Bioconversion of Pulp and Paper Mills Sludge and Prehydrolysate Stream into Ethanol and Cellulase Enzyme

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 CaCO3 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 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 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 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.