Tato diplomová práce se zabývá sledováním fermentačních postupů zaměřených na produkci ethanolu. Ethanol je produkován kvasinkami anebo bakteriemi Zymomonas mobilis. Zymomonas mobilis je gramnegativní bakterie, která se vyznačuje vysokým teoretickým výtěžkem ethanolu během fermentace a toleruje vysokou koncentraci ethanolu. Přestože je Zymomonas mobilis výborným producentem ethanolu, není vhodná pro konverzi biomasy, protože zkvašuje pouze glukosu, fruktosu a sacharosu. Tato práce se zabývá fermentačními testy bakterie rodu Zymomonas mobilis na širokém spektru substrátů za rozdílných podmínek. Byl sledován proces výroby ethanolu pomocí Zymomonas mobilis, konkrétně kmeny z České sbírky mikroorganismů CCM 2770, CCM 2771, CCM 3881 a CCM 3883. Porovnávány byly kultivace na různých typech medií (sacharosovém, fruktosovém, glukosovém a obilné zápary) při různých koncentracích uhlíkatého zdroje a za různých podmínek (aerobní, anaerobní). Ke stanovení obsahu cukru a lihu bylo použito HPLC. Ukázalo se, že jsou vhodné především kmeny CCM 3881 a CCM 2770, které narostly během 48 hodin a bylo u nich dosaženo nejlepších výsledků výtěžnosti ethanolu ( až 99 % teoretického výtěžku). Kmeny CCM 3883 a CCM 2771 rostly velmi pomalu, produkce ethanolu u kmenu CCM 2771 byla zanedbatelná. Jako nejlepší substrát se jeví glukosa,...

Ethanol, when mixed with gasoline, is an economical and environmentally friendly liquid fuel. Yeast cells under anaerobic conditions can ferment glucose to ethanol. However, glucose is expensive for industrial applications and starch is an economical alternative. Simultaneous cold starch hydrolysis and fermentation was investigated for ethanol production from wheat starch particles. With a view to minimize process costs while maintaining a satisfactory ethanol yield, both a recombinant yeast cell and an inexpensive medium were tested for their fermentation abilities. Initially, NRRL Y132 strain was compared to Muntons yeast for their abilities to produce ethanol from glucose. Both the wild-type and the recombinant NRRL Y132 strains were cultured on soluble starch to determine if the plasmid bearing strain could produce ethanol without the addition of -amylase. Finally, Muntons yeast was cultured on starch particles using both expensive and inexpensive media. Sequential hydrolysis and fermentation runs were performed using the inexpensive medium, with hydrolysis carried out at 30°C, 37.5°C, 45°C and 52.5°C. The wild-type, NRRL Y132 strain grew faster and produced more ethanol than Muntons yeast when cultured on glucose. Compared to the wild-type strain, the recombinant NRRL Y132 strain did not show enhanced ethanol production from soluble starch. The results of the simultaneous hydrolysis and fermentation runs showed that the ethanol yields for runs performed in expensive medium (0.41, 0.38 and 0.42 g ethanol / g glucose) were slightly lower than those for runs performed in the inexpensive medium (0.46, 0.44 and 0.43 g ethanol / g glucose). The growth rates for the expensive and inexpensive media runs were comparable. Hence, it was concluded that the inexpensive medium can be used for ethanol production from starch particles with good ethanol productivities. For the sequential hydrolysis and fermentation runs, it was observed that the growth rates (0.11, 0.10, 0.10 and 0.11 h-1) as well as the ethanol yields (0.44, 0.37, 0.44 and 0.39 g ethanol / g glucose) were similar in spite of the four different hydrolysis temperatures. Therefore, it was concluded that increasing the temperature above 30°C for enhancing starch particle hydrolysis does not increase fermentation productivity significantly.

