005; Spano et al., 
1978). 
Cellulase production in the microorganism is induced by crystalline cellulose and 
other known inducers (lactose and sepharose). Microbial uptake of the substrate is more 
complex than enzymatic hydrolysis of it. Although there is a similarity between the two 
cases, the microbial reaction is much more complex because the cellulase enzyme is 
excreted from it and works synergistically with the microorganism. The microbial 
process is also influenced by oxygen uptake in the bioreactor since this organism is 
highly aerobic, thus affected by oxygen mass transfer in the process of cell growth and 
enzyme production. Cellulase production is also accompanied by xylanase production. 
How the xylan content in the feedstock affects xylanase production is also an important 
issue in cellulase production.  
The best-known host culture in cellulose production is the softrot filamentous 
fungus -Trichoderma reesei. T. reesei holds the greatest potential for reducing the cost in 
the production of enzymes for lignocellulosic biomass conversion (Egyhazi et al. 2004; 
Gadgil et al. 1995; Juhasz et al., 2004; Kadam and Keutzer, 1995). T. reesei is relatively 
easy and inexpensive to cultivate and it is not easily susceptible to contamination 
(Penttil?, 1998).  It is a safe organism, because it is non-pathogenic and non-toxic to 
 51
healthy laboratory animals (Hjortkjaer et al., 1986) and to humans.  It does not produce 
mycotoxins or antibiotics under the conditions used for enzyme production (Nevalainen 
et al., 1998; Nevalainen et al., 1994) and it is a low-risk to microorganisms (Penttil?, 
1998).  T. reesei is used industrially for enzyme production (Esterbauer et al., 1991; 
Kubicek, 1992).  Most of the previous work is based on a variation of T. reesei-T. reesei 
Rut-C30 (ATCC-56765), which was isolated using a combination of ultraviolet 
irradiation and nitrosomethyl guanidine (NTG) by Montenecourt and Eveleigh 
(Montenecourt and Eveleigh, 1979).  The industrial strain, although not publically 
revealed, is also originated from this culture and modified genetically. 
In many investigations of cellulase production, soluble forms of modified 
cellulose and pure cellulose have been used as the carbon source. Mixtures of glucose 
and lactose have also been tested as a feasible substrate for cellulase production 
(Domingues et al., 2001). Pourquie (Pourquie et al., 1989) and Chaudhuri (Chaudhuri et 
al., 1993) reported cellulase production using lactose as the co-substrate. The soluble 
carbon sources are convenient to use and acceptable purely on a technical stand point. 
However the cost is prohibitively high. For many researchers, the preferred substrates 
have been pure commercial celluloses such as Solka Floc and Avicel (Mclean et al., 
1986; Shiang et al., 1991; Hayward et al., 2000; Yu et al., 1999). Hayward et al reported 
a cellulase yield above 150 FPU/g - cellulose and productivity above 55 FPU/L-hr 
obtained using Sloka Floc as the substrate. This appears to be the benchmark values of 
laboratory scale cellulase production. By fed-batch, relatively higher yield and 
productivity were achieved (Yu et al., 1998; Yu et al., 1999).  
 52
To convert a kraft process paper mill to an integrated biorefinery, the cellulase 
production has to be feasible, convenient, and economically viable. The substrate directly 
from paper and pulp industries could be considered. Sulfite wood pulp (Watson et al., 
1984), pretreated wood (Juhasz et al., 2005; Shin et al., 2000), sawdust (Lo et al., 2005), 
waste paper (Royer and Nakas, 1987; Chen and Wayman, 1991; Shin et al., 2000), waste 
cardboard (Szij?rt? et al., 2004), and paper sludge (Royer and Nakas, 1987; Maheshwari 
et al., 1994) could be used as a carbon source for cellulase production. In general, the 
performance of cellulase production with these substrates was far inferior to the cellulose 
production obtained with glucose and pure cellulose.  The individual results would vary 
depending upon what was used as the feedstock and what type of pretreatment had been 
applied.  Generally, the presence of acetic acid, furfural, and lignin in the pretreated 
biomass caused delays in enzyme production and this in turn inhibited the enzyme 
production by T. reesei (Vinzant et al., 1998; Hayward et al., 2000; Szengyel and Zacchi, 
2000; Bigelow and Wyman, 2002). Kraft paper sludge showed quite promising results. 
However, kraft paper sludge usually had high ash content, which is detrimental to cell 
growth. The solution to this problem is to remove as much of the ash as much as possible 
while retaining the carbohydrates (Wang et al., 2010). 
Using kraft pulp while on site has the benefit of providing a high quality carbon 
source for cellulase production. In alkaline delignification of biomass such as kraft 
pulping, lignin is predominantly degraded by various cleavage reactions of aryl ether 
linkages (Sj?str?m, 1993). The Kkraft pulping process also removes about 50% 
hemicellulose and brings about changes in the structure of fiber (Sj?str?m, 1993). The 
kraft pulp could encourage the growth of the T. reesei and allows greater production of 
 53
cellulases and faster secretion into the culture medium. Cellulase cost contributes 12.5?
43.4% to the total cost of cellulosic ethanol production when procured from external 
sources (Galbe and Zacchi, 2002; Ryu and Mandels, 1980; Wooley et al., 1999). 
However, the cost is drastically reduced once the in-house produced enzymes are used for 
saccharification purpose (Kadam, 1996). In addition, the extra-cellular enzymes are 
obtained from the fermenter broth, not inside the T. reesei cell body.  The crude enzyme 
solution (fermenter broth) can be used without further purification. 
The cost of bioethanol and cellulase production could be reduced if the immediate 
processing steps were minimized.  That is, a process can be economized to its maximum 
extent when the process steps are minimized, without compromising the quality of the 
final product.  Thus, process integration is a powerful tool in realizing the possible 
commercial production of bioethanol.  A bioethanol plant could be integrated within an 
operational kraft process paper mill.  This would save considerably on the transportation 
costs of moving the bulk paper sludge to a bioethanol plant. Kraft paper sludges work 
well in ethanol production process without pretreatment. The substrate of cellulase 
production can be obtained directly from a kraft paper mill itself.  In addition, in the kraft 
process paper mill, one of the by-products is a high-calorific-value lignin, which could be 
used to generate energy for supporting the bioethanol production and the cellulase 
process. 
 54
Table II-1. Composition of sludges and corn stover 
 
Primary sludge Recycle sludge Corn stover 
Glucan (%) 44.5 49.6 36.1 
Xylan (%) 9.9 13.7 21.4 
Lignin (%) 8.1 4.6 17.2 
Total Ash (%) 36.0 30.0 7.1 
Acid-soluble Ash (%) 26% 22% - 
Acid-insoluble Ash (%) 10% 8% - 
Others (%) 1.5 2.1 18.2 
Alkalinity 
(milliliters of 1 N H
2
SO
4
/10 g dry 
sludge) 
60 50 - 
 
a. All data in table are based on oven-dry original feedstocks 
b. Data of sludges are the mean value of triplicate (n=3; standard 
deviation<0.5 for glucan, xylan, and lignin)      
c. The composition of the corn stover was determined by the National 
Bioenergy Center, NREL, Golden, CO). 
 
 
 
 
 
 
 
 55
 
Fig. II-1. Bioethanol production process diagram (Biomass program, NREL, 
http://www1.eere.energy.gov/biomass/abcs_biofuels.html)  
 
 
 
 
 
 
 
 56
 
Fig.II-2. Pretreatment of biomass (Mosier et al, 2004) 
 
 
 
 57
 
Fig. II-3. Cellulose hydrolysis by cellulases from Trichoderma reesei. (Lynd et al., 2002) 
 
 
 
 
 
 
 
 
 
 
 58
 
 
Fig. II-4. Structure of xylan and the sites of attack by xylanolytic enzymes  
(Javier et al., 2007) 
 59
 
Fig. II-5. Mannanses attack on galactoglucomanna (Puls and Schuseil, 1993)       
 
 
 
 
 
 
 
 
 
 
 
 60
III. Bioconversion of Kraft Paper Mill Sludges to Ethanol by SSF and 
SSCF 
 
3.1 Abstract 
Paper mill sludge is a solid waste material composed of pulp residues and ash 
generated from pulping and paper making processes. The carbohydrate portion of the 
sludge has chemical and physical characteristics similar to pulp. Because of its high 
carbohydrate content and well dispersed structure, the sludges can be biologically 
converted to value-added products without pretreatment. In this study, two different types 
of paper mill sludges, primary sludge and recycle sludge, were evaluated as a feedstock 
for bioconversion to ethanol. The sludges were first subjected to enzymatic conversion to 
sugars by commercial cellulase enzymes. The enzymatic conversion was inefficient 
because of interference by ash in the sludges with the enzymatic reaction. The main cause 
was that the pH level is dictated by CaCO
3
 in ash, which is two units higher than the pH 
optimum of cellulase. To alleviate this problem, simultaneous saccharification and co-
fermentation (SSCF) using cellulase (Spezyme CP) and recombinant Escherichia coli 
(ATCC-55124), and simultaneous saccharification and fermentation (SSF) using 
cellulase and Saccharomyces cerevisiae (ATCC-200062) were applied to the sludges 
without any pretreatment. Ethanol yields of 75-81% of the theoretical maximum were 
obtained from the SSCF on the basis of total carbohydrates. The yield from the SSF was 
also in the range of 74-80% on the basis of glucan. The SSCF and SSF proceeded under 
 61
stable condition with the pH staying near 5.0, close to the optimum for cellulase. 
Decrease of pH occurred due to carbonic acid and other organic acids formed during
fermentation. The ash was partially neutralized by the acids produced from the SSCF and 
SSF and acted as a buffer to stabilize the pH during fermentation. When the SSF and 
SSCF were operated in fed-batch mode, the ethanol concentration in the broth increased 
from 25.5 and 32.6 g/L to 42 and 45 g/L, respectively. The ethanol concentration was 
limited by the tolerance of the microorganism in case of SSCF. The ethanol yield in fed-
batch operation dropped to 68% for SSCF and 70% for SSF. The high solids condition in 
the bioreactor appears to create adverse effects on the cellulase reaction. 
3.2 Introduction 
 
The pulp and paper industry generates 4-5 million tons of sludges per year in the 
US (Fan and Lynd, 2006a). The disposal of sludges by incineration or landfill causes 
substantial financial burden and is a source of various environmental problems. 
Incineration is a significant cost factor because of low energy efficiency caused by high 
moisture content (Fan and Lynd, 2006a; Scott et al., 1995; Sj?de et al., 2007). The fibers 
from the paper mill sludge have characteristics suitable for conversion to value-added 
products. Sludges from kraft paper mill have high carbohydrate and low lignin contents 
in comparison to natural lignocellulosic substrates. The composition of the organic 
portion of the sludge is very similar to that of commercial grade pulp: mostly cellulose 
with low amounts of hemicelluloses and lignin. It also has well-dispersed structure and
high surface area. It has been proven to be highly efficient for bioconversion to ethanol 
(Lark et al., 1997). Although it is projected that a majority of ethanol is to be produced 
 62
from natural lignocellulosic feedstock, paper sludge is a resource that may have 
opportunities for commercialization.  
As a feedstock for bioethanol, paper mill sludges offer two significant economic 
benefits. First, the cost of sludge is essentially zero and may even be a negative number 
considering that the cost of disposal is about $20 per wet ton (Randy Abston, Boise 
Paper, Jackson, AL, USA, personal communication, 2008). Second, elimination of 
pretreatment can significantly reduce the production cost of ethanol. Pretreatment opens 
up the biomass structure and makes it more amenable for enzymatic hydrolysis. It is also 
a major processing units in bioethanol plant accounting for about 30% of the total 
production cost of ethanol, second only to the raw material (Galbe and Zacchi, 2002). In 
alkaline delignification of biomass such as Kraft pulping, lignin is predominantly 
degraded by various cleavage reactions of aryl ether linkages (Sj?str?m, 1993). The Kraft 
pulping process also removes about 50% hemicellulose and brings about changes in the 
structure of fiber (Sj?str?m, 1993). The fibers in the sludge are short because they are 
screen rejects from paper machine and the digester, and are easily digested by cellulase 
enzymes (Fan and Lynd, 2006a; Fan and Lynd, 2006b; Scott et al., 1995; Jeffries and 
Schartman, 1999; Fan, et al., 2003).  
In this study, sludge generated from a paper mill was investigated as a feedstock 
for ethanol production. The main objectives of the investigation were to characterize the 
sludges in terms of composition and physical properties and to test the technical 
feasibility of converting the feedstock to ethanol without pretreatment. Bioconversion of 
pulp mill sludge has been previously investigated, wherein the pH of the sludge was pre-
adjusted by hydrochloric acid (Marques et al., 2008) or by sulfuric acid (Lynd et al., 
 63
2001). This strategy may encounter a problem when the sludge has a large amount of ash, 
mainly CaCO
3
, such as the one used in this work since the amount of acid to neutralize 
the ash would be excessive. SSF of paper mill sludge was also investigated by Fan, et al. 
(Fan and Lynd, 2006b), in which pH in the medium was adjusted by sulfuric acid. 
However, the effect of ash on the pH during the course of SSF was not addressed. The 
high ash content in paper mill sludges, specially CaCO
3
, causes several problems in the 
bioconversion process as it interferes with pH control, limits solid loading capacity in the 
bioreactor and reduces the efficiency of enzyme in a bioprocess. Additional technical 
issues associated with the contaminated substrate were also addressed in this 
investigation.  
3.3 Materials and Methods 
3.3.1 Materials 
Feedstock: The paper mill sludges were collected from a Kraft paper mill, Boise 
Paper Company (Jackson, AL, USA). There were two different types of sludges. One was 
primary sludge (PS), discharged from the paper machine and the digester, which was 
collected from the primary wastewater clarifier unit. The other one was recycle sludge 
(RS), which came from a reprocessing unit of recycle paper. Both were stored at 4o C. A 
sample quantity of wet hardwood pulp (HP) was also obtained from Boise Paper 
Company. It was used as a reference substrate in bioconversion studies. The paper sludge 
and hardwood pulp were analyzed for carbohydrates, moisture content, and ash content 
according to the National Renewable Energy Laboratory (NREL) standard procedure 
(NREL, 2008). HP was analyzed to contain 76.0% glucan, 21.1% xylan, and 2.9 wt. % 
unaccounted for (lignin, acetyl group, ash, and protein).  
 64
Enzymes: Cellulase enzyme (Spezyme CP, Lot No. 301-00348-257) was a kind 
gift from Danisco Genencor (Palo Alto, CA, USA) and its activity was measured to be 59 
FPU/mL. ?-glucosidase (Novozyme188, Cat. No. C-6150) was purchased from Sigma 
(St. Louis, MO, USA) and its activity was measured to be 750 CBU/mL.  
Microorganism: The microorganism used in the SSF was Saccharomyces 
cerevisiae ATCC-200062 (NREL-D5A). The growth media was YP medium, which 
contained 1% yeast extract (Sigma, Y-0500) and 2% peptone (Sigma, P-6588). 
Recombinant Escherichia coli ATCC-55124 (KO11) was for the SSCF experiments. This 
organism was grown in LB medium (Sigma, L-3152), which contained 1% tryptone, 
0.5% yeast extract, 1% NaCl, and 40 mg/L chloroamphenicol. 
3.3.2 Contaminant Microorganism Test 
A YPD agar (Sigma, Y-1500) or LB agar (Sigma, L-3027) plate was placed on a 
turntable, and 10 ?L of sludge solution was dispersed evenly on the plate. Tetracycline 
and cycloheximide were added to another plate for comparison purpose. The YPD or LB 
agar plate was then incubated at 30 ?C for 48 h. Samples from each flask were streaked 
on an YPD or LB agar plate to check microbial contamination. The gram stain was used 
to differentiate gram-positive and gram-negative bacteria. Colony forming unit (CFU) 
tests were performed to determine the concentration of microorganism cells. YPD or LB 
agar plates were prepared for this purpose by diluting the culture with sterile saline 
(0.89% NaCl solution) to obtain a spread plate cell count of 30-300 cells per plate.  
3.3.3 Digestibility Test 
The enzymatic digestibility of the sludge was determined according to NREL 
Chemical Analysis and Testing Standard Procedures (NREL, 2008). Screw-capped 250 
 65
mL Erlenmeyer flasks were used as the hydrolysis reactor. The sludge samples were 
suspended in DI water and put into the flasks to reach a total working volume of 100 mL 
with sludges loading such that the glucan content became 1% (w/v) in the reactor. Two 
different sterilization methods were applied. Sludges were steam sterilized by autoclaving 
at 121 ?C for 15 min, or tetracycline and cycloheximide were added into the reactor to 
maintain sterile condition. The enzymatic digestibility tests were carried out at 50 ?C in 
an incubator shaker (New Brunswick Scientific, Innova-4080) agitated at 150 RPM.  For 
hardwood pulp, enzymatic hydrolysis was carried out under three different conditions: 
pH 4.8 (0.05 M sodium citrate buffer), pH 7.0 (0.02 M sodium phosphate buffer), and 
with addition of CaCO
3
 to the level of 0.5 g/g-glucan matching that of sludge. Cellulase 
and ?-glucosidase were also added to maintain enzyme loading at 15 FPU and 30 CBU/g-
glucan, respectively. The cellulase enzyme was supplemented with ?-glucosidase at a 
ratio of 2 CBU/FPU. The enzymes were filter-sterilized. Hydrolyzate samples were taken 
at 6, 12, 24, 48, and 72 h and analyzed for glucose, xylose, and cellobiose. Total released 
glucose and cellobiose after 72 h of hydrolysis were used to calculate the enzymatic 
digestibility:  
100
)(_111.1
)(_053.1)(_cos
(%) ?
?
?+
=
gaddedGlucan
greleasedCellobiosegreleasedeGlu
ityDigestibil  
Hardwood pulp was put through the same procedure as a control. The 
digestibilities for xylan content were determined in a similar manner. For xylan 
digestibility, hydration factor of 1.136 was used in the equation. Reported digestibility 
value are the average of duplicate test with standard deviation (SD) smaller than 1.0. 
 66
3.3.4 Simultaneous Saccharification and Fermentation /Co-fermentation  
A 250-mL Erlenmeyer flask was used as the bioreactor. It was operated in an 
incubator shaker (New Brunswick Scientific, Innova-4080) at 37?C with 150 RPM with 
100 mL working volume. Sludges and growth medium were added such that the glucan 
content becomes 3% w/v or 6% w/v. Hardwood pulp was also put through the same 
procedure as the control. The sludge samples were steam sterilized at 121 ?C for 15 min. 
The growth media for SSF was YP medium. The growth media for SSCF was LB 
medium (Sigma, L-3152). For SSCF, 40 mg/L aseptic chloroamphenicol was added after 
autoclaving. 
SSF and SSCF of the sludges were carried out at 37 ?C without pH control. SSF 
and SSCF of the hardwood pulp were carried out at 37 ?C with pH 4.8 (0.05 M sodium 
citrate buffer) and pH 6 (0.05 M sodium phosphate buffer), respectively. Cellulase and ?-
glucosidase were also added to achieve enzyme loading at 15 FPU and 30 CBU/g-glucan, 
respectively. Optical density (OD) was measured by UV Spectrophotometer (BioTek 
Synergy HT Multidetection Microplate Readers) at 600 nm for yeast (NREL-D5A) and 
550 nm for recombinant E. coli (KO11). The initial OD after inoculation was 0.05, 
equivalent to 16 mg dry cell weight of KO11/L and 50 mg of NREL-D5A/L. In all of the 
microbial experiments, a sample from each flask was taken at the end of the run and 
streaked on an YPD plate to check for contamination and checked under an optical 
microscope. CFU tests were performed using YPD plates or LB agar to check 
microorganism viability. The SSF and SSCF experiments requiring pH monitoring were 
carried out using a 1-L bioreactor (Multifors IHORS HT 2?1L) with 400 mL working 
volume.  
 67
The ethanol yield was calculated as follows: 
100
0.511reactorin  (g)Sugar  Initial
reactorin  (g) produced Ethanol
 maximum] al theoreticof [% yield Ethanol ?
?
=  
Sugar is interpreted as glucose in the SSF and glucose plus xylose in the SSCF. 
The fed-batch experiments were started with 100 mL initial working volume and 
6% w/v glucan loading. Squeezed sludge cakes equivalent to 6 g-glucan were added to 
the bioreactors at desired times. The cellulase and ?-glucosidase were also added at 
beginning of fermentation process to achieve overall enzyme loading at 15 FPU and 
30 CBU/g-glucan based on total glucan. Addition of sludge was done at 24 h point for 
SSCF and at 24 and 48 h points for SSF. Samples were analyzed for glucose, xylose, 
organic acid, and ethanol by high pressure liquid chromatography (HPLC). Aseptic 
conditions were maintained in all of the microbial experiments. The fed-batch 
fermentation runs were made in triplicate.   
3.3.5 Analytical Methods 
The solid samples were analyzed for carbohydrates and Klason lignin following 
the NREL standard procedures (NREL, 2008). The moisture content was measured by an 
infrared moisture balance (Denver Instrument, IR-30). Sugars were determined by HPLC 
using a HPX-87P column. For the SSF or SSCF tests, BioRad-HPX-87H column was 
used for measurement of sugar, organic acid and ethanol. A refractive index detector was 
used with the HPLC. The acid-insoluble ash was determined following the Tappi test 
method (Test Method T 244 cm-99). Liquid sample analysis and ash determination were 
done in triplicate.  
The alkalinity of the sludge was determined by titration with 1N H
2
SO
4
 to pH 5.0 
and expressed in terms of milliliters of 1N H2SO4/10 g dry sludge.  
 68
The buffering capacity of the paper sludges was determined by titration. The 
experiments requiring pH monitoring were carried out using 1-L bioreactor (Multifors 
IHORS HT 2?1L) with 400 mL working volume at 37 ?C and 150 RPM. The sludges 
were added such that the glucan content becomes 3% (w/v). The sludge slurries were 
titrated with 20 g/L acetic acid. Deionized water was used as a reference. Where 
applicable, statistical computation including mean value and standard deviation was 
performed using Microsoft Office Excel 2003.   
3.4 Results and Discussion 
3.4.1 Composition  
The compositions of primary sludge and recycle sludge are shown in Table III-
1.The composition of corn stover is also provided as a reference. Paper mill sludges are 
high in glucan and low in lignin in comparison to corn stover. They contain a very high 
amount of ash, about one third of the total mass. The majority of ash content in sludge is 
from filler material added during the paper making process: clay, TiO
2
 and CaCO
3
. It is 
added as fine particles of 0.1 - 10 micrometer (Biermann, 1996). The ash was further 
analyzed to determine acid-soluble fraction and acid-insoluble fraction.  Based on the 
Boise Paper manufacturing process and the analysis data, we conclude that the acid-
soluble ash is mainly CaCO
3
, and acid- insoluble ash is mainly TiO
2
 and clay.  
3.4.2 Enzymatic Digestibility 
 
The enzymatic digestibility data of sludges and a reference substrate are 
summarized in Table III-2.  Hardwood pulp was taken as the reference substrate because 
the composition is similar to the organic portion of the sludges. Digestibility tests were 
made at two different temperatures of 37
o 
C and 50
o 
C. Although the optimum 
 69
temperature for enzymatic hydrolysis was 50
o 
C, tests at 37
o 
C were also made in 
consideration of SSF/SSCF investigated in this work. Digestibilities were determined for 
glucan and xylan separately. For sludges, tests were carried out without pH control 
because of the high ash content which could neutralize any acid. The pH of sludge 
digestion remained near neutral. For hardwood pulp, enzymatic hydrolysis was carried 
out under three different conditions: pH 4.8 (optimum for cellulase), pH 7.0, and with 
addition of CaCO
3
 to the level of 0.5 g/g-glucan matching that of sludge. The enzymatic 
digestion of sludges was dismal giving sugar yields of only 8 - 32% of theoretical 
maximum. The highest digestibility observed was that of hardwood conducted at pH 4.8 
and 50
o 
C which stood at 94.6% and 66.3% for glucan and xylan, respectively. The data 
clearly indicate that the digestibilities of sludges are far below to those of hardwood pulp 
tested at pH 4.8. Hardwood pulp tested at pH 7.0, although much lower than that tested at 
pH 4.8, was significantly higher than that tested with addition of CaCO
3
. These data 
collectively indicate that the inefficient digestion of sludges is mainly due to the presence 
of ash in the sludges. The high level of ash in sludge made it difficult to control pH 
because of the buffering action of CaCO
3
.  Also to be noted here is that the sugar yield 
obtained at 37
o 
C was higher than that obtained at 50
o 
C. This result is unexpected since 
50
o 
C is close to the optimum temperature for the cellulase enzymes. We have no clear 
explanation to offer at this time. It may be that the cellulose-enzyme reaction may be 
hampered by certain ionic components released from the sludges, and more of such 
components are released at high temperature.   
The cellulase enzyme used in the test was supplemented with ?-glucosidase at the 
level of 2 CBU/FPU, to suppress inhibition by cellobiose. Cellobiose was nonexistent in 
 70
the enzymatic hydrolysis of hardwood pulp at pH 4.8 with this enzyme formulation, but 
its accumulation was significant in all of the neutral pH runs. Cellobiose was higher also 
at 37 ?C, versus 50 ?C where two conditions were not favorable for fungal cellulases and 
?-glucosidase. Their activities peak at 50 ? 5 ?C and pH of 4.0 - 5.0 (Saddler and Gregg, 
1998). This perhaps is an indication that ?-glucosidase is more sensitive to pH and 
temperature than the other cellulose components. Marques et al. found the optimum 
condition of enzymatic hydrolysis of recycle sludge is at 35 ?C and pH 5.5. The recycle 
sludge used in their study was neutralized with hydrochloric acid before enzymatic 
hydrolysis (Marques et al., 2008).  
3.4.3 Microbial Contamination of Sludges 
Enzymatic hydrolysis of sludges conducted with addition of antibiotics without 
sterilization has shown very poor results giving digestibility of less than 10% (Table III-
2). To test bacterial contamination, sludge solution was sampled on YPD agar plate. After 
the incubation, the gram-negative bacteria and molds were identified from the gram stain 
test and the morphology of the microorganism. It appears that tetracycline and 
cycloheximide provide only a partial protection as we observed that some 
microorganisms still grew during the enzymatic hydrolysis. Steam sterilization of sludges 
was therefore necessary to eliminate the contamination problem.  
3.4.4 Simultaneous Saccharification and Fermentation 
Recognizing the difficulty of enzymatic hydrolysis of sludges, other means of 
improving the process were sought. The first was to remove the ash content of the 
sludges before subjecting them to bioconversion. This work is currently in progress in our 
laboratory. The second approach was to apply a different and more advanced 
 71
bioconversion scheme for the sludges, namely, the simultaneous saccharification and 
fermentation. The SSF is now considered as the norm for the bioconversion of 
lignocellulosic feedstocks for a number of reasons. One reason is that the enzymatic 
conversion becomes more efficient because of the inhibition of sugars to the enzyme can 
be removed. Simultaneous use of cellulase and microorganism makes the SSF proceed 
under sugar-limited conditions so that the sugar concentration in the bioreactor is kept 
below the inhibition threshold. According to Varga et al (Varga et al., 2002) product 
inhibition starts to occur at 15 g/L of glucose. In addition, the sugar yield obtained from 
the enzymatic digestion of sludges was very low since the pH of sludge digestion 
remained near neutral because of high ash content in the sludge. Some ethanologen 
microorganisms, especially yeast, have a fairly broad pH range, 3.0 - 6.0. The pH range 
for E.coli is near the neutral point. Therefore, the fermentation of sludges by ethanologen 
microorganisms could perform differently. 
With this understanding, a number SSF runs were made using the paper mill 
sludges without removing the ash. The enzyme mix and loading were identical to those of 
the digestibility test. S. cerevisiae, ATCC-200062 (NREL-D5A) was used for the 
conversion of glucose to ethanol. The SSF of sludges was run at 37
o 
C without pH 
control. Two different substrate loadings were applied: 3% and 6% glucan (w/v). The 
time-course results of the SSF run are shown in Fig.III-1, and the yield data are shown in 
Table III-3. The data summarized in Table III-3 shows that approximately 80% of the 
maximum theoretical yield is achieved with 3% glucan loading and 75% yield with 6% 
glucan loading, corresponding to ethanol concentration of 13.6 and 25.5 g/L, 
respectively. The yield of SSF is calculated on the basis of glucan in the sludge because 
 72
S. cerevisiae can only convert glucan, xylan being left unconverted. The 1:1 mixture of 
the two sludges gave about the same ethanol yield.  
The results show that conversion of sludges through SSF is far superior to 
enzymatic digestion. The 75-80% yield of ethanol from the SSF of the sludges is a drastic 
improvement over the 8-32% sugar yield obtained from the enzymatic digestion. It is 
surprising because the front part of the SSF is the enzymatic digestion. The profiles of 
glucose, xylose, and ethanol follow the pattern of well-behaving SSF (Fig. 1). The 
glucose concentrations remained below 0.1 g/L after 24 h signifying that the yeast was 
rapidly consuming any available glucose. The xylose concentrations remained unchanged 
after 24 h since the yeast NREL-D5A cannot convert xylose to ethanol. The two different 
sludges showed identical conversion pattern. Although the terminal ethanol yields were 
the same, hardwood pulp was converted somewhat faster than the sludges.  
The main reason for the improved performance of SSF appears to be the low pH 
level observed in the SSF. The SSF run started at a pH of 7.05 for primary sludge and 
7.12 for recycle sludge and decreased to pH of 5.73 and 5.69, respectively, a level close 
to the optimum for cellulase and for S. cerevisiae. After the initial drop, the pH stayed 
relatively constant throughout the SSF. Since pH is a critical factor in bioconversion of 
sludges, we investigated further on it to verify the reason for the pH drop. We have 
measured the level of acetic acid and lactic acid in our SSF experiments. The maximum 
acetic acid level was 1.8 g/L, and lactic acid was 2.8 g/L. We speculated that these acids 
may be the reason for the pH drop. These organic acids produced in quantities are not 
enough to dissolve all acid-soluble ash.  But they may interact with CaCO
3
 in the sludge 
forming buffers, Ca-acetate or Ca-lactate, eventually lowering the pH.  
 73
To test this possibility, we have carried out titrations adding acetic acid into 
deionized water and water-sludge mixtures, simulating the SSF condition. As shown in 
Fig. 2, pH drop in water was rather drastic changing from 7.0 to 3.5 with addition of 45 
mL of 20 g/L of acetic acid to 500 mL deionized water (equivalent to 4.5 g acetic acid 
per liter matching the total acid produced in the SSF).In the same test with water-sludge 
mixtures, the pH drop was much slower, decreasing from 7.0 to 6.6 with addition of same 
amount of acetic acid. The results indicate that acid production by yeast, although 
contributing to pH drop, cannot explain the entire pH drop in the SSF.  
Another reason one can deduce is carbon dioxide produced by yeast. It is 
produced in large quantities; about the same amount as ethanol, although most of it 
escapes from the broth during the SSF due to its low solubility in water. Carbon dioxide 
in the broth may exist in a supersaturated condition before it is released from the broth. 
One sample of supersaturated carbon dioxide is carbonated soft drinks, which show pH in 
the range of 2.5 - 4.2. The extent of supersaturation during fermentation can be quite high 
even under atmosphere pressure because it has to overcome the surface tension before it 
is released. If so, contribution of carbon dioxide to pH change is further increased to the 
level that it can explain the observed pH drop. Kraemer et al. found that CO
2
 was 
supersaturated during fermentative hydrogen production regardless of N
2
 sparging 
(Kraemer and Bagley, 2006). To what extent carbonic acid contributes to the pH drop of 
SSF is unknown at this time. The organic acid and carbonic acid may interact to give 
additive effects to account for the observed pH drop.  Whatever the reason may be, we 
emphasize that a normal operation of SSF of untreated paper mill sludge proceeds well 
 74
without pH control to give satisfactory performance as a process converting the feedstock 
to ethanol.  
3.4.5 Simultaneous Saccharification and Co-fermentation  
In order to utilize the xylan content in the sludge, another process scheme was 
investigated, namely, simultaneous saccharification and co-fermentation. This process 
uses recombinant E. coli ATCC
?
 55124 (KO11) in place of the yeast in SSF. This 
organism is known to convert xylose as well as glucose to ethanol with high efficiency 
(Ohta et al., 1991). The overall performance data and the time-course data of SSCF are 
presented in Table III-3 and Fig. III-3. The SSCF of sludges was also run at 37
o 
C without 
pH control. Two different substrate loadings were applied: 3% and 6% glucan (w/v). The 
ethanol yields obtained from SSCF are similar to those of SSF ranging from 75% to 80%. 
These yields are calculated on the basis of total carbohydrates (glucan and xylan) for 
obvious reason. The yields of SSCF were also calculated on the basis of glucan only for 
direct comparison with the SSF. The 3% glucan-based yield of ethanol was 98.3% for 
primary sludge and 98.9% for recycle sludge. 6% glucan-based yield of ethanol was 
95.8% for primary sludge and 96.3% for recycle sludge, corresponding to ethanol 
concentration of 32.5 and 32.7 g/L, respectively. In theory, this yield can surpass 100%. 
On the average, SSCF can produce 25% more ethanol from the same feedstock primarily 
because of the ability of KO11 to convert xylose to ethanol. The yields were slightly 
higher with 3% glucan loading than with 6% glucan loading. Elshafei et al. reported that 
substrate loading between 0.5-5% total solids in SSF was not affected by substrate or 
ethanol inhibition (Elshafei, et al., 1991). In this work, total solid loading was much 
 75
higher, ranging from 6% to14% (w/v). Here again the presence of high ash content in the 
sludges was not a problem in operation of the SSCF. 
`The time-course profiles of various observable parameters are shown in Fig. 3. 
There were discernable differences in sugar and pH profiles from those of SSF. The pH 
again dropped sharply in the early phase of the SSCF, and remained relatively constant 
afterwards. The SSCF was started at pH  7.0, decreased to pH of 5.93 for primary sludge 
and 5.89 for recycle sludge, which is close to the optimum for KO11 (pH of 6) but 
somewhat higher than those of SSF. The same argument made for the pH drop of SSF 
also applies here. During the SSCF, acetic acid and lactic acid were detected at the level 
of 3.0 and 1.0 g/L, respectively. The acid-soluble ash can dissolve under weak acidic 
condition and acts as a buffer. The ethanol conversion rate of hardwood pulp was faster 
than the sludges, but the terminal ethanol yields were essentially the same. The xylose 
concentration of both sludges decreased after 12 h. This is a positive proof that the xylan 
fraction is utilized in the SSCF by KO11. 
The profiles of glucose in Figures 1 and 3 showed that both SSF and SSCF 
proceed under a glucose-limited condition, which means the enzymatic reaction is the 
rate-limiting step. In the SSCF, xylose consumption occurred shortly after depletion of 
glucose. This is a classic example of diauxic consumption of substrates wherein glucose 
is preferred to xylose. The SSCF applies only to a feedstock that contains both glucan and 
xylan. It also requires cellulase enzyme that has built-in xylanase activity. Most of the 
commercial ?cellulases? including Spezyme CP have xylanase activity. The reacted 
residues after SSF and SSCF were analyzed for glucan and xylan contents (Table III-4). 
For both sludges, the SSF residues have higher unreacted glucan and xylan than SSCF 
 76
residues. The fact that two different microorganism are used has no bearing in this 
observation because the microbial actions are not the rate-controlling step. Therefore, the 
main difference between the two processes is that SSF has a buildup of xylose.  It is well 
known that xylose has an inhibitory effect on xylanase (Paul and Varma, 1990; Copa-
Pati?o et al., 1993; Rapp and Wagner, 1986). Low xylanase activity results in less xylan 
removal. Unreacted xylan in solid influences the glucan hydrolysis because xylan 
physically surrounds cellulose fibrils in the structure of lignocelluloses and becomes a 
barrier impeding glucanase action. It has indeed been proven that xylan removal 
improves the access of cellulase enzyme to cellulose and consequently the activity of 
cellulase (?hgren et al., 2007). It has also been reported that xylose has a direct inhibitory 
effect on cellulase (Xiao et al., 2004; Nigam and Prabhu, 1991 Kim and Lee, 2005).  
3.4.6 Fed-Batch Operation 
The concentration of ethanol in the bioreactor significantly affects the cost of the 
downstream separation process. The threshold level of ethanol concentration for 
economically feasible distillation is reported to be about 4 wt. % (Hahn-H?gerdal et al., 
2006; Wingren et al., 2003). We have attempted the fed-batch operation (intermittent 
input of feed) of SSF and SSCF in an effort to increase the terminal ethanol concentration 
in the bioreactor. The bioreaction was started with a sludge loading of 60 g-glucan/L. 
Additional feedstock of the same level was put into the reactor at 24 h point (one 
additional input) for SSCF and at 24 and 48 h points for SSF (two additional inputs). At 
these input points, the reaction has proceeded far enough to liquefy the dense solid slurry 
and retain fluidity high enough to accept additional solid feed. The ethanol production 
from the fed-batch runs are shown in Fig. 4. In the case of SSCF with one additional 
 77
feeding, ethanol concentration of 42 g/L was obtained at the end of the run. The yields 
were identical for both sludges. This is equivalent to ethanol yield of 68% on the basis of 
total sugar, which is substantially lower than the yield of single batch with 6% glucan 
loading. E. coli KO11 is known to utilize several pentoses and hexoses for producing 
ethanol with high metabolic yield (Ohta et al., 1991). However, its growth robustness and 
ethanol tolerance are relatively low compared to yeast (Dien et al., 2003). We believe it is 
the main reason for the low ethanol yield observed from the fed-batch SSCF. Additional 
buildup of ash from the sludge may also have played a role in lowering the ethanol yield. 
Xylose utilization in the fed-batch SSCF was somewhat inefficient compare to single-
feed runs, as evidenced by relatively high xylose concentration in the reactor which 
remained at 2 g/L even after 72 h. On the other hand, glucose had a short initial buildup 
and then remained below 0.1 g/L after 24 h. In the fed-batch SSF runs with two 
additional feedings, the final ethanol concentration of 45 g/L was achieved. The overall 
ethanol yield in the fed-batch SSF was approximately 70% based on glucan, which was 
also lower than the yield of single batch with only 6% glucan loading. In fed-batch SSF 
using yeast (ATCC-200062), ethanol concentration higher than SSCF was obtained, yet 
the overall yield was lower. Viability of the cells at the end of fed-batch SSF was tested 
by measurement of cell count on agar plate. The measured cell concentration of 5?10
7
 
CFU /mL indicated that the cells remained highly viable at the end of the operation. It 
appears that the cellulase enzyme somehow becomes less effective under high solid 
loading. The interference of high ash with the enzyme, unproductive binding with 
increased lignin content after successive feeding, product inhibition by ethanol and/or 
acids, and substrate inhibition by unreacted xylose are the potential reasons for the low 
 78
yield of the fed-batch SSF. Mass transfer resistance at the solid - liquid interface and 
within the solids may further increase the local concentration of some of the inhibitory 
components. The exact reasons and the mechanisms associated with these phenomena are 
yet to be determined.  
3.5 Conclusion 
Kraft paper mill sludges have features desirable for bioconversion to value-added 
products. The organic component of the sludges is essentially the same as commercial 
grade pulp, high in carbohydrates and low in lignin. Its small size and high surface area 
make it amenable for biological conversion without pretreatment. However, enzymatic 
hydrolysis of the sludges is inefficient because of the high ash content. The buffering 
action of the calcium carbonate in the ash keeps the pH near to the neutral point and two 
units higher than the optimum pH of cellulase.  
SSF based on Spezyme CP and S. cerevisiae (D
5
A), and the SSCF based on the 
same enzyme and the recombinant E. coli-KO11 performed well despite the high ash 
content. The main reason for the improved performance is the pH drop to 5.5 and 5.8 that 
occurred in the SSF and SSCF. The decrease of pH was caused by carbonic acid and 
organic acids produced during fermentation. The glucan-based ethanol yield for the 
primary sludge and recycle sludge in the SSF were 79.5% and 80.0%, respectively. In the 
SSCF using recombinant E. coli KO11, xylose was effectively utilized. The ethanol yield 
based on glucan and xylan was 80.1% and 77.1 %, respectively, for the primary sludge 
and recycle sludge. Fed-batch SSF and SSCF were attempted to increase the ethanol 
concentration. The ethanol concentration in the broth was increased to 42 and 45 g/L, for 
two-feed SSCF and three-feed SSF, respectively. The yields of the fed-batch runs were 
 79
lower at 68% for SSF and 70% SSCF. Further increase of ethanol concentration in the 
SSCF was difficult because of the low ethanol tolerance of E.coli KO11.  Yields are 
lower for multiple-feed fed-batch runs than the single-feed runs for both SSF and SSCF. 
The low conversion yield is related to the cellulase enzyme reaction rather than microbial 
reaction. High-solid conditions in the bioreactor and the product and substrate inhibition 
are believed to be the major factors suppressing the cellulase enzyme reaction. 
 
