Various parameters affecting the overall conversion of lignocellulosics to ethanol were studied. Of prime importance in a lignocellulosics to ethanol process is the pretreatment of the woody biomass. We have found that high levels of hydrolysis (92%) can be achieved with material that was acid soaked prior to steam-explosion and water extracted after steam explosion. This hydrolysis was accomplished through a combined hydrolysis and fermentation technique which produced, under optimum conditions, a broth containing 2.3% ethanol. Optimum conditions for the combined hydrolysis and fermentation process were found to be a culture pH of 4.8, a hydrolysis temperature of 45C and a fermentation temperature of 37C. Yeast extract was found to be inhibitory to the action of the cellulases and was only added following the hydrolysis step.
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The work described in this report covers four different topics. The first chapter describes preliminary work on acid hydrolysis of aspen wood using hydrogen fluoride (HF). This mode of acid hydrolysis was investigated because a thorough examination of the scientific literature indicated that this was one of the few chemical methods which could result in the almost complete hydrolysis of the carbohydrate component of wood to soluble oligosaccharides and monosaccharides. Although other methods of acid hydrolysis could release most of the cellulose and hemicellulose derived sugars, a large proportion of these sugars was further degraded to decomposition products. The HF work described in the first chapter is of a preliminary nature and was primarily carried out to verify the values published in previous papers and patents. The second chapter describes another method of acid hydrolysis which also looked as though it could liberate most of the sugar components of the hemicellulose and cellulose fractions without the resulting decomposition of the free sugars. This acid catalyzed organosolv (ACOS) treatment of aspenwood has been used in the past as a method for preparing pulps. The preliminary work outlined in this report describes some attempts at optimizing this process for sugar production rather than pulp production. The third chapter describes various fermentor runs in the Forintek 30L microferm fermentor. This work was carried out because previous data had indicated that the high cellulase values obtained in shake cultures were not reproduced when the same fungal stains were grown in the larger fermentor. Variations such as rate of agitation, degree of aeration, size of inoculum, etc., were to be studied to try to optimize larger scale production of cellulase enzymes. Chapter 4 is related to the first chapter on HF hydrolysis of wood. This chapter describes some preliminary attempts at assaying whether the oligosaccharides produced by HF hydrolysis can be utilized by fermentative organisms such as Saccharomyces cerevisiae and Zymomonas mobilis. This is important as efficient hydrolysis of lignocellulosic substrates to their component sugars will only be of use if the derived sugars can be fermented to products such as ethanol. Initial attempts were also made to try and further hydrolyze the oligosaccharides obtained by HF hydrolysis using the cellulases from various Trichoderma species. The work on the enzymatic hydrolysis of the HF oligosaccharides and the fermentability of the HF derived sugars produced very preliminary data and further work would have to be done before the results could be considered conclusive. The Appendix is also related to the first chapter on HF hydrolysis. It constitutes a review of the relevant literature.
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The microbial conversion of wood to sugars, liquid fuels and chemicals is highly dependant on the pretreatment method used. In the present report we have studied the pretreatment method of steam explosion as it pertains to the one-step or two-step conversion of wood to ethanol. Steam explosion was found to render the hemicellulose soluble in water, depolymerize the lignin and make the cellulose component more accessible to cellulase enzymes. A dilute presoaking of the aspenwood chips with H2SO4 greatly increased the solubility of the hemicellulose and selective extraction of steam exploded aspenwood produced substrates containing over 90% cellulose. The one-step coversion of cellulose toethanol was found to have major drawbacks notably the low ethanol and low substrate tolerance of Clostridium thermocellum. Attempts to overcome the first drawback by the adaptation of C. thermocellum to tolerate high ethanol levels was successful but did not result in increased ethanol yields. Attempts at increasing ethanol yields through the co-culture of C. thermocellum with either C. thermohydrosulfuicum or C. thermosaccharolyticum resulted in a 2 fold increase in ethanol levels. However, further attempts at increasing the substrate concentration above 1% were only partially successful and only extremely low levels of ethanol (<1%) could be produced from pretreated aspenwood using the one-stage process.
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Screening of highly cellulolytic fungi and the purification, identification and isolation of the cellulase and xylanase enzymes from these fungi as a prerequisite for future cloning studies
