Enhanced Cellulose Fermentation by an Asporogenous and Ethanol-Tolerant Mutant of Clostridium thermocellum

A mutant of Clostridium thermocellum isolated after UV mutagenesis and selection for resistance to fluoropyruvate was found to be asporogenous and ethanol tolerant. The mutant was also an ethanol hyperproducer, able to ferment 63 g of cellulose into 14.5 g of ethanol per liter of medium. The ratio of ethanol to total organic acids produced by the mutant was increased, and H₂ production was decreased. Culture conditions were optimized for ethanol production by the new strain.     

Synergistic Saccharification, and Direct Fermentation to Ethanol, of Amorphous Cellulose by Use of an Engineered Yeast Strain Codisplaying Three Types of Cellulolytic Enzyme

A whole-cell biocatalyst with the ability to induce synergistic and sequential cellulose-degradation reaction was constructed through codisplay of three types of cellulolytic enzyme on the cell surface of the yeast Saccharomyces cerevisiae. When a cell surface display system based on α-agglutinin was used, Trichoderma reesei endoglucanase II and cellobiohydrolase II and Aspergillus aculeatus β-glucosidase 1 were simultaneously codisplayed as individual fusion proteins with the C-terminal-half region of α-agglutinin. Codisplay of the three enzymes on the cell surface was confirmed by observation of immunofluorescence-labeled cells with a fluorescence microscope. A yeast strain codisplaying endoglucanase II and cellobiohydrolase II showed significantly higher hydrolytic activity with amorphous cellulose (phosphoric acid-swollen cellulose) than one displaying only endoglucanase II, and its main product was cellobiose; codisplay of β-glucosidase 1, endoglucanase II, and cellobiohydrolase II enabled the yeast strain to directly produce ethanol from the amorphous cellulose (which a yeast strain codisplaying β-glucosidase 1 and endoglucanase II could not), with a yield of approximately 3 g per liter from 10 g per liter within 40 h. The yield (in grams of ethanol produced per gram of carbohydrate consumed) was 0.45 g/g, which corresponds to 88.5% of the theoretical yield. This indicates that simultaneous and synergistic saccharification and fermentation of amorphous cellulose to ethanol can be efficiently accomplished using a yeast strain codisplaying the three cellulolytic enzymes.     

Synergistic saccharification, and direct fermentation to ethanol, of amorphous cellulose by use of an engineered yeast strain codisplaying three types of cellulolytic enzyme

A whole-cell biocatalyst with the ability to induce synergistic and sequential cellulose-degradation reaction was constructed through codisplay of three types of cellulolytic enzyme on the cell surface of the yeast Saccharomyces cerevisiae. When a cell surface display system based on alpha-agglutinin was used, Trichoderma reesei endoglucanase II and cellobiohydrolase II and Aspergillus aculeatus beta-glucosidase 1 were simultaneously codisplayed as individual fusion proteins with the C-terminal-half region of alpha-agglutinin. Codisplay of the three enzymes on the cell surface was confirmed by observation of immunofluorescence-labeled cells with a fluorescence microscope. A yeast strain codisplaying endoglucanase II and cellobiohydrolase II showed significantly higher hydrolytic activity with amorphous cellulose (phosphoric acid-swollen cellulose) than one displaying only endoglucanase II, and its main product was cellobiose; codisplay of beta-glucosidase 1, endoglucanase II, and cellobiohydrolase II enabled the yeast strain to directly produce ethanol from the amorphous cellulose (which a yeast strain codisplaying beta-glucosidase 1 and endoglucanase II could not), with a yield of approximately 3 g per liter from 10 g per liter within 40 h. The yield (in grams of ethanol produced per gram of carbohydrate consumed) was 0.45 g/g, which corresponds to 88.5% of the theoretical yield. This indicates that simultaneous and synergistic saccharification and fermentation of amorphous cellulose to ethanol can be efficiently accomplished using a yeast strain codisplaying the three cellulolytic enzymes.     

Efficient fermentation of Pinus sp. acid hydrolysates by an ethanologenic strain of Escherichia coli.

Process conditions for the acid hydrolysis of pine hemicellulose and cellulose have been described which provide a biocompatible sugar solution. By using an improved strain of recombinant Escherichia coli, strain KO11, hydrolysates supplemented with yeast extract and tryptone nutrients were converted to ethanol with an efficiency of 85% to over 100% on the basis of monomer sugar content (approximately 72 g/liter) and with the production of 35 g of ethanol per liter in 48 h. In the process described, approximately 347 liters of ethanol could be produced per dry metric ton of lignocellulose.     