 
 80
Table III-1 Composition of sludges and corn stover 
 
 
Primary sludge Recycle sludge Corn stover 
Glucan (%) 44.5 49.6 36.1 
Xylan (%) 9.9 13.7 21.4 
Lignin (%) 8.1 4.6 17.2 
Total Ash  (%) 36.0 30.0 7.1 
Acid-soluble Ash (%) 26 22 - 
Acid-insoluble Ash (%) 10 8 - 
Others (%) 1.5 2.1 18.2 
Alkalinity 
(milliliters of 1 N H
2
SO
4
/10 g dry 
sludge) 
60 50 - 
 
a. All data in table are based on oven-dry original feedstocks. 
b. Data of sludges are the mean value of triplicate (n=3; standard 
deviation<0.5 for glucan, xylan, and lignin).  
c.  The composition of the corn stover was determined by the National 
Bioenergy Center, NREL, Golden, CO). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 81
Table III-2 Enzymatic digestibility of paper sludges    
 
Sugar released at 72-h enzymatic 
hydrolysis (g/L) 
72-h digestibility 
(%) 
 
Temperature 
(?C) Glucose  Cellobiose Xylose Glucan Xylan 
50 0.6 0.3 0.2 8.3 7.9 
Primary sludge 
a
 37 2.5 1 0.8 32.0 31.7 
50 0.7 0.3 0.3 9.2 9.6 
Recycle sludge 
a
 37 2.4 0.9 0.9 30.2 28.7 
50 1.4 0.7 0.7 19.3 22.1 Hardwood pulp 
(HP) with  
0.5 g CaCO3/ g-
glucan 
a
 37 2.6 1 0.8 32.9 25.2 
50 10.5 0 2.1 94.6 66.3 HP with 0.05 M 
citrate buffer 
(pH=4.8) 
a
 37 9.1 0 1.8 82.0 56.8 
50 3.6 0.6 0.8 38.1 25.2 HP with 0.05 M 
phosphate buffer 
(pH = 7) 
a
 37 4.8 0.5 0.9 48.0 28.4 
Primary sludge 
b
 37 0.7 0.3 0.1 9.2 4.0 
Recycle sludge 
b
 37 0.6 0.3 0.1 8.3 3.2 
 
Data of sludges and hardwood pulp is the mean value of duplicate (n = 2; SD < 
1.0). 
a. Sterilization methods applied by autoclaving.  
b. Sterilization methods applied by antibiotics.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 82
Table III-3 Ethanol yield from SSF and SSCF of paper sludges  
 
Primary sludge (PS) Recycle sludge (RS) 
Mixture of PS and 
RS (1:1)  
 
6 % 
glucan 
loading 
3 % 
glucan 
loading 
6 % 
glucan 
loading 
3 % 
glucan 
loading 
6 % 
glucan 
loading 
3 % 
glucan 
loading 
SSF ethanol 
yield(%) 
a
 
74.5 79.5 75.0 80.0 74.6 80.0 
SSCF 
ethanol 
yield (%) 
b
 
78.0 80.1 75.1 77.1 77.0 80.5 
SSCF 
ethanol 
yield (%) 
c
 
95.8 98.3 96.3 98.9 94.5 98.8 
 
Data of sludges is the mean value of triplicate (n = 3; SD < 3.0). 
a. The ethanol yield of SSF based on glucan only. 
b. The ethanol yield of SSCF based on glucan and xylan. 
c. The ethanol yield of SSCF based on glucan only. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 83
Table III-4 Composition analysis of fermentation residues  
 
Primary sludge 
residue
 a
 
Recycle sludge 
residue
 a
 
Component (%) 
Primary 
sludge  
SSF
 b
 SSCF
 b
 
Recycle 
sludge 
SSF
 b
 SSCF
 b
 
Glucan 
44.5 
1.6 0.5 49.6 1.9 0.5 
Xylan 
9.9 
2.4 0.8 13.7 4.1 0.9 
 
Data of sludges is the mean value of triplicate (n = 3; SD < 0.5 for glucan and 
xylan).  
a. All data in table are based on oven-dry original sludges. 
b. SSF and SSCF were based on 3% (w/v) glucan loading. 
  
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 84
5
5.5
6
6.5
7
7.5
0 2040608010120
Time (h)
pH
 
0
2
4
6
8
10
12
14
0 20 40 60 80 100 120
Time (h)
E
t
ha
nol
 C
onc
e
n
t
r
a
t
i
o
n (
g
/
L
)
 
0
0.5
1
1.5
2
2.5
0 2040608010120
Time (h)
Gluc
os
e
 C
onc
e
n
t
r
a
t
io
n (
g
/L)
 
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 2040608010120
Time (h)
X
y
l
o
se Co
n
cen
t
r
at
i
o
n
 
(
g
/
L
)
 
 
Fig.III-1 Simultaneous saccharification and fermentation (SSF) of sludges by 
Saccharomyces cerevisiae (ATCC-200062)  
Squares with connecting lines represent primary sludge. Triangles with connecting lines 
represent recycle sludge. Filled triangles with connecting lines represent hardwood pulp. 
The data points represent average of triplicate runs. The pH was controlled at 4.8 for runs 
with hardwood pulp. Other conditions of the SSF were: 3% (w/v) glucan loading, 15 FPU 
Spezyme CP + 30 CBU of Novozyme-188/g-glucan. 
 
 85
3
4.5
6
7.5
0 5 10 15 20 25 30 35 40 45 50
20g/L HAc (mL)
pH
 
 
Fig.III-2 Buffer capacity titration curves with acetic acid for paper sludge slurries and 
deionized water 
Squares with connecting lines represent primary sludge. Triangles with connecting lines 
represent recycle sludge. Filled triangles with connecting lines represent water. 
The data points represent average of duplicate runs.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 86
5
5.5
6
6.5
7
7.5
0 2040608010120
Time (h)
pH
 
 
0
5
10
15
20
0 20 40 60 80 100 120
Time (h)
E
t
h
a
n
o
l
 Co
n
cen
t
r
at
i
o
n
 (
g
/
L
)
 
0
0.5
1
1.5
2
2.5
3
3.5
0 20 40 60 80 100 120
Time (h)
G
l
u
c
o
se Co
n
cen
t
r
at
i
o
n
 
(
g
/
L
)
 
0
0.5
1
1.5
2
2.5
0 2040608010120
Time (h)
X
y
lo
s
e
 C
o
nc
e
n
t
r
a
t
io
n 
(
g
/
L
)
 
 
Fig.III-3. Simultaneous saccharification and cofermentation (SSCF) of sludge by 
recombinant E.coli (KO11) 
Squares with connecting lines represent primary sludge. Triangles with connecting lines 
represent recycle sludge. Filled triangles with connecting lines represent hardwood pulp. 
The data points represent average of triplicate runs. The pH was controlled at 6.0 for runs 
with hardwood pulp. Other conditions of the SSCF were: 3% (w/v) glucan loading, 15 
FPU Spezyme CP + 30 CBU of Novozyme-188/g-glucan. 
 
 87
0
5
10
15
20
25
30
35
40
45
50
0 30 60 90 120 150
Time(h)
E
t
ha
nol
 C
onc
e
n
t
r
a
t
i
on (
g
/
L
)
Recycle Sludge-SSF
Primary Sludge-SSF
Primary Sludge-SSCF
Recycle Sludge-SSCF
  SSCF  Feeding
      
  SSF  Feeding
 
 
Fig.III-4. Fed-batch SSF and SSCF of sludges  
The data points represent average of triplicate runs. Other conditions of the SSF were: 
6% (w/v) glucan loading, 15 FPU Spezyme CP + 30 CBU of Novozyme-188/g-glucan. 
 
 88
IV. De-ashing Treatment of Paper Sludges and Enhancing Ethanl 
Production by SSF and SSCF of De-ashing Paper Sludge 
 
4.1 Abstract 
The previous study in our lab, the ?Bioconversion of Kraft Paper Mill Sludges to 
Ethanol by SSF and SSCF?, has demonstrated that paper sludges with high ash content 
work well in both Simultaneous Saccharification and Co-Fermentation (SSCF) and 
Simultaneous Saccharification and Fermentation (SSF). However, operations of using 
sludges with high ash content are difficult. In this work, de-ashing processes before SSCF 
and SSF were developed to overcome the difficulty. Low ash content sludges provide 
relatively higher ethanol yields, a result that was achieved with enzyme loading of 15 
FPU/g-glucan. Reduction in enzyme loading to 10 FPU/g-glucan decreased the ethanol 
yield, but a 70% yield was still obtained. The effect of different levels of solid loading in 
the fermentation test was also investigated. In addition, different low-cost fermentation 
nutrients were evaluated for their effectiveness in the fermentation of paper sludges to 
produce ethanol. When the SSCF and SSF were operated in fed-batch mode by using 
low-ash content sludges, the ethanol concentration in the broth was increased to 48 g/L 
and 60 g/L, respectively.  
4.2 Introduction 
Feedstock and the enzymes were identified as two primary cost items in the 
biomass conversion processes (Nguyen and Saddler, 1991; Aden and Foust, 2009). In our
 89
previous study, we have demonstrated that the SSF based on Spezyme CP and 
Saccharomyces cerevisiae (NREL-D5A), and the SSCF based on the same enzyme and 
the recombinant  Escherichia coli -KO11 performed reasonably well in bioconversion of 
untreated kraft paper sludges to ethanol in spite of the high ash content (Kang et 
al.,2010). SSCF performed with 13.5% solid loading of primary paper sludge resulted 
ethanol concentration of 3.25 wt. %; and SSF with the same feed resulted ethanol 
concentration 2.65 wt. %. Higher ethanol concentration is achieved with high solid 
loading. However, operation of high solid with high ash content creates extremely high 
viscosity causing difficulty in mixing and pumping. High viscosity and non-Newtonian 
behavior of the broth require strong agitation in order to provide adequate mixing during 
fermentation. The ash content comes primarily from paper filler materials such as clay 
(kaolin) and CaCO
3
, etc.  In our previous study, we found that the sludge hampers the 
enzymatic reaction primarily by the buffering action of the CaCO
3 
in the ash, which drifts 
the pH away from the optimum value for cellulase (Kang et al., 2010). Nikolov et al., 
(2000) reported that fillers and other additives in the paper form an adhesive ?envelope? 
around the cellulose fiber to obstruct the access of the enzymes to the cellulose substrate. 
Clay minerals are known to form aggregates with organic molecules, and the formation 
of enzyme-clay complexes could alter the level of enzyme activity (Tietjen and Wetzel, 
2003; Cabezas et al., 1991; Haska, 1981). Additional evidences were also found that clay 
had an inhibitory effect on cellulase activity (Hamzehi and Pflug, 1981; Pflug, 1982; 
Tothill et al., 1993). The high ash content in paper mill sludges also limits the cellulose 
loading capacity in the bioreactor since it lowers the cellulosic portion of the feedstock. 
Much of these problems can be alleviated by de-ashing of the sludges. 
 90
A number of pulp and paper mills have implemented reclamation of fiber, fillers, 
or both from the sludges to reuse the separated materials and also as a means to reduce 
sludge volume in the pulp and papermaking processes (Hardesty and Beer, 1993; 
Rosenqvist, 1978; and Soderhjelm, 1976). Several different methods were used to recover 
the fiber and/or fillers from paper sludge (Leuthold and Leuthold, 1996; Maxham, 1994; 
Simpson and Lam, 1998). The equipment used to separate fiber or fillers from sludge 
varies but it can be as conventional as screens and cleaners (Dorica and Simandl, 1995). 
Reclamation and reuse of sludge in pulp and papermaking processes, however, has not 
been practiced widely because of the concern that it may lower the quality of the final 
product. In normal process of de-ashing, the purity of the fiber has inverse relationship 
with the recovery yield of the fiber. For bioconversion purpose, the de-ashing operation 
needs to be optimized in order to recover as much fiber as possible while keeping the ash 
content low enough to be accepted as a fermentation feedstock. Various chemical 
treatments were also used for de-ashing (Lynd et al., 2001; Marques et al., 2008). 
However, they are not deemed appropriate for application in bioconversion feedstock 
because of high processing cost and safety issues. 
Fermentation nutrient is a significant factor in bioconversion of lignocellulosic 
biomass to ethanol. Expensive complex nutrients, such as yeast extract and peptone, are 
utilized in most laboratory studies, the cost of which is prohibitively high for commercial 
production. Corn steep liquor is a byproduct of corn wet-milling and has been used as a 
fermentation nutrient in several different commercial fermentation processes (Kadam and 
Newman, 1997; Lawford and Rousseau, 1997; Amartey and Jeffries, 1994; Tang et al., 
2006; Underwood et al., 2002).  
 91
This investigation was undertaken to improve the process of converting the kraft 
paper mill sludges to ethanol from the level we have previously developed in our 
laboratory. The focus of the work was on three main issues. The first was to develop a 
method to de-ash kraft paper mill primary sludge. The second was to ascertain the effects 
of de-ashing on the efficiency of bioconversion to ethanol. The third was to improve the 
process economics deploying low-cost fermentation nutrients. As tools of bioconversion 
tests, the Simultaneous Saccharification and Co-Fermentation (SSCF) and Simultaneous 
Saccharification and Fermentation (SSF) were operated under batch and fed-batch 
modes. Special attention was paid on the product yield, concentration, and enzyme 
dosage in order to assess the overall process performance. 
4.3 Materials and Methods 
4.3.1 Materials 
Feedstocks: The paper primary sludge (PS) was collected from the primary 
wastewater clarifier unit of a Kraft paper mill, Boise Paper Company (Jackson, AL, 
USA). The PS was washed by tap water three times and further thickened to 30% 
consistency using a vacuum filter, and stored at 4
o 
C. A sample quantity of wet hardwood 
pulp (HP) was also obtained from Boise Paper Company. It was used as a reference 
substrate in bioconversion studies. The paper sludge and hardwood pulp were analyzed 
for carbohydrates, moisture content, and ash content according to the National 
Renewable Energy Laboratory (NREL) standard procedure (NREL, 2008). Hardwood 
pulp was analyzed to contain 76.0% glucan, 21.1% xylan, and 2.9 wt. % unaccounted for 
(lignin, acetyl group, ash, and protein). Primary sludge was analyzed to contain 44.5 % 
glucan, 9.9 % xylan, 8.1% lignin and 36.0 wt. % ash, which includes 26.0% acid-soluble 
 92
ash and 10.0% acid-insoluble ash. De-ashing was done by treating by screening. It was 
then thickened to about 30% consistency using a vacuum filter.  The screening procedure 
was that of Dorica et al., (Dorica and Simandl, 1995) with a slight modification that CO
2
 
was used instead of air. One liter of resuspended paper sludge with 3% consistency was 
placed in a 2 L beaker and mixed at 300 RPM using a laboratory stirrer (LR400A, Fisher 
Scientific) for 30 minutes with carbon dioxide bubbles through two glass tubings, and put 
through 100 mesh screen. This procedure was repeated three times and thickened to about 
45% consistency using a vacuum filter. The resulting solid was referred as S-PS hereafter 
and was subjected to bioconversion tests. The PS after the two consecutive screenings 
was referred to as S-PS-1 and the PS after three consecutive screenings referred to as S-
PS-2 (Fig.IV-1.). S-PS-1 was analyzed to contain 64.8% glucan, 13.5% xylan, 5.6% 
lignin and 14.0 wt. % ash. S-PS-2 was analyzed to contain 71.2% glucan, 14.8% xylan, 
6.2% lignin and 6.1 wt. % ash. 
Enzymes: Cellulase enzyme (Spezyme CP, Lot No. 301-00348-257) was a kind 
gift from Genencor-Danisco (Palo Alto, CA, USA). The specific activity of Spezyme CP 
was 59 FPU/mL, as determined by NREL standard procedure (NREL, 2008). ?-
glucosidase (Novozyme188, Cat. No. C-6150) was purchased from Sigma (St. Louis, 
MO, USA). Its specific activity was measured to be 750 CBU/mL.  
Microorganism: The microorganism used in the SSF was Saccharomyces 
cerevisiae ATCC-200062 (NREL-D
5
A). This organism was grown on YPD agar plates 
containing solid YPD medium, which contained 2.0% peptone (Sigma, P-6588), 1.0% 
yeast extract (Sigma, Y-0500), 1.5% agar (Sigma, A-1296) and 2.0% (w/v) glucose 
(Sigma, G-8270). The recombinant Escherichia coli ATCC-55124 (KO11) was used for 
 93
the SSCF. This organism was grown on LB agar plates containing solid LB medium 
(Sigma, L-3152) , which contained 1% tryptone, 0.5% yeast extract, and 1% NaCl, 
supplemented with 2% xylose (Sigma, X1500), 1.5% agar (Sigma, A-1296) and 40 mg/L 
or 600 mg/L chloroamphenicol (Fluka, 23275) (Yomano et al.,1998). 
4.3.2 Colony Forming Unit Test 
The colony forming unit (CFU) tests were performed to determine the viable cell 
population. The YPD or LB agar plate was prepared for this purpose diluting the culture 
with sterile saline (0.89% NaCl solution) to obtain a spread plate cell count of 30-300 
cells/plate. 
4.3.3 Simultaneous Saccharification and Fermentation /Co-fermentation  
A 250 mL Erlenmeyer flask was used as a bioreactor with 100 mL total working 
volume. It was operated in an incubator shaker (New Brunswick Scientific, Innova-4080) 
at 37 ?C. Sludges and growth medium were added such that the glucan content becomes 
3% or 6% (w/v).  Hardwood pulp (HP-I) and hardwood pulp with 20% CaCO
3
 based on 
dry basis of hardwood pulp (HP-II) were used as control and put through the same 
bioconversion procedures. The sludge samples were steam sterilized at 121 ?C for 15 
min. The growth media for SSF was YP medium. The growth media for SSCF was LB 
medium (Sigma, L-3152). For SSCF, 40 mg/L of chloroamphenicol was added after 
autoclaving to prevent bacterial contamination (Yomano et al., 1998).  
The SSF and SSCF of hardwood pulp were carried out at initial pH of 4.8 in 0.05 
M sodium citrate buffer and pH 6.0 in 0.05 M sodium phosphate buffer, respectively, 
without further control of pH during fermentation.  Three levels of cellulase loadings 
were applied: 5, 10 and 15 FPU /g-glucan in paper sludge. The cellulase enzyme was 
 94
supplemented with ?-glucosidase at a ratio of 2 CBU/FPU. The optical density (OD) was 
measured by a UV Spectrophotometer (BioTek Synergy HT Multidetection Microplate 
Readers) at 600 nm for yeast (NREL-D
5
A) and 550 nm for Recombinant E. coli (KO11). 
The initial OD after inoculation was 0.05, equivalent to 16 mg dry cell weight of KO11/L 
and 50 mg of NREL-D
5
A/L. In all of the microbial experiments, a sample from each 
flask was taken at the end of the run and streaked on an YPD plate to check for 
contamination. The presence of contaminants was also checked under an optical 
microscope. CFU tests were performed to check microorganism viability. The SSF and 
SSCF experiments requiring pH monitoring were carried out using a 1-L bioreactor 
(Multifors IHORS HT 2?1L) with 400 mL working volume.  
The ethanol yield was calculated as follows: 
100
0.511reactorin  (g)Sugar  Initial
reactorin  (g) produced Ethanol
 maximum] al theoreticof [% yield Ethanol ?
?
=  
Sugar is interpreted as glucose in the SSF and glucose plus xylose in the SSCF. 
The fed-batch experiments were started with 100 mL initial working volume, and 
squeezed sludge cakes were added to the bioreactor at the desired time to achieve the 
total glucan content of 6% (w/v). Addition of sludge was done at the 24 h for SSCF and 
at 12, 24 and 48 h for SSF. The cellulase and ?-glucosidase were also added to maintain 
overall enzyme loading at 10 FPU and 20 CBU/g-glucan. Samples were analyzed for 
glucose, xylose, organic acid, and ethanol by HPLC. Aseptic conditions were maintained 
in all of the microbial experiments. The fed-batch fermentation runs were made in 
triplicates.  
The fermentation runs for the low-cost medium study were operated in an 
incubator shaker (New Brunswick Scientific, Innova-4080) at 37 ?C with 150 RPM. The 
 95
cellulase and ?-glucosidase were also added to maintain enzyme loading at 10 FPU and 
20 CBU/g-glucan.  Two growth media were used for the SSF test of screen de-ashed 
paper sludges: (I) ?YP medium? containing 10 g/L yeast extract (Difco, Detroit, MI) and 
20 g/L peptone (Difco), and (II) ?low-cost medium? containing 0.45% by volume corn 
steep liquor (CSL) (Sigma C4648, 50% solid) and 5 mM MgSO
4
 as described by 
Newman et al. (Kadam and Newman, 1997). 
For the SSCF test of screening de-ashed paper sludges, seven growth media were 
used: (A) a ?LB medium? containing 5 g/L yeast extract (Difco, Detroit, MI) and 10 g/L 
peptone (Difco), and  (B) 1.0% CSL by volume (Sigma C4648, 50% solid) without 
mineral supplement; (C) 1.0% CSL by volume with mineral supplement as described by 
Martinez et al. (Martinez, A. et al. 1999); (D) 3.0% CSL by volume without mineral 
supplement; (E) 3.0% CSL by volume with mineral supplement; (F) 5.0 % CSL by 
volume without mineral supplement ; (G) 5.0 % CSL by volume with mineral 
supplement. Mineral supplements per liter for CSL + M medium were: 1 g of KH
2
PO
4
, 
0.5 g of K
2
HPO
4
, 3.1 g of (NH
4
)
2
SO
4
, 0.4 g of MgCl
2
?6H
2
O and 20 mg of FeCl
3
?6H
2
O. 
4.3.4 Analytical Methods 
The solid samples were analyzed for carbohydrates and Klason lignin following 
the NREL CAT standard procedures (NREL, 2008). The moisture content was measured 
by an infrared moisture balance (Denver Instrument, IR-30). Sugars were determined by 
HPLC using an HPX-87P column. For the SSF or SSCF tests, a BioRad-HPX-87H 
column was used for measurement of sugar, organic acid and ethanol. A refractive index 
detector was used with the HPLC. The acid-insoluble ash was determined following the 
Tappi test method (T 244 cm-99). Liquid sample analysis and ash determination were 
 96
done in triplicates. Where applicable, statistical data including mean value and standard 
deviation were computed using Microsoft Office Excel 2003.   
4.4 Results and Discussion 
4.4.1 De-ashing of Sludges by Screen-Washing 
The PS was further analyzed for the ash contents to give 26% acid soluble 
fraction and 10% acid-insoluble fraction based on oven-dry untreated sludge. In reference 
to the Boise Paper making process and the analysis of data, we conclude that acid soluble 
ash is primarily from CaCO
3
, and acid- insoluble ash is from TiO
2
 and clay.  Part of the 
insoluble ash content was physically associated with the fibers concurring to the findings 
of Middleton and Scallan (1991). The suspended fine particles not associated with fibers 
are presumed to pass through the screen with the filtrate. Separation of fiber or fillers 
from sludge has been practiced in pulp and paper industry using commercially available 
screens and cleaners (Dorica and Simandl, 1995). Table IV-1 shows the glucan/xylan 
content and their loss in the screening process. With S-PS-1, the glucan/xylan loss was 
11.2%/17.2% when the ash removal was 76.4%. As indicated by the data on screening of 
S-PS-2, increase of ash removal caused increase of the glucan/xylan loss; glucan/xylan 
loss corresponding to 90.6% ash removal was 25.6%/34.3%.   The CO
2
 bubbles enhance 
the extent of separation of the ash from the fibers. The gas bubbles rise to the top surface 
carrying the particle of low density, such as fine fiber, to the surface of the liquid phase. 
Carbon dioxide
 
was used in place of air since a large amount of it is produced during the 
fermentation process, and it is also a by-product in the manufacture of lime from calcium 
carbonate during the kraft chemical recovery process (Biermann, 1996). Use of CO
2
 has 
additional benefit that it increases the dissolution of CaCO
3
. The solubility of CO
2 
in the 
 97
water is much higher than air (88 mL CO
2
/100 mL of water at 20 ?C) and its aqueous 
solution is carbonic acid, a weak unstable acid. The water insoluble calcium carbonate in 
the presence of water and carbon dioxide dissolves as water soluble calcium bicarbonate 
which is a compound existing only in solution. Fig.IV-2.A through C shows increasing 
degree of separation after successive treatments of mechanical means (screening).  
4.4.2 SSF and SSCF of Screened De-ashing of Sludges  
Data on S-PS-1 and PS in Table IV-2 clearly indicate that ash removal increases 
the ethanol fermentation yield. The ethanol fermentation yields of the S-PS-2 with the 
lowest ash content at very low enzyme loading of 5 FPU cellulase /mL were higher than 
those of the original sludge at medium enzyme loading of 10 FPU cellulase /mL. The 
adverse effect of ash in the sludge is primarily on the enzymatic reaction part of the 
SSF/SSCF rather than the microbial reaction. A number of researchers found that various 
components in the ash interfere with cellulase reaction (Nikolov et al., 2000; Tietjen and 
Wetzel, 2003; Cabezas et al., 1991; Haska, 1981; Hamzehi and Pflug, 1981; Pflug, 1982; 
Tothill et al., 1993). Improvement of yield by de-ashing is therefore due to improvement 
in the enzymatic hydrolysis. It is quite obvious that de-ashing removed the chemicals that 
inhibit enzymatic hydrolysis and/or fermentation process. The fact that SSF/SSCF 
proceeds under glucose limited condition (Fig. IV-3 and IV-4) further proves that the 
hydrolysis is the rate-limiting step of the SSF/SSCF. On the other hand, ash removal 
causes loss of fine fibers, therefore loss the glucan/xylan. As shown in the case of S-PS-2, 
90% removal of ash accompanied loss of glucan/xylan as high as 25.6%/34.3% during 
the screen de-ashing.  Considering large glucan/xylan loss during the screening of S-PS-
2, and that only 6% difference in ethanol yield between the two enzyme loading of 15 
 98
and 10 FPU cellulase /g-glucan), S-PS-1 was used in the subsequent bioconversion tests 
in which medium enzyme loading  of 10 FPU cellulase/g-glucan was applied.  
Ash removal also decreases the total bulk solid loading for a given glucan content 
in the feedstock. As an example, for 6% glucan loading, the total solid loading of the 
original primary sludge, S-PS-1 and S-PS-2   were 13.5 wt.%, 9.3 wt.% and 8.4 wt.%, 
respectively. Fermentation of high viscosity broth requires strong agitation to provide 
adequate mixing and mass transfer. Agitation intensity as it relates to solid loading thus 
became of our interest. When sludge and enzyme loadings increase, the viscosity of the 
reaction mixture increases accordingly. Fermentation results indicate that the dense 
sludges (high solid loading) require longer time for liquefaction (breakdown of viscosity). 
The ethanol yields from fermentation were also lower at higher solid loading (Table IV-
3). With high solid loading (21.6% w/v for PS), the enzymatic hydrolysis was insufficient 
to liquefy the sludge, resulting in a very low ethanol yield (data not shown here). In a 
separate research in our laboatory, we found that the binding capacity of cellulase 
decreased when the substrate concentration increased (Wang et al., 2011). This has a 
negative effect on the productivity because the enzymatic hydrolysis is the rate-limiting 
step in the SSF. There are a number of reports that suggests agitation enhances the 
hydrolysis yield of cellulosic substrates. The literature information along these lines 
collectively indicate that agitation enhances the adsorption of cellulase to the substrate, 
therefore increasing the activities of the endoglucanases and the cellobiohydrolases 
(Cavaco-Paulo and Almeida, 1994; Cavaco-Paulo et al.,1996; Cavaco-Paulo et al.,1998; 
and Sakata et al.,1985). Most of data in Table IV-4, except the ethanol yield from SSCF 
of PS at relatively high agitation (250 RPM),  agree clearly show that agitation has a 
 99
positive effect on ethanol production in the SSF and SSCF, which agree with the previous 
findings. Enhanced interaction between enzyme and the solid substrates of sludge due to 
high mixing is a plausible reason for improvement of ethanol production. In addition, 
agitation may also improved mass transfer of sugar into microorganisms.   
However, the ethanol yield from SSCF of PS at relatively high agitation (250 
RPM) was lower than the medium agitation (150 RPM) (Table IV-4). It was probably 
due to the fact that the E.coli is less viable under high agitation as measured by CFU 
tests. It is well known that growth robustness of E.coli is low compared to yeast (Dien et 
al., 2003). The yeast used in this work is very robust and can withstand high agitation 
under anaerobic condition. It was reaffirmed by our CFU tests that showed no observable 
damage after the SSF. Mechanical damage of the E.coli by shear stress could result in a 
slower growth under high agitation. Glass bead milling is frequently used for cell lysis 
for production of intracellular products from microorganisms, in which the harvested 
cells are agitated with glass beads in a closed chamber (Kura and Sch?tte, 1987). In our 
sludge processing, 4.9% (w/v) ash content was present in the fermentation broth of 6% 
(w/v) glucan loading of untreated PS. This ash may have exerted the same effect as glass 
beads. 
4.4.3 Evaluation of Low-cost Fermentation Medium for Fermentation 
The yeast extract and peptone are high quality nitrogen sources containing 
vitamin B complex and amino acids, yet the cost is prohibitively high to be used as 
nutrient for industrial fermentation. Replacement of the laboratory medium with practical 
nutrients is one of the major technical hurdles to be cleared for the development of 
industrial fermentation from laboratory work. The fermentation route to produce ethanol 
 100
from biomass is no exception. Substitution of the laboratory medium with alternative 
inexpensive nitrogen source medium, however, has often resulted slow rates and low 
yield in ethanol production.  Corn steep liquor (CSL), which is low in carbohydrates and 
rich in proteins, is considered as a suitable replacement for yeast extract in fermentation 
media (Thomsen, 2006). It is currently used widely in industrial fermentation processes.   
A number of SSF runs were made using screen de-ashed paper mill sludges 
without pH control. The enzyme loadings were: 10 FPU/g-glucan and 20 CBU/g-glucan. 
Saccharomyces cerevisiae, ATCC-200062 (NREL-D
5
A) was used for the conversion of 
glucose to ethanol. Two different substrate loadings were applied: 3% and 6% glucan 
(w/v). The ethanol yield of S-PS-1 from the SSF with lean medium (II) was 71.1% of 
theoretical maximum with 6% glucan loading. It is very close to the ethanol yield 
achieved with the rich medium (I) -72.8% under the same conditions. This is in 
agreement with the previous findings that CSL is a good substitute for yeast extract and 
peptone (Kadam and Newman, 1997; Amartey and Jeffries, 1994; and Tang et al., 2006).  
In our previous study (Kang et al., 2010), the ash in the sludge, especially calcium 
carbonate, was partially neutralized by the acids produced from the SSCF and SSF and 
acted as a buffer to stabilize the pH during fermentation. After screening, S-PS-1 still 
contained 8.0% acid-soluble ash, which is primarily CaCO
3. 
The time-course results of 
the SSF run are shown in Fig. IV-3.
 