Ethanol, when mixed with gasoline, is an economical and environmentally friendly liquid fuel. Yeast cells under anaerobic conditions can ferment glucose to ethanol. However, glucose is expensive for industrial applications and starch is an economical alternative. Simultaneous cold starch hydrolysis and fermentation was investigated for ethanol production from wheat starch particles. With a view to minimize process costs while maintaining a satisfactory ethanol yield, both a recombinant yeast cell and an inexpensive medium were tested for their fermentation abilities. Initially, NRRL Y132 strain was compared to Muntons yeast for their abilities to produce ethanol from glucose. Both the wild-type and the recombinant NRRL Y132 strains were cultured on soluble starch to determine if the plasmid bearing strain could produce ethanol without the addition of -amylase. Finally, Muntons yeast was cultured on starch particles using both expensive and inexpensive media. Sequential hydrolysis and fermentation runs were performed using the inexpensive medium, with hydrolysis carried out at 30°C, 37.5°C, 45°C and 52.5°C. The wild-type, NRRL Y132 strain grew faster and produced more ethanol than Muntons yeast when cultured on glucose. Compared to the wild-type strain, the recombinant NRRL Y132 strain did not show enhanced ethanol production from soluble starch. The results of the simultaneous hydrolysis and fermentation runs showed that the ethanol yields for runs performed in expensive medium (0.41, 0.38 and 0.42 g ethanol / g glucose) were slightly lower than those for runs performed in the inexpensive medium (0.46, 0.44 and 0.43 g ethanol / g glucose). The growth rates for the expensive and inexpensive media runs were comparable. Hence, it was concluded that the inexpensive medium can be used for ethanol production from starch particles with good ethanol productivities. For the sequential hydrolysis and fermentation runs, it was observed that the growth rates (0.11, 0.10, 0.10 and 0.11 h-1) as well as the ethanol yields (0.44, 0.37, 0.44 and 0.39 g ethanol / g glucose) were similar in spite of the four different hydrolysis temperatures. Therefore, it was concluded that increasing the temperature above 30°C for enhancing starch particle hydrolysis does not increase fermentation productivity significantly.

Fourteen varieties of wheat grown in western Canada were assessed for differences in starch content and structure. Physicochemical properties of starch such as amylopectin to amylose ratio, starch granule morphology, and thermal and pasting properties were all found to vary significantly between varieties. Enzymatic susceptibility was measured using industrial α-amylase to hydrolyze gelatinized starches and resultant reducing sugar content ranged from ~407−500mg glucose equivalents per gram starch, indicating different patterns of oligosaccharide chain lengths present after hydrolysis. During fermentation striking differences in glucose generation were observed, the high glucose cohort averaged 1.21 g/g-starch for the initial time point, compared to a range of 0.83−1.05 g/g-starch for the low glucose cohort. In general, the pattern of glucose generation appears to be consistent with ethanol and biomass production. Correlating structural attributes with fermentation performance suggests that amylopectin to amylose ratio is the most predictive factor in the pattern of wheat starch hydrolysis.

Fourteen varieties of wheat grown in western Canada were assessed for differences in starch content and structure. Physicochemical properties of starch such as amylopectin to amylose ratio, starch granule morphology, and thermal and pasting properties were all found to vary significantly between varieties. Enzymatic susceptibility was measured using industrial α-amylase to hydrolyze gelatinized starches and resultant reducing sugar content ranged from ~407−500mg glucose equivalents per gram starch, indicating different patterns of oligosaccharide chain lengths present after hydrolysis. During fermentation striking differences in glucose generation were observed, the high glucose cohort averaged 1.21 g/g-starch for the initial time point, compared to a range of 0.83−1.05 g/g-starch for the low glucose cohort. In general, the pattern of glucose generation appears to be consistent with ethanol and biomass production. Correlating structural attributes with fermentation performance suggests that amylopectin to amylose ratio is the most predictive factor in the pattern of wheat starch hydrolysis.

Doctor of Philosophy / Department of Biological & Agricultural Engineering / Donghai Wang / Grain sorghum is the second major starch-rich raw material (after corn) for bioethanol production in the United States. Most sorghum feedstock for bioethanol production is normal non-tannin sorghum. Waxy sorghum and tannin sorghum are rarely used due to lack of scientific information about waxy sorghum fermentation performance and the way to increase fermentation efficiency of tannin sorghum. The main objectives of this study were to investigate the fermentation performance of waxy sorghum and to improve fermentation efficiency of tannin sorghum using techniques such as germination and ozonation treatments. The ethanol fermentation performance on both waxy sorghum and tannin sorghum were evaluated using a dry grind ethanol fermentation procedure. Fermentation efficiencies of tested waxy sorghum varieties ranged from 86 to 93%, which was higher than normal (non-waxy) sorghum varieties. The advantages of using waxy sorghums for ethanol production include less energy consumption, higher starch and protein digestibility, shorter fermentation time, and less residual starch in distillers dried grains with solubles (DDGS). Results from germination study showed germination significantly increased fermentation efficiency of tannin sorghum. The laboratory results were further confirmed by those from five field-sprouted grain sorghum samples. Significantly increased free amino nitrogen (FAN) contents in sprouted sorghum samples accelerated the ethanol fermentation process. Results from both laboratory-germinated and fieldsprouted samples demonstrated that germination not only increased fermentation efficiency (higher than 90%) but also reduced fermentation time by about 50%, which could result in energy saving and increased production capacity without additional investment. The excellent performance of sprouted sorghums may provide farmers a new market for field-sprouted sorghum (poor quality as food or feed) in a bad year. A previous study showed ozone had a strong connection to degradation of lignin macromolecules. The hypothesis was that ozone treatment may also reduce tannin activity and increase fermentation efficiency of tannin sorghum. Results showed that the ethanol production performance (ethanol yield, fermentation efficiency, and fermentation kinetics) of the ozone-treated, tannin sorghum flours was significantly improved compared with the untreated control. The other effects of ozonation on sorghum flour include pH value decrease, discoloration, and inactivation of tannin. In summary, these studies showed sorghum, no matter it was waxy, field-sprouted, or tannin sorghum, can be an excellent feedstock for ethanol production.