Efficient fermentation of Pinus sp. acid hydrolysates by an ethanologenic strain of Escherichia coli.

Process conditions for the acid hydrolysis of pine hemicellulose and cellulose have been described which provide a biocompatible sugar solution. By using an improved strain of recombinant Escherichia coli, strain KO11, hydrolysates supplemented with yeast extract and tryptone nutrients were converted to ethanol with an efficiency of 85% to over 100% on the basis of monomer sugar content (approximately 72 g/liter) and with the production of 35 g of ethanol per liter in 48 h. In the process described, approximately 347 liters of ethanol could be produced per dry metric ton of lignocellulose.     

Efficient ethanol production from glucose, lactose, and xylose by recombinant Escherichia coli.

Lactose and all of the major sugars (glucose, xylose, arabinose, galactose, and mannose) present in cellulose and hemicellulose were converted to ethanol by recombinant Escherichia coli containing plasmid-borne genes encoding the enzymes for the ethanol pathway from Zymomonas mobilis. Environmental tolerances, plasmid stability, expression of Z. mobilis pyruvate decarboxylase, substrate range, and ethanol production (from glucose, lactose, and xylose) were compared among eight American Type Culture Collection strains. E. coli ATCC 9637(pLO1297), ATCC 11303(pLO1297), and ATCC 15224(pLO1297) were selected for further development on the basis of environmental hardiness and ethanol production. Volumetric ethanol productivities per hour in batch culture were 1.4 g/liter for glucose (12%), 1.3 g/liter for lactose (12%), and 0.64 g/liter for xylose (8%). Ethanol productivities per hour ranged from 2.1 g/g of cell dry weight with 12% glucose to 1.3 g/g of cell dry weight with 8% xylose. The ethanol yield per gram of xylose was higher for recombinant E. coli than commonly reported for Saccharomyces cerevisiae with glucose. Glucose (12%), lactose (12%), and xylose (8%) were converted to (by volume) 7.2% ethanol, 6.5% ethanol, and 5.2% ethanol, respectively.     

Efficient ethanol production from glucose, lactose, and xylose by recombinant Escherichia coli

Lactose and all of the major sugars (glucose, xylose, arabinose, galactose, and mannose) present in cellulose and hemicellulose were converted to ethanol by recombinant Escherichia coli containing plasmid-borne genes encoding the enzymes for the ethanol pathway from Zymomonas mobilis. Environmental tolerances, plasmid stability, expression of Z. mobilis pyruvate decarboxylase, substrate range, and ethanol production (from glucose, lactose, and xylose) were compared among eight American Type Culture Collection strains. E. coli ATCC 9637(pLO1297), ATCC 11303(pLO1297), and ATCC 15224(pLO1297) were selected for further development on the basis of environmental hardiness and ethanol production. Volumetric ethanol productivities per hour in batch culture were 1.4 g/liter for glucose (12%), 1.3 g/liter for lactose (12%), and 0.64 g/liter for xylose (8%). Ethanol productivities per hour ranged from 2.1 g/g of cell dry weight with 12% glucose to 1.3 g/g of cell dry weight with 8% xylose. The ethanol yield per gram of xylose was higher for recombinant E. coli than commonly reported for Saccharomyces cerevisiae with glucose. Glucose (12%), lactose (12%), and xylose (8%) were converted to (by volume) 7.2% ethanol, 6.5% ethanol, and 5.2% ethanol, respectively.     

Characterization of and Ethanol Hyper-production by Clostridium thermocellum I-1-B