During the fermentation of S-PS-1, HP-I and HP-II , the pH quickly dropped and 
reached the final values (at 120 h) of 5.7, 4.3 and 5.6, respectively. The decrease of pH 
occurred due to carbonic acid (CO
2
 dissolved in water) and other organic acids formed 
during fermentation. This pH drop by CO
2
 and organic acid is counteracted by the 
 101
buffering action of ash in the S-PS-1, calcium carbonate in the HP, and CSL in the lean 
medium to reach the respective final pH values in the bioreactor. Since CSL contains 
proteins, peptides and amino acids, the addition of CSL may strengthen the buffering 
capacity of the medium (Stanburg and Whitaker, 1984).  
In general, the activity of Saccharomyces cerevisiae is stable under neutral or 
slightly acidic and anaerobic conditions. Under the anaerobic conditions, the intracellular 
pH of Saccharomyces cerevisiae is usually maintained between 5.5 and 5.75 when the 
external pH is 3.0 (Imai and Ohno, 1995a) or between 5.9 and 6.75 when the external pH 
is between 6.0 and 10.0 (Imai and Ohno, 1995b). The various enzymes involved in yeast 
metabolism of sugars are located within the yeast cell, the enzyme activity is unaffected 
if the internal pH of the yeast cell is stable. The ethanol yields of S-PS-1, HP-I, and HP-II 
with 3% glucan loading are essentially the same: 73.7%, 73.7%, and 72.4%, respectively. 
This yield level is also very close to that from fermentation of hardwood pulp under pH 
4.8 (73.0%). Our previous study (Kang et al., 2010) has shown that although the terminal 
ethanol yields were the same, hardwood pulp was converted faster than the untreated 
sludges. After screen de-ashing, S-PS-1 attained almost identical ethanol producing rate 
as that of HP. 
In order to utilize the xylan content in the sludge, the SSCF was also performed. 
In this process, recombinant E.coli ATCC 55124 (KO11) was used in place of the yeast. 
This organism is known to convert xylose as well as glucose to ethanol with high 
efficiency (Ohta et al., 1991). This strain has performed well with high metabolic yields 
in rich media. However, the yields of both strains declined in a minimal media (Martinez 
et al., 1999; York and Ingram., 1996). The poor performance in a minimal media may be 
 102
attributed to NADH-mediated inhibition of citrate synthase, limiting the availability of 
glutamate, a protective osmolyte (Underwood et al. 2002a and 2002b). In this work, 
different media were tested for their performance in ethanol fermentation by E.coli KO11 
from the screen de-ashed paper sludges. The ethanol yield data are shown in Table IV-5. 
The yields were calculated on the basis of total carbohydrates (glucan and xylan). The 
yields of SSCF were also calculated on the basis of glucan only for direct comparison 
with the SSF.  On the average, the SSCF produced 15% more ethanol than the SSF from 
the same feedstock because KO11 can convert both glucose and xylose to ethanol.  
Our initial approach was to use CSL as a source of protein and vitamin, NH
4
Cl as 
an inorganic nitrogen source with other minerals. For 1% CSL supplementation, addition 
of minerals increased the ethanol yield from 53.1% to 57.4%. At CSL levels above 1%, 
supplemention minerals showed no significant improvement in ethanol production. With 
1% CSL and very low amount of yeast extract (0.05%) (medium H), the ethanol yield 
reached 73.5%, which was almost identical to that of rich medium.  Although the 
terminal ethanol yields were almost identical, S-PS-1 in the rich medium was converted 
faster than that in medium H. The 3% CSL alone (medium F) appears to provide 
sufficient nutrients to achieve ethanol yield of 71.2 %, which is comparable to that from 
rich medium A (73.6%). There was no apparent improvement in ethanol yields with CSL 
level above 3%. These results support the findings of previous studies that the 
recombinant strain of E. coli KO11 does not produce ethanol efficiently without the 
addition of large amounts of complex nutrients (Asghari et al., 1996; York and Ingram, 
1996). It is also noteworthy that the initial cell density is low, 16 mg dry cell weight of 
KO11/L. In this study, CSL was tested as a nutritional supplement for E.Coli-KO11. The 
 103
3% CSL was shown to meet the nutrient requirement, enough to attain cell growth to the 
level obtainable from rich medium A. The economic feasibility of using medium G and H 
in this process is yet to be investigated. There are some technical issues concerning the 
use of CSL as fermentation media; 1) need for sterilization to remove bacterial 
contaminants, 2) difficulty of product separation caused by impurities in the broth that are 
originated from CSL; 3) increase of waste treatment burden due to high BOD in the spent 
fermentation liquor, and 4) inconsistent composition of CSL that varies with the source 
(Lee, 2005; Lawford and Rousseau, 1997). Handling of CSL is not easy because it is 
highly viscous and tends to gelate turning into sticky liquid. In view of the 
aforementioned problems associated with CSL, the subsequent tests were performed 
using medium H containing low CSL (1%) and low amount of yeast extract (0.05%). The 
time-course profiles of various observable parameters of SSCF based on medium H are 
shown in Fig. IV-4. 
Of our keen interest here was to observe how the pH profiles vary with different 
nutrient media and substrates.  The pH dropped sharply in the early phase of the SSCF 
due to production of carbonic acid (CO
2
 dissolved in water) and other organic acids, and 
remained relatively constant afterwards. The SSCF was started with pH 7, but decreased 
to 5.9 for S-PS-1. In the HP-I run, however, the pH quickly dropped from 6.8 to 4.2, 
similar to the pH profile of the SSF process. Much like CSL, the yeast extract and 
peptone also have a certain level of buffering capacity (Terzaghi and Sandine, 1975; 
Hugo and Lund, 1968). However, at low level, their buffer capacity was not high enough 
to significantly affect the pH behavior of the SSCF of the HP-I. Presence of CaCO
3
 
enhances the buffering capacity of the medium. The pH of SSCF of HP-II with CaCO
3
 
 104
dropped from 6.8 (0 h) to 5.8 (120 h), which is similar to the pH profile of the SSCF of S-
PS-1. Ethanologenic derivatives of E. coli B function efficiently between pH 5.8 and pH 
7.5 (Beall et al., 1991). Often pH 6.0 is used as a practical optimum because it lowers 
solubilization of CO
2
 (Moniruzzaman et al., 1998). The pH in the fermentation HP-II 
remained below 5 after 6 h. Insufficient use of xylose, even glucose, and low ethanol 
yield indicates that E. coli KO11 was inhibited under low pH. Moniruzzaman et al also 
found that only 2 h exposure at pH 3 could cause long delays in cell growth and low 
ethanol yield (Moniruzzaman et al., 1998). 
4.4.4 Fed-batch Operation 
The concentration of ethanol in the bioreactor is a factor significantly affecting 
the cost of the downstream separation process. Alcohol concentration of 40 g/L has been 
mentioned as a target value in biomass fermentations (Hohmann and Rendleman, 1993). 
We attempted the fed-batch operation (intermittent input of feed) of SSF and SSCF in an 
effort to increase the terminal ethanol concentration in the bioreactor. The bioreaction 
was started with the initial sludge loading of 60 g-glucan/L. Additional feedstock of the 
same level was put into the reactor at 24 h for SSCF and at 12, 24 and 48 h for SSF. At 
these input points the reaction has proceeded far enough to liquefy the dense solid slurry 
and retain fluidity high enough to accept additional solid feed. The ethanol production 
from the fed-batch runs are shown in Fig.IV-5. 
In the case of SSCF with one additional feeding, ethanol concentration of 47.8 
g/L was obtained at the end of the run. This is equivalent to an ethanol yield of 70% on 
the basis of total sugar with 10% w/v total glucan loading, which was slightly lower than 
the yield of single-batch with 6% glucan loading. However, it was still higher than the 
 105
ethanol yield of 68% from untreated PS even with a lower enzyme loading and a low-cost 
medium. This is a significant benefit in the SSF gained specifically by de-ashing of the 
sludge. The reason for the low ethanol yield observed from the fed-batch SSCF is that the 
ethanol tolerance of E. coli KO11 is relatively low compared to yeast (Dien et al., 2003, 
Kang et al., 2010). In the fed-batch SSF runs with three additional feedings, the final 
ethanol concentration of 60 g/L was achieved, which was much higher than that that of 
the fed-batch SSF of untreated primary sludge, 45 g/L, with two additional feedings. The 
overall ethanol yield in the fed-batch SSF was approximately 70% based on glucan -
15%w/v (23.1%w/v total solid loading), which was also lower than the yield of single 
batch with 6% glucan loading, yet comparable to that of the fed-batch SSF of untreated 
PS with a lower enzyme loading and a low-cost medium. The yield and product 
concentration data collectively indicate that the removal of ash and other extraneous 
components has positive effects on the bioconversion process.  
4.5 Conclusion 
Kraft paper mill sludges have features desirable for bioconversion to value-added 
products. Without any cleaning, the sludges can be converted to ethanol by SSF or SSCF 
with reasonable efficiency (Kang et al., 2010). The bioconversion processes, however, 
can be significantly improved by de-ashing and deinking of the sludges. The major 
benefits of de-ashing are: (1) the ethanol yields in the SSF or SSCF are significantly 
improved, (2) the solid loadings into the bioreactor are increased, the latter leading to 
higher ethanol concentration. The ethanol yields for the de-ashed primary sludge (S-PS-
1) reached 72.8% and 73.6% of the theoretical maximum for SSF and SSCF, 
respectively.  It is particularly notable that these yields were achieved with relatively low 
 106
enzyme loading of 10 FPU/g-glucan and 20 CBU/g-glucan. The often used laboratory 
fermentation media, yeast extract and peptone can be replaced by CSL or at least 
supplemented at a reduced level, without adversely affecting the ethanol yield. Fed-batch 
SSF and SSCF were attempted to increase the ethanol concentration. The ethanol 
concentration in the broth was increased to 47.8 g/L and 60 g/L, for 2-feed SSCF and 4-
feed SSF, respectively.  
 107
     Table IV-1 Composition of de-ashed sludges 
 (%) PS S-PS-1
d
 S-PS-1
e
 S-PS-2 
d
 S-PS-2
 e
 
Glucan 44.5 38.4 39.5 32.4 33.1 
Xylan 9.9 7.8 8.2 5.9 6.5 
Ash 36.0 10.7 8.5 4.5 3.4 
Glucan loss - 13.7 11.2 27.2 25.6 
Xylan loss - 21.2 17.2 40.4 34.3 
Ash removal - 70.3 76.4 87.5 90.6 
 
a. The data of glucan, xylan and ash in table are based on oven-dry untreated 
primary sludge. 
b. The data of glucan loss, xylan loss and ash removal in table are based on 
the initial glucan, xylan and ash of oven-dry untreated primary sludge. 
c. All Data are the mean value of duplicate (n=2; standard deviation<1.0). 
d. Without carbon dioxide. 
e. With carbon dioxide. 
 108
Table IV-2 Ethanol yield from SSF and SSCF of treated paper sludges  
 
Data of sludges are the mean value of duplicate (n=2; standard deviation<0.1). 
a. The ethanol yield of SSF based on glucan only. 
b. The ethanol yield of SSCF based on glucan and xylan. 
c. The ethanol yield of SSCF based on glucan only. 
d. SSF and SSCF based on 6% (w/v) glucan loading/ 100 mL working 
volume. 
 
Enzyme 
loading 
15 FPU of Cellulase + 30 
CBU of ?-glucosidase /g-
glucan 
10 FPU of Cellulase + 20 
CBU of ?-glucosidase/g-
glucan 
5 FPU of Cellulase + 10 
CBU of ?-glucosidase/g-
glucan 
 
P.S. S-PS-1 S-PS-2 P.S. S-PS-1 S-PS-2 P.S. S-PS-1 S-PS-2 
SSF 
Ethanol 
Yield(%) 
a
 
74.5 77.6 81.2 67.1 72.8 75.7 60.5 64.5 70.2 
SSCF 
Ethanol 
Yield(%) 
b
 
78 80.4 82.8 68.2 73.6 76 59.6 65.1 71.8 
SSCF 
Ethanol 
Yield(%) 
c
 
95.8 97.5 100.4 83.7 89.3 92.2 73.2 78.9 87.1 
 109
Table IV-3 Ethanol yield from SSF and SSCF of paper sludges at different solid loading  
Sludge PS S-PS-1 
Solid loading (%w/v) 6.7 13.5 20.2 4.6 9.3 13.9 
Glucan loading (%w/v) 3.0 6.0 9.0 3.0 6.0 9.0 
SSF Ethanol Yield(%) a 68.8 66.1 - 74.3 72.8 71 
SSCF Ethanol Yield(%) b 72.4 69.8 - 75.1 73.6 70.8 
SSCF Ethanol Yield(%) c 87.8 85.7 - 91.1 89.3 85.9 
 
a. Simultaneous Saccharification and Fermentation (SSF) based on glucan 
only. 
b. Simultaneous Saccharification and co-Fermentation (SSCF) based on 
glucan and xylan. 
c. Simultaneous Saccharification and co-Fermentation (SSCF) based on 
glucan. 
d. Data of sludges are the mean value of duplicate (n=2; standard 
deviation<0.1). 
e. Enzyme loading, 10 FPU of Cellulase + 20 CBU of ?-glucosidase /g-
glucan. 
 110
Table IV-4 Ethanol yield from SSF and SSCF of sludges at different agitation intensity  
Sludge PS S-PS-1 
Agitation intensity (rpm) 250 150 100 250 150 100 
SSF Ethanol Yield(%) a 67.7 66.1 58.4 74.4 72.8 69.0 
SSCF Ethanol Yield(%) b 64.4 69.8 61.1 74.9 73.6 70.1 
SSCF Ethanol Yield(%) c 79.1 85.7 75.0 90.9 89.3 85.0 
 
a. Simultaneous Saccharification and Fermentation (SSF) based on glucan 
only. 
b. Simultaneous Saccharification and co-Fermentation (SSCF) based on 
glucan and xylan. 
c. Simultaneous Saccharification and co-Fermentation (SSCF) based on 
glucan. 
d. Data of sludges are the mean value of duplicate (n=2; standard 
deviation<0.1). 
e. Enzyme loading, 10 FPU of Cellulase + 20 CBU of ?-glucosidase /g-
glucan. 
f. 6% (w/v) glucan loading / 100 mL working volume. 
 111
Table IV-5 Ethanol yield from SSCF of de-ashed paper sludges on different medium 
Medium 
Yeast 
Extract 
(%w/v) 
Peptone 
(%w/v) 
CSL 
(%v/v) 
Mineral 
Supplement 
SSCF 
Ethanol 
Yield(%) 
a
 
SSCF 
Ethanol 
Yield(%) 
b
 
A 0.5 1 0 0 73.6 89.3 
B 0 0 1 0 53.1 64.4 
C 0 0 1 Yes 57.4 69.6 
D 0 0 2 0 64.4 78.1 
E 0 0 2 Yes 65.6 79.6 
F 0 0 3 0 72.1 87.5 
G 0 0 3 Yes 71.2 86.4 
H 0.05 0 1 0 73.5 89.2 
 
All Data are the mean value of duplicate (n=2; standard deviation<1.0). The 
Enzyme loading was: 10 FPU of Cellulase + 20 CBU of ?-glucosidase /g-glucan. 
a. The ethanol yield of SSCF based on glucan and xylan. 
b. The ethanol yield of SSCF based on glucan only. 
c. SSCF based on 6% (w/v) glucan loading. 
 
 
 
 
 112
 
 
 
 
 
 
 
 
 
Fig.IV-1. De-ashing process of the primary sludge. 
CO
2
 
CO
2
 
100 mesh Sieve 
S-PS-1 
3% paper sludge slurry 
CO
2
 
CO
2
 
 113
  
 
   
Fig.IV-2. Scanning electron micrograph (SEM) of de-ashed and untreated sludge 
A. untreated primary sludge (?2000).  
B. primary sludge after two consecutive screenings (?2000).  
C. primary sludge after two consecutive screenings (?2000).  
 114
4
4.5
5
5.5
6
6.5
7
7.5
0 30 60 90 120
Time (h)
pH
 
0
2.5
5
7.5
10
12.5
0 30 60 90 120
Time (h)
E
t
ha
nol
 Conc
e
n
t
r
a
t
i
on 
(g/
L
)
 
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 30 60 90 120
Time (h)
G
l
uc
o
s
e
 Conc
e
n
t
r
a
t
i
on (
g
/
L
)
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 30 60 90 120
Time (h)
X
y
lo
s
e
 C
o
n
c
e
n
tr
at
io
n
 (
g
/L
)
 
Fig.IV-3. Simultaneous Saccharification and Fermentation (SSF) of de-ashed Sludges by 
Saccharomyces cerevisiae (ATCC-200062)  
Squares represent de-ashed primary sludge (S-PS-1). Triangles represent hardwood pulp 
(HP-I). Filled triangles represent hardwood pulp with 20% CaCO3 on dry basis of 
hardwood pulp (HP-II). 
The data points represent the average of triplicate runs. The pH was not controlled. Other 
conditions of the SSF were: 3% (w/v) glucan loading, 37?C, 10 FPU Spezyme CP + 20 
CBU of Novozyme-188/g-glucan. 
 115
4
4.5
5
5.5
6
6.5
7
7.5
0 30 60 90 120
Time (h)
pH
 
0
2
4
6
8
10
12
14
16
0 30 60 90 120
Time (h)
E
t
ha
nol
 Conc
e
n
t
r
a
t
i
on (g/
L
)
 
0
3
6
9
12
15
0 30 60 90 120
Time (h)
G
l
uc
os
e
 Conc
e
n
t
r
a
t
i
on (g/
L
)
 
0
1
2
3
4
5
6
0 30 60 90 120
Time (h)
X
y
lo
s
e
 C
o
n
c
e
n
tr
at
io
n
 (
g
/L
)
 
Fig.IV-4. Simultaneous saccharification and co-fermentation (SSCF) of de-ashed sludges 
by Escherichia coli KO11 (ATCC-55124) 
Squares lines represent de-ashed primary sludge (S-PS-1). Triangles represent hardwood 
pulp (HP-I). Filled triangles represent hardwood pulp with 20% CaCO3 on the basis of 
dry hardwood pulp (HP-II). 
The data points represent the average of triplicate runs. The pH was not controlled. Other 
conditions of the SSF were: 3% (w/v) glucan loading, 37?C, 10 FPU Spezyme CP + 20 
CBU of Novozyme-188/g-glucan. 
 
 116
0
10
20
30
40
50
60
70
0 30 60 90 120 150
Time (h)
E
t
ha
nol
 Con
c
e
n
t
r
a
t
i
o
n
 (
g
/
L
)
S-PS-1-SSF
S-PS-1-SSCF
SSF Feeding
SSCF Feeding
 
Fig.IV-5. Fed-Batch SSF and SSCF of S-PS-1 
The data points represent the average of triplicate runs. Other conditions of the SSF were: 
6g initial and additional feeding of glucan loading (9.3 g dry basis S-PS-1), 37?C, and 10 
FPU Spezyme CP + 20 CBU of Novozyme-188/g-glucan. Total glucan loading for SSCF 
was 10% w/v and for SSF was 15% w/v. 
 117
V. Cellulase Production from Unbleached Karft Hardwood Pulp and 
Paper Sludge 
 
5.1 Abstract 
Cellulase on-site production can be a supplementary unit in a kraft paper mill 
production system. By using cellulase and Saccharomyces cerevisiae on kraft paper 
sludge, which is waste from the kraft pulp making process, it is possible to produce 
bioethanol. It is feasible and convenient to integrate these two processes without any 
impact on the production of paper goods and on the production of bioethanol. The 
cellulase enzyme was produced by Trichoderma reesei Rut C-30 and was investigated in 
a 3 L fermenter for its productivities and titers.  Experiments were conducted over a 
range of possible pH values to determine the impact upon T. reesei Rut C-30 growth and 
cellulase production. Different concentrations of unbleached kraft processed hardwood 
pulp were examined to determine the maximum levels of cellulase activity obtainable. 
The highest titer of 7.5 FPU/mL was obtained when using 4% (w/v) hardwood pulp. 
Paper sludge was also considered as a potential feedstock for cellulase production. 
However, it also has a high ash content which is detrimental to cell growth. It is desirable 
to remove ash as much as possible from the sludge, while retaining carbohydrates. In this 
study, the de-ashed sludge pre-processed via physical and chemical treatment was used as 
substrates for cellulase production and ethanol fermentation. The cellulase enzyme
 118
produced from de-ashed sludge exhibited cellulase activity as high as 8.0 FPU/mL. The 
sugar and ethanol yields using the crude enzyme solutions in the enzymatic hydrolyisis 
and fermentation test were compared with a commercially available enzyme, Spezyme
CP. In addition, cellulase produced from both substrates showed slightly higher xylanase 
activity. 
5.2 Introduction 
The bioconversion of paper sludge to bioethanol is an intriguing possibility in the 
wood- based biorefinery industry.  In our previous research (Kang et al., 2010), we found 
that ethanol production could be a supplementary part of an integrated biorefinery in a 
kraft process paper mill, while still preserving the main products, paper and paper related 
products. One of the bottlenecks in the production of bioethanol is the high cost from the 
usage of the enzymes utilized in the hydrolysis step (Steele et al., 2005; Spano et al., 
1978). The cost of bioethanol and cellulase production could be reduced by simplifying 
the processing steps. The operating cost and capital investment of this supplementary unit 
can be reduced by sharing the existing facilities of the pulp mill thus utilizing the utilities 
and the power that may be available at low cost from the pulp mill operation.    
The soft-rot filamentous fungus, Trichoderma reesei, holds the greatest potential 
for reducing the cost in the production of enzymes for lignocellulosic biomass conversion 
(Egyhazi et al. 2004; Gadgil et al. 1995; Juhasz et al., 2004; Kadam and Keutzer, 1995).  
T. reesei Rut-C30, a mutant of T. reesei, was isolated using a combination of ultraviolet 
irradiation and nitrosomethyl guanidine (NTG) by Montenecourt and Eveleigh 
(Montenecourt and Eveleigh, 1979).  Most previous works are based on this mutant. 
 119
The current laboratory investigations primarily have been devoted to reduce the 
cost of enzyme production. Carbon source is one of the main factors in microbial 
cellulase production (Ryu and Mandels, 1980; Campbell and Laherrehe, 1998). In order 
to convert a kraft paper mill to an integrated biorefinery, cellulase production using 
carbon sources directly from the kraft mill could be feasible, convenient, and 
economically viable.  Sulfite wood pulp (Watson et al., 1984), pretreated wood (Juhasz et 
al., 2005; Shin et al., 2000), sawdust (Lo et al., 2005), waste paper (Royer and Nakas, 
1987; Chen and Wayman, 1991; Shin et al., 2000), waste cardboard (Szij?rt? et al., 2004) 
and paper sludge (Royer and Nakas, 1987; Maheshwari et al., 1994) could be used as 
carbon sources for cellulase production. Shin et al. reported production of cellulase by T. 
reesei Rut C-30 on various lignocellulosic substrates (the newspaper sludge, office paper 
sludge, and steam-exploded woods treated in various ways). The steam-exploded wood 
showed the best properties for substrate in cellulase production. Chen and Wayman used 
waste newspaper and sorbose in the T. reesei Rut C-30 cellulase production and partially 
enzymatic saccharified newspaper was found to be most effective. The enzymatic 
hydrolyzate of acid pretreated wastepaper was also used as soluble-inducing substrate for 
cellulase production in continuous culture of T. reesei Rut C-30 (Ju and Afolabi, 1999). 
Chahal used NaOH treated chemithermo mechanical pulp as a carbon source in the solid-
state fermentation for the T. reesei Rut C-30 cellulase production (Chahal et al, 1985).  
Using kraft pulp on site has the benefit of providing a high quality carbon source 
for cellulase production. The alkaline delignification of wood causes the lignin 
degradation predominantly by the cleavage of lignin-hemicellulose bonds (Wang et al., 
1967).  In addition, delignification causes changes in the structure of the cellulose fiber 
 120
(Lewin and Roland, 1971). One advantage of using kraft pulp is that the fungus can 
easily assimilate the substrate and quickly initiates the cellulase production. Kraft paper 
maybe a good substrate for cellulase production because the fibers in the sludge are fine 
kraft pulp because they are screen rejects from paper machine and the digester, and are 
easily digested by cellulase enzymes (Fan and Lynd, 2006a; Fan and Lynd, 2006b; Scott 
et al., 1995; Jeffries and Schartman ,1999; Fan, et al.,2003).  However, using paper mill 
sludge as a source for cellulase production is rarely studied. Maheshwari et al used paper 
mill sludge as a potential source for cellulase production, in which a mixed cultivation of 
T. reesei QM 9123 and Aspergillus niger was used (Maheshwari et al., 1994) , however, 
scaling up cellulase fermentation in bioreactors and properties of the cellulase produced 
from sludge have not been well studied. 
There are several factors which govern the cellulase production. pH is one of the 
main factors that has a significant role in cellulase production (Ryu and Mandels, 1980; 
Xiong et al., 2004; Mukhopadhyay and Nandi, 1999; Wayman and Chen, 1992; Kadam 
and Keutzer, 1995). The impact of carbon source on cellulase production has been well 
documented. In addition to pH, this study also investigated on how different 
concentrations of unbleached kraft processed hardwood pulp could have an impact upon 
the efficiency of cellulase enzyme production by T. reesei. The production of cellulase 
using industrial wastes, de-ashed and chemical treated paper mill sludge, as the main 
carbon source, was also investigated. The objective of this study was to evaluate the 
paper mill sludge for its applicability in economical cellulase production. The enzymatic 
hydrolysis experiments were intended to be performed by using cellulase produced by 
 121
both substrate and by Spezyme CP at the same enzyme loading based on the substrate 
(filter paper units (FPU)/g of cellulose). 
5.3 Materials and Methods 
5.3.1 Materials 
Feedstock: The unbleached hardwood pulp and primary paper sludge were 
collected from a kraft paper mill (Boise Paper Company Jackson, AL, USA). Primary 
sludge (PS), discharged from the paper machine and the digester, was collected from the 
primary wastewater clarifier unit. Both were stored at 4
o 
C. The paper sludge and 
hardwood pulp were analyzed for carbohydrates, moisture content, and ash content 
according to the standard biomass analytical procedure of the National Renewable 
Energy Laboratory (NREL) (NREL, 2008). Hardwood pulp was analyzed to contain 
76.0% glucan, 21.1% xylan, and 2.9 wt. % unaccounted for (lignin, acetyl group, ash, and 
protein). Unbleached hardwood pulp was ground in a Oster 14-Speed Blender, and then 
milled in an IKA MF 10 basic mill (IKA Werke Gmbh & Co., Satufen, Germany) to give 
a particle size lower than 20 mesh. Primary paper sludge was analyzed to contain 44.5 % 
glucan, 9.9 % xylan, 8.1% lignin and 36.0 wt. % ash, which includes 26.0% acid-soluble 
ash and 10.0% acid-insoluble ash. De-ashing was done by treating by screening 
according to the previou chapter. The deashed paper sludge was analyzed to contain 
64.8% glucan, 13.5% xylan, 5.6% lignin and 14.0 wt. % ash. De-ashed sludge was 
leached with 1 N sulfuric acid and further water washing until pH around 6.0. Acid 
leaching sludge was analyzed to contain 72.6% glucan, 15.8% xylan, 4.4% lignin and 4.7 
wt. % ash.  The acid leaching sludge was leached with 1 N sodium hydroxide and further 
 122
water washing until pH around 7.5. Alkaline leaching sludge analyzed to contain 74.5% 
glucan, 15.0% xylan, 3.7% lignin and 3.8 wt. % ash. 
Enzymes: The cellulase enzyme (Spezyme CP, Lot No. 301-00348-257) was a 
kind gift from Danisco Genencor (Palo Alto, CA, USA). The activity of Spezyme CP was 
59 FPU/mL, as determined by NREL standard procedure (NREL, 2008). 
Microorganism: The microorganism used in the Simultaneous Saccharification 
and Fermentation (SSF) was Saccharomyces cerevisiae ATCC-200062 (NREL-D5A). 
The growth media was YP medium, which contained 1% yeast extract (Sigma, Y-0500) 
and 2% peptone (Sigma, P-6588). 
T. reesei Rut C-30 (ATCC 56765) was used in the study of cellulase production T. 
reesei was grown on potato dextrose agar (Difco Lab., Detroit, USA) slants. 5 days after 
inoculation at 28 ?C, the greenish conidia were harvested and suspended in sterile water. 
The spore suspension was ready for inoculation to the media of cellulase production.  
Seed culture of  T. reesei was grown in a 500 mL Erlenmeyer flask on a medium 
containing: 2 g/L KH
2
PO
4
, 1.4 g/L (NH
4
)
2
SO
4
; 0.3 g/L MgSO
4
; 0.005 g/L FeSO
4
; 0.075 
g/L MnSO
4
; 0.0015 g/L ZnSO
4
; 0.002 g/L CoCl
2
; 0.3 g/L CaCl
2
; 0.3 g/L urea; 0.015 g/L 
Tween 80; 0.1 g/L peptone; 5g/L glucose; 10 g/L unbleached kraft hardwood pulp. 
Silicone sponge closures (part 2004-00005; Bellco Glass, Vineland, NJ) were used to cap 
the flasks. 
5.3.2 Cellulase Production 
A seed culture containing 150 mL of medium in a 500 mL Erlenmeyer flask was 
inoculated with 150 ?L of spore suspension (107 spores/mL) and incubated at 28 ?C on a 
rotary shaker at 180 rpm. After two days of pre-cultivation, a 150 mL inoculum from the 
 123
seed culture was inoculated into the production medium in order to initiate the cellulase 
production. The scale up enzyme production experiments were performed in a 3 L 
fermenter (New Brunswick, USA) with an operating volume of 1.5 L. The temperature, 
pH, agitation, aeration and dissolved O
2
 were automatically monitored and controlled in 
this fermenter. The composition of production medium for cellulase was the same as that 
of the corresponding growth medium, except that it was supplemented with different 
concentration of hardwood pulp (from 10 to 50 g/L) or different concentration of de-
ashed sludge instead of 10 g/L grounded hardwood pulp and 5 g/L glucose as the carbon 
source. Sterile diluted (1:10) antifoam (BYK-019 water defoamer) was added manually 
to control foaming. Temperature was maintained at 28 ?C. The pH was allowed to drop 
from initial pH 6.0 to three different values, 3.5, 4.0 and 5.0, after which it was adjusted 
not to fall below those values. The pH was controlled by the automatic addition of 
NH
4
OH, which also supplied the nitrogen necessary for the synthesis of excreted 
proteins. Dissolved O
2
 was maintained above 25% of the saturation by varying the 
agitation and aeration rates. Approximately 15 mL of fermentation broth were taken 
aseptically from the vessel, and it was immediately subjected to phase separation. 
Supernatants collected by centrifugation (5600 ? g, 10 min) were assayed for reducing 
sugar content, dry cell weight, extracellular protein, and cellulase activity. 
5.3.3 Enzyme Analysis 
Cellulase activity of the samples was determined as filter paper activity (FPA) 
expressed in filter paper units (FPU) according to Mandels? procedure (Mandels et al., 
1976). 3,5- dinitrosalicylic acid (DNS) method  (Miller ,1959) was used to estimate the 
reducing sugar released in 60 minutes from a mixture of 0.5 mL appropriately diluted 
 124
enzyme solution (supernatant of culture broth), 1 mL 0.1 M acetate buffer (pH 4.8), and 
50 mg Whatman No.1 filter paper (Whatman Lab., Hillsboro, USA), incubated at 50 ?C. 
One FPU was defined as the amount of enzyme that releases 1 ?mol glucose/min. based 
on 2 mg glucose released in 60 minutes, according to National Renewable Energy Lab 
procedure (NREL, 2008). Activities were reported as FPU/mL. Carboxymethylcellulase 
(CMCase; endoglucanase) activity was determined by measuring the release of reducing 
sugars from 1% carboxymethylcellulose (Sigma) at 50 ?C and pH 4.8 (0.1 M acetate 
buffer) for 10 min. (Wood and Bhat, 1988).  
The xylanase activity was determined by following the release of reducing sugars 
from a 1% birchwood xylan (Sigma) solution at 50 ?C for 5 min (Bailey et al., 1992). 
Xylanase activity was defined as the amount of enzyme that released either 1 ?mol of 
glucose or xylose as reducing sugar equivalents per min.  
The ?-Glucosidase activity was determined by measuring the release of 
nitrophenol from 4-nitrophenyl-?-D-glucopyranoside (Sigma) for 15 minutes at 50 ?C 
and pH 4.8 (0.05 M acetate buffer) (H?gerdal et al., 1979). The reaction was stopped by 
the addition of 1 M Na
2
CO
3
, and the absorbance was measured at 410 nm from a 
nitrophenol standard curve. One unit of activity was defined as the amount of enzyme 
that released 1 ?mol of 4-nitrophenol per min.  
Extracellular protein was assayed according to Bradford?s method (Bradford, 
1976), using bovine serum albumin as the standard. Dry cell weight (DCW) was assayed 
according to Morikawa?s method (Moedcawa et al., 1985), measuring optical density 
after treatment with 1 N perchloric acid. One gram of mycelial dry weight per liter 
corresponded to 0.65x absorbance at 260 nm.         
 125
5.3.4 Enzymatic Hydrolysis Test 
The enzymatic digestibility of the sludge was determined according to NREL 
Chemical Analysis and Testing Standard Procedures (NREL, 2008). Two different 
substrates, unbleached hardwood pulp and dilute-acid-pretreated corn stover (NREL) 
were each tested for enzymatic hydrolysis. The dilute-acid pretreated corn stover had the 
composition of: 60.0% glucan, 2.5% xylan, 33.6% Klason lignin, 1.2% acid soluble 
lignin, and 5.7 wt. % unaccounted for acetyl group, ash, and protein. The enzyme loaded 
was 15 FPU/g-glucan of Spezyme CP or the cellulase produced in this work from both 
substrates. Screw-capped 250 mL Erlenmeyer flasks were used as the hydrolysis reactor. 
The samples were suspended in DI water, and put into the flasks to reach a total volume 
of 100 mL such that the glucan content of the substrates become 1% (w/v) in the reactor. 
The enzymatic digestibility tests were carried out at 50?C or 36?C, and pH 4.8 using 0.05 
M sodium citrate buffer in an incubator shaker (New Brunswick Scientific, Innova-4080) 
agitated at 150 rpm. The hydrolyzate samples were taken at 6, 12, 24, 48, and 72 h, and 
analyzed for glucose, xylose, and cellobiose. Total released glucose and cellobiose after 
72 h of hydrolysis were used to calculate the enzymatic digestibility:   
100
)(_111.1
)(_053.1)(_cos
(%) ?
?
?+
=
gaddedGlucan
greleasedCellobiosegreleasedeGlu
ityDigestibil  
The digestibilities for xylan contents were also determined in a similar manner. 
For xylan digestibilities, hydration factor of 1.136 was used in the equation. 
5.3.5 Simultaneous Saccharification and Fermentation (SSF) 
A 250 mL Erlenmeyer flask was used as the bioreactor. It was operated in an 
incubator shaker (New Brunswick Scientific, Innova-4080) at 37?C with 150 rpm with 
100 mL working volume. Sludges and growth medium were added such that the glucan 
 126
content becomes 3% or 6% (w/v). The hardwood pulp also underwent the same 
procedure as a control. The sludge samples were steam sterilized at 121 ?C for 15 min. 
The growth media for SSF was YP medium, which contained 1% yeast extract (Sigma, 
Y-0500) and 2% peptone (Sigma, P-6588).  
The SSF of sludges was carried out 37 ?C without pH control. The cellulase 
enzyme loading was 15 FPU of Enzyme from both substrates or Spezyme CP and 30 
CBU of Novozyme-188/g-glucan. Optical density (OD) was measured by UV 
Spectrophotometer (BioTek Synergy HT Multidetection Microplate Readers) at 600 nm 
for yeast (NREL-D5A). The initial OD after inoculation was 0.05, equivalent to 50 mg of 
NREL-D5A/L. In all of the microbial experiments, a sample from each flask was taken at 
the end of the run and streaked on an YPD plate to check for contamination. Presence of 
contaminants was also checked under an optical microscope. The ethanol yield was 
calculated as follows: 
100
0.511reactorin  (g)Sugar  Initial
reactorin  (g) produced Ethanol
 maximum] al theoreticof [% yield Ethanol ?
?
=
 
