The objective of the present dissertation is to investigate the bioprocess for the production of acetone-butanol-ethanol (ABE) from lignocellulosic feedstocks via ABE fermentation with solvent-producing Clostridium acetobutylicum ATCC 824. The first part of this dissertation focuses on the use of natural herbaceous feedstocks (corn stover, switchgrass, etc.) as the starting materials. To serve as carbon source for Clostridium, pretreatment and enzymatic hydrolysis, as prerequisites for downstream fermentation, are necessarily required to degrade the polymeric carbohydrate into monomer sugars. In this dissertation, alkaline pretreatment is chosen as the pretreatment method among various strategies as it is capable of preserving most of the hexose and pentose in the feedstocks, both of which can be catabolized by the culture for solvent production. It was found that supplementation minute quantity of poly-diallyl-dimethyl-ammonium chloride (polyDADMAC) in alkaline pretreatment led to slightly negative effect on delignification but enhanced enzymatic hydrolysis of pretreated solids quite significantly (e.g., 14-25% increase in glucan digestibility). Structural characterization of pretreated solids with SEM, XRD and BET indicated that polyDADMAC addition substantially disrupted and swelled the crystallized cellulose fibers. As supportive control, lignin-free cellulose was treated with hot water with and without addition of polyDADMAC. It was shown that addition of polyDADMAC in hot-water treatment enhanced enzymatic hydrolysis of lignin-free cellulose likewise. Therefore, the effect of polyDADMAC on alkaline pretreatment was speculated to be by direct interaction with cellulosic fiber, rather than with lignin or other extraneous components. The possible mechanism behind such a disruption was also discussed.
Following the alkaline pretreatment, the acquired solid was found to be inhibitory to ABE fermentation. An in situ detoxification method with the addition of non-ionic surfactant Tween 80 was therefore applied to improve the fermentability of the solids. Both simultaneous saccharification and fermentation (SSF) and separate hydrolysis and fermentation (SHF) were applied for solvent production with Clostridium acetobutylicum (ATCC-824). Fermentation with Avicel and pure sugars as substrate was also carried out control. The results from SSF of Avicel indicated that enzyme loading played a key role in the bioconversion yields. SSF of alkali-pretreated switchgrass could be as efficient as SSF of Avicel and addition of Tween 80 was found to enhance the bioconversion yield quite significantly. However, attempts at SSF were strongly limited by solid loading with 5 wt. % as maximum level. With an enzyme loading of 15 FPU/g glucan, SSF of 5% well-pretreated switchgrass produced 12.2 g/L of total solvents, the level of which was far from the inhibition threshold for this specific culture. Therefore, SHF was further applied and produced 14.3 g/L of solvents. The bioconversion features of SSF and SHF are also compared in conjunction with the overall bioconversion yield and productivity.
The second part of this dissertation aims to investigate the technical feasibility of utilizing the waste materials in the pulp and paper industry as feedstock for bio-butanol production. The concept of integrating of the biorefinery to the pulping industry has been proposed to diversify the product portfolio and generate extra revenue for the pulp mills. In a Kraft pulping process, woodchips are fractionated into major product of pulp and a number of byproducts (extractives, hemicellulose-degraded carbohydrates and lignin) ended up as a complex mixture in the black liquor. The hemicellulose and lignin in the black liquor is usually incinerated in the recovery boiler for power generation. In consideration of the heating value of hemicellulose (13.6 MJ/kg) is rather low in comparison with that of lignin (27.0 MJ/kg), the hemicellulose represents a highly underutilized sugar source. In this part of the dissertation, the hemicellulose pre-hydrolysate is investigated as liquid sugar feedstock for production of acetone, butanol and ethanol by ABE fermentation. For this purpose, hot-water treatment is applied to Poplar (hardwood) and Southern pine (softwood) to extract the hemicellulose portion. The acquired hemicellulose prehydrolysate was analyzed to contain, in addition to the carbohydrates mostly in the form of oligosaccharides, various degradation compounds. The toxicity test with model degradation compounds indicates phenolic compounds exert tremendous inhibition on the cell growth. Therefore, detoxification is required prior to fermentation. Adsorption with activated carbon is found to greatly reduce the phenolic content. Upon detoxification, simultaneous saccharification and fermentation (SSF) of concentrated poplar prehydrolysate with 43.3 g/L of sugar produced a total of 10.8 g/L ABE giving a solvent yield of 0.25 (g-solvent/g-sugar). Comparatively, SSF of concentrated southern pine prehydrolysate resulted an ABE production of 13.2 g/L and productivity of 0.28 (g-solvent/g-sugar). The details of hot-water extraction conditions, performance of detoxification as well as the fermentation profiles are discussed.
In the paper making process, the poor-quality and short fibers in the wood pulp, which are hardly to be retained on the fiber screens and paper machines, are discharged into the waste stream as paper mill sludge (PS). PS is investigated as a feedstock for acetone-butanol-ethanol (ABE) production by simultaneous saccharification and fermentation (SSF). As a waste material in the pulp mills, sludge typically contains, in addition to the carbohydrates, considerable portion of inorganic ashes originated from the fillers (clay, TiO2 and CaCO3) added in the paper making process, exhibiting tremendous inhibition effects on enzymatic hydrolysis. Therefore, bioconversion of sludge requires partial removal of ash to enhance its enzymatic digestibility. Enzymatic hydrolysis was found to be the rate-limiting step in the ABE production by SSF. A total of 16.4-18.0 g/L of ABE solvents were produced in the SSF of de-ashed PS with solid loading of 6.3-7.4% and enzyme loading of 10-15 FPU/g-glucan, and the final solvent yield reached 0.27 g/g sugars. These levels of solvent in SSF are comparable to those of glucose and cellulose controls, approaching to the upper limits. No pretreatment and pH control are needed in ABE fermentation of paper mill sludge, which makes it an attractive feedstock for production of butanol.