Ethanol can be used as a complete fuel or as an octane enhancer, and has the advantages of being renewable and environmentally friendly. Ethanol produced by a fermentation process, generally referred to as bioethanol, is considered to be a partial solution to the worldwide energy crisis. Traditionally, industrial bioethanol fermentation involves two major steps: starch hydrolysis and fermentation. Since the key microorganism, Saccharomyces cerevisiae, lacks amylolytic activity and is unable to directly utilize starch for proliferation and fermentation, it requires intensive amount of energy and pure starch hydrolyzing enzymes to gelatinize, liquefy and dextrinize the raw starch before fermentation. It has been suggested that genetically engineered yeast which expresses amylolytic enzymes could potentially perform simultaneous starch hydrolysis and fermentation. This improvement could greatly reduce the capital and energy costs in current bioethanol producing plants and make bioethanol production more economical. In this project, a novel yeast strain of Saccharomyces cerevisiae was genetically engineered in such a way that barley alpha-amylase was constitutively expressed and immobilized on the yeast cell surface. This particular alpha-amylase was selected based on its superior kinetic properties and its pH optimum which is compatible with the pH of yeast culture media. The cDNA encoding barley Ñ-amylase, with a secretion signal sequence, was fused to the cDNA encoding the C-terminal half of a cell wall anchoring protein, alpha-agglutinin. The fusion gene was cloned downstream of a constitutive promoter ADH1 in a yeast episomal plasmid pAMY. The pAMY harbouring yeast showed detectable amylolytic activity in a starch plate assay. In addition, alpha-amylase activity was detected only in the cell pellet fraction and not in the culture supernatant. In batch fermentation studies using soluble wheat starch as sole carbon source, even though pAMY harbouring yeast was able to hydrolyse soluble starch under fermentation conditions, no ethanol was produced. This was probably due to insufficient alpha-amylase activity which resulted from the enzyme being anchored on the cell wall by alpha-agglutinin. Further research using alternative cell surface anchoring system might be able to produce yeast with industrial applications.

Ethanol can be used as a complete fuel or as an octane enhancer, and has the advantages of being renewable and environmentally friendly. Ethanol produced by a fermentation process, generally referred to as bioethanol, is considered to be a partial solution to the worldwide energy crisis. Traditionally, industrial bioethanol fermentation involves two major steps: starch hydrolysis and fermentation. Since the key microorganism, Saccharomyces cerevisiae, lacks amylolytic activity and is unable to directly utilize starch for proliferation and fermentation, it requires intensive amount of energy and pure starch hydrolyzing enzymes to gelatinize, liquefy and dextrinize the raw starch before fermentation. It has been suggested that genetically engineered yeast which expresses amylolytic enzymes could potentially perform simultaneous starch hydrolysis and fermentation. This improvement could greatly reduce the capital and energy costs in current bioethanol producing plants and make bioethanol production more economical. In this project, a novel yeast strain of Saccharomyces cerevisiae was genetically engineered in such a way that barley alpha-amylase was constitutively expressed and immobilized on the yeast cell surface. This particular alpha-amylase was selected based on its superior kinetic properties and its pH optimum which is compatible with the pH of yeast culture media. The cDNA encoding barley Ñ-amylase, with a secretion signal sequence, was fused to the cDNA encoding the C-terminal half of a cell wall anchoring protein, alpha-agglutinin. The fusion gene was cloned downstream of a constitutive promoter ADH1 in a yeast episomal plasmid pAMY. The pAMY harbouring yeast showed detectable amylolytic activity in a starch plate assay. In addition, alpha-amylase activity was detected only in the cell pellet fraction and not in the culture supernatant. In batch fermentation studies using soluble wheat starch as sole carbon source, even though pAMY harbouring yeast was able to hydrolyse soluble starch under fermentation conditions, no ethanol was produced. This was probably due to insufficient alpha-amylase activity which resulted from the enzyme being anchored on the cell wall by alpha-agglutinin. Further research using alternative cell surface anchoring system might be able to produce yeast with industrial applications.