Sugar is interpreted as glucose in the SSF. 
Fed-batch fermentation was carried out at 37? C in a 250 mL Erlenmeyer flask. 
Experiments were started with 100 mL initial working volume, and squeezed sludge 
cakes were added to the bioreactor at desired time to achieve the total glucan content of 
6% (w/v). Addition of sludge was done at the 24 h point for the SSF. Cellulase enzymes 
were also added to achieve overall enzyme loading at 15 FPU and 30 CBU/g glucan. 
Samples were analyzed for glucose, xylose, organic acid, and ethanol by HPLC. Aseptic 
 127
conditions were maintained in all of the microbial experiments. The fed-batch 
fermentations runs were made in triplicates.   
5.3.6 Analytical Methods 
The solid samples were analyzed for carbohydrates and Klason lignin in 
triplicates following the NREL standard procedures (NREL, 2008). The moisture content 
was measured by an infrared moisture balance (Denver Instrument, IR-30). Sugars were 
determined by HPLC using an HPX-87P column. For the SSF tests, a BioRad-HPX-87H 
column was used for measurement of sugar, organic acid and ethanol. A refractive index 
detector was used in the HPLC. Liquid sample analysis was done in triplicates. The 
element analysis was done by Soil Testing Laboratory in Auburn University. The metals 
were analyzed with a Varian Vista Axial ICP. Where applicable, statistical computation 
including mean value and standard deviation was performed using Microsoft Office 
Excel 2003.   
5.4 Results and Discussion 
5.4.1 Effect of Different pH on Cellulase Production from Hardwood Pulp 
pH has a major impact on the amount of enzymes produced by T. reesei. A 
combination of pH values and type of carbon sources will result in different yields of 
cellulose. For example, fermentation under pH 3.0 to 4.0 gave the best result on pure 
cellulose carbon source (Ryu and Mandels, 1980), while the optimum pH was 4.0 to 5.0 
on lactose carbon source. Higher initial pH turned to be better when working with 
lignocelluloses (Xiong et al., 2004). R. Doppelbauer et al. (R. Doppelbauer et al., 1987) 
found a constant pH 6.0 during the fermentation gave optimal yields in the case of wheat 
straw, waste paper and sulfite pulp as substrates. 
 128
In this study, the pH was allowed to drop from an initial pH of approximately 6.0 
to three different values of 3.5 (Condition I), 4.0 (Condition II), and 5.0 (Condition III).  
Once the desired ?floor? pH was reached, the environment was adjusted to prevent any 
further decline. 
Fig.V-1 shows the pH of cultivation strongly influenced the production of 
cellulase.  The highest cellulase activity was obtained at Condition I.  With the increase 
in pH the cellulase activity decreased gradually.  The cellulase activity reached its 
maximum of 4.6 FPU/mL for Condition I at 96 h, 4.2 FPU/mL for Condition II at 96 h 
and 3.5 FPU/mL for Condition III at 84 h, respectively.  These are shown as in Fig.V-1.  
Table V-1 shows these values in a tabular form. 
During the first day of the fermentation process, a rapid drop in the pH was 
observed.  Sternberg reported the phenomenon and ascribed this to liberation of the 
hydrogen ions, which resulted from the depletion of the inorganic nitrogen source 
(Sternberg, 1976). A rapid drop in the dissolved oxygen (DO) level and an increase in the 
agitation and the aeration rates (data not shown here) were also observed.  To address 
this, the DO level was kept above 25% of the saturation value by varying the agitation 
and the aeration rates in response to changes in the amount of DO. These issues merely 
demonstrated that the fungal growth started immediately after inoculation.  To confirm 
this educated guess, measurements of the reducing sugars in the hardwood pulp were 
taken.  The results showed that the concentration of reducing sugars was less than 1.5 g/L 
during whole fermentation process, which indicated that fungus used most of the 
reducing sugars. 
 129
Enzyme secretion displayed a lag-phase of about 24 h at a pH of 5.0.  After that, 
the rate of enzyme secretion rapidly increased and most of the enzyme secretion was 
obtained during the stationary phase.  At the stationary phase, the DO level was still 
controlled at 25%, however, the air flow rate decreased slightly during fermentation as 
compared to the air flow rate during the cell growth phase.  About 10% of cellulase was 
continuing to be excreted when the pH was increased from 3.5 to 5.4 under Condition I 
from 72 h to 96 h.  About 5 % of FPU was continuing to be excreted when the pH 
increased from 4.0 to 5.4 under Condition II from 72 h to hour 84 h.  It is probable that 
the lower cellulase amount came from being physically adsorbed on the remaining 
fermentation solids, which mainly consisted of fungal mycelium. The pH values 
increased from 5.0 to 6.7 during 72 h to hour 96 h.  The DCW decreased during this time, 
which may be due to autolysis of the fungus at the higher pH. The cellulase activity 
decreased after 84 h under Condition II and after 72 h under Condition III, which may be 
due to the deactivation of the enzyme as a result of the higher pH.  Another possibility for 
the reduced cellulase activity could be proteolysis of cellulase, which tends to occur in 
the late station stage.   
Observations were made of the cellulase production growth profile.  It was found 
that the time required for the fungus to reach maximum cell concentration was 
approximately 36 h under Condition I and Condition II, and 48 h under Condition III.  It 
is interesting to note that under Condition III, the DCW was much higher than the other 
conditions. Reducing sugars were measured at the same time and it was noticed that 
under Condition I, the amount was low, i.e. less than 1 g/L concentration of reducing 
sugars.  At the same time, maximum DCW was 3.8 g/l.  Although the pH was relatively 
 130
low at this condition, it was still high enough for the fungus to grow and to take part in 
the fermentation.  Under Condition II, clearly the fungus was able to grow better, the 
maximum DCW increased to 4.4 g/L (see Fig.V-1). Under Condition III, the maximum 
DCW was 5.8 g/L. 
The optimal pH for enzymatic hydrolysis of cellulose is 4.8 (Ghose, 1987).  Some 
researchers used the pH of 4.8 for their cellulase studies (Olsson et al., 2003), but 4.8 is 
not the optimal pH when the carbon source is hardwood pulp. 
Although T. reesei grew faster at the higher pH (Condition III), the obtained final 
cellulase activities were 20% lower.  The reason may be that lower pH produces slow 
growth and prolongs the period of enzyme production. Inside the cell, where the pH is 
neutral, immediate dissociation of the acids occurs, causing a decrease in the intracellular 
pH, which can inhibit cell growth. Because these compounds are weak acids, the 
concentration of the associated acids increased and thus inhibited cell growth.  If the pH 
beyond the cell?s walls is low, then the growth is inhibited even more (Lambert and 
Stratford, 1999). It is known that microorganisms often have multiple systems for a 
single function. Therefore, different metabolic pathways could be used depending on the 
pH conditions. The level of cellulase increase in acidic medium suggests that T.reesei 
cells may use different metabolic pathways depending on the pH of the extracellular 
environments. Sternberg et al. (1976) suggested that the fungi should be grown in an 
environment where the pH values are sub-optimal for enzymatic hydrolysis in order to 
gain benefits such as reduction in the efficiency of cellulose with the reduced 
accumulation of extracellular sugars and reduction in the impact of catabolite repression 
of enzyme synthesis.  
 131
In addition to cellulase, a certain amount of xylanase and glucosidase are also 
produced from T. reesei. With hardwood pulp containing 20% xylan, higher ?-
glucosidase and xylanase activities were expected to be achieved than from pure 
cellulose.  Chaudhary and Tauro (Chaudhary and Tauro et al., 1982) found that xylan (a 
good carbon source) positively affects the production of ?-glucosidase, and Bailey 
(Bailey et al., 1993) found that xylan was an effective inducer of xylanases. The 
measurements supported this expectation. The highest ?-glucosidase and xylanase 
activities were obtained under Condition III.  When the pH was reduced, the ?-
glucosidase and xylanase activities were gradually decreased.  Other researchers had 
similar results (Tangnu et al., 1981; Bailey et al., 1993).  Xiong found that at a low pH 
cellulase was prevalent and at a high pH xylanase was prevalent.  However, they found 
that cellulase activities did not change much when the pH values were in the range of 4.0 
to 5.5 and they noticed that the cellulase activities actually decreased significantly when 
the pH was 3.5 (Xiong et al., 2004).  The different results might be due to the usage of 
lactose as a carbon source (Xiong et al., 2004).  The results are presented in Table V-1. 
5.4.2 Effect of Different Hardwood Pulp Concentration 
Previous work indicates that higher concentration of the carbon source resulted in 
higher enzyme activities (Esterbauer et al., 1991; Tangnu et al., 1981; Yu et al., 1998). 
Fig.V-2. shows the impact of different hardwood pulp concentrations on cellulase 
production in the 3 L fermentor. 
Enzyme formation commenced about one day after inoculation.  The maximum 
enzyme activity was reached after 4 to 5 days of incubation.  The pH was allowed to drop 
from the initial pH value of 6.0 to 3.5 and was adjusted not to fall below this value.  The 
 132
pH values increased after 3 to 5 days of incubation (data not shown here).  As shown in 
Fig.V-2., the enzyme activities and DCW increased in proportion to substrate 
concentration, up to 40 g/L. The highest productivity was also obtained at substrate 
concentration of 40 g/L , however, yield was highest by using 20 g/L hardwood pulp as a 
substrate (see Table V-2). This result agreed with previous studies (Esterbauer et al., 
1991; Tangnu et al., 1981; Yu et al., 1998).  A maximum of 7.5 FPU/mL was found when 
the concentration is 40 g/L of hardwood pulp, which is comparable to the results with 
pure cellulose such as Solka Floc (Velkovska et al., 1997; Domingues et al., 2000) and 
the results with glucose or lactose (Ahamed and Vermette, 2008; Domingues et al., 
2001). 
Several studies speculated that lignin on the fiber surface would impair inter-fiber 
bonding due to the hydrophobic nature of lignin (Shao and Li, 2006; Li and Reeve, 
2002). Since chemical pulping dissolves the lignin and other materials of the inter-fiber 
matrix material and dissolves most of the fiber wall lignin, the pulp fibers become more 
hydrophilic and acquire surface groups that are capable of hydrogen bonding resulting in 
improved inter-fiber bonding. Therefore, the adjacent fibers in the pulp tend to held 
together by the hydrogen bond formation between the cellulosic surfaces (Sixta, 2006; 
Nissan and Batten, 1990).  In this context, it seems that further increase in substrate 
concentration would result in decreased production levels due to the creation of very 
thick slurry.  As expected, at 50 g/L concentration of hardwood pulp, the cellulase 
activity markedly decreased since the mixture was too thick to achieve effective mixing 
or aeration.  The fungus growth rate was also found to be low.  As Fig.V-2 shows, at 50 
g/L hardwood pulp concentration, the fungus grew more slowly and maximum DCW was 
 133
lower than with other concentrations.  Schugerl (Schugerl et al., 1981) reported that high 
viscosity unfavorably influences mixing and oxygen transfer.  When higher viscosity 
mixtures are used, higher power inputs are required to achieve similar levels of mixing in 
the gas/liquid interfacial area. The stirring efficiency is reduced since the impeller is more 
easily flooded.  The mass transfer coefficient decreases. Researchers found that with 
better designs in the aerator and mixer stages in a larger scale fermentor, higher cellulase 
activity could be obtained as a result of the more efficient oxygen mass transfer.  Any 
such design would avoid oxygen supplementation since any operation that involved 
oxygen supplementation would make the final products more expensive (Hayward et al, 
2000). 
5.4.3 Enzyme Production from De-ashed and Chemical Treated Primary Sludge  
The composition analysis of the primary sludge indicates that the hemicellulose 
and lignin content is low compared to other raw materials.  This property of the sludge 
should be favorable for cellulase production. Unfortunately, high ash content is 
detrimental to the growth of microorganisms. Moreover, all residues retained from each 
grade of operation in the paper making process made a great negative effect on the 
cellulase production. In our study, using original untreated sludge as substrate, the fungus 
was not able to grow in the fermentation medium. Apparently ash and some other 
chemicals in the sludge greatly inhibit the cellulase production, even the cell growth. So, 
it is desirable to remove ash from the feedstock, while retaining carbohydrates.  
However, cellulase activity measured for the production culture in a 3-L 
fermenter was still low (less than 2 FPU/mL) by using de-ased sludge with ash content of 
14.0% (data not shown here). It is worth noting that an increase in the substrate 
concentration from 25 g/L to 35 g/L did not improve, but instead inhibit the cellulase 
 134
production. All these results indicated that the primary sludge, with high cellulose content 
due to hemicellulose and lignin removed in pulping process, was not effective in the 
cellulase production. The cellulase production was inhibited by the by-products generated 
during the paper and pulping process. Ash and some other insoluble chemicals such as 
clay, CaCO
3
 are really detrimental to the microorganism and inhibitory against the 
cellulase production. Further treatment of sludge is required to enhance the cellulase 
production. 
De-ashed sludge was leached with 1 N sulfuric acid and further water washing. In 
this case, CaCO
3
, clay could be partly removed. Cellulase production with acid treated 
sludge as the substrate performed better than the previous runs (Fig.V-3). The cellulase 
production commenced within 24 hr, which indicated the lag phase was shortened due to 
the better substrate and cell growth. The Filter paper activity and productivity reached its 
maximum of 3.2 FPU/mL and 37.5 FPU/L?h at 96h, respectively.  
Seeking further improvement of cellulase production, the acid treated sludge was 
treated with 1% (w/v) NaOH solution and further water washing. The ash in the alkaline 
treated sludge was further decreased to 3.8 %, the glucan content was increased as shown 
in Table V-3.  Most of the inhibitory chemicals against the cellulase production such as 
alulminum and copper were further removed by this NaOH based treatment. This further 
treatment provided the sludge good characteristics as carbon source for cellulase 
production. The strain grew well, and produced 8 FPU/mL of cellulase at 132h with 35g-
glucan/L (47g/L sludge) concentration (Fig.V-4).  The yield of cellulase enzyme and 
productivity reached 307 FPU/g of glucan and 60.6 FPU/L?h, respectively. And the 
specific cellulase activity was 8 FPU/ mg protein. Table V-3 shows the activities of 
 135
cellulolytic and hemicellulolytic enzymes of the fermentation broth. The cellulase titers, 
productivities of all runs above were summarized in Table V-4.  
The specific cellulase activity was obtained from 40 g/L hardwood pulp is 7.7 
FPU/mg protein. Although the protein content of enzyme was measured by the Bradford 
method, without TCA precipitation, the value was much higher than the reported specific 
activity of commercial cellulases. These numbers, however, do not necessarily reflect the 
molecular activities of the respective enzymes. It may have more to do with the fact that 
some of the commercial enzymes contain non-enzyme proteins mixed in as a nutrient 
during fermentation.  In the cellulase production from both substrates, nitrogen sources 
were mostly inorganic salts, therefore, background protein levels were very low in the 
broth. Additionally, it suggests a possibility of developing a simpler purification process.  
This inhibitory effect may be due to metal ions in the sludge. Stutzenberger and 
Sterpu found that the metals in municipal refuse compost, such as aluminium and 
calcium, appeared to specifically inhibit cellulase production at high concentration 
(Stutzenberger and Sterpu, 1978). The data in this study agreed with it. The calcium 
content was reduced from 8.9 % (untreated sludge) to 4.7% (deashed sludge), 1.5% (acid 
treated sludge) and 1.1 wt. % (alkaline treated sludge). The aluminum content was 
reduced from 0.55 % (untreated sludge) to 0.44 % (deashed sludge), 0.37% (acid treated 
sludge) and 0.003 wt. % (alkaline treated sludge). 
5.4.4 Enzymatic Hydrolysis Test 
Performance of hydrolysis tests were conducted for hardwood pulp enzyme (HP-
enzyme) and sludge enzyme using two different substrates: Corn stover pretreated by 
dilute-acid and kraft processed hardwood pulp. The same hydrolysis test was done for 
 136
Spezyme CP (Genencor, Lot No. 301-00348-257) for comparison purposes.  A glucan 
loading of 1% (w/v) and 15 FPU/g-glucan of enzyme loading were used.  The hydrolysis 
profiles are shown in Fig.V-5. Only data that were from the hydrolysis and fermentation 
tests by using HP-enzyme are shown here, since both enzymes produced from hardwood 
pulp enzyme (HP-enzyme) and sludge enzyme had the same performance. 
In side by side comparison between HP-enzyme and Spezyme CP for enzyme 
hydrolysis of pretreated corn stover, the yield and the glucose profile were almost equal, 
91.0% for Spezyme CP and 90.2% for HP-enzyme.  Since both enzymes were 
supplemented without ?-glucosidase, the difference in gross activity indicates that there is 
a very slight difference in endo and exo-glucanase activities between the two. 
The same tests were repeated using hardwood pulp as a substrate. The results 
were slightly different from the previous test as shown in Fig.V-5. The HP-enzyme 
showed higher overall glucose yield than Spezyme CP. The 72 h yields were 88.2% (1% 
glucan loading) for HP-enzyme and 85.2% (1% glucan loading) for Spezyme CP.  The 
major difference between the two substrates is the xylan content: 2.5% in acid-treated 
corn stover versus 21.1% xylan in hardwood pulp. We speculate that the difference in 
yield has to do with the xylanase activity. This was confirmed by the xylan digestibility 
data from the same experiments and the xylanase activity of the two enzymes.  The xylan 
digestibility profiles for the two enzymes are shown in Fig.V-5.  The results confirmed 
that HP-enzyme has higher xylanase activity than Spezyme CP.  The high xylanase 
activity of HP-enzyme may result from the fact that hardwood pulp has relatively high 
xylan (21.1%) which may have acted as an effective inducer for xylanase production by 
T. reesei.  Other researchers have proposed that xylan removal improves the access of 
 137
cellulase enzyme to cellulose and thus improves cellulose activity (?hgren et al., 2007).  
Furthermore, it was also found that xylose has a direct inhibitory effect on cellulase 
production (Xiao et al., 2004; Nigam and Prabhu, 1991; Todorovic and Grujic, 1987; 
Kim and Lee, 2005). 
5.4.5 SSF Experiments 
Batch and fed-batch SSF experiments that used paper sludge as the substrate were 
performed over a 5-day period and ethanol concentration was monitored as a function of 
time.  Fig.V-6. shows the ethanol data obtained from the SSF tests. In the batch SSF 
experiments, the 96 h measurement for a cellulase from the hardwood pulp loading of 15 
FPU/g-glucan revealed that the ethanol concentrations had reached 25.2 g/L, which is 
similar to what was obtained when using a commercial cellulase (25.1 g/L).  The 
commercial cellulase had almost the same amount of ethanol yield, which was 74% 
(based on 6% (w/v) glucan). 
In the fed-batch SSF experiments, the ethanol concentration could achieve 45 g/L, 
however only 68% yield was obtained (data not shown).  The low conversion is attributed 
to the cellulase enzyme reaction rather than to the microbial reaction.  Saccharomyces 
cerevisiae (D
5
A) is very robust and has a high ethanol tolerance. We believe that the 
high-solid conditions in the bioreactor and in the product plus substrate inhibition are 
major factors for suppressing the cellulase enzyme reaction. 
The projected cellulase plant is a low-investment supplementary unit that can be 
built within a kraft paper mill. On?site cellulase production can be promoted as a stand-
alone unit or as a unit within an ethanol production system supplying the enzyme in-
house, thus reducing the overall production cost.   
 138
5.5 Conclusion 
To reduce the cost of pretreatment and to simplify the bioconversion process, 
unbleached kraft processed hardwood pulp and treated paper sludge were tested as 
feedstocks for production of cellulase enzymes using T. reesei Rut C-30.  The T. reesei 
strains can produce cellulase enzymes in an efficient manner.  The cellulase production 
by this scheme yielded 246.7 FPU/g-glucan of hardwood pulp, and a productivity of 56.8 
FPU/L.h.  The titer of cellulase produced from 4% hardwood pulp loading was 7.5 
FPU/mL, which is comparable to those produced from pure cellulosic substrates.  The 
cellulase production yield of 307 FPU/g glucan of sludge, and productivity of 60.6 
FPU/L h were achievable from alkaline treated sludge. The titer of enzyme was 8.0 
FPU/mL. The specific activities of cellulase from both substrates were higher than that of 
the commercial cellulases. The main reason is that cellulase was produced here using 
inorganic salts as a main nitrogen source, thereby resulting in very little non-enzyme 
proteins in the broth. Cellulase produced from has both substrates has higher xylanase 
activity than Spezyme CP. No difference was found between cellulase produced from 
unbleached hardwood pulp and Spezyme CP in their performance in the ethanol 
production from primary paper sludge. 
 139
Table V-1. Enzyme activities and productivity obtained in the cultivation of T. reesei Rut 
C-30 on 20 g/L hardwood pulp under different pH control conditions 
pH control 
conditions 
Maximum 
FPA 
Time (hr) 
FPA 
(FPU/mL)
Productivity 
(FPU/L/h) 
Yield 
(FPU/g) 
?-Glucosidase  
(pNPG U/mg) 
Xylanase 
(IU/mg)
Condition I 
96 4.6 47.9 230 
0.35 49.3 
Condition II 96 4.2 43.8 140 0.39 56.1 
Condition 
III 84 3.5 41.7 87.5 0.51 78.9 
 
 140
Table V-2. Enzyme activity, yield and productivity obtained in the cultivation of T. reesei 
Rut C-30 on different concentration of hardwood pulp as substrates 
Hardwood pulp 
concentration (g/L) 
Time (h) FPA 
(FPU/mL)
Productivity 
(FPU/L/h) 
Yield 
(FPU/g glucan) 
20 96 4.6 47.9 302.6 
30 120 6 50.0 263.2 
40 132 7.5 56.8 246.7 
50 120 4.5 37.5 118.4 
 
 141
Table V-3. Enzyme activities obtained in the cultivation of T. reesei Rut C-30 on 40 g/L 
hardwood pulp 
Filter Paper Activity 
( FPU/mL) 
? ? Glucosidase 
(pNPG U/mg) 
Xylanase Activity 
(IU/mg) 
CMCase Activity 
(IU/mg) 
7.5 0.4 156.2 4.2 
 
 142
Table V-4. Specific activities of cellulolytic and henicellulolytic enzymes of fermentation 
broth of alkaline treated sludge 
Filter Paper 
Activity 
( FPU/mL) 
? ? Glucosidase 
(pNPG U/mg) 
Xylanase 
Activity 
(IU/mg) 
CMCase Activity 
(IU/mg) 
8.0 0.5 170.2 4.4 
 
 
 
 143
Table V-5. Cellulase activity and productivity of T. reesei Rut C-30 in 3-L fermenter 
using acid and alkaline treated paper mill sludge as substrates 
 FPA 
(FPU/mL) 
Productivity 
(FPU/L/h) 
Yield 
(FPU/g glucan) 
Acid Treated Sludge 3.2 37.5 176.3 
Alkaline Treated Sludge 8.0 60.6 306.8 
 
 
 144
 
Condition I 
0
1
2
3
4
5
6
7
2040608010
Time (h)
pH
,
 D
C
W
 (g/
L
),
 
R
e
duc
i
ng S
uga
r
 (g/
L
)
0
1
2
3
4
5
F
P
A
 (
F
P
U
/m
l)
 
Condition II
0
1
2
3
4
5
6
7
2040608010
Time (h)
p
H
, D
C
W
 9
g
/L
)
,
 
R
e
duc
i
ng S
uga
r
 (
g
/
L
)
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
F
P
A
 (
F
PU
/ml)
 
Condition III
0
1
2
3
4
5
6
7
8
2040608010
Time (h)
p
H
, D
C
W
 (
g
/L
)
,
 
R
e
duc
i
ng S
uga
r (
g
/
L
)
0
1
2
3
4
F
P
A
 (
F
P
U
/m
l)
 
Fig.V-1. Cellulase production by T. reesei Rut C-30 under different pH control conditions 
Squares with connecting lines represent Dry Cell Weight (DCW, g/L). Triangles with 
connecting lines represent Reducing Sugar (g/L). Filled triangles with connecting lines 
represent pH. Filled squares with connecting lines represent Filter Paper Activity (FPA, 
FPU/ml).
 145
A 
0
1
2
3
4
5
6
0 153045607590105120135
Time (h)
DCW
 (
g
/
l
)
20g/L
30g/L
40g/L
50g/L
 
B 
0
1
2
3
4
5
6
7
8
0 153045607590105120135
Time (h)
FP
A
 (
F
P
U
/
m
l
)
20g/L
30g/L
40g/L
50g/L
 
Fig.V-2. Cellulase production by T. reesei Rut C-30 at different concentration of 
hardwood pulp 
 146
0
0.5
1
1.5
2
2.5
3
3.5
0 20 40 60 80 100 120
Time (h)
F
i
l
t
er
 
p
a
p
er a
c
t
i
v
i
t
y
 (
F
P
U
/
m
L
R
e
d
u
c
in
g s
u
gar
 (
g
/
L
)
 
  
   
  
S
o
l
ubl
e
 pr
o
t
e
i
n (g
/
L
)
Reducing sugar (g/L)
Filter paper activity (FPU/ml)
Soluble protein (g/L)
 
Fig.V-3. Cellulase production by T. reesei using acid leached sludge 
 147
0
1
2
3
4
5
6
7
8
9
0 2040608010120140
Time (h)
F
ilt
e
r
 p
a
p
e
r
 a
c
t
i
v
i
t
y
 (
F
P
U
/m
L
)
Re
d
u
c
i
n
g s
u
gar
 (
g
/
L
)
        
So
l
ubl
e
 pr
o
t
e
i
n (
g
/
L
)
Reducing sugar (g/L)
Filter paper activity (FPU/ml)
Soluble protein (g/L)
 
Fig.V-4. Cellulase production by T. reesei using alkaline treated sludge 
 
 148
 
1% w/v pretreated corn stover loading
0.0%
10.0%
20.0%
30.0%
40.0%
50.0%
60.0%
70.0%
80.0%
90.0%
100.0%
0244872
Time (h)
D
i
ge
s
t
ibilit
y
 (
%
)
Glucan Diestibility Spezyme CP
Xylan Digestibility Spezyme CP
Glucan Diestibility HP-Enzyme 
Xylan Digestibility HP-Enzyme 
 
1% w/v hardwood pulp loading
0.0%
10.0%
20.0%
30.0%
40.0%
50.0%
60.0%
70.0%
80.0%
90.0%
100.0%
0244872
Time (h)
D
i
g
e
s
t
i
b
il
it
y
 (
%
)
Glucan Diestibility Spezyme CP
Xylan Digestibility Spezyme CP
Glucan Diestibility HP-Enzyme 
Xylan Digestibility HP-Enzyme 
 
A B 
Fig.V-5. Enzymatic hydrolysis of different substrates by HP-enzyme and Spezyme CP 
 149
0.0
5.0
10.0
15.0
20.0
25.0
30.0
2040608010
Time (h)
E
t
ha
no
l
 C
o
nc
en
t
r
at
i
o
n
 (
g
/
L
)
Spezyme CP
HP-Enzyme
 
Fig.V-6. Ethanol production from untreated primary sludge by HP-enzyme and Spezyme 
CP 
 
 150
VI. Ethanol Production from the Mixture of Hemicellulose 
Prehydrolysate and Paper Sludge, Integrated with Hemicellulose Pre-
extraction of Southern Pine 
 
6.1 Abstract 
Most of the hemicellulose fraction of pulp mill feedstock (softwood or hardwood) is 
released into black liquor during the pulping process. The black liquor is combusted to 
recover chemicals and to generate steam and electricity. It is technically feasible to 
recover this fraction of carbohydrate and enhance its value by converting it into value-
added products. Hemicellulose is selectively converted to soluble sugars (termed as 
prehydrolysate) by treating it with hot water. The sugars produced from pre-hydrolysis 
process are mixtures of pentose, hexose, and their oligomers. In this study, pectinase and 
Saccharomyces cerevisiae were used to convert the prehydrolysate into ethanol. The 
prehydrolysate produced from wood also contains toxins, primarily lignin and sugar 
degradation products, which strongly inhibit microbial reaction. De-toxification of the 
prehydrolysates was done by overliming (addition of excess CaO). When hydrolysate is 
obtained by treating wood, the total sugar concentration is below 4 wt. %. Consequently, 
when the hydrolysate is used as a fermentation substrate, the ethanol concentration is less 
than 2%, which is far below the level acceptable as distillation feed. Use of the mixture of
 151
prehydrolysate and pulp mill sludges as the fermentation feed, however, can increase the 
product concentration. In bioconversion of sludge, a certain amount of water is added to 
attain fluidity required for Simultaneous Saccharificiaton and Fermentation (SSF) 
operation. In this study, prehydrolysate, in place of water, was added into the bioreactor 
along with the sludge. Using this procedure, there was a net increase of total sugar 
concentration in the bioreactor above that of the base case, which led to an increase in 
product concentration. The experimental data detailing the proposed bioprocess 
converting the mixed-feed to ethanol are presented in this paper. 
6.2 Introduction 
While the commercialization of bio-ethanol production is still emerging from the 
cradle, another industry is struggling in the United States, namely the pulp and paper 
industry. The combination of reduced demand for paper in the domestic market along 
with the increased supply of cheap foreign paper, primarily from Asia, is causing great 
difficulty for the pulp and paper industry in the United States, forcing the pulp and paper 
industries to find new opportunities in order to improve their competitiveness. An 
integrated bio-refinery which can extract hemicellulose while preserving cellulose for 
pulp production can be developed. Such a process can transform the pulp and paper 
industry into a competitive and innovative one, which would allow significant economic 
growth through new value-added products (i.e. ethanol), while still preserving the main 
product-paper. 
In the United States, 80% of total pulping capacity is chemical pulp. 
Approximately 98% of chemical pulp capacity is kraft pulping while the remaining 2% is 
sulfite pulping (American Forest & Paper Association, 2002). Eighty percent (80%) of 
 152
the hemicellulose fraction of pulp mill feedstock (softwood or hardwood) is released into 
black liquor during the kraft pulping process. The black liquor is burned to recover 
chemicals and generate steam and electricity. Since the heating value of hemicellulose 
(13.6 MJ/kg) is only half of that of lignin (27 MJ/kg), and it is technically difficult to 
separate this hemicellulose part from alkaline black liquor before it is burned to generate 
heat and power (Sjostrom, 1993), pre-extracting the hemicellulose prior to the pulping 
process for producing high value products has the potential to improve the overall 
economics of the process.  
It is technically feasible to remove part of the hemicellulose before pulping. 
Hemicellulose is selectively converted to soluble sugars (termed as prehydrolysate) in 
treatment with hot water (Yoon et al., 2008; Yoon et al., 2010 ), diluted acid (Springer 
and Harris, 1982) or mild alkaline hydrolysis (Al-dajani and Tschirner, 2008). Hot water 
extraction is attractive for wood hemicellulose removal due to savings in the cost of 
mineral acid or alkaline. The sugars produced from prehydrolysis contain mixtures of 
pentose, hexose, and their oligosaccharides.  
Hemicellulose prehydrolysate can be converted to ethanol from lignocellulosic 
biomass through hydrolysis and downstream fermentation processes (Walton et al., 
2010a). Besides ethanol, other value added chemicals are also produced from 
hemicellulose prehydrolysate (Walton et al, 2010b; Soderqvist et al., 2001). 
Oligosaccharides may further prove to be a source of higher value-added products, such 
as animal feed additives (Davis et al., 2002; Fernandez et al., 2002).  
The major softwood hemicellulose is an O-acetyl-galactoglucomannan (15-25% 
of wood) which is beta-1,4-linked mannose and glucose residues in the ratio 3:1 often 
 153
with alpha-1,6-galactose as side group (Timell, 1967; Ethier et al.,1998;Meier, H. 1961; 
Katz, 1965; Lindberg et al., 1973). Because softwood hydrolysates contain much more 
mannose than xylose, there is less need for a xylose-fermenting organism when this raw 
material is used. Regarding softwood hydrolysates, S. cerevisiae has a great advantage 
over Ethanologenic Bacteria, such as, E-coli, since S. cerevisiae has higher tolerance to 
toxin and ethanol. Saccharomyces cerevisiae was used to convert the hexose in the 
prehydrolysate into ethanol in this study. The prehydrolysate produced from 
lignocellulosic biomass contains some toxic compounds such as furan, organic acids, and 
various phenolic compounds that inhibit microbial fermentation to obtain the desired 
products (Klinke et al. 2004; Palmqvist and Hahn-H?gerdal 2000). The prehydrolysate, 
therefore, has to be detoxified for efficient conversion to ethanol. Several detoxification 
processes can be used for removing the inhibitory compounds from wood hydrolysates, 
such as over liming (Martinez A et al, 2001), ammonium hydroxide detoxification 
(Alriksson et al, 2005), enzymatic detoxification   (J?nsson et al., 1998), ion exchange 
resins (Fein et al., 1984), active charcoal (Roberto et al., 1991), extraction (Fein et al., 
1984), and adsorption (Roberto et al., 1991). The detoxification of pine wood 
prehydrolysate was developed in this study. 
To avoid excessive damage to the fiber quality, hemicellulose can not be totally 
removed. Therefore, the pre-extraction process gave a low concentration of sugars. If the 
prehydrolysate is used as a fermentation substrate, the ethanol concentration is less than 
2.0%, which is far below the level acceptable as distillation feed. In bioconversion of the 
sludge, the sludge needs to be diluted with water to attain the fluidity required for the 
fermentation operation. In this study, prehydrolysate, in place of water, was added into 
 154
the bioreactor along with the paper sludge. Under the proposed scheme, there is a net 
increase of total sugar concentration in the bioreactor compared to prehydrolysate alone, 
thereby reducing the cost of ethanol distillation. 
6.3 Materials and Methods 
6.3.1 Materials 
Feedstock: Fresh southern pine chips were obtained from Rock-Tenn Company in 
Demopolis, Alabama. Chips with major defects including bark, knots, and decayed parts 
were removed prior to screening on a chip class laboratory screen equipped with a stack 
from top to bottom of 45-mm round screens, 8-mm bar screens, 6-mm bar screens, and 4-
mm round screens. The wood fraction passing 45-mm round screens and 8-mm bar 
screens and retained on 6-mm bar screens was collected, well mixed, and air-dried before 
use. 
The paper primary sludge (PS) was collected from the primary wastewater 
clarifier unit of a kraft paper mill, Boise Paper Company (Jackson, AL, USA). The sludge 
was washed with tap water three times and was further thickened to 38% consistency 
using a vacuum filter, and stored at 4
o 
C.  The primary sludge was analyzed for 
carbohydrates, moisture content, and ash content according to the National Renewable 
Energy Laboratory (NREL) standard procedure (NREL, 2008). The primary sludge was 
analyzed and found to contain 44.5 % glucan, 9.9 % xylan, 8.1% lignin and 36.0 wt. % 
ash, which includes 26.0% acid-soluble ash and 10.0% acid-insoluble ash.  
Enzymes: Cellulase enzyme (Spezyme CP, Lot No. 301-00348-257, 59 FPU/mL, 
123mg protein/mL), Xylanase (Multifect Xylanase, Lot No. 301-04021-015; 42 mg 
protein/mL), Pectinase (Multifect Pectinase FE, Lot No. A21-03356-001, 82 mg 
 155
protein/mL), and measurements of their protein content/ specific activities were 
generously provided by Genencor Division of Danisco US, Inc. (Palo Alto, CA, USA). ?-
glucosidase (Novozyme 188, Cat. No. C-6150, 665 CBU/mL, 140mg protein/mL) was 
purchased from Sigma (St. Louis, MO, USA). The activities present in commercial 
enzyme preparations are shown in Table VI-1 which is based on the publication by Dien 
et al (Dien et al., 2008; Berlin et al., 2007).    
Microorganism: The microorganism used in the SSF was Saccharomyces 
cerevisiae ATCC-200062 (NREL-D5A). The growth medium was YP medium, which 
contained 1% yeast extract (Sigma, Y-0500) and 2% peptone (Sigma, P-6588).  
6.3.2 Pre extraction  
Hot water has proved to be an effective solvent for the extraction of hemicellulose 
from wood (Werpy and Petersen,2004).  Three stage extractions were done to increase 
the sugar content in the extracted liquor and to simulate the continuous extraction 
process. The extractions were conducted using a 500 mL cylindrical bomb digester that 
was placed inside a computer-profiled M/K laboratory digester filled with water as a heat 
transfer fluid. In every water-extraction stage, seventy grams of oven-dried untreated 
softwood chips were used in the bomb digester at a liquor-to-wood ratio is 5.8 to 1. The 
digester temperature was ramped from room temperature to a preset maximum 
temperature of 170 ?C at a rate of 3.2 ?C per minute. At the end of the digester operation, 
each bomb was then quenched in a cold water bath. Extraction times at the preset 
extraction temperature (170 ?C) varied from 0 to 90 min to attain various wood weight 
loss levels ranging from 0 to 15% based on oven-dry wood. After completion of water 
extraction, extract was drained from the wood chips (70% of the total liquor) and 
 156
collected to be used for chemical analysis. The liquor used in the first stage was 
deionized (DI) water. The liquor used in the second stage was mixed with the extract 
liquor from the first stage extraction (70% volume of the total first stage liquor) and 
additional DI water to make up lost volume (30% volume of the total first stage liquor) 
since the extracted wood chip absorbed around 30% volume of total liquor. The liquor 
used in the third stage was mixed with the extract liquor from the second stage extraction 
(70% volume of the total second stage liquor) and DI water (30% volume of the total 
second stage liquor). As wood chips are extracted, their weight can be expected to 
decrease due to components dissolved and diffused from the wood into the extraction 
liquor (termed as prehydrolysate). The wood weight loss data were calculated from the 
difference between the weight of fresh wood chips and that of thoroughly washed wood 
residue based on its dry weight. The sugar contents of the extract were determined 
according to methods published by the National Renewable Energy Laboratory (NREL, 
2008). The total amount of cellulose, and hemicelluloses, which led to these sugar 
concentrations, were then calculated using the following equations described by van 
Heiningen et al. (Yoon and van Heiningen, 2010b). 
)
180
162
(*
b
Man
)
180
162
(*  Cellulose ?= Glu
 
Where b= 4.15 (This is the average value for number of mannose units per glucose unit in 
hemicellulose of pine/spruce wood, based on Janson (Janson, 1974) : 
CelluloseManGluGalXylAra ?++++= )
180
162
(*)()
150
132
(*)(  oseHemicellul  
In order to determine the intensity of the pre extraction, the combined effects of time and 
temperature were measured in terms of the H-factor (H), defined as:    
 157
 dT
t
))
T
16.113
20.43((exp H
0
?=
?
 
Where t is the time (hours) needed for achieving the desired temperature (T). 
Table VI-2 shows the first stage of water-extraction condition, weight loss of 
wood chips, and the concentrations of glucose, xylose, galactose, arabinose, and 
mannose. The water-extracted wood chips were used in the subsequent treatment and 
kraft pulping experiments (Yoon et al., 2010). 
6.3.3 Composition Analysis of Prehydrolysate    
The liquid samples were analyzed for carbohydrates following the NREL standard 
procedures (NREL, 2008). The extracted liquor was centrifuged to separate the solid part 
from the liquid. Then, the solid-free liquid was incubated for 1 h with 4% sulfuric acid at 
121?C in an autoclave along with sugar recovery standards determined according to 
methods published by the National Renewable Energy Laboratory (NREL, 2008). The 
liquid was neutralized with CaCO
3
, and the total amount of sugar monomers was 
analyzed by high pressure liquid chromatography (HPLC). The concentration of 
oligosaccharides with a degree of polymerization larger than 2 was calculated as follows: 
11.1
)/(___)/(___
)/(_
LgHydrolysisAcidBeforeHexoseLgHydrolysisAcidAfterHexose
LgOligomerHexose
?
=
136.1
)/(___)/(___
)/(_
LgHydrolysisAcidBeforePentoseLgHydrolysisAcidAfterPentose
LgOligomerPentose
?
=
 
Hexose oligomer here is interpreted as mannose oligomer (Man 2), galalactose oligomer 
(Gal 2), and glucose oligomer (Glu 2). Pentose oligomer here is interpreted as arabinose 
oligomer (Ara 2) and xylose oligomer (Xyl 2). 
Hexose here is interpreted as mannose (Man), galactose (Gal), and glucose (Glu). 
Pentose here is interpreted as arabinose (Ara), and xylose (Xyl). 
 158
 
The concentration of acetyl group linked with carbohydrate was calculated as follows:
 
394.1
)/(____)/(____
)/(_
LgHydrolysisAcidBeforeAcidAceticLgHydrolysisAcidAfterAcidAcetic
LgGroupAcetyl
?
=
 
6.3.4 Contaminant Microorganism Test 
A YPD agar (Sigma, Y-1500) plate was placed on a turntable, and 10 ?L of 
sludge solution was dispersed evenly on the plate. The YPD agar plate was then 
incubated at 30 ?C for 48 h. Samples from each flask were streaked on an YPD agar plate 
to check for microbial contamination. The gram stain was used to differentiate gram-
positive and gram-negative bacteria. Colony forming unit (CFU) tests were performed to 
determine the concentration of microorganism cells. YPD agar plates were prepared for 
this purpose by diluting the culture with sterile saline (0.89% NaCl solution) to obtain a 
spread plate cell count of 30-300 cells per plate.  
6.3.5 Treatment of Prehydrolysate with CaO 
Prehydrolysate (100 mL) was held at either 25 ?C or 60 ?C in an incubator shaker 
(New Brunswick Scientific, Innova-4080) with 150 RPM during the rapid addition of 
anhydrous CaO (Fisher Scientific) until pH around 10.0. After 30 min of incubation, the 
hydrolysate was cooled to ambient temperature in a second water bath, followed by pH 
readjustment to 5.5 with 85% phosphoric acid and used immediately for analyses or in 
fermentations without further storage. Approximately 80% of the treated hydrolysate was 
decanted for use in fermentation experiments. Residual suspended particulates were 
removed by centrifuge at 5000 RPM for 5 min.  
6.3.6 Enzymatic Hydrolysis Test            
The enzymatic digestibility of the extracted liquor was determined according to 
NREL Chemical Analysis and Testing Standard Procedures (NREL, 2008). To each 20 
 159
mL glass scintillation vial, 0.5 mL 1 M sodium citrate buffer (pH 4.5), 0.1 mL of a 2% 
sodium azide solution was added to prevent the growth of organisms during the digestion. 
The extracted liquor was neutralized by calcium carbonate until pH reached 5.0, and then 
9.4 mL prehydrolysate was added to the 20 mL glass scintillation vial. A 100 uL enzyme 
solution was diluted by DI water so that the enzyme was added to maintain enzyme 
loading at 25 mg protein/g-mannose oligomer. It was operated in an incubator shaker 
(New Brunswick Scientific, Innova-4080) at 50 ?C with 150 RPM.  The samples were 
taken at 6, 12, 24, 48 h and analyzed for glucose, xylose, galactose and mannose. The 
sugar digestibilities of released sugars, such as glucose, xylose, galactose and mannose, 
were used to calculate the enzymatic digestibility of sugar respectively:   
100
)(__
)(_
(%)_ ?=
gaddedSugarTotal
greleasedSugar
ityDigestibilSugar  
Reported digestibility values are the average of duplicate tests with standard 
deviation (SD) smaller than 1.0. 
6.3.7 Separation Hydrolysis and Fermentation (SHF) and SSF of Prehydrolysate   
In the SHF process, the treated prehydrolysate was subjected to enzymatic 
hydrolysis first for 48 h at 50 ?C and then to the fermentation step at 32 ?C. The SSF 
process was carried out at 37 ?C. A 20 mL serum bottle was used as the bioreactor. To 
each serum bottle, 9.3 mL treated or untreated extracted liquor was added. The samples 
were steam sterilized at 121 ?C for 15 min. Then 0.1 mL enzyme solution was diluted by 
DI water in such a way that the enzyme was added to maintain enzyme loading at 25 mg 
protein/g-mannose oligomer. Our previous study shows that acid soluble ash in the paper 
sludge, such as calcium carbonate, could act as a buffer to stabilize the pH during 
fermentation (Kang et al., 2010). Therefore, calcium carbonate was used in the SSF 
 160
process of softwood prehydrolysate. Fermentation was carried out under two different 
conditions: 0.5mL 1M sodium citrate buffer (pH 4.5) for SHF and 0.5 mL deionised 
water with addition of the CaCO
3
 to the level of 0.05 g/10mL for SSF. To each serum 
bottle, 0.1mL concentrated yeast was added along with the hydrolysate discussed above 
to make the initial yeast concentration of each sample 50 mg/L. The serum bottles were 
then sealed with rubber septa, vented by a needle. It was operated in an incubator shaker 
(New Brunswick Scientific, Innova-4080) at 36 ?C with 150 RPM. Hydrolyzate samples 
were taken at 6, 12, 24, 48 h and analyzed for glucose, xylose, galactose, arabinose, 
mannose and ethanol. In all of the microbial experiments, a sample from each flask was 
taken at the end of the run and streaked on an YPD plate to check for contamination and 
checked under an optical microscope. CFU tests were performed using YPD agar plates 
to check microorganism viability. A mixture of 0.2 mL of the aliquots and the yeast was 
transferred into a 1.5 mL centrifuge tube and centrifuged at 5000 RPM for 5 min. After 
the centrifuge, 0.1 mL of the supernatant was withdrawn and mixed with 0.9 mL DI 
water for chemical analysis). Aseptic conditions were maintained in all of the microbial 
experiments 
The ethanol yield was calculated as follows: 
100
0.511reactorin  (g)Sugar  Total Initial
reactorin  (g) produced Ethanol
 maximum] al theoreticof [% yield Ethanol ?
?
=
 
Sugar is interpreted as hexose in the reactor. 
Reported digestibility values are the average of duplicate test with standard 
deviation (SD) smaller than 1.0. 
 161
6.3.8 SSF of Paper Primary Sludge with or without Prehydrolysate 
The SSF test of the sludge was determined according to NREL Chemical Analysis 
and Testing Standard Procedures (NREL, 2008). Screw-capped 250 mL Erlenmeyer 
flasks were used as the hydrolysis reactor. The sludge samples were suspended in DI 
water or prehydrolysate and put into the flasks to reach a total working volume of 100 
mL with sludges loading such that the glucan content became 6% (w/v) in the reactor. 
Samples were steam sterilized by autoclaving at 121 ?C for 15 min. The test was 
conducted in an incubator shaker (New Brunswick Scientific, Innova-4080) at 37?C with 
150 RPM. The growth medium for SSF was YP medium. SSF of the sludges was carried 
out at 37 ?C without pH control. Cellulase and ?-glucosidase were also added to achieve 
enzyme loading at 15 FPU and 30 CBU/g-glucan, respectively. Multifect Pectinase FE 
was added as 25 mg protein/g-mannose oligomer for the SSF of paper primary sludge 
with prehydrolysate. For comparison, the same amount of Multifect Pectinase FE was 
also added for the SSF of only paper primary sludge. Optical density (OD) was measured 
by UV Spectrophotometer (BioTek Synergy HT Multidetection Microplate Readers) at 
600 nm for yeast (NREL-D
5
A). The initial OD after inoculation was 0.05, equivalent to 
50 mg of NREL-D
5
A/L. In all of the microbial experiments, a sample from each flask 
was taken at the end of the run and streaked on a YPD plate to check for contamination 
and checked under an optical microscope. CFU tests were performed using YPD agar 
plates to check microorganism viability.  
The ethanol yield was calculated as follows: 
100
0.511reactorin  (g)Sugar  Total Initial
reactorin  (g) produced Ethanol
 maximum] al theoreticof [% yield Ethanol ?
?
=
 
 162
Sugar is interpreted as hexose in the SSF. 
Samples were analyzed for glucose, xylose, organic acid, and ethanol by high 
pressure liquid chromatography (HPLC). Aseptic conditions were maintained in all of the 
microbial experiments.  
6.3.9 Analytical Methods 
The solid samples were analyzed for carbohydrates, acid insoluble lignin (AIL) 
and acid soluble lignin (ASL) following the NREL standard procedures (NREL, 2008). 
The moisture content was measured by an infrared moisture balance (Denver Instrument, 
IR-30). Sugars were determined by HPLC using a BioRad-HPX-87P column. Ethanol, 
HMF, levulinic acid, acetic acid and Furfural were determined by HPLC using a BioRad-
HPX-87H column. A refractive index detector was used with the HPLC. The acid-
insoluble ash was determined following the Tappi test method (Test Method T 244 cm-
99). Liquid sample analysis and ash determination were done in triplicate. The total 
phenolics in the prehydrolysate before and after treatment were determined using Folin-
Ciocalteu method as described by Kujala et al. (2000). 
Where applicable, statistical computation, including mean value and standard 
deviation, was performed using Microsoft Office Excel 2003.   
6.4 Results and Discussion 
6.4.1 Pre-extraction of Softwood 
Water under high pressure and high temperature (160-180?C) was used 
selectively to remove the hemicelluloses from the biomass (Springer and Harris, 1982). 
As wood chips were extracted, their weight decreased as components dissolved and 
diffused from the wood into the extraction liquor (termed as prehydrolysate). The weight 
 163
loss increased with extraction time from almost 4% at the mildest extraction condition (0 
min, 115 H-factor hours) to about 15% at the most severe condition (80 min, 1356 H-
factor hours) (Table VI-2). With the rising of the H-factor, the extracted amount of 
hemicellulose increased up to about 11%. In the case of the extracted amount of cellulose, 
however, only a small trace (0.03%) was detected over a higher H-factor of 1300 h. This 
indicates that there is almost no change in the cellulose content of wood chips because 
the sugar contribution is dominated by hemicellulose within this weight loss range. Total 
sugar content represents the sum of cellulose and hemicelluloses dissolved at a given 
weight loss. The purpose of pre-extraction is to extract hemicellulose while still 
preserving cellulose for quality pulp production. To avoid damaging the fiber quality, 
cellulose was not to be removed at all and hemicellulose was not to be totally removed. 
Therefore, the extraction time of 66 minutes was chosen to extract as much hemicellulose 
as possible without damaging fiber in this experiment. To increase the sugar content in 
the extracted liquor and simulate the continuous extraction, a specified time was used in 
the second and third stages of water extraction. The goal was to extract 10% 
hemicellulose from fresh wood. 
6.4.2 Composition of Softwood Prehydrolysate 
During the pre-extraction process, the hydronium ion from water ionization 
causes the cleavage of the acetyl group, which forms acetic acid acting as a catalyst for 
the hydrolysis of glycosidic bonds in hemicellulose. The hemicellulose chain is 
decomposed first into oligosaccharides and then into monomers. Hemicellulose sugars 
can degrade into furfural, hydroxymethylfurfural (HMF), and some organic acids, such as 
levulinic acid. 
 164
Since 70% of liquor used in the second and third stages was derived directly from 
the last stage, the sugar and organic compounds content of the liquor did increase 
substantially with each stage (Table VI-3 and VI-4). However, there were not much 
difference between the three stages in the resulting pH of the pre-hydrolyzate: 3.50, 3.43 
and 3.39, respectively, in spite of the concentration of acetic acid increasing. The pH did 
not decrease much possibly because ash in the wood is mainly an inorganic salt, which 
dissolved in the water during extraction to form a buffer. In addition, the sugar oligomers 
released during pre-extraction of pine wood were acetylated. Only part of acetyl groups 
in the original wood was released as free acetic acid in the prehydrolysate (Table VI-4), 
possibly due to the moderate stability of ester linkage towards acid.  
The pre-extraction converted most of the hemicellulose (10% based on dry wood) 
to oligomers and monomers. It can be seen from Table VI-3 that most of the dissolved 
sugars were already present in oligomer form rather than as monomers. However, the 
proportion of oligomers was significant and dependent on the type of sugar (Table VI-3). 
As anticipated, the oligomer/monomer ratio was higher for the hexose which had 
previously been shown to be more resistant to acid-catalyzed degradation (BeMiller, 
1967). The arabinose substituents in the arabinoglucuronxylan ? another major 
hemicellulose in softwood which is extremely labile toward acid hydrolysis, were almost 
completely depolymerized during pre- extraction. 
Furfural and HMF can be produced in the hemicellulose extraction process and 
acid hydrolysis, due to the acidic condition and the intense pressure (Nguyen, et al., 
1998). The increase of HMF and furfural (Table VI-4) shows that hexose and pentose 
 165
underwent intense degradation with each additional stage of extraction as solution acidity 
slightly increases.  
Some studies have suggested that covalent linkages between lignin and 
hemicellulose exist in native wood (Bj?rkman, 1957; Erikkson and Lindgren, 1977). 
These structures are typically referred to as lignin-carbohydrate complexes (LCC). The 
lignin is covalently bound to the hemicellulose which, in turn, is bound to cellulose 
through extensive hydrogen bonding. Lignin?carbohydrate bonds and some inter-unit 
lignin bonds, mainly derived from the benzyl alkyl ether type, may be cleaved during 
water pre-hydrolysis. Acetic acid catalyzes the hydrolysis of the ?-ether linkages in lignin 
resulting in the formation of different phenolic products from lignin. Part of the dissolved 
lignin precipitated during the cooling of the prehydrolysate and appeared to be very fine. 
This lignin was separated from the prehydrolysate by centrifugation and denoted as a 
solid fraction. The compositions of the solid fraction (Table VI-5) contained about 6% 
(w/w) carbohydrate and 85% lignin, which show that this part of lignin compounds was 
released in LCC form. Pre-extraction could facilitate subsequent alkaline delignification 
because of the partial hydrolytic degradation of lignin compounds, the cleavage of alkali-
stable carbohydrate?lignin bonds, and the improvement in the accessibility of the cooking 
liquor (Sixta, Herbert (2006) Handbook of pulp, Wiley-VCH, Weinheim). 
6.4.3 Enzymatic Hydrolysis of Softwood Prehydrolysate 
Secondary hydrolysis of softwood prehydrolysate to monosaccharides can be 
accomplished by enzymatic or diluted acid hydrolysis. However, dilute acid hydrolysis 
produces toxins that negatively affect biocatalyst growth and metabolism (Klinke, et al., 
2004). These toxins can be minimized by the enzymatic hydrolysis process which is also 
 166
more environmentally friendly. Moreover, the enzymatic hydrolysis process reduces the 
consumption of acids and alkalis (Hashimoto and Nakata, 2003). In this study, four 
different commercial enzymes were tested, Cellulase enzyme (Spezyme CP), Multifect 
Xylanase, Multifect Pectinase FE, and ?-glucosidase (Novozyme 188), based on the same 
loading protein number (25 mg protein/g mannose oligomer) to evaluate their abilities to 
undergo hydrolysis of softwood prehydrolysate. The results are shown in Fig.VI-1.  
There was relatively low sugar yield by using Spezyme CP and Novozyme-188. 
Over 70% xylose yield was achieved using Multifect Xylanase since it has the highest 
xylanase specific activity among the four enzymes. Multifect Pectinase FE, however, 
appeared to be the most effective. Adding Multifect Pectinase FE significantly improved 
the yields of glucose, galactose and mannose.  
It was speculated that adding pectinase might aid saccharification of 
hemicellulose oligomer because these types of preparations also demonstrated 
hemicellulase related activities, even though only traces of pectin substances are found in 
the cell walls of pine wood. In addition, Berlin et al. found the Multifect Pectinase FE 
shows relatively high specific activity of mannase (3.0 U/mg protein), compared to 
Multifect Xylanase, Spezyme CP and Novozyme- 188 (Berlin  et al.,2007).   From Table 
VI-1, it can be seen that Multifect Pectinase FE also shows relatively high specific 
activity of ?-Glucosidase, Xylanase, ?-Xylosidase, and ?-Galactosidase.  
Similar to the cellulolytic enzymes, synergistic action also occurs in the mannan 
structure as a result of a variety of main- and side-chain-cleaving enzymes.  Due to the 
complexity of the galactoglucomannan structure, both main-and side-chain cleaving 
enzyme activities are required to hydrolyze the oligomer into monomer sugars (Filhom, 
 167
1998). For instance, ?-Glucosidase, an exo-type enzyme, hydrolyzes 1,4-?-D-
glucopyranose at the non-reducing end of the oligosaccharides released from 
glucomannan and galactoglucomannan by ?-mannanase. A side-chain-cleaving enzyme, 
?-Galactosidase, catalyzes the hydrolysis of ?-1, 6-linked D-galactopyranosyl side chains 
of galactomannan and galactoglucomannan (Moreira and Filho, 2008). 
The highest galactose yield (over 70%) achieved by using Multifect Pectinase FE 
also showed the synergistic action of different enzymes. Even though Novozyme-188 has 
the highest ?-Galactosidase specific activity among four enzymes, only 50% galactose 
yield was reached by using ?-glucosidases (Novozyme-188). Clarke et al. also found that 
the galactose release from softwood pulp is enhanced by the presence of mannanase in 
combination with ?-galactosidase (Clarke et al., 2000). Over 60% xylose yield was also 
achieved, even though Multifect Pectinase FE has slightly lower specific activity of 
xylanase than that of Spezyme CP, and 30 times lower than that of Multifect Xylanase. 
However, Multifect Pectinase FE contains the highest ?-Xylosidase activity. It was 
suggested that supplementation of ?-xylosidase improved glucose release during 
hydrolysis by decreasing the accumulation of xylose oligomers, which are found to 
inhibit cellulase activity (Kumar and Wyman, 2009). 
6.4.4 De-toxification and SHF of Softwood Prehydrolysate 
Detoxfication of lignocellulosic hydrolysates by adding CaO or Ca(OH)
2
 to 
increase pH to 10 (over liming) followed by pH readjustment to 5.5 with acid was 
reported to remove certain inhibitory compounds generated in the hydrolysis process 
(Larsson  et al.,1999).  
 168
Two different detoxification conditions were performed in this study: temperature 
at 25 ?C or 60 ?C at 30 min with 10 g CaO/L followed by pH readjustment to 5.5 with 
72% sulphuric acid.   
None of the organic acids concentrations was affected by either condition. 
Phenolic compounds, HMF and furfural were partially removed. A small decrease in 
concentration of total sugars (8% to 12%) and fermentable sugars (glucose and mannose, 
6% to 9%) was observed because of dilution with acid when pH was adjusted to 5.5 and 
because of sugar degradation under alkaline condition. Overall, at a relatively high 
temperature (60 ?C), the concentrations of all compounds decrease slightly more than at 
25 ?C, as shown in Fig.VI-2. 
The treated prehydrolysate was subjected first to enzymatic hydrolysis by 
Multifect Pectinase FE for 48 h at 50 ?C and then to the fermentation step at 32 ?C. The 
overall performance data and the time-course data of SHF are presented in Fig. VI-3. The 
concentration of xylose, galactose and arabinose remained unchanged during the 
fermentation process (data not shown). Although galactose is also a hexose, the 
concentration of galactose remained unchanged during the fermentation process. In the 
yeast Saccharomyces cerevisiae, the flux through the galactose utilization pathway is 
threefold lower than the rate of glucose utilization (Ostergaard et al., 2000). Therefore 
only the uptake of glucose and mannose was calculated in ethanol yield. 
Excellent ethanol production was observed after detoxification. The ethanol yields 
based on initial fermentable sugars (glucose and mannose) were 83.9% and 85.0% at 48 
h, respectively for the detoxification condition of the pre-hyrdrolysate at 25 ?C and 60 
?C, compared to only 56.9% ethanol yield for the untreated prehydrolysate. The ethanol 
 169
concentrations at 96 h increased from 6.0 to 10.4 after 25 ?C and to 10.6 g/L after 60 ?C 
detoxification condition. Although the loss of fermentable sugars is more at 60 ?C than at 
25 ?C, the former temperature was more appropriate for detoxification since it removed 
more HMF, furfural and phenolic compounds. In addition, it is more convenient and 
economical to do detoxification at the higher temperature because of the time and energy 
consumed in cooling down the prehydrolysate from the pre-extraction temperature (170 
?C) to room temperature, such as 25 ?C. Therefore 60 ?C was used during the 
detoxification process for the rest of the study. 
It was very interesting to see that the sugars concentrations from enzymatic 
hydrolysis also increased after detoxification. The glucose concentrations at 48 h 
increased from 4.1 to 4.3 after 25 ?C and 4.4 g/L after the 60 ?C detoxification condition. 
The mannose concentrations at 48 h increased from 10.8 to11.3 after 25 ?C and 11.5 g/L 
after the 60 ?C detoxification condition. The study also found the enzymatic yields of all 
five sugars increased after detoxification under different enzymes, such as Multifect 
Xylanase, Spezyme CP and Novozyme -188 (data not shown here). Apparently, the 
toxins in the prehydrolysate are inhibitory to enzymes. 
The glucose was consumed quickly within 12 h even for untreated pre- 
hydrolysate. However, the mannose consumption rate was much slower for untreated 
hydrolysate. Most glucose could be consumed within 12 h, while mannose needed a 
longer time, about 24 h. Within the first 12 h of fermentation of glucose and mannose, 
glucose tended to be used preferentially compared to mannose by Saccharomyces 
cerevisiae in the prehydrolysate. It has also been reported that Saccharomyces cerevisiae 
would consume glucose faster than mannose (Smith et al., 1997).  
 170
The inhibition of ethanol fermentation was most likely not from furfural and HMF, 
because the concentration of furfural and HMF after detoxification were less than 1 g/L 
and 0.6 g/L, respectively. Low concentrations of furfural and HMF do not affect the 
fermentation of lignocellulosic hydrolysate (Tu et al., 2009;   Taherzadeh et al., 1997). 
Saccharomyces cerevisiae can convert furfural and HMF to their corresponding alcohols 
via NADH-dependent alcohol dehydrogenase (Palmqvist et al., 1999). Although different 
conditions were used in this study, similar results were determined, that after 24 h of 
fermentation the furfural and HMF were quickly consumed by yeast in the fermentation. 
The organic acids, acetic, levulinic and formic acid, inhibit yeast fermentation by 
reducing biomass formation and ethanol yields (Larsson et al., 1999). The concentration 
of organic acids did not change after detoxification. However, Van Zyl et al. (1991) 
found that the degree of inhibition caused by acetic acid depended not only on its 
concentration, but also on the pH of the medium. When the pH of the medium is low, 
acetic acid (pKa=4.75 at 25 ?C), formic acid ( pKa =3.75 at 20 ?C) and levulinic acid 
(pKa=4.66 at 25 ?C) appear in the undissociated form, diffuse across the plasma 
membrane, and dissociate due to higher intracellular pH, thus decreasing the cytosolic pH 
(Pampulha  et al.,1989). As a consequence, the internal pH drops, thus inhibiting cell 
activity and even causing the death of cell. According to the Henderson?Hasselbalch 
equation, at relatively high pH, the concentration of the undissociated form is much lower 
than that at low pH. Therefore the inhibition of organic acids could be lower at relatively 
higher pH when the pH was adjusted to 5.5 while fermenting above pH 7 is unfavourable 
for ethanol production. 
 171
Phenolic compounds could be the major inhibitors in the hydrolysate. Phenolic 
compounds are known to increase biological membrane fluidity and can cause loss of 
cellular integrity, thereby affecting its role in acting as selective barriers and enzyme 
matrices (Heipieper et al., 1991; Heipieper et al., 1994). 
6.4.5 SSF of Softwood Prehydrolysate   
SSF of prehydrolysate after detoxfication was also investigated in this study.  SSF 
combines enzymatic hydrolysis and fermentation in one step. This process can greatly 
reduce the product inhibition to enzymes, which in turn increases sugar production rates, 
concentrations, and yields, and also decreases process time. There is a resulting cost 
saving due to the reduced number of vessels required compared to SHF process.  
The profile of ethanol production with SSF is shown in Fig.VI-4. The observed 
trends for sugar consumption and ethanol production in the SSF process of softwood 
prehydrolysate were similar to the SHF process of prehydrolysate. In the SHF, the 
fermentation of this prehydrolysate produced ethanol at a concentration of 10.51 g/L after 
a total reaction time of 72 h (hydrolysis 48 h and fermentation 24 h) and 10.56 g/L after 
96 h (hydrolysis 48 h and fermentation 48 h), respectively, for 84.6% and 85.0% of the 
ethanol yield. The slightly lower ethanol yield of 82.6% and ethanol concentration of 
10.3 g/L was reached with SSF at 48 h, however there was a gain of 24 h using SSF 
compared with SHF.  
6.4.6 SSF of Prehydrolysate with Paper Sludge 
The ethanol concentration is slightly over 1 % for either SHF or SSF processes of 
prehydrolysate, which is far below the acceptable level as distillation feed. The 
concentration of ethanol in the bioreactor significantly affects the cost of the downstream 
 172
separation process.  Use of the prehydrolysate and pulp mill sludges mixture as the 
fermentation feed can increase the product concentration. Our previous study 
demonstrated that the ethanol production based on Spezyme CP and S. cerevisiae (D
5
A) 
performed well by using untreated kraft paper sludges (Kang et al., 2010). The profile of 
ethanol production of SSF of prehydrolysate with paper sludge is also shown in Fig.VI-4. 
The bioreaction was started with a total sludge loading of 150 g/L, which was 66.75 g-
glucan/L. The ethanol concentration increased from 10.3 g/L (prehydrolysate only) and 
27.7 g/L (paper sludge only) to 31.0 g/L (mixture of prehydrolysate and paper sludge). It 
was noticed that the ethanol yield of the mixture - 70.1% was lower than that of paper 
sludge (73.2%) or prehydrolysate (82.6%). The cellulase enzyme becomes less effective 
by inhibition of ethanol (Wu and Lee, 1997), phenolic compounds (Ximenes et al., 2010), 
furan compounds (Hodge et al., 2008), sugar monomers and sugar oligomers (Xiao et al., 
2004; Nigam and Prabhu, 1991; Kim and Lee, 2005; Kumar and Wyman, 2009).  
6.5 Conclusion 
It is technically feasible to remove part of the hemicellulose before pulping. 
Hemicellulose prehydrolysate can be converted to ethanol from lignocellulosic biomass 
through detoxification, enzymatic hydrolysis and downstream fermentation processes. 
Detoxfication removed the part of toxin which strongly inhibits microbial action, and 
therefore increased the efficiency of ethanol conversion. Different commercial enzyme 
preparation has a different effect on the hydrolysis of prehydrolysate. The Multifect 
Pectinase FE was shown to be most efficient. The overliming detoxification increased 
microbial ethanol conversion. The ethanol yield based on initial fermentable sugars was 
83.9% and 85.0%, respectively for 25 and 60 ?C detoxfication treatment. The 
 173
detoxfication treatment also increased the enzymatic yield of prehydrolysate. In the SHF 
of this prehydrolysate, the ethanol concentration of 10.51 g/L and the ethanol yield of 
84.6% were reached after a total reaction time of 72h (hydrolysis 48 h and fermentation 
24h). The slightly lower ethanol yield of 82.6% and the ethanol concentration of 10.3g/L 
were reached with SSF for 48 h, however there is a gain of 24 h using SSF compared 
with SHF Use of the mixture of prehydrolysate and kraft pulp mill sludges as the 
fermentation feed was attempted to increase the ethanol concentration. The ethanol 
concentration in the broth was increased to 31.5 g/L. The yield of the mixture run was 
lower at 71.5%. The low conversion yield is related to the cellulase enzyme reaction 
rather than microbial reaction. 
 174
Table VI-1. Enzyme activities detected in commercial enzyme preparations  
Activity (units/mg) Spezyme 
CP 
Novozyme 
188 
Multifect 
Xylanase 
Multifect 
Pectinase FE 
Cellulase (FPU)a 0.47 0.06 0.02 0.05 
?-Glucosidase 1.04 4.75 0.85 4.22 
Xylanase (OSX) b 21.32 0.88 600.07 20.29 
?-Arbinofuransoidase 0.18 0.21 0.22 22.71 
?-Xylosidase 0.06 0.12 0.54 2.27 
?-Galactosidase 0.00 0.83 0.06 0.39 
Feruloyl esterase 0.00 0.00 0.00 0.12 
p-Coumaroyl esterase nm c nm c 0.03 0.26 
 
a. Filter paper units. 
b. Oat spelt xylan. 
c. Not measured. 
 175
Table VI-2. Sugar content of first stage prehydrolysate at different time periods 
Water Extraction at 170 
?C 
Total Sugars in Prehydrolysate 
(g/L)  
Polysaccharid
es (% dry 
basis) 
Time 
(min) 
H Wt. 
Loss 
(%) 
Glu Xyl Gal Ara Man C H 
0 115 4.24 0.33 0.74 0.91 0.53 1.81 0.00 2.24 
11 277 5.83 0.56 1.20 1.18 0.69 2.39 0.00 3.12 
25 483 8.57 1.07 2.23 1.79 1.05 4.47 0.00 5.50 
45 839 11.64 1.60 3.13 2.53 1.48 6.69 0.00 8.00 
66 1124 14.00 1.79 3.55 2.81 1.57 9.60 0.00 10.03 
80 1356 14.94 2.40 4.05 3.11 1.83 9.71 0.03 10.91 
 
All data in the table represent the mean values of duplicate runs (n=2; standard 
deviation<0.5).?Total Sugars? in the prehydrolysate were present as monomers. 
C: Cellulose.  
H: Hemicellulose. 
 176
Table VI-3. Sugar content of three stage prehydrolysates 
 Monomer Sugars in 
Prehydrolysate (g/L) 
Oligomer Sugars in Prehydrolysate 
(g/L) 
Total 
Sugars
(g/L) 
 Glu Xyl Gal Ara Man Glu2 Xyl2 Gal2 Ara2 Man2  
1st 
Stage  0.40 1.14 0.73 1.46 0.55 1.60 2.46 2.07 0.27 8.15 19.32 
2 nd 
Stage 1.07 2.65 1.59 2.27 1.60 2.88 3.02 3.60 0.33 12.97 31.99 
3rd 
Stage 2.41 3.76 2.61 3.54 3.20 2.97 2.65 3.93 0.10 15.53 40.70 
 
All data in the table represent the mean value of duplicate runs (n=2; standard 
deviation<0.5). 
 177
Table VI-4. Organic compounds content of three stage prehydrolysates 
(g/L) Acetic acid Acetyl group Levulinic acid HMF Furfural 
1st Stage  0.75 0.25 0.07 0.20 0.37 
2nd Stage  1.58 0.37 0.12 0.57 0.98 
3rd Stage 2.61 0.48 0.20 1.02 1.41 
 
All data in the table represent the mean value of duplicate runs (n=2; standard 
deviation<0.5). 
 178
Table VI-5. Composition of solid fraction in three stage prehydrolysates 
 Solid 
Fraction 
Conc.(g/L) 
Glu 
(Wt%) 
Xyl 
(Wt%) 
Gal 
(Wt%) 
Ara 
(Wt%) 
Man 
(Wt%) 
AIL 
(Wt%) 
ASL 
(Wt%) 
1st Stage  5.5 2.0 2.1 0.3 0.2 1.8 84.7 2.0 
2nd Stage 4.6 1.8 2.0 0.4 0.2 2.0 85.1 2.4 
3rd Stage 4.2 2.1 2.4 0.4 0.2 2.1 84.5 2.7 
 
All data in table are the mean value of duplicate runs (n=2; standard 
deviation<0.5). 
 179
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Cellulase
Spezyme CP
?-Glucosidase
Novozyme 188
Multifect
Xylanase
Multifect
Pectinase FE
48h
 en
z
y
m
a
t
i
c d
i
g
est
i
b
i
l
i
t
y/%
Glucose Xylose Galactose Mannose
 
Fig.VI-1. Different commercial enzyme preparation undergo hydrolysis of softwood 
prehydrolysate 
The data represent the average of duplicate runs. The enzyme loading was 25 mg 
protein/g-mannose oligomer. 
 180
 
0
10
20
30
40
50
60
70
80
90
100
Total Sugar Mannose +
Glucose
Acetic Acid Levulinic
acid
HMF Furfural Total
Soluble
Phenolic
%
untreated 25 ?C 60 ?C
 
Fig.VI-2. Composition change of prehydrolysate after de-toxification  
The data represent average of duplicate runs and are expressed as percentage of the 
concentration of compounds after detoxification. 
 181
0
2
4
6
8
10
12
02040608010
Time (h)
E
t
ha
nol
 C
onc
.
 (
g
/
L
)
0
1
2
3
4
5
2040608010
Time (h)
G
l
uc
os
e
 C
onc
.
 (
g
/
L
)
 
0
2
4
6
8
10
12
14
16
2040608010
Time (h)
M
a
nnos
e
 C
onc
.
 (
g
/
L
)
 
Fig.VI-3. Separation hydrolysis and fermentation (SHF) of prehydrolysate by Multifect 
Pectinase FE and Saccharomyces cerevisiae (ATCC-200062) 
Squares with connecting lines represent prehydrolysate after 60 ?C detoxification 
treatment. Triangles with connecting lines represent prehydrolysate after 25 ?C 
detoxification treatment. Filled triangles with connecting lines represent untreated pre- 
hydrolysate. The data points represent the average of duplicate runs. 
 182
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
0 24487296120
Time (h)
E
t
ha
ni
l
 Y
i
e
l
d (%)
 
0
5
10
15
20
25
30
35
0 24487296120
Time (h)
E
t
ha
nol
 C
onc
.
 
(
g
/
L
)
 
Fig.VI-4. Simultaneous saccharification and fermentation (SSF) of prehydrolysate by 
Saccharomyces cerevisiae (ATCC-200062) and Multifect Pectinase FE 
Filled triangles with connecting lines represent paper sludge with prehydrolysate. 
Triangles with connecting lines represent paper sludge only. Squares with connecting 
lines represent prehydrolysate only. The data points represent the average of duplicate 
runs.  
 
 183
VI. Future Work 
The focus of this dissertation was to study the technical feasibility of establishing 
a biorefinery based on existing paper mills especially those of kraft paper mills. The 
concept of a wood based-biorefinery is to use all components of wood to make pulp and 
paper, while also creating transportation fuels, power, and chemicals. A wood based 
biorefinery could transform the pulp and paper industry, making it sustainable, 
competitive, and innovative. It would promote significant economic growth through new 
value-added products based on existing byproducts or waste at pulp and paper mills (i.e. 
hemicelluloses, black liquor, paper sludge and wood chip rejects), while still preserving 
the main paper and paper-related products. Ethanol production from paper sludge and 
hemicellulose prehydrolysate, and cellulase production from paper sludge and hardwood 
pulp have been investigated. 
The following points are suggested for the future study aimed at establishing a 
biorefinery based on existing paper mills: 
? Butanol production from paper sludge: Butanol can be derived from 
lignocellulosic materials, such as paper sludge. Butanol is considered to be a 
better biofuel than bioethanol because it's less corrosive and has a higher caloric 
value, giving it a higher energy value. Like bioethanol, butanol is being 
considered as an additive to gasoline. Certain clostridial species are well known 
for their ability to produce butanol via ABE fermentation, such as, wild-type
 184
strain Clostridium acetobutylicum (ATCC 824), Clostridium acetobutylicum M5, 
Clostidiumbeijerinckii NCIMB 8052 and Clostidium beijerinckii BA101 (Parekh 
and Blaschek, 1999; Ezeji et al., 2007; Lee et al.,2009; Gu et al., 2009). In the 
future research, Clostridium acetobutylicum (ATCC 824) will be used to convert 
paper sludge and the prehydrolysate into butanol.  
? Lactic acid production from hemicellulose prehydrolysate prehydrolysate: Lactic 
acid (LA) is another value added product which could be produced in the paper 
mills with applications in food industry, cosmetics, polymers, and 
pharmaceuticals.  Recently, the manufacture of biodegradable polylactate 
polymers, an environment-friendly alternative to conventional non-biodegradable 
plastics derived from petrochemicals has, received much attention. Presently, 90% 
of the total LA produced annually worldwide is based on microbial fermentation 
of starch-derived glucose or sucrose (Litchfield, 1996) and the remainder is 
produced by the hydrolysis of lactonitrile (Nolasco-Hipolito et al., 2002). With 
the concern on feedstock cost, the use of lignocellulosic materials, hemicellulose 
hydrolyzate and paper sludge as an inexpensive carbon source for LA production 
have been pursued (Garde et al., 2002; Lee et al., 2004; Neureiter et al., 2004). 
Bioconversion of the sugars in the hemicellulose prehydrolysate to LA would be 
helpful in improving the economics of biorefinery. 
? Study of lignin-carbohydrate complex (LCC) from hemicellulose prehydrolysate: 
Lignin can be partly removed from wood during the hot water pre-hydrolysis 
process. However, lignins are rarely isolated as pure materials, and are always 
associated with carbohydrate linkages to varying extents as lower molecular 
 185
weight segments or lignin-carbohydrate complex (LCC). LCC components 
present in hemicellulose prehydrolysate liquor could be characterized by using 
Nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy 
(FTIR), HPLC, thermal analysis, elemental analysis, atomic absorption 
spectroscopy, and treatment with different commercial enzymes. These diverse 
experimental techniques could give us a deeper insight into the structures and 
properties of LCC and will be valuable in evaluating LCC for new applications. In 
addition, identification and tracking the release of LCC would provide the 
information of basic mechanism during hot water pre-hydrolysis. 
 