The anaerobic, cellulolytic bacterium Clostridium thermocellum formed biofilms on cellulose consisting of a single layer of cells which did not secrete an extracellular polymeric matrix. Sporulation occurred under normal growth and was believed to assist with biofilm translocation to new substrates. Although the cell-substrate distance was less than 210 nm, the biofilm layer lost up to 29% of hydrolyzed oligomeric products when reactors were loaded with extreme concentrations of cellulose (up to 200 g/litre). This effect was much less severe at lower cellulose concentrations. Of the total cellulose carbon, 4% (gC/gC) was utilized for cell mass production and up to 75% was converted into primary metabolites (ethanol, acetic acid, lactic acid, carbon dioxide). Increasing the starting cellulose concentration shifted the ethanol-to-acetic acid ratio from 0.91 g/g to 0.41 g/g. Such high substrate loadings and metabolite shifts have not been previously reported and may be of interest for consolidated bioprocessing strategies. Cellulose conversion was initially limited by microbial growth, with a biofilm development rate estimated at 0.46h-1 to 0.33h-1 and where up to 20% of the substrate was consumed. Subsequently, substrate-limited conditions determined the rate kinetics. Surface accessibility for microbial colonization was the dominant rate limiting factor, while mass imposed constraints very late towards the end-point fermentation. CO2 was found to be an excellent reporter molecule for cellulose consumption and biofilm growth. Online CO2 tracking may also be used to assess the digestibility of substrates with unknown surface properties. A mathematical model that described biofilm growth, substrate consumption and product formation was found to have an excellent fit with experimental data of CO2 production which reinforced the previous findings on the cellulolytic biofilm form and function. Together, these results demonstrate that biofilms are undeniably the key to understanding the effective microbial conversion of cellulosic substrates.

Xylose is a pentose sugar that forms a substantial proportion of the monosaccharides released from lignocellulosic biomass after hydrolysis. Therefore, the economic and commercial viability of biofuel production from lignocellulosic material via microbial fermentation relies upon maximising the metabolism of monosaccharides like xylose. As such, second generation biofuels are becoming a focus of biofuel innovation because of the depleting fossil fuel reserves and increasing levels of carbon emissions. Even so the majority of current biofuel production uses glucose as a carbon source from corn, wheat or sugar cane. This conflicts with food production and has prompted the food versus fuel debate. The introduction of xylose metabolising pathways into current biofuel production microorganisms like yeast, which cannot utilize xylose, would allow xylose use from lignocellulosic biomass. The xylose-reductase (XR) pathway from fungi utilise the xylose reductase, xylose dehydrogenase and xylulokinase genes, whereas the xylose isomerase (XI) pathway from bacteria consists of xylose isomerase and xylulokinase. In this project plasmid constructs containing the two pathways were successfully introduced into yeast. The genes were further integrated into specific chromosomal sites for comparison. Depending on the type of media used, some xylose uptake and ethanol production could be demonstrated for some of these strains, but overall levels of xylose use did not reach a level likely to impact upon commercial biofuel production. As a result, several strategies were investigated with a view to increasing xylose metabolism and ethanol production from the strains. Alterations were made to the cassette design for the xylose enzyme genes, such as gene promoter replacement or removal of the epitope tag. A pentose specific transporter, GXF1, from Candida tropicalis was also introduced. However, none of these strategies improved xylose use. A further approach, which led to minor increases in xylose metabolism, was deletion of the PHO13 gene, which is thought to impact upon expression of pentose phosphate genes. One further goal of this work was to investigate whether xylose metabolism could be connected to butanol production, as butanol has superior properties as a biofuel in yeast. Unfortunately, butanol was not detected from heterologous butanol producing strains bearing the plasmid based XI pathway, presumably because the growth and health of these strains was quite poor. Overall this project has demonstrated that S. cerevisiae is able to metabolise hemicellulosic xylose to ethanol using heterologous pathways, however, the very low levels generated mean that a great deal of genetic and metabolic engineering would be required for optimisation of biofuel production for commercial viability.