 
 186
Bibliography 
Abston, R., 2008. Boise Paper, Jackson, AL, Personal Communication 
Aden, A., Foust, T., 2009. Technoeconomic Analysis of the Dilute Sulfuric Acid and 
Enzymatic Hydrolysis Process for the Conversion of Corn Stover to Ethanol. 
Cellulose. 16(4), 535-545. 
Agosin, E., Monties, B., Odier, E., 1985. Structural changes in wheat straw components 
during decay by lignin-degrading white-rot fungi in relation to improvement of 
digestibility for ruminants. J. Sci. Food Agric. 36, 925?935. 
Ahamed,A.,Vermette,P., 2008.Culture-based strategies to enhance cellulase enzyme 
production from Trichoderma reesei RUT-C30 in bioreactor culture conditions. 
Biochem. Eng. J.40(3), 399-407. 
Alabama Forest Commission , 2009. Woody biomass energy opportunities in Alabama. 
Available in, 
http://www.forestry.state.al.us/Biomass/Woody%20Biomass%20Energy%20Opp
ortunities%20in%20Alabama.pdf 
Al-dajani,W.W., Tschirner, U.W., 2008. Pre-extraction of hemicelluloses and subsequent 
kraft pulping?part I: alkaline extraction. Tappi ,6, 3?8. 
Alen, R., 2000. Basic chemistry of wood delignification. Published in cooperation with 
the Finnish Paper Engineers Association and TAPPI. 58-104. 
Allen, S.G., Schulman, D., Lichwa, J., Antal, M.J.,2001. A comparison of aqueous and 
dilute-acid single-temperature pretreatment of yellow poplar sawdust. Ind. & eng. 
chem. res. 40(10),2352-2361. 
Alkasrawi,M.,Eriksson,T.,Borjesson,J.,Wingren,A., Galbe, M., Tjerneld,F.,2003. The 
effect of Tween-20 on simultaneous saccharification and fermentation of 
softwood to ethanol. Enzyme Microb. Technol.33(1), 71-78. 
Alriksson.B., Horv?th,I.S., Sj?de,A., Nilvebrant,N.O., J?nsson,L.J., Ammonium 
hydroxide detoxification of spruce acid hydrolysates.Appl. Biochem. Biotechnol. 
124(1-3), 911-922. 
 187
Amartey,S., Jeffries,T.W.,1994.Comparison of corn steep liquor with other nutrients in 
the fermentation of d-xylose by Pichia stipitis CBS 6054. Biotechnol. Lett. 16, 
211?214. 
American Forest & Paper Association, 2002. Paper, Paperboard & Wood Pulp: 2002 
Statistics, Data through 2001. 
Andreotti, R. E., Mandels, M.,Roche, C.,1977. in Bioconversion of Cellulosic Substrates 
into Energy, Chemicals and Microbial Protein: Proceedings of Bioconversion 
Symposium. Ghose, T.K., (ed.) Indian Institute of Technology, Delhi. 
Applegarth, D. A., Dutton, G. G. S., 1965. Ion ex- change chromatography of 
hemicelluloses. Tappi 48, 204. 
Arato, C., Pye, E.K. and Gjennestad, G., 2005. The lignol approach to biorefining of 
woody biomass to produce ethanol and chemicals. Appl. Microbiol. Biotechnol. 
121, 871-882. 
Asghari,A., Bothast,R.J., Doran,J.B., Ingram,L.O., 1996. Ethanol production from 
hemicellulose hydrolysates of agricultural residues using genetically engineered 
Escherichia coli strain KO11. J. Ind. Microbiol. Biotechnol.16(1), 42-47. 
Aziz, S., Sarkanen, K.,1989. Organosolv pulping a review Tappi  72(3), 169-175. 
Azzam, A.M. ,1989.Pretreatment of cane bagasse with alkaline hydrogen peroxide for 
enzymatic hydrolysis of cellulose and ethanol fermentation. J. Environ. Sci. Heal. 
24, 421-433. 
Bailey, M.J., Biely, P., Poutanen, K., 1992. Interlaboratory testing of methods for asssay 
of xylanase activity. J. Biotechnol. 23, 257?270. 
Bailey,M.J., Buchert,J., Viikari, L.,1993.Effect of pH on production of xylanase by 
Trichoderma reesei on xylan- and cellulose-based media. Appl. Microbiol. 
Biotechnol. 40, 224?229. 
Bailey, M. J., Siika-aho, M., Valkeaj?rvi, A., Penttil? ,M. E.,1993. Hydrolytic properties 
of two cellulases of Trichoderma reesei expressed in yeast. Appl. Biochem. 
Biotechnol. 17,65-76. 
Ballesteros, I., Ballesteros, M., Cabanas, A., Carrasco, J. , Martin,C., Negro, M.J., 
Saez,F., Saez ,R.,1991. Selection of thermotolerant yeasts for simultaneous 
saccharification and fermentation (SSF) of cellulose to ethanol. Appl. Biochem. 
Biotechnol.28/29, 307?315. 
Bajpai, P., 2004. Biological bleaching of chemical pulps. Crit. Rev. Biotechnol., 24, 1?
58. 
 188
Baptista, C., Belgacem, M.N., Duarte, A.P.,2004.The effect of surfactants on kraft 
pulping of Pinus pinaster, Appita J. 57 (1), 35-39. 
Basaglia, M. , Concheri, G., Cardinali, S., Pasti-Grigsby, M.B., Nuti, M.P., 1992. 
Enhanced degradation of ammonium-pretreated wheat straw by lignocellulolytic 
Streptomyces spp. Can. J. Microbiol. 38(10).1022-1025. 
Beall, D.S., Ohta, K., Ingram,L.O., 1991. Parametric studies of ethanol production from 
xylose and other sugars by recombinant Escherichia coli. Biotechnol. Bioeng. 38, 
296?303. 
Beardmore, D. H.,Fan, L. T.,Lee, Y. H. ,1980. Gamma-ray irradiation as a pretreatment 
for the enzymatic hydrolysis of cellulose. Biotechnol. Lett. 2(10), 435-438. 
BeMiller, J.N., 1967. Acid-catalyzed hydrolysis of glycosides. Adv. Carbohydr. Chem. 
22, 25?108. 
Ben-Ghedalia, D., Miron, J., 1981.The effect of combined chemical and enzyme 
treatment on the saccharification and in vitro digestion rate of wheat straw. 
Biotechnol. Bioeng. 23, 823?831.  
Ben-Ghedalia, D.,Shefet, G., 1983. Chemical treatments for increasing the digestibility of 
cotton straw. J. Agric. Sci. 100, 393?400. 
Berlin, A., Gilkes,N., Kilburn,D., Bura,R., Markov,A., Skomarovsky,A., Okunev, O., 
Gusakov,A., Maximenko,V., Gregg,D., Sinitsyn,A., Saddler,J., 2005a. Evaluation 
of novel fungal cellulase preparations for ability to hydrolyze softwood 
substrates?Evidence for the role of accessory enzymes. Enzyme Microb. 
Technol., 37,175?184. 
Berlin, A., Gilkes,N., Kurabi,A., Bura,R., Tu,M., Kilburn,D., Saddler,J., 2005b. Weak 
lignin-binding enzymes?A novel approach to improve the activity of cellulases 
for hydrolysis of lignocellulosics. Appl. Biochem. Biotechnol.,121?124,163?170. 
Berlin, A., Gilkes,N., Kilburn,D., Maximenko,V., Bura,R., Markov,A., Skomarovsky,A., 
Okunev, O., Gusakov,A., Gregg,D., Sinitsyn,A., Saddler,J., 2006. Evaluation of 
cellulase preparations for hydrolysis of hardwood substrates. 
Appl.Biochem.Biotechnol., 129?132, 528?545. 
Berlin, A., Maximenko, V., Gilkes,N., Saddler,J., 2006. Optimization of enzyme 
complexes for lignocellulose hydrolysis. Biotechnol. Bioeng. 97,287-296. 
Bhat, M.K.,2000. Cellulases and related enzymes in biotechnology. Biotechnol. Adv. 18, 
355?383. 
Biely, P. ,1985. Microbial xylanolytic systems. Trends Biotechnol. 3, 286?290. 
 189
Biely, P., Vrsanska, M., Tenkanen, M., Kluepfel, D., 1997. Endo-?-1, 4-xylanase 
families: differences in catalytic properties. J. Biotechnol. 57(3) ,151-166. 
Biermann, C. J.,1996. Handbook of Pulping and Papermaking, 2
nd
 ed. Academic Press, 
San Diego. 
Bigelow, M., Wyman, C. E., 2002. Cellulase production  on bagasse pretreated with hot 
water. Appl. Biochem. Biotechnol. 98-100, 921-934. 
Biley,J.E., Ollis,D.F., 1986. Biochemical engineering Fundamentals, 2nd, Mcgraw Hill 
Book Co,New York. 
Biely, P.,1985. Microbial xylanolytic systems. Trends Biotechnol. 3, 286?290. 
Bjerre, A.B., Olesen, A.B., Fernqvist, T., 1996. Pretreatment of wheat straw using 
combined wet oxidation and alkaline hydrolysis resulting in convertible cellulose 
and hemicellulose. Biotechnol. Bioeng. 49, 568?577. 
Bj?rkman, A., 1957. Studies on Finley Divided Wood Part 3. Extraction of Lignin-
Carbohydrate Complexes with Neutral Solvents. Svensk Papperst. 60 (7), 243-
251. 
B?rjesson,J., Peterson,R., Tjerneld,F.,2007. Enhanced enzymatic conversion of softwood 
lignocellulose by poly(ethylene glycol) addition. Enzyme Micro. Technol.,40, 
754?762. 
Boussaid, A.L., Esteghlalian, A.R., Gregg, D.J., Lee, K.H., Saddler, J.N., 2000. Steam 
pretreatment of Douglas-fir wood chips - Can conditions for optimum 
hemicellulose recovery still provide adequate access for efficient enzymatic 
hydrolysis. Appl. Biochem. Biotechnol.  84-6, 693-705. 
Bradford, M. M., 1976. A rapid and sensitive method for quantification of microgram 
quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 
72, 248-254. 
Buchert, J., Salminen, J., Siika-aho, M., Viikari, L., 1993.The role of T. reesei xylanase 
and mannanase in treatment of softwood kraft pulp prior to bleaching. 
Holzforschung,47, 413-478. 
Buckeridge, M. S., Pessoa, dos Santos, H., Tine, M. A. S., 2000. Mobilisation of storage 
cell wall polysaccharides in seeds. Plant Physiol. Biochem. 38, 141?156. 
Buhner J, Agblevor FA. 2004. Effect of detoxification of dilute-acid corn fiber 
hydrolysate on xylitol production. Appl. Biochem. Biotechnol. 119, 13-30. 
Burns, D.S., Ooshima, H., Converse, A.O., 1989. Surface area of pretreated 
lignocellulosics as a function of the extent of enzymatic hydrolysis. Appl. 
Biochem. Biotechnol. 20?21, 79?94. 
 190
Cabezas, M.J., Salvador, D. and Sinisterra, J.V., 1991.  Stabilization-activation of 
pancreatic enzymes adsorbed on to a sepiolite clay. J. Chem. Tech. Biotechnol. 
52, 265?274.  
Campbell, C.J., Laherrehe, J. H., 1998. The end of cheap oil. Sci. Am. 3, 60 ? 65. 
Casebier, R.L., Hamilton, J.K., Hergert, H.L., 1969. Chemistry and mechanism of water 
prehydrolysis of southern pine wood. Tappi, 52, 12, 2369?2377. 
Castanon,M., Wilke,C.R.,1981. Effects of the surfactant Tween 80 on enzymatic 
hydrolysis of newspaper. Biotechnol. Bioeng., 23, 1365?1372. 
Caufield, D.F., Moore, W.E., 1974. Effect of varying crystallinity of cellulose on 
enzymic hydrolysis. Wood Science, 6, 4, 375?379. 
Cavaco-Paulo, A.,Almeida, L., 1994. Cellulase hydrolysis of cotton cellulose: the effects 
of mechanical action, enzyme concentration and dyed substrates. Biocatalysis.10, 
353?360. 
Cavaco-Paulo, A., Almeida, L., Bishop, D.P., 1996. Effects of agitation and 
endoglucanase pre-treatment on the hydrolysis of cotton fabrics by a total 
cellulase. Text. Res. J. 66(5), 287?294. 
Cavaco-Paulo, A., Almeida, L., Bishop, D.P., 1998. Hydrolysis of cotton fabrics by 
engineered cellulases from Trichoderma reesei. Text. Res. J. 68 (4), 273?280. 
Caylak,B.,Vardar,S.F.,1996. Comparison of different production processes for 
bioethanol. Turk. J. Chem., 22,351?359. 
Chahal, D.S.,1985. Solid-state fermentation with Trichoderma reesei for cellulase 
production. Appl. Environ. Microbiol. 49(1),205?210. 
Chandakant,P., Bisaria,V.S.,1998. Simultaneous bioconversion of cellulose and 
hemicellulose to ethanol. Crit. Rev. Biotechnol.18(4), 295-331. 
Chandler, J.A., Jewell, W.J., Gossett, J.M., Van Soest, P.J., Robertson, J.B., 1980. 
Predicting methane fermentation biodegradability. Biotechnol. Bioeng. Sym. No. 
10, 93-107. 
Chang,M.M.,Chou,Y.C.T.,Tsao,G.T.,1981.structure Pretreatment and Hydrolysis of 
Cellulose. Adv.Biochem.Eng.,20,15-42. 
Chang, V.S., Holtzapple, M.T., 2000.Fundamental factors affecting biomass enzymatic 
reactivity. Appl. Biochem. Biotechnol. 84/86, 5?37. 
Chang, V.S., Nagwani, M., Kim, C.H. and Holtzapple, M.T., 2001. Oxidative lime 
pretreatment of high-lignin biomass - Poplar wood and newspaper. Appl. 
Biochem. Biotechnol. 94(1),1-28. 
 191
Chanzy, H., Henrisaat, B., Vuong, R., 1984. Colloidal gold labelling of 1,4-?-D-glucan 
cellobiohydolase adsorbed on cellulose substrates. FEBS Lett., 172, 193-197. 
Chanzy, H., Imada, K., Mollard, A., Vuong, R., Barnoud, F., 1979.Crystallographic 
aspects of sub-elementary cellulose fibrils occurring in the wall of rose cells 
cultured in vitro. Photoplasma, 100, 3?4, 303?316. 
Chapin, F. S., Matson, P. A., Mooney, H. A., 2002. Principles of Terrestrial Ecosystem 
Ecology; SpringerVerlag,New York. 
Chaudhary K, Tauro P., 1982. Selective induction of ?-glucosidase in Trichoderma 
reesei by xylan. Eur. J. Appl. Microbiol. Biotechnol. 15, 185?187. 
Chen, H. Z., Hayn, M., Esterbauer, H.,1992. Purification and characterization of two 
extracellular ?-glucosidases from Trichoderma reesei. Biochim. Biophys. Acta 
1121, 54-60. 
Chen, S., Wayman, M., 1991. Cellulase production induced by carbon sources devired  
from waste newsprint. Process Biochem. 26, 93-100. 
Chesson, A., Gardner ,P.T., Wood, T.J., 1997. Cell wall porosity and available surface 
area of wheat straw and wheat grain fractions. J. Sci. Food Agric.,75(3),289?295. 
Chum, H.L., Johnsoon, D.K., Black, S., 1988. Organosolv pretreatment for enzymatic 
hydrolysis of poplars: 1. enzyme hydrolysis of cellulosic residues. Biotechnol. 
Bioeng. 31, 643?649. 
Chum, H.L., Johnson, D.K., Black, S.K., 1990. Organosolv Pretreatment for Enzymatic-
Hydrolysis of Poplars .2. Catalyst Effects and the Combined Severity Parameter. 
Ind. Eng. Chem. Res. 29(2),156-162. 
Clark, D.P.., 1989. The fermentation pathways of Escherichia coli. FEMS Microbiol. 
Rev. 5, 223?234. 
Clarke, A.J., 1997. Interactions and associations, Chapter 3 In: Biodegradation of 
cellulose: Enzymology and Biotechnology. Technomic publishing co., Inc., 
Lancaster, Basel. 
Clarke, J. H., Davidson, K., Rixon, J. E., Halstead, J. R., Fransen, M. P., and Gilbert, H. 
J., 2000. A comparison of enzyme aided bleaching of softwood paper pulp using 
combinations of xylanase, mannanase and alpha-galactosidase. Appl. Microbiol. 
Biotechnol. 53, 661?667. 
Clark, T.A., McDonald, A.G., Senior, D.J., Mayers, P.R., 1990. Mannanase and xylanase 
treatment of softwood chemical pulps: effects on pulp properties and 
bleachability. In: Kirk, K.T., Chang, H.-M. (Eds.), Biotechnology in Pulp and 
Paper Manufacture. Butterworth-Heinemann,Boston, Pulp and Paper 
Manufacture, Butterworth-Heinemann. 
 192
Closset, G., 2004. Annex XV Targets Barriers to Black liquor Gasification, Solutions, 39-
41. 
Closset, G., Raymond, D., Thorp,B., 2005. The Integrated Forest Products Biorefinery. 
An Agenda 2020 Program. Available: 
http://www.agenda2020.org/PDF/IFPB_Brochure.pdf 
Converse, A.O., Matsuno, R., Tanaka,M., Taniguchi,M.,1988. A model for enzyme 
adsorption and hydrolysis of microcrystalline cellulose with slow deactivation of 
the adsorbed enzyme. Biotechnol. Bioeng. 32, 38?45. 
Converse, A.O., Ooshima,H.,Burns,D.S.,1990. Kinetics of enzymatic hydrolysis of 
lignocellulosic materials based on surface area of cellulose accessible to enzyme 
and enzyme adsorption on lignin and cellulose. Appl. Biochem. Biotechnol., 
24/25,67-73. 
Copa-Pati?o, J.L., Kim,Y.G. and Broda, P.,1993. Production and initial characterization 
of the xylan-degrading system of Phanerochaete chrysosporium. Appl. Microb. 
Biotechnol. 40(1), 69-76. 
Coughlan, M.P., Hazlewood, G.P., 1993. ? -1,4-D-Xylan-degrading enzyme systems: 
biochemistry,molecular biology and applications. Appl. Biochem. Biotechnol. 17, 
259?289. 
Coughlan, M.P., Tuohy, M.G., Edivaldo Filho, X.F., Pulsf,J., Claeyssens,M., 
Vrsanska,M.,1993. Enzymological aspects of microbial hemicellulases with 
emphasis on fungal systems. in Hemicellulose and Hemicellulases, Portland 
Press. 
Dadi, A.P., Varanasi, S., Schall, C.A.,2006. Enhancement of cellulose saccharifi cation 
kinetics using an ionic liquid pretreatment step. Biotechnol Bioeng. 95, 904?910. 
Dale,B.,Henk,L.,Shianga,M.,1984. Fermentation of lignocellulosic materials treated by 
ammonia freeze-explosion. Dev. Ind. Microbiol. 26,223-233. 
Dale, B.E., Moreira, M.J., 1982. A freeze explosion technique for increasing cellulose 
hydrolysis. Biotechnol. Bioeng.12, 31?43. 
Davis,M.E., Maxwell, C.V., Brown, D.C., de Rodas,B.Z., Johnson,Z.B., Kegley, E.B., 
Hellwig,D.H., Dvorak,R.A., 2002. Effect of dietary mannan oligosaccharides 
and(or) pharmacological additions of copper sulfate on growth performance and 
immunocompetence of weanling and growing/finishing pigs. J. Anim. Sci. 80, 
2887-2894. 
Desai, S.G., Converse,A.O.,1997. Substrate reactivity as a function of the extent of 
reaction in the enzymatic hydrolysis of lignocellulose. Biotechnol. Bioeng., 56 
,650?655 
 193
Deshpande, M.V., 1992. Ethanol Production From Cellulose by Coupled 
Saccharification/Fermentation Using Saccharomyces cerevisiae and Cellulase 
Complex From Sclerotium Rolfsii UV-8 Mutant. Appl. Biochem. Biotechnol., 36, 
227?234.  
Dhawan, S., Kaur, J.,2007.  Microbial Mannanases: An Overview of Production and 
Applications. Crit. Rev. Biotechnol. 27(4), 197-216. 
Dien,B.S., Cotta,M.A. and Jeffries,T.W., 2003.Bacteria engineered for fuel ethanol 
production: Current status. Appl. Microb. Biotechnol. 63 (3), 258?266.  
Dien,B.S., Ximenes, E.A.,O'Bryan, P.J.,Moniruzzaman, M.,Li, X., Balan, V., Dale,B., 
Cotta, M.,2008. Enzyme characterization for hydrolysis of AFEX and liquid hot-
water pretreated distillers' grains and their conversion to ethanol. Bioresource 
Tech. 99, 5216?5225. 
Domalski, E.S., Milne T.A., ed.1987. Thermodynamic Data for Biomass Materials and 
Waste Components. The ASME Research Committee on Industrial and Municipal 
Wastes, New York: The American Society of Mechanical Engineers. 
Domingues, F.C., Queiroz, J.A., Cabral, J.M.S.,  Fonseca, L.P., 2000. The influence of 
culture conditions on mycelial structure and cellulase production by Trichoderma 
reesei Rut C-30. Enzyme Microb.Technol. 26, 394?401. 
Domingues, F.C., Queiroz, J.A., Cabral, J.M.S.,  Fonseca, L.P., 2001. Production of 
cellulases in batch culture using a mutant strain of Trichoderma reesei growing on 
soluble carbon source, Biotechnol. Lett. 23,771?775. 
Doppelbauer, R., Esterbauer, H., Steiner, W., Lafferty, R. M., Steinmuler, H., 1987. The 
use of lignocellulosic wastes for production of cellulase by Trichoderma reesei. 
Appl. Microbiol. Biotechnol. 26, 485?494. 
Dorica , J. and Simandl, J.,1995. Separation of fiber and ash in deinking effluents?a case 
study, Tappi ,78, 109?116. 
Draude, K.M., Kurniawan,C.B., Duff, S.T.B.,2001.  Effect of oxygen delignification on 
the rate and extent of enzymatic hydrolysis of lignocellulosic material, Bioresour. 
Technol.,79, 113?120. 
Duff,S.J.B., Murray,W.D., 1996. Bioconversion of forest products industry waste 
cellulosics to fuel ethanol: a review. Bioresour. Technol. 55, 1?33. 
Duffaud,G.D., McCutchen,C.M., Leduc,P., Parker,K.L., Kelly, R.M.,1997.  Purification 
and characterization of extremely thermostable ?- mannanase, ?-mannosidase, 
and ?-galactosidase from the hyperthermophilic Eubacterium Thermotoga 
neapolitana 5068. Appl. Environ. Microbiol.,63,169?177. 
 194
Dunlap,C.E., Thomson,J.,Chiang,L.C.,1976. Treatment processes to increase cellulose 
microbial digestibility. AICHE, Symp.,Ser.,72(158),58. 
Eckert,C., Liottaabc, C., Ragauskasb,A., Hallettac, J., Kitchensac,C., Hillac,E., Draucker, 
L., 2007. Tunable solvents for fine chemicals from the biorefinery. Green Chem. 
9,545-548. 
Egyhazi, A., Dienes, D., Reczey, K., Simandi, B., 2004. Examination of cellulase enzyme 
production by Trichoderma reesei rut C30 using supercritical carbon dioxide cell 
disruption. Chem. Biochem. Eng. Q. 18(3), 257-261. 
Ethier,N.,Talbot,G.,Sygusch,J.,1998. Gene Cloning, DNA Sequencing, and Expression of 
Thermostable -Mannanase from Bacillus stearothermophilus. Appl. Environ. 
Microbiol., 64(11), 4428?4432. 
Elshafei, A.M., Vega, L.L., Kalsson, K.T., Clausen, E.C. and Gaddy, J.L., 1991. The 
saccharification of corn stover by cellulase from Penicillium funiculosum. Biores. 
Technol. 35, 73-80. 
Energy Information Administration,2007.Energy Consumption by Sector, 1949?2006. 
Energy Information Administration. 2007-01. Available at: 
http://www.eia.doe.gov/aer/pdf/pages/sec2_4.pdf. 
Environmental Paper Network,2007. The State of the Paper Industry: Monitoring the 
Indicators of Environmental Performance. Available at:  
http://www.greenbiz.com/resources/resource/the-state-paper-industry-monitoring-
indicators-environmental-performance. 
Eriksson,K.E., Blanchette, R.A.,Ander,P., 1990. Microbial and enzymatic degradation of 
wood and wood components. Springer Series in Wood Science, Springer-
Verlag,Berlin. 
Erikkson, O. and Lindgren, B.O., 1977. About the Linkage Between Lignin and 
Hemicelluloses in Wood. Svensk Papperst. 80 (2), 59-63. 
Eriksson, T.,Karlsson,J.,Tjerneld,F.,2002a. A model explaining declining rate in 
hydrolysis of lignocellulose substrates with cellobiohydrolase I (cel7A) and 
endoglucanase I (cel7B) of Trichoderma reesei, Appl. Biochem. Biotechnol . 101, 
41?60. 
Eriksson, T., Boerjesson,J., Tjerneld,F.,2002b. Mechanism of surfactant effect in 
enzymatic hydrolysis of lignocellulose. Enzyme Microb. Technol. 31(3),353-364. 
Esterbauer H, Steiner J, Labudova I, Hermann A, Hayn M.,1991. Production of 
Trichoderma cellulase in laboratory and pilot scale. Biores. Technol. 51?65. 
 195
Ezeji,T., Qureshi,N., and Blaschek, H.P. (2007) Butanol production from agricultural 
residues: Impact of degradation products on Clostridium beijerinckii growth and 
butanol fermentation. Biotechnol Bioeng. 97(6),1460-9. 
Fan, L.T., Lee, Y.H., Gharpuray, M.M., 1982. The nature of lignocellulosics and their 
pretreatments for enzymatic hydrolysis. Adv. Biochem. Eng. 23, 157?187. 
Fan, L.T., Lee. Y.H., Beardmore. D.H., 1980. The influence of major structural features 
of cellulose on rate of enzymatic hydrolysis.Biotechnol. Bioeng. 23, 419-424. 
Fan, L.T., Lee. Y.H., Beardmore. D.H., 1981. Mechanisms of the enzymatic hydrolysis 
of cellulose: Effects of major structural features of cellulose on enzymatic 
hydrolysis. Biotechnol. Bioeng. 22, 177?199. 
Fan, Z., Lynd, L. R., 2006a. Conversion of paper sludge to ethanol. I: Impact of feeding 
frequency and mixing energy characterization. Bioprocess Biosyst Eng. 30(1), 27-
34. 
Fan,Z., Lynd, L.R., 2006b. Conversion of paper sludge to ethanol, II: process design and 
economic analysis. Bioprocess Biosyst. Eng. 30(1), 35-45. 
Fan, Z., South, C., Lyford, K., Munsie, J. , Walsum, P. and Lynd, L. R., 2003. 
Conversion of paper sludge to ethanol in a semicontinuous solids-fed reactor.  
Bioprocess Biosyst Eng. 26(2), 93-101. 
Fein, J.E. , Tallim,R., Lawford, G.R., 1984. Evaluation of d-xylose fermenting yeasts for 
utilization of a wood-derived hemicellulose hydrolysate. Can. J. Micrbiol. 30 , 
682?690. 
Feist, W.C., Springer, E.L., Hajny, G.J., 1974. Storing wood chips at elevated 
temperatures.  Effect of alkali pretreatment. Tappi, 57, 8, 112?114. 
Feldman, D., 2002. Lignin and its polyblends ? a review. In ?Chemical, Modification, 
Properties, and Usage of Lignin? (T. Q. Hu, ed.), Kluwer Academic/Plenum 
Publishers, New York. 
Fengel, D., Wegener, G., 1984.Wood:  Chemistry, Ultrastructure, Reactions. de Gruyter, 
Berlin, Germany.  
Fernandez,F., Hinton,M., Van Gils,B.,2002. Dietary mannan-oligosaccharides and their 
effect on chicken caecal microflora in relation to Salmonella Enteritidis 
colonization. Avian Pathol. 31, 49-58. 
Fernandez-Bolanos, J., Felizon, B., Heredia, A., Jimenez, A., 1999. Characterization of 
the lignin obtained by alkaline delignification and of the cellulose residue from 
steam-exploded olive stones. Biores. Technol. 68, 121?132. 
 196
Filho, E.X.F ., 1998. Hemicellulases and biotechnology. In: Pandalai SG (ed) Recent 
research developments in microbiology. Research Signpost, Trivandrum. 
Filho, E.X.F., Puls,J.,Coughlan,M.P.,1993. Physicochemical and catalytic properties of a 
low-molecular-weight endo-1,4- beta -D-xylanase from Myrothecium verrucaria. 
Enzyme Microb.Technol.,(15)66,535-540. 
Foreman, P.K., Brown, D., Dankmeyer,L., Dean,R., Diener, S., Dunn-Coleman,N.S., 
Goedegebuur,F., Houfek,T., England,G., Kelley,A., Meerman,H., Mitchell,T., 
Mitchinson,C., Olivares, H., Teunissen, P., Yao, J., Ward, M.,2003. 
Transcriptional regulation of biomassdegrading enzymes in the filamentous 
fungus Trichoderma reesei. J. Biol. Chem. 278(34),31988?31997. 
Foster, B.L., Dale, B.E., Doran-Peterson, J.B., 2001. Enzymatic hydrolysis of ammonia-
treated sugar beet pulp, Appl. Biochem. Biotechnol. 91 (3), 269?282. 
Franco, P. F., Ferreira, H. M., Filho, E. X., 2004. Production and characterization of 
hemicellulase activities from Trichoderma harzianumstrain T4. Biotechnol. Appl. 
Biochem., 40, 255?259. 
Gadgil, N.J., Daginawala, H.F., Chakrabarti,T., Khanna, P.,1995. Enhanced Cellulase 
Production by a Mutant of Trichoderma-Reesei. Enzyme Microb.Technol.17(10), 
942-946. 
Galbe, M. and Zacchi, G., 2002. A review of the production of ethanol from softwood. 
Appl. Microbiol. Biotechnol. 59, 618?628. 
Garde A, Jonsson G, Schmidt AS, Ahring BK, 2002. Lacticacid production from wheat 
straw hemicellulose hydrolyzate by Lactobacillus pentosus and Lactobacillus 
brevis. Bioresource Technology 81: 217?223. 
Garrote, G., Dominguez, H., Paraj? J.C., 2002. Autohydrolysis of corncob: study of non-
isothermal operation for xylooligosaccharide production. J. Food Eng. 52, 211?
218. 
Ghose, T.K., 1987. Measurement of cellulase activities. Pure Appl. Chem. 59,257?268. 
Ghosh, T.K., Roychoudhury, P.K., Ghosh, P., 1984. Simultaneous saccharification and 
fermentation (SSF) of lignocellulosics to ethanol under vacuum cycling and step 
feeding. Biotechnol. Bioeng. 26, 377?381. 
Gilarranz, M.A., Rodriguez, F., Oliet, M., 2000. Lignin behavior during the autocatalyzed 
methanol pulping of Eucalyptus globulus - Changes in molecular weight and 
functionality. Holzforschung. 54(4), 373-380. 
Gilkes, N. R., Henrissat, B., Kilburn, D. G., Miller, R. C., Jr., Warren, R. A.,1991. 
Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and 
enzyme families. Microbiol. Rev. 55, 303?315. 
 197
Glasser, W.G., Northey, R.A., Schultz, T.P., 2000. Lignin: Historical, Biological, and 
Materials Perspectives. American Chemical Society, Washington, D.C. 
Goldstein,I.S.,1981. In organic chemicals from biomass,CRC press, Inc., Boca Raton,FL. 
Gollapalli, L.E., Dale, B.E. and Rivers, D.M., 2002. Predicting digestibility of ammonia 
fiber explosion (AFEX)-treated rice straw. Appl. Biochem. Biotechnol. 98, 23-35. 
Gong, C. S., Ladisch, M.R.,Tsao,G.T.,1977. Cellobiase from Trichoderma viride: 
purification, properties, kinetics, and mechanism. Biotechnol. Bioeng. 19, 959-
981. 
Gong, C.S., Cao, N.J.,Du,J., Tsao,G.T.,1999. Ethanol production from renewable 
resources, Adv. Biochem. Eng. Biotechnol. 65,207?241. 
Gunata, Z., Vallier, M. J.,1999. Production of a highly glucose-tolerant extracellular ?-
glucosidase by three Aspergillus strains. Biotechnol. Lett. 21,219-223. 
Grethlein, H., 1985. The effect of pore size distribution on the rate of enzymatic 
hydrolysis of cellulosic substrates. Biores. Technol. Feb, 155?160. 
Grethlein, H.E., Allen, D.C., Converse, A.O.,1984. A comparative study of the enzymatic 
hydrolysis of acid-pretreated white pine and mixed hardwood. Biotechnol. 
Bioeng. 26(12), 1498 ? 1505. 
Grethlein, H.E., Converse, A.O., 1991. Common Aspects of Acid Prehydrolysis and 
Steam Explosion for Pretreating Wood. Biores. Technol. 36(1),77-82. 
Gritzali, M.,  Brown, R.D.Jr., 1979. The cellulase system of Trichoderma. The 
relationship between purified extracellular enzymes from unduced or 
cellulosegrown cells. Adv. Chem. Ser. 81, 237.260. 
Grohmann, K., 1993. Simultaneous saccharification and fermentation of cellulosic 
substrates to ethanol. In: Bioconversion of Forest and Agricultural Plant Residues. 
Biotechnology in Agriculture No. 9. C.A.B. International, Wallingford, UK, 183-
209.  
Grohmann, K., Mitchell, D.J., Himmel, M.E., Dael, B.E., Schroeder, H.A., 1989. The 
role of ester groups in resistance of plant cell wall polysaccharides to enzymatic 
hydrolysis. Appl. Biochem. Biotechnol. 20/21, 45?61. 
Grohmann, K., Torget, R.W., Himmel, M.E., 1986. Dilute acid pretreatment of biomass 
at high solid concentrations. Biotechnol. Bioeng. Symp. 17, 135?151. 
Grohmann, K., Torget, R.W., Himmel, M.E., 1985. Optimization of dilute acid 
pretreatment of biomass. Biotechnol. Bioeng. Symp. 15, 59?80. 
 198
Grous, W.R., Converse, A.O., Grethlein, H.E., 1986. Effect of Steam Explosion 
Pretreatment on Pore-Size and Enzymatic-Hydrolysis of Poplar. Enzyme Microb. 
Technol. 8(5), 274-280. 
Gu, Y., Li, J., Zhang, L., Chen, J., Niu, L., Yang, Y., Yang, S., and Jiang, W. (2009) 
Improvement of xylose utilization in Clostridium acetobutylicum via expression 
of the talA gene encoding transaldolase from Escherichia coli. J Biotechnol. 
143(4):284-7 
G?bitz,G.M., Hayn,M., Sommerauer,M., Steiner,W., 1996. Mannandegrading enzymes 
from Sclerotium rolfsii: characterization and synergism of two endo ?-
mannanases and ?-mannosidase. Biores. Technol., 58,127?135. 
Gupta, R., Kim, T.H., Lee, Y.Y.,2008. Substrate Dependency and Effect of Xylanase 
Supplementation on Enzymatic Hydrolysis of Ammonia-Treated Biomass. Appl. 
Biochem. Biotechnol. 148(1-3), 59-67. 
Haab, D., Hagspiel, K., Szakmary, K., Kubicek, C. P., 1990. Formation of extracellular 
proteases from Trichoderma reesei QM9414 involved in cellulase degradation. J. 
Biotechnol. 16, 187?198. 
H?gerdal B., Harris H., Pye E.K., 1979. Association of beta-glucosidase with intact cells 
of Thermoactinomyces. Biotechnol. Bioeng. 21(3), 345-55. 
Hahn-H?gerdal, B., Galbe, M., Gorwa-Grauslund, M.F., Lid?n, G.and Zacchi, G.,2006. 
Bio-ethanol-the fuel of tomorrow from the residues of today. Trends Biotechnol. 
24(12), 549-556.  
Hahn-H?gerdal, B., Jeppsson, H., Olsson, L., Mohagegi, A., 1994. An Interlaboratory 
comparison of the performance of ethanol producing micro-organisms in a 
xylose-rich acid hydrolysate. Appl. Microbiol. Biotechnol. 41, 62?72. 
Hamzehi,E., Pflug, W., 1981. Sorption and Binding Mechanism of Polysaccharide 
Cleaving Soil Enzymes by Clay Minerals. Z. Pflanzenernaehr. Bodenkd. 144, 
505?513. 
Handford, M. G., Baldwin, T. C., Goubet, F., 2003. Localization and characterization of 
cell wall mannan polysaccharides in Arabidopsis thaliana. Planta. 218, 27?36. 
Hansen, C. M., Bj?rkman, A.,1998. The ultrastructure of wood from a solubility 
parameter point of view. Holzforschung 52 (4), 335-344. 
Hardesty, K.L. and Beer ,E.H., 1993. Drying and recycling of primary sludge at 
Champion International, Tappi ,76, 207?211. 
Hartman,J., Albertsson,A.C., Lindblad, M.S., Sj?berg, J., 2006. Oxygen barrier materials 
from renewable sources: Material properties of softwood hemicellulose-based 
films. J. Appl. Polym. Sci. 100(4), 2985-2991. 
 199
Hashimoto, T., Nakata,Y., 2003. Synergistic degradation of arabinoxylan with ?- L-
arabinofuranosidase, xylanase and ?-xylosidase from soy sauce koji mold, 
Aspergillus oryzae, in high salt condition. J. Biosci. Bioeng. 95(2), 164 ? 169. 
Haska, G., 1981. Activity of bacteriolytic enzymes adsorbed to clays. Microbial Ecology. 
7, 331-341. 
Hayward, T.K., Hamilton, J., Tholudur, A., McMillan, J.D., 2000.  Improvements in titer, 
productivity, and yield using solka-floc for cellulase production. Appl. Biochem.  
Biotechnol. 84/86, 859-874.  
Heipieper, H.J., Keweloh, H., Rehm, H.J.,1991. Influence of phenols on growth and 
membrane permeability of free and immobilized Escherichia coli. Appl. Environ. 
Microbiol. 57(4), 1213?1217. 
Heipieper, H.J., Weber, F.J., Sikkema, J., Keweloh, H., de Bont,J.A.M., 1994. 
Mechanisms of resistance of whole cells to toxic organic solvents. Trends 
Biotechnol. 12(10), 409?415. 
Helle, S.S., Duff,S.J.B., Cooper, D.G.,1993. Effect of surfactants on cellulose hydrolysis. 
Biotechnol. Bioeng., 42, 611?617. 
Hjortkjaer, R.K., Bille-Hansen, V., Hazelden, K.P., McConville, M., McGregor, D.B., 
Cuthbert, J.A., Greenough, R.J., Chapman, E., Gardner, J.R. and Ashby, R. 1986. 
Safety evaluation of Celluclast, an acid cellulase derived from Trichoderma 
reesei. Fd Chem Toxicol. 24(1), 55-63. 
Ho,N.W.Y., Chen,Z., Brainard,A.P., 1998. Genetically engineered Saccharomyces yeast 
capable of effective cofermentation of glucose and xylose. Appl. Environ. 
Microbiol. 64 (5),1852?1859. 
Hodge, D.B., Karim,M.N., Schell,D.J., McMillan,J.D. ,2008. Soluble and insoluble solids 
contributions to high-solids enzymatic hydrolysis of lignocellulose. Bioresour. 
Technol. 99, 8940-8948. 
Hohmann, N., Rendleman, C.M., 1993. Emerging technologies in ethanol production. US 
Department of Agriculuture Information Bulletin Number 663, 1-17. 
Holtzapple,M.T.,1993. Chapters cellulose, hemicelluloses, and lignin. In:  
Macrae,R.,Robinson,R.K., Sadler,M.J., Editors, Encyclopedia of Food Science, 
Food Technology, and Nutrition, Academic Press, London. 
Holtzapple, M., Cognate, M., Shu, Y., Hendrichson, C., 1990. Inhibition of T. reesei 
cellulase by sugars and solvents. Biotechnol. Bioeng. 36, 275?287. 
Holtzapple, M.T., Humphrey, A.E., 1984. The Effect of Organosolv Pretreatment on the 
Enzymatic-Hydrolysis of Poplar. Biotechnol. Bioeng. 26(7), 670-676. 
 200
Horecker, B.L., 1962. Pentose Metabolism in Bacteria. Wiley, New York.  
Hoshino E., Kanda T., Sasaki Y., Nisizawa K., 1992. Adsorption mode of exo-cellulases 
and endo-cellulases from irpex-lacteus (Polyporus, Tulipiferae) on cellulose with 
different crystallinities. J. Biochem.,600?605. 
Hoshino E., Sasaki Y., Okazaki M., Nisizawa K.,Kanda T., 1993. Mode of action of exo-
type and endo-type cellulases from irpex-lacteus in the hydrolysis of cellulose 
with different crystallinities. J. Biochem., 114, 230?235. 
Hoshino E., Shiroishi M., Amano Y., Nomura M., Kanda T., 1997. Synergistic actions of 
exo-type cellulases in the hydrolysis of cellulose with different crystallinities. J. 
Ferment. Bioeng.,84, 300?306. 
Hsu, T.A. (Editor), 1996. Pretreatment of biomass. Handbook on bioethanol:production 
and utilization. Taylor & Francis, Washington. 
Huang, X.L., Penner, M.H.,1991. Apparent substrate inhibition of the Trichoderma reesei 
cellulase system. J. Agric. Food Chem., 39, 2096?2100. 
Hugo, W.B., Lund,B.M.,1968. The effect of supplementation of growth media with yeast 
extract on dehydrogenases activity of  Escherichia Coli. J. appl. Bact. 21 (2), 249-
256. 
Ilm?n,M., Thrane,C., Penttil?,M.,1996. The glucose repressor gene cre1 of Trichoderma: 
isolation and expression of a full-length and a truncated mutant form. Mol. Gen. 
Genet. 251, 451?460. 
Imai, T., Ohno,T., 1995a. The relationship between viability and intracellular pH in the 
yeast Saccharomyces cerevisiae. Appl. Environ. Microbiol.61, 3604?3608. 
Imai, T., Ohno,T., 1995b. Measurement of yeast intracellular pH by image processing 
and the change it undergoes during growth phase. J. Biotechnol.38, 165?172 
Inderwildi, O. R., King, D. A., 2009. Quo Vadis Biofuels. Energy Environ. Sci.2,343 
Ingram, L.O., Lai,X., Moniruzzaman,M., Wood,B.E., York, S.W.,1997. Fuel ethanol 
production from lignocellulose using engineered bacteria. In Saha,B.D., 
Woodward, J., editors. Fuel and chemicals from biomass. ACS Symp Ser 666, 
Washington, DC, 57?73. 
Iyer, P.V., Wu, Z.-W., Kim, S.B., Lee, Y.Y., 1996. Ammonia Recycled percolation 
Process for Pretreatment of Herbaceous Biomass. Appl. Biochem. Biotechnol. 57-
58, 121?132. 
Janson, J.,1974. Analytik der Polysaccharide in Holz und Zellstoff. Faserforshung 
Textiltechnik 25, 375. 
 201
Javier,P.F.I., ?scar,G., Sanz-Aparicio,J.,D?az,P., 2007. Xylanases: Molecular Properties 
and Applications, in Industrial Enzymes Structure, Function and Applications by 
Polaina,J., MacCabe,A.P., Springer Netherlands. 
Jayme, G. ,P. K. Tio. 1968. Infrarotspektroskopische. Untersuchungen an 
Polysacchariden und ihren Derivaten. Das Papier 22, 322?328. 
Jeffries, T.W., Schartman R., 1999. Bioconversion of Secondary Fiber Fines to Ethanol 
Using Counter-Current Enzymatic Saccharification and Co-Fermentation.  Appl. 
Biochem.  Biotechnol. 78 (1-3), 435-444. 
J?nsson, L.J., Palmqvist,E., Nilvebrant, N. O., Hahn-H?gerdal, B., 1998. Detoxification 
of wood hydrolysates with laccase and peroxidase from the white-rot fungus 
Trametes versicolor, Appl. Microbiol. Biotechnol. 49 ,691?697. 
Ju, L.K., Afolabi, O.A.,1995. Wastepaper hydrolysate as soluble inducing substrate for 
cellulase production in continuous culture of Trichoderma reesei, Biotechnol. 
Progr. 15(1), 91-97. 
Juhasz, T., Szengyel, Z., Szijarto, N., Reczey, K., 2004. Effect of pH on cellulase 
production of Trichoderma reesei RUT C30. Appl. Biochem. Biotechnol. 113-
116, 201-211.  
Juhasz, T., Szengyel, Z., Reczey, K., Aiika-Aho, M.,Viikari, L.,2005. Characterization of 
cellulases and hemicellulases produced by Trichoderma reesei on various carbon 
sources. Process Biochem. 40, 3519-3525. 
Kadam, K. L., 1996. Cellulase production. In Wyman,C. E. (Ed.), Handbook on 
bioethanol: Production and utilization Chapter 11, Washington, DC: Taylor and 
Francis. 
Kadam, K.L., Keutzer, W.J., 1995. Enhancement in Cellulase Production by 
Trichoderma-Reesei Rut-C30 Due to Citric-Acid. Biotechnol Lett. 17(10), 1111-
1114. 
Kadam, K. L., Newman, M. M.,1997. Development of a Low-cost Fermentation Medium 
for Ethanol Production from Biomass. Appl. Microbiol. Biotechnol. 47, 625-629. 
Kadam, K. L., Schmidt,S.L.,1997.  Evaluation of Candida acidothermophilum in ethanol 
production from lignocellulosic biomass. Appl. Microbiol. Biotechnol. 48,709?
713. 
Kadla, J.F., Kubo, S., Venditti, R.A., Gilbert, R.D., Compere, A.L.,Griffith,W., 2002. 
Lignin-based carbon fibers for composite fiber applications. Carbon 40,2913-
2920. 
Kalman, G., Varga, E., Reczey, K., 2002. Dilute Sulfuric Acid Pretreatment of Corn. 
Stover at Long Residence Times. Chem.Biochem. Eng. 16(4), 151-157. 
 202
Kamm,B., Kamm,M., 2004. Principles of biorefineries. Appl. Microbiol. Biotechnol. 64, 
137-145. 
Kaar, W.E., Holtzapple, M.T.,2000. Using lime pretreatment to facilitate the enzymic 
hydrolysis of corn stover. Biomass Bioenergy 18(3),189-199. 
Kang,L., Wang,W., Lee,Y.Y.,2010. Bioconversion of kraft paper mill sludges to ethanol 
by SSF and SSCF. Appl.Biochem. Biotechnol.161(1-8),53-66. 
Kasahara, K., Sasaki, H., Donkai, N., Yoshihara, T., Takagishi, T., 2001. Modification of 
tencel with treatment of ferric sodium tartrate complex solution I. Effect of 
treatment condition. Cellulose, 8, 23?28. 
Katz, G., 1965.  The location and significance of the O-acetyl groups in a glucomannnan 
from Parana pine. Tappi,48, 34-41. 
Kilpel?inen, I., Xie,H., King,A., Granstrom,M., Heikkinen,S., Argyropoulos,D., 2007. 
Dissolution of wood in ionic liquids. J. Agric. Food Chem. 55, 9142-9148. 
Kim, H.,Hong,J.,2001. Supercritical CO2 pretreatment of lignocellulose 
enhancesenzymatic cellulose hydrolysis. Bioresour. Technol. 77(2),139-144. 
Kim,J.W., Mazza,G.,2008. Optimization of phosphoric acid catalyzed fractionation and 
enzymatic digestibility of flax shives, Ind. Crops Prod. 28(3), 346-355. 
Kim, S.B., Lee, Y.Y., 1996. Fractionation of Herbaceous Biomass by Ammonia-
Hydrogen Peroxide Percolation Treatment. Appl. Biochem. Biotechnol. 57?58, 
147?156. 
Kim, J.S., Lee, Y.Y., Park, S.C., 2000. Pretreatment of wastepaper and pulp mill sludge 
by aqueous ammonia and hydrogen peroxide. Appl. Biochem. Biotechnol. 84-86, 
129?139. 
Kim, S., Dale, B.E., 2004. Global potential bioethanol production from wasted crops and 
crop residues. Biomass Bioenergy. 26, 361-375. 
Kim, S., Holtzapple, M.T., 2005. Lime pretreatment and enzymatic hydrolysis of corn 
stover. Bioresour. Technol. 96(18),1994-2006. 
Kim, S. B., Moon, N. K., 2003. Enzymatic digestibility of used newspaper treated with 
aqueous ammonia?hydrogen peroxide solution. Appl. Biochem. Biotechnol. 105?
108, 365?373. 
Kim, T.H., Lee, Y.Y., 2005a. Pretreatment and fractionation of corn stover by ammonia 
recycle percolation process. Bioresour. Technol. 96(18),2007- 2013. 
Kim, T.H., Lee, Y.Y., 2005b. Pretreatment of Corn Stover by Soaking in Aqueous  
Ammonia Process.  Appl. Biochem.  Biotechnol. 121/124, 1119-1131. 
 203
Kirk, T.K., 1975.Lignin-degrading enzyme system.Biotechnol Bioeng Symp. (5),139-
150. 
Kleinert, T.N., 1974. Organosolv pulping with aqueous alcohol. Tappi 57(8),99-102. 
Kleman-Leyer, K.M., Siika-Aho,M.,Teeri,T.T.,Kirk,T.K., 1996. The Cellulases 
Endoglucanase I and Cellobiohydrolase II of Trichoderma reesei Act 
Synergistically To Solubilize Native Cotton Cellulose but Not To Decrease Its 
Molecular Size. Enzyme Microb. Technol. 62(8), 2883-2887. 
Klinke, H.B., Thomsen, A.B., Ahring, B.K.,2004. Inhibition of ethanol-producing yeast 
and bacteria by degradation products produced during pre-treatment of biomass. 
Appl. Microbiol. Biotechnol.  66(1),10-26. 
Kong, F., Engler, C.R., Soltes, Ed.J., 1992. Effects of cell-wall acetate, xylan backbone, 
and lignin on enzymic hydrolysis of aspen wood. Appl. Biochem. Biotechnol. 34, 
23-35. 
Koullas, D.P., Christakopoulos, P.F., Kekos, D., Macris, B.J., Koukios, E.G., 1990. 
Effect of cellulose crystallinity on the enzymic hydrolysis of lignocellulosics by 
Fusarium oxysporum cellulsases. Cellulose Chem. Technol. 24, 469?474. 
Knappert, D., Grethlein, H., Converse, A., 1980. Partial acid hydrolysis of cellulosic 
materials as a pretreatment for enzymatic hydrolysis. Biotechnol. Bioeng. 22, 
1449-1463. 
Kraemer, J.T., Bagley,D.M., 2006. Supersaturation of dissolved H2 and CO2 during 
fermentative hydrogen production with N2 sparging. Biotechnol Lett. 28,1485?
91. 
Kristensen,J.B., B?rjesson,J.,Bruun,M.H., Tjerneld,F., Jorgensen,H.,2007. Use of surface 
active additives in enzymatic hydrolysis of wheat straw lignocellulose, Enzyme 
Microb. Technol., 40, 888?895. 
Kubicek,C.P., 1992. The cellulase proteins of Trichoderma reesei: structure, multiplicity, 
mode of action and regulation of formation. Adv. Biochem. Eng. Biotechnol. 45, 
1?27. 
Kujala T.S., Loponen, J.M., Klika, K.D., Pihlaja, K., 2000. Phenolics and betacyanins in 
red beetroot (Beta vulgaris) root: Distribution and effect of cold storage on the 
content of total phenolics and three individual Compounds. J. Agric. Food Chem. 
48, 5388-5342. 
Kumar,R., Wyman,C.E.,2008. Effect of enzyme supplementation at moderate cellulase 
loadings on initial glucose and xylose release from corn stover solids pretreated 
by leading technologies, Biotechnol. Bioeng.102(2), 457 ? 467. 
 204
Kura,M., Sch?tte,H.,1987. Purification of proteins and the disruption of microbial cells. 
Biotechnol. Prog. 3,31?42. 
Kyriacou, A., Neufeld, R.J., MacKenzie, C.R., 1989. Reversibility and competition in the 
adsorption of Trichoderma reesei cellulase components. Biotechnol. Bioeng. 33, 
631?637. 
Ladisch, M.R., 1989. Hydrolysis, in Biomass Handbook, Hall,C.M., Kitani, O.(eds), 
Gordon and Breach, London. 
Ladisch, M.R., Ladisch, C.M., Tsao, G.T.,1978. Cellulose to sugars: new path gives 
quantitative yield. Science 201,743-745. 
Ladisch, M.R., Lin, K.W., Voloch, M., Tsao, G.T., 1983. Process considerations in the 
enzymatic hydrolysis of biomass. Enzyme Microb. Technol. 5, 2, 82?102. 
Lambert,R.J., Stratford, M.,1999. Weak-acid preservatives: modelling microbial 
inhibition and response. J.Appl. Microbiol. 86, 157?164. 
Langsford, M.L., Singh, G.B., Moser, B., Miller, R.C.Jr., Warren, R.A.J., Killburn, D.G., 
1987. Glycosylation of bacterial cellulases prevent proteolytic cleavage between 
functional domains. FEBS Lett., 225, 163-167. 
Lark, N., Xia, Y., Qin, C-G.,Gong, C.S., and Tsao, G.T., 1997. Production of ethanol 
from recycled paper sludge using cellulase and yeast Kluveromyces marxianus. 
Biomass Bioenergy 12,135. 
Larsson, S., Palmqvist, E., Hahn-H?gerdal, B., Tengborg, C., Stenberg, K., Zacchi, G., 
Nilvebrant, N.O., 1998. The generation of fermentation inhibitors during dilute 
acid hydrolysis of softwood. Enz. Microb. Technol. 24, 151?159. 
Larsson, S., Palmqvist, E., Hahn-H?gerdal, B., Tengborg, C., Stenberg, K., Zacchi, G., 
1999. The generation of fermentation inhibitors during dilute acid hydrolysis of 
softwood. Enzyme Microbial. Technol. 24, 151?159. 
Larsson, S., Reimann, A., Nilvebrant, N. O., Jonsson, L. J.,1999. Comparison of different 
methods for the detoxification of lignocellulose hydrolysates of spruce. Appl. 
Biochem.Biotechnol. 77?79, 91?103. 
Laser, M., Schulman, D., Allen, S.G.,Lichwa, J., Antal, M.J., Jr., Lynd, L.R.,2002. A 
comparison of liquid hot water and steam pretreatments of sugar cane bagasse for 
bioconversion to ethanol. Bioresource Technol. 2002, 81, 33-44. 
Laureano-Perez ,L., Teymouri,F., Alizadeh,H., Dale,B.E.,2005. Understanding factors 
that limit enzymatic hydrolysis of biomass Appl. Biochem. Biotechnol. 1081-
1099. 
 205
Lawford, H. G., Rousseau J. D., 1997. Corn steep liquor as a cost-effective nutrition 
adjunct in high-performance Zymomonas ethanol fermentations. Appl. Microbiol. 
Biotechnol. 63-65,287-304. 
Lawford, H. G., Rousseau J. D., 1998. Improving fermentation performance of 
recombinant Zymomonas in acetic acid-containing media. Appl. Biochem. 
Biotechnol., 70?72,161?172. 
Lawford, H. G., Rousseau J. D., McMillan,J.D. ,1997. Optimisation of seed production 
for a simultaneous saccharification cofermentation biomass- to-ethanol process 
using recombinant Zymomonas. Appl. Biochem. Biotechnol.,63? 65,269 ?286. 
Lechner, B. E., Papinutti, V. L.,2006. Production  of lignocellulosic enzymes during 
growth and fruiting of the edible fungus Lentinus tigrinus on  wheat   straw.  
Process Biochem. 41(3), 594-598. 
Lee, J. Y. , Jang, Y. S. , Lee, J. , Papoutsakis, E. T. and Lee, S. Y. (2009) Metabolic 
engineering of Clostridium acetobutylicum M5 for highly selective butanol 
production. Biotechnol. J., 4 , 1432-1440. 
Lee, K., 2005. A media design program for lactic acid production coupled with extraction 
by electrodialysis. Bioresour, Technol. 96, 1505?1510. 
Lee, S.-M., Koo, Y.-M., and Lin, J., 2004, Production of lactic acid from paper sludge by 
simultaneous saccharification and fermentation, Advances in Biochemical 
Engineering/Biotechnology, 87. 173-194 
Lee,W.G., Lee,J.S., Lee,J.P., Shin,C.S., Kim,M.S., Park, S.C., 1996. Effect of surfactants 
on ethanol fermentation using glucose and cellulosic hydrolyzates. Biotechnol. 
Lett. 18(3),299-304. 
Lehtio, J., Sugiyama, J., Gustavsson, M., Fransson, L., Linder, M., Teeri, T. T., 2003.The 
binding specificity and affinity determinants of family 1 and family 3 cellulose 
binding modules. Proceedings of the National Academy of Sciences  USA 100, 
484-489. 
Lehninger,A.L., Nelson,D.L.,Cox,M.M.,2004.Lehninger Principles of Biochemistry. 4th, 
W. H. Freeman &Co. 
Leitten,C.F., Griffith,W.L.,Compere,A.L.,Shaffer,J.T.,2002. in 2002 Future Car Congress 
Proceedings, Society of Automotive Engineers, paper 2002-01-1907. 
Leuthold, D.A. and Leuthold, M.A.,1996. Method of dry separating fibers from paper 
making waste sludge and fiber product thereof. U.S. Patent 5,527,432. 
Lewin, M., Roldan, L.G., 1971. The effect of liquid anhydrous ammonia in the structure 
and morphology of cotton cellulose. J. Polymer Sci. Part C, 36, 213?229. 
 206
Li, K., Reeve, D. W.,2002. The Origins of Kraft Wood Fibre Surface Lignin, J. Pulp Pap. 
Sci., 28(11),369-373. 
Liepman, A.H., Nairn,C.J., Willats, W.G.T., S?rensen, I., Roberts, A.W., Keegstra 
,K.,(2007. Functional genomic analysis supports conservation of function among 
cellulose synthase-like A gene family members and suggest diverse roles of 
mannans in plants. Plant Physiol. 143:,1881?1893. 
Lin, K.W., Ladisch, M.R., Voloch, M., Patterson, J.A., Noller ,C.H., 1985.Effects of 
pretreatments and fermentation on pore size in cellulosic materials. Biotechnol 
Bioeng. 27,1427?1433 . 
Lindberg, B., Resell, K. G., Svensson, S., 1973. Positions of the O-acetyl groups in pine 
glucomannan. Svensk. Paperstid. 76, 383-384. 
Lindblad,M.S., Ranucci,E., Albertsson,A.C.,2001. Biodegradable polymers from 
renewable sources, new hemicellulose-based hydrogels. Macromol. Rapid 
Commun. 22, 962?967. 
Litchfield, J. H., 1996, Microbiological production of lactic acid. Advances in Applied 
Microbiology, 42, 45-95 
Lo, C. M., Zhang, Q., Lee, P., Ju, L. K.,2005. Cellulase production by Trichoderma 
reesei using sawdust hydrolysate. Appl. Biochem. Biotechnol. 121/124, 561-573.   
Lora,J.H., Glasser,W.G., 2002. Recent industrial applications of lignin: a sustainable 
alternative to nonrenewable materials. J. Polym. Environ. 10,39-48. 
Lora, J.H., Wayman, M., 1978. Delignification of hardwoods by autohydrolysis and 
extraction. Tappi, 61, 47-50. 
Lynd, L. R., Elander, R. T., Wyman, C. E., 1996. Likely features and costs of mature 
biomass ethanol technology. Appl. Biochem. Biotechnol., 57-58,741-761. 
Lynd,L.R., Lyford, K., South,C.R., van Walsum, P., and Levenson, K., 2001. Evaluation 
of paper sludge for amenability to enzymatic hydrolysis and conversion to 
ethanol. TAPPI ,84, 50. 
Lynd, L.R., van Zyl, W. H., McBride, J.E., Laser, M., 2005. Consolidated bioprocessing 
of cellulosic biomass: an update. Curr. Opin. Biotechnol., 16, 577-583. 
Lynd,L.R., Weimer,P.J.,van Zyl,W.H., Pretorius,I.S., 2002. Microbial Cellulose 
Utilization: Fundamentals and Biotechnology.  Microbiol Mol.Biol . Revs. , 
66(3), 506-577. 
Lynd,.L.R., Wyman,C.E., Gerngross,T.U.,1999. Biocommodity Engineering. Biotechnol. 
Prog., 15, 777-793. 
 207
Maheshwari, D. K., Gohede, S., Paul, J., Varma, A.,1994. Paper mill sludge as a potential 
source for cellulase production by Trichoderma reesei QM 9213 and Aspergillus 
niger using mixed cultivation. Carbohydr. Polym. 23, 161-163. 
Maheswari, D.K., Jahan, H., Paul, J., Varma, A., 1993. Wheat Straw, a Potential 
Substrate for Cellulase Production Using Trichoderma-Reesei. W. J. Microbiol. 
Biotechnol. 9(1),120-121. 
Mandels,M.,Hontz,L.,Nystrom,J., 1974. Enzymatic hydrolysis of waste cellulose, 
Biotechnol. Bioeng. 15, 1471-1493. 
Mandels, M., Andreotti, R., Roche, C.,1976. Measurement ofsaccharifying cellulase. 
Biotechnol. Bioeng. Symp. 6(6), 21?33. 
Mansfield, S.D., Meder,R.,2003.  Cellulose hydrolysis ? the role of monocomponent 
cellulases in crystalline cellulose degradation. Cellulose, 10(2),159-169. 
Mansfield, S.D., Saddler, J.N., 2003. Applications of enzymes to lignocellulosics, (ACS 
Symposium Series Vol. 855). Washington, DC: Oxford University Press. 
Marchildon,L., Castro,C.C., Lapointe,M.W., Daneault,C., 1993. Evaluation of carbon-
dioxide as a carrier gas in flotation de-inking. Tappi.76(3), 155-159. 
Marques, S., Alves, L., Roseiro J.C. and G?rio, F.M., 2008. Conversion of recycled paper 
sludge to ethanol by SHF and SSF using Pichia stipitis.  Biomass Bioenergy , 32, 
400?406. 
Martin, N.,Anglani,N.,Einstein,D.,Khrushch,M., Worrell,E., Price, L.K., 2000. 
Opportunities to Improve Energy Efficiency and Reduce Greenhouse 
GasEmissions in the U.S. Pulp and Paper Industry. 
Martin,C., J?nsson, L.J.,2003. Comparison of the resistance of industrial and laboratory 
strains of Saccharomyces and Zygosaccharomyces to lignocellulose-derived 
fermentation inhibitors. Enz. Microbial. Technol. 32, 386?395. 
Mart?nez, A.T., Gonz?lez, A.E., Valmaseda, M., Dale, B.E., Lambregts, M.J., Haw, 
J.F.,1991. Solid-State Nmr-Studies of Lignin and Plant Polysaccharide 
Degradation by Fungi. Holzforschung. 45, 49-54. 
Martinez,A., Rodriguez,M.E., Wells, M.L., York,S.W., Preston,J.F., Ingram,L.O., 2001. 
Detoxification of dilute acid hydrolysates of lignocellulose with lime. Biotechnol. 
Prog. 17(2),287?293. 
Martinez,A., York, S.W., Yomano, L.P., Pineda,V.L., Davis, F.C., Shelton, J.C., 
Ingram,L.O., 1999. Biosynthetic burden and plasmid burden limit expression of 
chromosomally integrated heterologous genes (pdc, adhB) in Escherichia coli. 
Biotechnol. Prog. 15, 891?897. 
 208
Matsumura, Y., Sudo, K., Shimizu, K., 1977. Enzymatic hydrolysis of Wood II. Effect of 
grinding and alkali treatment on hydrolysis of woods by Trichoderma viride 
cellulase. Mokuzai Gakkaishi, 23, 11, 562?570. 
Maxham, J.V., 1994. Conversion of pulp and paper mill waste solids to a papermaking 
filler product. U.S. Patent 5,332,474. 
McAloon, F., Taylor, W., Yee, K., Ibsen, Wooley, R., 2000. Determining the Cost of 
Producing Ethanol from Corn Starch and Lignocellulosic Feedstocks,  National 
Renewable Energy Laboratory Report. available from, 
http://www.nrel.gov/docs/fy01osti/28893.pdf 
McCrae, S.I., Leith, K.M., Gordon, A.H., Wood, T.M., 1994. Xylan-Degrading Enzyme-
System Produced by the Fungus Aspergillus-Awamori - Isolation and 
Characterization of a Feruloyl Esterase and a p-Coumaroyl Esterase. Enzyme 
Microb.Technol.(16)10, 826-834. 
Mcelroy, A. K., 2006. Quest Beyond Corn and Sugarcane. Ethanol Producer Magazine, 
12,121-129. 
McGinnis, G.D., Wilson, W.W., Mullen, C.E., 1983. Biomass pretreatment with water 
and high pressure oxygen. The wet-oxidation process. Ind. Eng. Chem. Prod. Res. 
Dev. 22, 2, 352?357. 
McMillan, J.D., 1994. Pretreatment of lignocellulosic biomass. In: Himmel,M,E,, 
Baker,J.O., Overend,R.P., Enzymatic Conversion of Biomass for Fuels 
Production, ACS Symposium Series, vol. 556. ACS, Washington, D.C. 
Meier, H., 1961. Isolation and characterization of an acetyl glucomannan from pine 
(Pinus Glues&is). Acta Chem.Stand.,15, 1381-1385. 
Mensink, M., Axeg?rd, P., Karlsson, M., McKeough, P., Westenbroek, A., Petit-Conil, 
M., Eltrop,L., Niemel?,K., 2007. A bio-solution to climate change. Final report of 
the Biorefinery Taskforce to the Forest-based Sector Technology Platform, 
Available at: 
www.forestplatform.org/easydata/customers/ftp/files/New_files/FTP_biorefinery_
report_part1.pdf 
Merivuori, H., Sands, J.A., Montenecourt, B.S., 1985. Effects of tunicamycin on 
secretion and enzymatic activities of cellulase from Trichoderma reesei, Appl. 
Microbiol. Sci., 23, 60-66. 
Mes-Hartree, M., Hogan, C.M., Saddler, J.N., 1984. Conversion of pretreated 
lignocellulosic substrates to ethanol using a two stage process. Can. Bioenergy 
R&D semin., Elsevier Appl. Sci. London, UK., 5th, 469?472. 
 209
Meunier-Goddik, Penner, M.H., 1999. Enzyme-catalyzed saccharification of model 
celluloses in the presence of lignacious residues, J. Agric. Food Chem. 47, 346?
351. 
Middleton, S. R., Scallan, A. M.,1991.A kinetic-model for the adsorption of fillers by 
pulp fibers. J.Pulp Paper Sci. 17(4), J127?J133. 
Miller, G. L., 1959. Use of Dinitrosalicylic Acid Reagent for Determination of. Reducing 
Sugar. Anal. Chem. 31, 426 ? 428. 
Miller, T.L.,Churchill, B.W., 1986. Substrates for large-scale fermentations. In: Demain, 
A.L. and Solomon, L.A., Editors, 1986. Manual of Industrial Microbiology and 
Biotechnology, American Society for Microbiology, Washington, DC. 
Mischak, H., Hofer, F., Messner, R., Weissinger,E., Hayn,M., Tomme,P., Esterbauer, H., 
Ku?chler,E., Claeyssens ,M., Kubicek,C. P., 1989. Monoclonal antibodies against 
different domains of cellobiohydrolase I and II from Trichoderma reesei. 
Biochim. Biophys. Acta. 900, 1?7. 
Mishra,S.P.,2000. A Textbook of Fibre Science and Technology, New Age International. 
Moedcawa, Y., Kawamori,Y., Ado,Y. ,Shinsha,Y.,Oda,F., Takasawa,S., 1985. 
Improvement of cellulase production in Trichoderma reesei. Agric.Biol.Chem. 
49, 1869-1871. 
Mohan,D.,Pittman,C.U., Steele,P.H.,2006. Pyrolysis of wood/biomass for bio-oil: a 
critical review. Energy Fuels ,20, 848?889. 
Mohagheghi., A., Tucker, M., Grohmann, K., Wyman, C.E.,1992. High Solids 
Simultaneous Saccharification And Fermentation Of Pretreated Wheat Straw To 
Ethanol. Appl. Biochem. Biotechnol. 33, 67?81. 
Moniruzzaman, M., York, S.W., Ingram,L.O., 1998. Effects of process errors on the 
production of ethanol by Escherichia coli KO11. J Ind. Microbiol. Biotechnol. 20, 
281?286. 
Montague, L., 2003. Lignin Process Design Confirmation and Capital Cost Evaluation. 
NREL/SR-510-31579. 
Montenecourt,B.S., Eveleigh,D.E., 1979.Selective screening methods for the isolation of 
high yielding cellulase mutants of Trichoderma reesei. Adv Chem Ser 181, 289-
301. 
Montiel, M.D., Rodr?guez, J., P?rez-Leblic, M.I., Hern?ndez, M., Arias, M.E., Copa-
Pati?o, J.L.,1999. Screeening of mannanase in actinomycetes and their potential 
application in the biobleaching of pine kraft pulps. Appl. Microbiol. Biotechnol. 
52, 240?245. 
 210
Mooney, C.A., Mansfield, S.D., Touhy, M.G., Saddler, J.N., 1998. The effect of initial 
pore volume and lignin content on the enzymatic hydrolysis of softwood. Biores. 
Technol. 64, 113?119. 
Moreira, L. R. S., Filho, E. X. F.,2008. An overview of mannan structure and mannan-
degrading enzyme systems. Appl. Microbiol. Biotechnol. 79(2), 165-78. 
Moritz, J. W., Duff, S. J. B. 1996. Simultaneous saccharification and extractive 
fermentation of cellulosic substrates.  Biotechnol. Bioeng., 49, 504?511. 
Mosier,N., Wyman, C., Dale, B.E., Elander, R., Lee, Y.Y., Holtzapple ,M., Ladisch, M., 
2005. Features of promising technologies for pretreatment of lignocellulosic 
biomass. Bioresour. Technol. 96,673-686. 
Mukhopadhyay,S.N.,Malik,R.K.,1980, Increased production of cellulase of Trichoderma 
sp. by pH cycling and temperature profiling . Biotechnol.Bioeng.22, 2237-2250. 
Mukhopadhyay, S., Nandi, B., 1999. Optimization of cellulase production by 
Trichoderma reesei ATCC 26921 using a simplified medium on water hyacinth 
biomass.  J. Sci. Ind. Res. 58, 107?111. 
Mussatto, S.I., Dragone,G., Roberto,I.C., 2005. Kinetic behavior of Candida 
guilliermondii yeast during xylitol production from brewer's spent grain 
hemicellulosic hydrolysate. Biotechnol. Prog. 21(4),1352-1356. 
Neely, W.C., 1984. Factors affecting the pretreatment of biomass with gaseous ozone. 
Biotechnol. Bioeng. 20, 59?65.  
Neilson, M.J., Shafizadeh, F., Aziz, S. and Sarkanen, K.V., 1983. Evaluation of 
Organosolv Pulp as a Suitable Substrate for Rapid Enzymatic-Hydrolysis. 
Biotechnol. Bioeng. 25(2), 609-612. 
Neuman,R.P., Walker,L.P.,1992. Solution exclusion from cellulose in packed columns; 
process modeling and analysis. Biotechnol. Bioeng., 40,226? 234. 
Neureiter, M.; Danner, H.; Madzingaidzo, L.; Miyafuji, H.; Thomasser, C.; Bvochora, J.; 
Bamusi, S.; Braun, R., 2004. Lignocellulose feedstocks for the production of 
lactic acid. Chemical and Biochemical Engineering Quarterly , 18(1), 55-63 
Nevalainen H, Neethling D. The safety of Trichoderma and Gliocladium In: Harman GE, 
Kubicek CP, editors. Trichoderma andGliocladium. Bristol, PA, Taylor&Francis, 
1998.  
Nevalainen, H., Suominen, P., Taimisto, K. 1994. On the safety of Trichoderma reesei. J. 
Biotechnol. 37,193-200. 
 211
Nguyen, Q. A., Saddler, J. N., 1991. An integrated model for the technical and economic 
evaluation of an enzymic biomass conversion process. Biores. Technol. 35, 275-
282. 
Nguyen, Q.A.,Tucker, M. P., Boynton, B. L.,Keller, F. A.,Schell, D. J.,1998.Dilute acid 
pretreatment of softwoods. Appl. Biochem. Biotechnol. 70-72, 77-87. 
Nguyen, Q.A.,Tucker, M. P.,Keller,F.A., Beaty,D.A., Connors,K.M., Eddy,F.P.,1999.  
Dilute acid hydrolysis of softwoods. Appl. Biochem. Biotechnol.77-9,133-142. 
Nguyen, Q.A., Tucker, M.P., Keller, F.A., Eddy, F.P., 2000. Two-stage dilute-acid 
pretreatment of softwoods. Appl. Biochem. Biotechnol.84-6,561- 576. 
Nidetzky,B., Claeyssens,M.,1994. Specific quantification of Trichoderma reesei 
cellulases in reconstituted mixtures and its application to cellulose-cellulose 
binding studies. Biotechnol. Bioeng. 44, 961?966. 
Nidetzky, B., Steiner ,W. , Claeyssens, M.,1994. Cellulose hydrolysis by the cellulases 
from Trichoderma reesei: adsorptions of two cellobiohydrolases, two 
endocellulases and their core proteins on filter paper and their relation to 
hydrolysis. Biochem. J., 303, 817?823. 
Nigam, P., Prabhu, K.A., 1991, Influence of sugars on the activity of cellulase system 
from two basidiomycetes cultures. J. Basic Microb. 31(4), 279?283. 
Nikolov, T., Bakalova, N., Petrova, S., Benadova, R., Spasov, S., and Kolev, D., 2000, 
An effective method for bioconversion of delignified waste cellulose fibers from 
the paper industry with a cellulase complex. Bioresour. Technol. 71, 1-4. 
Nilvebrant, N.O., Reimann, A., Larsson, S. and J?nsson, L.J., 2001. Detoxification of 
lignocellulose hydrolysates with ion exchange resins. Appl. Biochem. Biotechnol. 
91?93,35?49. 
Nissan,A.H.,Batten,G.L.,1990. On the primacy of the hydrogen bonds in paper 
mechanics. Tappi 73 , 159?164. 
Nolasco-Hipolito, C., Matsunaka, T., Kobayashi, G., Sonomoto, K., Ishizaki,A.,2002. 
Synchronized fresh cell bioreactor system for continuous l-(+)-lactic acid 
production using Lactococcus lactis IO-1 in hydrolysed sago starch, J. Biosci. 
Bioeng. 93, 281?287. 
 
NREL, 2008.  Laboratory Analytical Procedure (LAP), National Renewable Energy 
Laboratory,Golden,CO. Available from: 
http://www.nrel.gov/biomass/analytical_procedures.html. 
 212
?hgren, K.,Bura, R.,Saddler,J.N., Zacchi,G.,2007. Effect of hemicellulose and lignin 
removal on enzymatic hydrolysis of steam pretreated corn stover.  Biores. 
Technol.  98, 2503?2510. 
Ohta,K., Beall,D.S., Mejia,J.P., Shanmugam,K.T., Ingram,L,O., 1991. Metabolic 
engineering of Klebsiella oxytoca M5A1 for ethanol production from xylose and 
glucose. Appl. Environ. Microbiol. 57(4), 893?900. 
Ohta, K., Beall, D.S., Mejia, J.P., Shanmugam, K.T., Ingram, L.O., 2004. Genetic 
improvement of Escherichia coli for ethanol production: Chromosomal 
integration of Zymonomas mobilis genes encoding pyruvate decarboxylase and 
alcohol dehydrogenase II. Appl. Environ. Microbiol. 57, 893?900. 
Olden, K.B., Bernard, B.A., Humphries, M.J, Yeo, T.-K., White, S.J., Newton, S.A., 
Bauer, H.C., Parent, J.B., 1985. Function of glycoportein glycans.Trends 
Biochem. Sci., 10, 78-81. 
Olsson, L., Christensen, T.M.I.E., Hansen, K.P., Palmqvist, E.A., 2003.Influence of the 
carbon source on production of cellulases, hemicellulases and pectinases by 
Trichoderma reesei Rut C-30. Enzyme Microb. Technol. 33, 612?619. 
Olsson, L.,Hahn-H?gerdal, B., Fermentative performance of bacteria and yeasts in 
lignocellulose hydrolysates. Process Biochem., 1993, 28,249?257.  
Olsson, L.,Hahn-H?gerdal, B., Zacchi,G.,1995. Kinetics of ethanol production by 
recombinant Escherichia coli KO11. Biotech. Bioeng., 45,356 ?365. 
Ooshima, H., Burns,D.S., Converse,A.O.,1990. Adsorption of cellulase from 
Trichoderma reesei on cellulose and lignacious residue in wood pretreated by 
dilute sulfuric acid with explosive decompression, Biotechnol. Bioeng. 36, 446?
452. 
Ooshima, H., Sakata, M., Harano,Y.,1986. Enhancement of enzymatic hydrolysis of 
cellulose by surfactant. Biotechnol. Bioeng. 28,1727?1734. 
Ostergaard, S., Roca,C.,R?nnow, B.,Nielsen,J., Olsson,L., 2000. Physiological studies in 
aerobic batch cultivations of Saccharomyces cerevisiae strains harboring the 
MEL1 gene. Biotechnol. Bioeng. 68, 252-259. 
Otter, D.E., Munro, P.A., Scott ,G.K., Geddes, R.,1989. Desorption of Trichoderma 
reesei cellulase from cellulose by a range of desorbents, Biotechnol. Bioeng. 34, 
291?298. 
Palmqvist, E, Almeida, J. S., Hahn-H?gerdal, B.,1999. Influence of furfural on anaerobic 
glycolytic kinetics of Saccharomyces cerevisiae in batch culture, Biotechnol. 
Bioeng. 62, 447-454. 
Palmqvist, E, Hahn-H?gerdal, B.,2000a.Fermentation of lignocellulosics hydrolysates. I: 
 213
inhibition and detoxification Bioresour. Technol. 74, 17-24. 
Palmqvist, E, Hahn-H?gerdal, B.,2000b. Fermentation of lignocellulosics hydrolysates. 
II: inhibitors and mechanisms of inhibition, Bioresour.Technol. 74, 25-33. 
Palonen, H., 2004. Role of lignin in the enzymatic hydrolysis of lignocellulose. VTT 
Publications, 520,1-80. 
Palonen, H., Tenkanen, M., Linder, M.,1999. Dynamic interaction of Trichoderma reesei 
cellobiohydrolases Cel6A and Cel7A and cellulose at equilibrium and during 
hydrolysis. Appl. Environ. Microbiol. 65, 5229?5233. 
Palonen, H., Tjerneld, F., Zacchi, G., Tenkanen, M., 2004. Adsorption of Trichoderma 
reesei CBH I and EG II and their catalytic domains on steam pretreated softwood 
and isolated lignin. J. Biotechnol. 107(1), 65-72. 
Pampulha, M.E., Loureiro-Dias, M.C.,1989. Combined effect of acetic acid, pH and 
ethanol on intracellular pH of fermenting yeast. Appl. Microbiol. Biotechnol. 20, 
286?293. 
Pan, X.J., Zhang, X., Gregg, D.J., Saddler, J.N., 2004. Enhanced enzymatic hydrolysis of 
steam-exploded Douglas fir wood by alkali-oxygen post-treatment. Appl. 
Biochem. Biotechnol.113-116, 1103-1114. 
Pan, X.J., Arato, C., Gilkes,N., Gregg,D.J., Mabee,W., Pye,E.K., Xiao,Z., Zhang,X., 
Saddler,J.N.,2005. Biorefining of softwoods using ethanol organosolv pulping - 
preliminary evaluation of process streams for manufacture of fuel-grade ethanol 
and co-products. Biotech. Bioeng. 90 (4), 473-481. 
Parekh ,M. and Blaschek,H.P. (1999) Butanol production by hypersolvent-producing 
mutant Clostridium beijerinckii BA101 in corn steep water medium containing 
maltodextrin.  Biotechnol. Letters , 21, 45-48 
Park, J.W., Takahata,Y., Kajiuchi,T., Akehata,T., 1992. Effects of nonionic surfactant on 
enzymatic hydrolysis of used newspaper. Biotechnol. Bioeng. 39, 117?120. 
Park, S.C., Kademi,A., Baratti ,J.C.,1993. Alcoholic fermentation of cellulose 
hydrolysate by Zymomonas mobilis. Biotechnol. Lett.,15, 1179? 1184. 
Paul,J. and Varma,A.K.,1990. Influence of sugars on endoglucanase and.beta.-xylanase 
activities of a Bacillus strain. Biotechnol.Lett.12 (1), 61-64. 
Pemberton, M.S., Brown, R.D.Jr., Emert, G.H., 1980. The role of b-glucosidase in the 
bioconversion of cellulose to ethanol. Can. J. Chem. Eng. 58, 723?729. 
Penner, M.H., Liaw, E. T.,1994. Kinetic consequences of high ratios of substrate to 
enzyme saccharification systems based on Trichoderma cellulase. In: Himmel, 
 214
M.E., Baker,J.O. , Overend, R.P., Editors, Enzymatic Conversion of Biomass for 
Fuels Production, American Chemical Society, Washington, D.C. 
Penttil?, M.,1998. Heterologous protein production in Trichoderma. In: Harman, G., 
Kubicek, C., (eds.) Trichoderma and Gliocladium, Enzymes, biological control 
and commercial applications, Vol. 2, Laylor & Francis Ltd., London, UK, 365-
381. 
Penttil?, M., Nevalainen, H., R?tt?, M., Salminen, E., Knowles, J., 1987. A versatile 
transformation system for cellulolytic filamentous fungus Trichoderma reesei.  
Gene. 61(2), 155-64. 
Perez, J. A.,Gonzalez, A., Oliva, J. M.,Ballesteros, I.,Manzanares, P. ,2007. Effect of 
process variables on liquid hot water pretreatment of wheat straw for 
bioconversion to fuel-ethanol in a batch reactor. J. Chem. Technol. Biotechnol. 
82, 929?938. 
Petkowicz, C.L.O., Reicher, F., Chanzy, H., Taravel, F.R., Vuong, R., 2001. Linear 
mannan in the endosperm of Schizolobium amazonicum. Carbohydr Polym 
44,107?112. 
Pflug W., 1982. Effect of clay minerals on the activity of polysaccharide cleaving soil 
enzymes. Z. Planzenernaehr. Bodenkd.145, 493-502. 
Philippidis, G.P., 1996.Cellulose bioconversion technology. In: Wyman, C.E., Editor, 
Handbook on Bioethanol: Production and Utilization, Taylor & Francis, 
Washington, DC, 253?285. 
Philippidis, G.P., Smith, T.K., 1995. Limiting Factors in the simultaneous 
saccharification and fermentation process for conversion of cellulosic biomass to 
fuel ethanol. Appl. Biochem. Biotechnol.51/52,117-124. 
Philippidis, G., Smith, T. K.,Wyman, C. E., 1993.Study of the enzymatic hydrolysis of 
cellulose for production of fuel ethanol by the simultaneous saccharification and 
fermentation process. Biotechnol. Bioeng. 41, 846?853. 
Pirkkalainen K., Vainio U., Kisko K., Elbra T., Kohout T., Kotelnikova,N.E., 
Serimaa,R.,2007. Structure of nickel nanoparticles in a microcrystalline cellulose 
matrix studied using anomalous small-angle x-ray scattering. J. Appl. 
Crystallogr.40, 489 ? 494. 
Puls,J., Schuseil,J., 1993. Chemistry of hemicellulose: relationship between 
hemicellulose structure and enzyme required for hydrolysis. In: Coughlan,M.P., 
Hazlewood,G.P. (eds) Hemicellulose and hemicellulases. Portland, London, 1?27. 
Puri, V.P., 1984. Effect of crystallinity and degree of polymerization of cellulose on 
enzymatic saccharification. Biotechnol. Bioeng. 26, 1219?1222. 
 215
Ragauskas, A.J., Nagy, M., Kim, D.H., Eckert, C.A., Liotta, C.L., 2006. From wood to 
fuels: Integrating biofuels and pulp production. Ind. Biotechnol. 2(1), 55-65. 
Rapp, P. and Wagner, F.,1986. Production and Properties of Xylan-Degrading Enzymes 
from Cellulomonas uda. Appl. Environ. Microbiol. 51(4), 746?752. 
Ravinder, T., Ramesh, B., Seenayya, G., Reddy, G., 2000. Fermentative production of 
acetic acid from various pure and natural cellulosic materials by Clostridium 
lentocellum SG6. World J. Microbiol. Biotechnol., 16(6), 507- 512. 
Reczey, K., Brumbauer, A., Bollok, M., Szengyel, Z., Zacchi,G., 1998. Use of 
hemicellulose hydrolysate for ?-glucosidase fermentation. Appl. Biochem. 
Biotechnol. 70-72,225-235. 
Reddy,N., Yang,Y., 2005. Biofibers from agricultural byproducts for industrial 
applications. Trends Biotechnol. 23, 22-27.  
Reese, E.T., Siu, R.G.H., Levinson,H.S.,1950. 1950. Biological degradation of soluble 
cellulose derivatives. J Bacteriol., 59,485. 
Reichert, W.M., Visser, A.E., Swatloski, R.P., Spear, S.K.,Rogers, R.D., 2001. 
Derivatization of chitin in room temperature ionic liquids. Abstracts of Papers, 
222nd ACS National Meeting, Chicago, IL, United States. 
Roberto, I.C., Lacis, L.S., Barbosa, M., De Mancilha, I.M.,1991. Utilization of sugar cane 
bagasse hemicellulosic hydrolysate by Pichia stipitis for  the production of 
ethanol. Process Biochem . 26,15?21 
Robinson, J., Keating, J.D., Boussaid, A., Mansfield, S.D., Saddler, J.N., 2002. The 
influence of bark on the fermentation of Douglas-fir whitewood pre-hydrolysates. 
Appl. Biochem. Biotechnol. 59(4-5), 443-448. 
Roelofsen, P.A.,1959.The plant cell-wall, Encyclopedia of Plant Anatomy, Berlin ?
Nikolasse. 
Rogers,P.L., Lee,K.J., Tribe,D.E.,1979.Kinetics of alcohol production by Zymomonas 
mobilis at high sugar concentrations. Biotechnol. Lett.165?170. 
Rosenqvist, G.V.,1978. Use of primary wastewater-treatment sludge in manufacture of 
printing paper at Kymi-Kymmene, Pap Puu-Pap Tim, 60, 205?207.  
Rowell, R. M., 2005. Handbook of wood chemistry and wood composites, New York: 
CRC Press. 
Royer,J.C., Nakas, J.P.,1987. Production of mycelial protein and hydrolytic enzymes 
from paper mill sludges by cellulolytic fungi. J. Ind. Microbiol. Biotechnol. 
1987,2(1), 9-13. 
 216
Rughani, J., Mcginnis, G.D., 1989. Combined Rapid Steam Hydrolysis and Organosolv 
Pretreatment of Mixed Southern Hardwoods. Biotechnol. Bioeng. 33(6),681-686. 
Ryu, D.D.Y., Mandels, M., 1980. Cellulases - Biosynthesis and Applications. Enzyme 
Microb. Technol. 2(2), 91-102. 
Saddler, J.N. and Gregg, D.J., 1998. Ethanol production from forest product wastes. in 
Forest products biotechnology (Bruce,A. and Palfreyman, J. W.), Taylor & 
Francis, London. 
Saha, B.C., Iten, L.B., Cotta, M.A., Wu, Y.V., 2005a. Dilute acid pretreatment, 
enzymatic saccharification, and fermentation of rice hulls to ethanol. Biotechnol. 
Progr. 21(3), 816-822. 
Saha, B.C., Iten, L.B., Cotta, M.A., Wu, Y.V., 2005b. Dilute acid pretreatment, 
enzymatic saccharification and fermentation of wheat straw to ethanol. Process 
Biochem. 40(12), 3693-3700. 
Sakata,M., Ooshima, H., Harano,Y., 1985.Effects of agitation on enzymatic 
saccharification of cellulose. Biotechnol. Lett. 689-694. 
Sarkanen, S., Teller, D.C., Hall, J., Mccarthy, J.L., 1981. Lignin .18. Associative Effects 
among Organosolv Lignin Components. Macromolecules. 14(2),426-434. 
Sarvari Horvath, I., Franz?n, C.J., Taherzadeh, M.J., Niklasson, C., Lid?n, G., 2003. 
Effects of furfural on the respiratory metabolism of Saccharomyces cerevisiae in 
glucose-limited chemostats. Appl. Environ.Microbiol. 69, 4076-4086. 
Sasaki, T., Tanaka, T., Nanbu, N., Sato, Y., Kainuma, K., 1979. Correlation between X-
ray diffraction measurements of cellulose crystalline structure and the 
susceptibility of microbial cellulase. Biotechnol. Bioeng. 21, 1031?1042. 
Sattler, W., Esterbaue, H., Glatter, O., Steiner, w.,1989. The effect of enzyme 
concentration on the rate of hydrolysis of cellulose.Biotechnol. Bioeng . 33,1221?
1234. 
Steiner, W., Sattler ,W., Esterbaue, H.,1988. Adsorption of Trichoderma reesei cellulase 
on cellulose: experimental data and their analysis by different equations. 
Biotechnol. Bioeng. 32 , 853?865. 
Sawada, T., Nakamura, Y., Kobayashi, F., Kuwahara, M., Watanabe, T., 1995. Effects of 
fungal pretreatment and steam explosion pretreatment on enzymatic 
saccharification of plant biomass. Biotechnol, Bioeng. 48, 719?724. 
Schell, D. J., Farmer, J., Newman, M.,cMillan, J. D., 2003. Dilute sulfuric acid 
pretreatment of corn stover in pilot-scale reactor. Investigation of yields, kinetics, 
and enzymatic digestibilities of solids. Appl. Biochem. Biotechnol.105-108, 69-
85. 
 217
Schell, D., Nguyen, Q., Tucker, M. and Boynton, B., 1998. Pretreatment of softwood by 
acid-catalyzed steam explosion followed by alkali extraction. Appl. Biochem. 
Biotechnol.70-72, 17-24. 
Schell, D. J., Walter, P. J., Johnson, D. K., 1992. Dilute sulfuric acid pretreatment of corn 
stover at high solids concentrations. Appl. Biochem. Biotechnol.34-35, 659-665. 
Sch?nberg,C., Oksanen,T.,Suurn?kki,A.,Kettunen,H., Buchert,J.,2001. The Importance 
of Xylan for the Strength Properties of Spruce Kraft Pulp Fibres. Holzforschung, 
55(6), 639?644. 
Schugerl, K., 1981. Oxygen transfer into highly viscous media. Adv. Biochem. Eng. 19, 
71?174. 
Schwald, W., Brownell, H.H., Saddler, J.N., 1988. Enzymatic hydrolysis of steam treated 
aspen wood: Influence of partial hemicellulose and lignin removal prior to 
pretreatment. J. Wood Chem. Tech. 8, 4, 543?560. 
Scott, G.M., Abubakr S., and Smith, A., 1995.  Sludge Characteristics and Disposal 
Alternatives for the Pulp and Paper Industry.  Proceedings of the 1995 
international environmental conference May 7?10, Tappi Press, Atlanta, GA , 
269?279. 
Scott, R.W., 1989. Influence of cations and borate on the alkaline extraction of xylan and 
glucomannan from pine pulps , J. Appl. Polym. Sci. 38(5), 907-914. 
Sears, K. D., Beelik, A., Casebier, R. L., Engen, R. J., Hamilton, J. K., Hergert, H. 
L.,1971. Southern pineprehydrolyzates: Characterization of polysaccharides and 
lignin fragments. J. Polym. Sci. Polym. Symp. 36(1),425?443. 
Sedlak, M., Ho, N., 2004. Production of ethanol from cellulosic biomass hydrolysates 
using genetically engineered saccharomyces yeast capable of cofermenting 
glucose and xylose. Appl. Biochem. Biotechnol. 114(1-3), 403-416. 
Service, R.F., 2007. Cellulosic ethanol: Biofuel researchers prepare to reap a new harvest, 
Science, 315,1488-1491. 
Sewalt,V.J.H., Glasser,W.G., Beauchemin,K.A.,1997. Lignin impact on fiber 
degradation. 3. Reversal of inhibition of enzymatic hydrolysis by chemical 
modification of lignin and by additives. J. Agri. Food Chem.,45 (5),1823?1828. 
Shah,M.M.,Song,S.K., Lee,Y.Y, Torget,R.,1991. Effect of pretreatment on simultaneous 
saccharification and fermentation of hardwood into Acetone/Butanol. Appl. 
Biochem. Biotechnol. 28-29(1), 99-109. 
Shahbazi, A., Li, Y.B., Mims, M.R., 2005. Application of sequential aqueous steam 
treatments to the fractionation of softwood. Appl. Biochem. Biotechnol. 121, 973-
987. 
 218
Shao, Z., Li, K,2006, Surface Lignin and Interfibre Bonding. J. Wood Chem. Technol. 
26(3), 231-244. 
Shevchenko, S.M., Paszner, L., Saddler, J.N., 2001a.  Treatment of organosolv and steam 
explosion lignins with molecular hydrogen iodide: A structural probe. Cem. 
Concr. Compos.35(3-4), 295-318. 
Shevchenko, S.M., Chang, K., Dick, D.G., Gregg, D.J., Saddler, J.N., 2001b.Structure 
and properties of lignin in softwoods after SO2-catalyzed steam explosion and 
enzymatic hydrolysis. Cem. Concr. Compos. 35(5-6), 487-502. 
Shimizu, K., 2001. Chemistry of hemicelluloses; in: Wood and Cellulosic Chemistry. 2nd  
edition, Eds. Hon, D.N.S., Shiraishi , N., Marcel Dekker Inc., New York, USA, 
177-214. 
Shin, C.S., Lee, J.P., Lee, J.S., Park, S. C.,2000. Enzyme production of Trichoderma 
reesei Rut C-30 on various lignocellulosic substrates. Appl. Biochem. Biotechnol. 
84/86, 237-245. 
Sidiras, D., Koukios, E., 2004. Simulation of acid-catalysed organosolv fractionation  of 
wheat straw. Bioresour. Technol. 94(1),91-98. 
Simpson C.W. and Lam R.,1998. Method for forming pulp from processed recycled 
fibers. U.S. Patent 5,772,847. 
Sintsyn, A.P., Gusakov, A.V., Vlasenko, E.Y., 1991. Effect of structural and physico-
chemical features of cellulosic substrates on the efficiency of enzymatic 
hydrolysis. Appl. Biochem. Biotechnol. 30, 43?59. 
Sixta, H., 2006. Handbook of pulp, Wiley-VCH, Weinheim 
Sj?de, A., Alriksson B., J?nsson L.J,, Nilvebrant, N., 2007. The potential in bioethanol 
production from waste fiber sludges in pulp mill-based biorefineries.  Appl. 
Biochem.  Biotechnol. 136?140, 327-337. 
Sj?str?m, E., 1993. Wood Chemistry: Fundamentals and Applications. 2
nd
 ed. Academic 
Press, Espoo, Finland. 
Smith, M. T., Cameron, D. R., Duff, S. J. B., 1997. Comparison of industrial yeast strains 
for fermentation of spent sulphite pulping liquor fortified with wood hydrolysate, 
J. Ind. Microbiol. Biot. 18, 18-21. 
Smook, G.A. 1992. Handbook for Pulp and Paper Technologists. 2nd edition. Angus 
Wilde Publications, Vancouver, B.C., and Bellingham,Washington. 
Soderhjelm,L., 1976. Possible uses for fibrous sludges from pulp and paper industry, Pap 
Puu-Pap Tim,58, 620?627. 
 219
Song,T., Pranovich,A.,Sumerskiy,I.,Holmbom,B., 2008. Extraction of 
galactoglucomannan from spruce wood with pressurised hot water. 
Holzforschung. 62(6), 659-66. 
Sousa, J., Torriani, J., Luengo, C., Fusco, J., Cukierman,A.L., 1991. Microporosity and 
surfaces areas study of pine charcoal by SAXS and CO2 adsorption techniques. J. 
Appl. Crystallogr.,34, 803?808. 
South, C.R., Hogsett, D.A. and Lynd, L.R., 1993. Continuous Fermentation of Cellulosic 
Biomass to Ethanol. Appl. Biochem.  Biotechnol., 39,587-600. 
Spano, L., Allen,A., Tassinaris, T., Mandels, M., Ryu, D.D.Y., 1978. Reassessment of 
economic of cellulose process technology for production of ethanol from 
cellulose. Proc. Second Fuels from Biomass Symp. 671?684. 
Spindler, D.D., Wyman, C.E., Grohmann, K., Mohagheghi,A.,1989.  Simultaneous 
saccharification and fermentation of pretreated wheat straw to ethanol with 
selected yeast strains and ?-glucosidase supplementation. Appl. Biochem.  
Biotechnol., 21(1),529-540. 
Sprenger, G.A.,1996. Carbohydrate metabolism in Zymomonas mobilis: a catabolic 
highway with some scenic routes. FMS Microbiol. Lett., 145, 301?307. 
Springer, E. L., Harris, J. F., 1982. Prehydrolysis of aspen wood with water and  with 
dilute aqueous sulfuric acid, Svensk Papperst., 85, 152-154. 
Stals,I.,Sandra,K.,Geysens,S.,Contreras,R.,Van Beeumen,J., Claeyssens,M.,2004. Factors 
influencing glycosylation of Trichoderma reesei cellulases. I: Postsecretorial 
changes of the O- and N-glycosylation pattern of Cel7A. Glycobiology, 14(8), 
713-724. 
Stanburg, P. F., Whitaker,A., 1984. Principles of fermentation technology. Pergamon 
Press, Oxford, 78?82. 
Steele, E., Raj, S., Nghiem, J., Stowers, M., 2005. Enzyme recovery and recycling 
following hydrolysis of ammonia fiber explosion-treated corn stover. Appl. 
Biochem.  Biotechnol. 121, 901-910. 
Stenberg, K., Bollok, M., Reczey, K., Galbe, M., Zacchi, G., 2000. Effect of substrate 
and cellulase concentration on simultaneous saccharification and fermentation of 
steam-pretreated softwood for ethanol production. Biotechnol. Bioeng., 68, 205?
210.  
Sternberg, D., 1976. Production of cellulase by Trichoderma. Biotechnol. Bioeng. Symp. 
6, 35?53. 
Sternberg, D.,Vijayakumar,P.,Reese.E.T., 1977. ?-Glucosidase: microbial production and 
effect on enzymatic hydrolysis of cellulose. Can. J. Microbiol. 23,139-147. 
 220
Stutzenberger,F., Sterpu,I. 1978. Effect of municipal refuse metals on cellulase 
production by Thermomonospora curvata. Appl Environ Microbiol. 36(1): 201?
204. 
Sullivan,J.T., Hershberger,T.V.,1959. Effect of Chlorine Dioxide on Lignin Content and 
Cellulose Digestibility of Forages , Science, 130,1252. 
Sun, Y.,Cheng, J.J., 2005. Dilute acid pretreatment of rye straw and bermudagrass for 
ethanol production. Bioresour. Technol. 96(14),1599-1606. 
Suominen, P., M?ntyl?, A., Karhunen, T., Hakola, S., Nevalainen, H., 1993. High 
frequency one-step gene replacement in Trichoderma reesei. II. Effects of 
deletions of individual cellulase genes. Mol. Gen. Genet. 241, 523-530. 
Suto, M.,Tomita, F., 2001. Induction and catabolite repression mechanisms of cellulase in 
fungi. J. Biosci. Bioeng. ,92(4),305-311. 
Szczodrak,J., Targonski,Z.,1989.  Simultaneous saccharification and fermentation of 
cellulose: effect of ethanol and cellulases on particular stages. Acta Biotechnol., 
9,555?564. 
Szengyel,Z., Zacchi,G.,2000.Effect of acetic acid and furfural on cellulase production of 
Trichoderma reesei RUT C30. Appl. Biochem.  Biotechnol. 89(1),31-42.  
Szij?rt?, N., Faigl, Zs., M?zes, M., Bers?nyi, A., R?czey, K., 2004. Cellulase 
fermentation on a novel substrate (waste cardboard) and subsequent utilization of 
home-produced cellulase and commercial amylase in a rabbit feeding trial. Ind. 
Crops Prod. 20, 49-57. 
Taherzadeh,M.J., Eklund, R., Gustafsson,L., Niklasson,C., Lid?n,G.,1997. 
Characterization and fermentation of dilute-acid hydrolyzates from wood, Ind. 
Eng. Chem.Res. 36, 4659-4665. 
Taherzadeh,M.J., Gustafsson,L., Niklasson,C., Lid?n,G., 2000a.Inhibition effects of 
furfural on aerobic batch cultivation of Saccharomyces cerevisiae growing on 
ethanol and/or acetic acid. J. Biosci. Bioeng. 90,374?380. 
Taherzadeh,M,J,, Gustafsson,L., Niklasson,C., Lid?n,G.,2000b. Physiological effects of 
5-hydroxymethylfurfural on Saccharomyces cerevisiae. Appl. Microbiol. 
Biotechnol.2000b,74(1),17-24. 
Tanahashi, M., Takada, S., Aoki, T., Goto, T., Higuchi, T., Hanai, S.,1983. 
Characterization of explosion wood. 1. Structure and physical properties. Wood 
Research, 69, 36?51. 
Tang, Y., An, M., Liu, K., Nagai, S., Shigematsu, T., Morimura, S., Kida, 
K.,2006.Ethanol production from acid hydrolysate of wood biomass using the 
 221
flocculating yeast Saccharomyces cerevisiae strain KF-7. Process Biochem. 41, 
909?914. 
Tangnu, S.K., Blanch, H.W., Wilke, C.R., 1981. Enhanced production of cellulase, 
hemicellulase, and ?-glucosidase by Trichoderma reesei (Rut C-
30).Biotechnol.Bioeng.23, 1837-1849. 
Taniguchi, M., Suzuki, H., Watanabe, D., Sakai, K., Hoshino, K., Tanaka, T., Evaluation 
of pretreatment with Pleurotus ostreatus for enzymatic hydrolysis of rice straw. J. 
Biosci. Bioeng. 100(6), 637-643. 
Tappi, Acid-Insoluble Ash in Wood, Pulp, Paper, and Paperboard, Test Method T 244 
cm-99 
Tarkow, H., Feist, W.C., 1969. Cellulases and their applications. A Mechanism for 
Improving the Digestibility of Lignocellulosic Materials with Dilute Alkali and 
Liquid Ammonia. Washington. 
Teixeira,L.C., Linden,J.C.,Schroeder,H.A.,2000. Simultaneous saccharification and 
cofermentation of peracetic acid-pretreated biomass. Appl. Biochem.  Biotechnol. 
84-86 , 111-127. 
Tengborg, C., Galbe,M., Zacchi,G., 2001. Reduced inhibition of enzymatic hydrolysis of 
steam pretreated softwood. Enzyme Microb. Technol. 28, 835?844. 
Tenkanen,M., Thornton, J.,Viikari, L.,1995a. An acetylglucomannan esterase of 
Aspergillus oryzae; purification, characterization and role in the hydrolysis of O-
acetyl-galactoglucomannan. J. Biotechnol.,42(3),197-206. 
Tenkanen, M., Buchert, J., Viikari, L., 1995b. Binding of hemicellulases on isolated 
polysaccharide substrates. Enzyme Microb. Biol. 17, 499-505. 
Terzaghi, B. E., Sandine, W. E.,1975. Improved medium for lactic streptococci and their 
bacteriophages. Appl. Microbiol. 29, 807-813. 
Teymouri,F.,Laureano-Perez,L.,Alizadeh,H.,Dale,B.E.,2005. Optimization of the 
ammonia fiber explosion (AFEX) treatment parameters for enzymatic hydrolysis 
of corn stover. Bioresour. Technol.96 (18), 2014-2018. 
Thompson, D. N., Chen, H. C., and Grethlein, H. E.,1992. Comparison of. Pretreatment 
Methods on the Basis of Available Surface Area. Biores.Technol., 39,155?163. 
Thomsen,M.H., 2005.Complex media from processing of agricultural crops for microbial 
fermentation. Appl Microbiol Biotechnol. 68, 598-606. 
Thornton, J., Ekman,R.,Holmbom,B., Orsa,F.,1994. Polysaccharides Dissolved from 
Norway Spruce in Thermomechanical Pulping and Peroxide Bleaching. J. Wood 
Chem. Technol. 14(2), 159 ? 175. 
 222
Thring, R.W., Chorent, E., Overend, R., 1990. Recovery of a solvolytic lignin: effects of 
spent liquor/acid volume ration, acid concentration and temperature. Biomass 23, 
289?305. 
Tietjen,T., Wetzel, R.G., 2003. Extracellular enzyme?clay mineral complexes: enzyme 
adsorption, alteration of enzyme activity, and protection from photodegradation. 
Aquat. Ecol. 37,331?339. 
Tillman, L.M., Lee, Y.Y.,1990.Pulping of southern pine under low-water alkaline 
conditions using supercritical carbon dioxide-sulfur dioxide mixtures. Tappi. 
73(7), 140-146. 
Timell, T.E., 1967. Recent progress in the chemistry of wood hemicelluloses. Wood Sci. 
Technol. 1, 1, 45?70.  
Todorovic, R. and Grujic, S., 1987, Effect of pretreatment and reaction products on 
hydrolysis of wheat straw by Trichoderma harzianum C1R1 enzymes. Microbios 
Letters, 34(134), 71?78. 
Tomme, P., Warren, R. A. J., Miller, R. C. J., Kilburn, D. G., Gilkes, N. R.,1995.  in 
Enzymatic Degradation of Insoluble Polysaccharides (Saddler, J. N. , Penner, M., 
eds), Vol. 618 , 142-163, American Chemical Society, Washington, D. C. 
Tonn, S.T., Philippidis,G.P., Ho,N.Y., Chen, Z., Brainard,A., Lumpkin,R.E., Riley, 
C.J.,1997. Enhanced cofermentation of glucose and xylose by recombinant 
Saccharomyces yeast strains in batch and continuous operating modes. Appl. 
Biochem. Biotechnol., 63? 65,243?255. 
Torget,R.,Walter,P., Himmel,M.,Grohmann,K.,1991. Diluted-acid pretreatment of corn 
residues and short-rotation woody crops, Appl. Biochem. Biotechnol. 28/29, 75?
86. 
Tothill,I.E., Best,D.J., and Seal,K.J., 1993. Detection of cellulolytic enzymes in 
waterborne paint. Int. Biodeterioration Biodegrad. 31(2), 115-128. 
Tsao, G.T. ,1978. Cellulosic Material as a Renewable Resource. Process Biochem. 
13(10),12-14. 
Tu, M. B., Zhang, X., Paice, M., McFarlane, P., Saddler, J. N., 2009. Effect of  
Surfactants on Separate Hydrolysis Fermentation and Simultaneous 
Saccharification Fermentation of Pretreated Lodgepole Pine. Biotechnol. Progr. 
25, 1122-1129. 
Tucker, M.P., Kim, K.H., Newman, M.M., Nguyen, Q.A., 2003. Effects of temperature 
and moisture on dilute-acid steam explosion pretreatment of corn stover and 
cellulase enzyme digestibility. Applied Biochemistry and Biotechnology. 
105,165-177. 
 223
Tutus, A., Eroglu,H.,2003. Practical solution to silica problem in straw pulping.Appita J., 
56, 111-115. 
Tutus, A., Eroglu,H.,2004. An alternative solution to the silica problem in wheat straw 
pulping. Appita Journal, 57, 214-217. 
Um, B.H., van Walsum, G.P.,2009. Acid Hydrolysis of Hemicellulose in Green Liquor 
Pre-Pulping Extract of Mixed Northern Hardwoods. Appl. Biochem. Biotechnol. 
153(1-3), 127-138. 
Underwood,S.A., Buszko, M.L., Shanmugam,K.T., Ingram, L.O.,2002a.Flux through 
citrate synthase limits the growth of ethanologenic Escherichia coli KO11 during 
xylose fermentation. Appl.Environ.Microbiol. 68(3):1071-81. 
Underwood, S.A., Zhou,S., Causey, T. B., Yomano, L. P., Shanmugam,K.T., 
Ingram,L.O., 2002b. Genetic changes to optimize carbon partitioning between 
ethanol and biosynthesis in ethanologenic Escherichia coli. Appl. 
Environ.Microbiol. 68 , 6263?6272. 
Vallander, L., Eriksson, K.E., 1985. Enzymic saccharification of pretreated wheat straw. 
Biotechnol. Bioeng. 27, 650?659. 
van Maris,A.J., Abbott,D.A., Bellissimi,E., van den Brink,J., Kuyper,M., Luttik,M.A., 
Wisselink,H.W., Scheffers,W.A., van Dijken,J.P., Pronk,J.T.,  2006, Alcoholic 
fermentation of carbon sources in biomass hydrolysates by Saccharomyces 
cerevisiae : current status. Antonie van Leeuwenhoek , 90(4),391-418. 
Van Soest, P.J. 1994. The Nutritional Ecology of the Ruminant, 2nd edition. Cornell 
University Press. Ithaca, NY. 
Varga, E., Szengyel, Z., Reczey, K., 2002. Chemical Pretreatments of Corn Stover for 
Enhancing Enzymatic Digestibility. Appl. Biochem.  Biotechnol. 98/100, 73-87.  
Varga, E., Szengyel, Z., Reczey, K., 2004. Optimization of steam pretreatment of corn 
stover to enhance enzymatic digestibility. Appl. Biochem.  Biotechnol. 
113/116,509-523.  
Velkovska,S., Marten, M.R., Ollis, D.F., 1997. Kinetic model for batch cellulase ... 
Trichoderma reesei RUT C30. J. Biotechnol. 54, 83?94. 
Vidal, P.F., Molinier, J., 1988. Ozonolysis of lignin ? improvement of in vitro 
digestibility of poplar sawdust. Biomass 16, 1?17. 
Viikari, L., Kantelinen, A., Sundquist, J., Linko, M., 1994. Xylanases in bleaching: from 
an idea to the industry. FEMS Microbiol. Rev. 13, 335?350.  
 224
Vinzant, T.B., Adney, W.S., Decker, S.R., Baker, J.O., Kinter, M.T., Sherman, N.E., Fox, 
J.W., Himmel, M.E., 2001. Fingerprinting Trichoderma reesei hydrolases in a 
commercial cellulase preparation. Appl. Biochem. Biotechnol .91/93, 99? 107. 
Walseth, C.S., 1952. The influence of the fine structure of cellulose on the action of 
cellulases. Tappi, 35, 233?238. 
Walther ,T., Hensirisak, P., Agblevor, F.A., 2001. The influence of aeration and 
hemicellulosic sugars on xylitol production by Candida tropicalis. Biores. 
Technol. 76,213-220. 
Walton, S.,L., van Heiningen, A., van Walsu, G.,P.,2010a. Inhibition effects on 
fermentation of hardwood extracted hemicelluloses by acetic acid and sodium. 
Biores. Technol. 101(6), 1935-1940. 
Walton, S.,L., Bischoff,K.M., van Heiningen, A., van Walsu, G.,P., 2010b.Production of 
lactic acid from hemicellulose extracts by Bacillus coagulans MXL-9. J. Ind. 
Microbiol.Biotechnol. 1367-5435 
Wang, P. Y., Bolker, H. I., Purves, C. B., 1967. Uronic acid ester groups in some 
softwoods and hardwoods. Tappi 50(3), 123- 124. 
Wang,W., Kang,L.,Lee,Y.Y.,2010. Production of Cellulase from Kraft Paper Mill Sludge 
by Trichoderma Reesei Rut C-30. Appl. Biochem. Biotechnol. 161,382-94. 
Wang, W., Kang, L., Wei, H., Arora, R., Lee, Y.Y.(2011) Appl. Biochem. Biotechnol. 
Study on the Decreased Sugar Yield in Enzymatic Hydrolysis of Cellulosic 
Substrate at High Solid Loading. (Accepted) Available at: www.springerlink.com 
Watanabe, T., Sato, T., Yoshioka, S. , Koshijima, T. , Kuwahara,M., 1992. Purification 
and properties of Aspergillus niger ?-glucosidase. Eur. J. Biochem. 209, 651-659. 
Watson,T.G.,Nelligan,I.,Lessing,L.,1984. Cellulase production by Trichoderma reesei 
(RUT-C30) in fed-batch culture. Biotechnol. Lett .6(10),667-672 .  
Wayman M.,Chen S., 1992. Cellulase production by Trichoderma reesei using whole 
wheat flour as a carbon source. Enzyme Microb.Technol. 14, 825-831. 
Wayman, M., Parekh, S.R., 1990. Biothechnology of Biomass Conversion. Open 
University Press, Milton Keynes, U.K.,  
Wei,C.J.,Cheng,C.Y.,1985. Effect of hydrogen peroxide pretreatment on the structural 
features and the enzymatic hydrolysis of rice straw. Biotechnol. Bioeng. 27(10), 
1418 ? 1426. 
Weil, J.R., Brewer, M., Hendrickson, R., Sarikaya, A., Ladisch, M.R., 1998. Continuous 
pH monitoring during pretreatment of yellow poplar wood sawdust by pressure 
cooking in water. Appl. Biochem. Biotechnol. 70?72, 99?111. 
 225
Weimer, P.J., Chou, Y.-C.T., Weston, W.M., Chase, D.B., 1986. Effect of supercritical 
ammonia on the physical and chemical structure of ground wood. Biotechnol. 
Bioeng. Symp. 17, 5?18. 
Weimer, P.J., Lopez-Guisa, J.M., French, A.D., 1990. Effect of cellulose fine structure on 
kinetics of its digestion by mixed ruminal microorganisms in vitro. Appl. Environ. 
Microbiol., 56, 2421 ? 2419. 
Wenzl, H. F. J.,1970. The Chemical Technology of Wood, Academic Press, New York. 
Werpy, T., Petersen, G., 2004. Top Value-added Chemicals from Biomass, Volume 1: 
Results of Screening for Potential Candidates from Sugars and Synthesis Gas. 
Pacific Northwest National Laboratory, U.S. Department of Energy, USA. 
Available: http://www1.eere.energy.gov/biomass/pdfs/35523.pdf 
Willfor, S.,Holmbom, B.,2004. Isolation and characterisation of water soluble 
polysaccharides from Norway spruce and Scots pine.Wood Sci. Technol. 38, 173-
179. 
Wingren, A., Galbe, M.and Zacchi, G.,2003. Techno-economic evaluation of producing 
ethanol from softwood: comparison of SSF and SHF and identification of 
Bottlenecks. Biotechnol Prog. 19, 1109?17. 
White, P., Johnson, L.A. (EDs), 2003. Corn: Chemistry and Technology. 2nd Edition, 
American Association of Cereal Chemists. 
Wong,K.K.Y., Saddler,J.N.,1993. Applications of hemicellulases in the food, feed, and 
pulp and paper industries. In: Coughlan MP, Hazlewood GP (eds) Hemicellulose 
and hemicellulases. Portland, London. 
Wood, T. M., Bhat, K. M., 1988. Methods for measuring cellulase activities. Meth. 
Enzymol.160, 87?112. 
Wooley, R., Ruth, M., Glassner, D., Sheehan, J., 1999.Process design and costing of 
bioethanol technology: A tool for determining the status and direction of research 
and development. Biotechnol. Progr. 15, 794?803. 
Wu,J., Ju , L.K.,1998. Enhancing enzymatic saccharification of waste newsprint by 
surfactant addition. Biotechnol. Prog. 14 , 649?652. 
Wu,M.M., Chang,K., Gregg,D.J.,Boussaid,A., Beatson,R.P., Saddler,J.N.,1999. 
Optimization of steam explosion to enhance hemicellulose recovery and 
enzymatic hydrolysis of cellulose in softwoods. Appl. Biochem. Biotechnol. 77-
9,47-54. 
Wu, Z., Lee,Y.Y., 1997. Inhibition of the enzymatic hydrolysis of cellulose by ethanol. 
Biotechnol. Lett .19(10), 977?979. 
 226
Wyman, C.E., 1996. Handbook on Bioethanol: Production and Utilization. Taylor & 
Francis, Washington D.C.,  
Wyman,C.E., Dale,B.E., Elander,R.T., Holtzapple ,M., Ladisch, M.R., Lee, Y.Y., 2005. 
Coordinated development of leading biomass pretreatment technologies. Biores. 
Technol. 96(18),1959-1966. 
Wyman,C.E., Spindler,D.D., Grohmann,K.,1992. Simultaneous saccharification and 
fermentation of several  lignocellulosic feedstocks to fuel ethanol. Biomass 
Bioenerg., 3, 301?307. 
Xia, L.M., Shen, X.L., 2004. High-yield cellulase production by Trichoderma reesei ZU-
02 on corn cob residue. Biores. Technol. 91(3), 259-262. 
Xiao, Z., Zhang, X., Gregg, D.J., Saddler, J.N., 2004. Effects of sugar inhibition on 
cellulases and ?-glucosidase during enzymatic hydrolysis of softwood substrates. 
Appl. Biochem. Biotechnol. 113/116, 1115?1126. 
Ximenes,E., Kim,Y., Mosier,N., Dien,B., Ladisch,M.,2010. Inhibition of cellulases by 
phenols, Enzyme Microb. Technol. 46, 170?176. 
Xiong, H., von Weymarn, N., Leisola, M., Turunen, O., 2004. Influence of pH on the 
production of xylanases by Trichoderma reesei Rut C-30. Proc. Biochem. 39,729-
733. 
Yan, T. R., Lin, C. L.,1997. Purification and characterization of a glucose-tolerant ?-
glucosidase from Aspergillus niger CCRC 31494. Biosci. Biotechnol. Biochem. 
61, 965-970. 
Yang, B., Boussaid, A., Mansfield, S.D., Gregg, D.J., Saddler, J.N., 2002. Fast and 
efficient alkaline peroxide treatment to enhance the enzymatic digestibility of 
steam-exploded softwood substrates. Biotech. Bioeng. 77(6),678-684. 
Yang, B., Wyman, C.E.,2006. BSA treatment to enhance enzymatic hydrolysis of 
cellulose in lignin containing substrates.Biotechnl. Bioeng., 94 (4), 611?617. 
Yomano, L. P., York, S. W.,Ingram, L.O.,1998. Isolation and characterization of ethanol 
tolerant mutants of Escherichia coli KO11 for fuel ethanol production. J. Ind. 
Microbiol. 20, 132-138. 
Yomano, L. P., York, S. W.,Zhou,S., Shanmugam,K.T., Ingram, L.O., 2008. Re-
engineering Escherichia coli for ethanol production, Biotechnol. Lett. 30(12), 
2097-2103. 
Yoon, H.H., 1998. Pretreatment of lignocellulosic biomass by autohydrolysis and 
aqueous ammonia percolation. Korean J. Chem. Eng. 15, 6, 631?636. 
 227
Yoon, S. H., Cullinan, H.T., Krishnagopalan,G.A., 2010a. Oxidative-reductive 
Modification of Alkaline Pulping of Southern Pine Integrated with Hydrothermal 
Pre-extraction of Hemicelluloses. Ind. Eng. Chem. Res., 49 (13), 5969?5976. 
Yoon, S. H. and van Heiningen, A., 2010b.  Green liquor extraction of hemicelluloses 
from southern pine in an Integrated Forest Biorefinery. J. Ind. Eng. Chem.,16 (1), 
74-80. 
Yoon, S. H., Macewan, K., van Heiningen, A., 2008. Hot-water pre-extraction  from 
loblolly pine (Pinus taeda) in an integrated forest products biorefinery, Tappi J. 
7,436, 27-32. 
York, S.W., Ingram, L.O., 1996. Ethanol production by recombinant Escherichia coli 
KO11 using crude yeast autolysate as a nutrient supplement. Biotechnol. Lett. 18, 
683?688. 
Yu,X., Yun, H.S., Koo, Y.M.,1998. Production of Cellulase by Trichoderma reesei Rut 
C30 in a Batch Fermenter. J. Microbiol. Biotechnol. 8(6), 575 -580. 
Zaldivar,J., Ingram, L.O.,1998. Effect of organic acids on the growth and fermentation of 
ethanologenic Escherichia coli LY01. Biotechnol. Bioeng.  66(4), 203 ? 210. 
Zanzi,R., Sjostrom,K.,Bjornbom,E.,2002. Rapid pyrolysis of agricultural residues at high 
temperature. Biomass Bioenergy, 23, 357?366. 
Zhang, M., Eddy, C., Deanda, K., Finkelstein, M., Picataggio, S., 1995. Metabolic 
engineering of a pentose metabolism pathway in ethanologenic Zymononas 
mobilis. Science, 267, 240?243. 
Zhang, Y. H. P., Ding, S. Y., Mielenz, J. R., Cui, J. B., Elander, R. T. , Laser, M., 
Himmel, M. E., McMillan, J. R., Lynd, L. R.,2007. Fractionating recalcitrant 
lignocellulose at modest reaction conditions. Biotechnol. Bioeng. 97,214-223. 
Zhang,Y.H.P., Lynd, L.R., 2004. Toward an Aggregated Understanding of Enzymatic 
Hydrolysis of Cellulose: Noncomplexed Cellulase Systems. Biotechnol. Bioeng., 
797-824. 
Zhao, X. Wang, L., Liu, D., 2007. Effect of several factors on peracetic acid pretreatment 
of sugarcane bagasse for enzymatic hydrolysis, J. Chem. Tech. Biotech..82(12) , 
1115-1121. 
Zheng, Y., Lin, H.-M., Tsao, G.T., 1998. Pretreatment for cellulose hydrolysis by carbon 
dioxide explosion. Biotechnol. Prog. 14, 890?896.