An ethanol hyper-producing clostridial strain, I-1-B, was isolated from Shibi hot spring, Kagoshima prefecture and identified as Clostridium thermocellum based on morphological and physiological proper­ ties. The carbohydrates used as energy sources were glucose, fructose, cellobiose, cellulose and esculin. Fermentation products were ethanol, lactate, acetate, formate, carbon dioxide, and hydrogen. The optimum, maximum, and minimum temperature for growth are about 60, 70, and 47°C, respectively. Optimum pH for growth is about 7.5, and growth occurs at starting pH between 6.0 and 9.0. I-1-B strain has strong tolerance for ethanol and hyper ethanol-productivity. Ethanol concentrations causing 50%. decrease of growth yield are 27 and 16g/liter for I-1-B and ATCC27405 of C. thermocellum, respectively. The organism was cultured on a medium containing 80 g/liter cellulose at 60°C for 156 h. The culture was fed with a vitamin mixture containing vitamin B₁₂ and mineral salts solution at intervals. In this culture the organism produced 23.6 g/liter (512mM) ethanol, 8.5 g/liter (94mM) lactate, 2.9 g/liter (48mM) acetate, and 0.9 g/liter (20mM) formate. The molar ratio of ethanol to total acidic products was 3.2. The ethanol productivity of the strain I-1-B is superior to any of the wild and mutant strains of C. thermocellum so far reported.     

Bio-ethanol Production from Green Onion by Yeast in Repeated Batch

Considered to be the cleanest liquid fuel, bio-ethanol can be a reliable alternative to fossil fuels. It is produced by fermentation of sugar components of plant materials. The common onions are considered to be a favorable source of fermentation products as they have high sugar contents as well as contain various nutrients. This study focused on the effective production of ethanol from Green onion (Allium fistulosum L.) by the yeast “Saccharomyces cerevisiae” in repeated batch. The results showed that the total sugar concentration of onion juice was 68.4 g/l. The maximum rate of productivity, ethanol yield and final bio-ethanol percentage was 7 g/l/h (g ethanol per liter of onion juice per hour), 35 g/l (g ethanol per liter of onion juice) and 90 %, respectively.     

Effect of Sugar Concentration in Jerusalem Artichoke Extract on Kluyveromyces marxianus Growth and Ethanol Production

The effect of inulin sugars concentration on the growth and ethanol production by Kluyveromyces marxianus UCD (FST) 55-82 was studied. A maximum ethanol concentration of 102 g/liter was obtained from 250 g of sugars per liter initial concentration. The maximum specific growth rate varied from 0.44 h⁻¹ at 50 g of sugar per liter to 0.13 h⁻¹ at 300 g of sugar per liter, whereas the ethanol yield remained almost constant at 0.45 g of ethanol per g of sugars utilized.     

Direct and Efficient Production of Ethanol from Cellulosic Material with a Yeast Strain Displaying Cellulolytic Enzymes

For direct and efficient ethanol production from cellulosic materials, we constructed a novel cellulose-degrading yeast strain by genetically codisplaying two cellulolytic enzymes on the cell surface of Saccharomyces cerevisiae. By using a cell surface engineering system based on α-agglutinin, endoglucanase II (EGII) from the filamentous fungus Trichoderma reesei QM9414 was displayed on the cell surface as a fusion protein containing an RGSHis6 (Arg-Gly-Ser-His₆) peptide tag in the N-terminal region. EGII activity was detected in the cell pellet fraction but not in the culture supernatant. Localization of the RGSHis6-EGII-α-agglutinin fusion protein on the cell surface was confirmed by immunofluorescence microscopy. The yeast strain displaying EGII showed significantly elevated hydrolytic activity toward barley β-glucan, a linear polysaccharide composed of an average of 1,200 glucose residues. In a further step, EGII and β-glucosidase 1 from Aspergillus aculeatus No. F-50 were codisplayed on the cell surface. The resulting yeast cells could grow in synthetic medium containing β-glucan as the sole carbon source and could directly ferment 45 g of β-glucan per liter to produce 16.5 g of ethanol per liter within about 50 h. The yield in terms of grams of ethanol produced per gram of carbohydrate utilized was 0.48 g/g, which corresponds to 93.3% of the theoretical yield. This result indicates that efficient simultaneous saccharification and fermentation of cellulose to ethanol are carried out by a recombinant yeast cells displaying cellulolytic enzymes.     

A study of producing ethanol from cellulose using Clostridium thermocellum

The feasibility of producing ethanol in a continuous system from cellulose using Clostridirrrn thermocellum was investigated. This anaerobic and therniophilic bacterium was able to degrade cellulose directly into ethanol with acetic acid, hydrogen. and carbon dioxide as by-products of this fermentation. The fermentation was first carried out in a batch mode to study the effects of buffers, temperature, and agitation on microbial growth and ethanol production. From the compounds used to control pH. sodium bicarbonate had the most preferred effects on generation time and ethanol production. As expected, there was a positive relationship between temperature and growth rate. On the other hand, agitation did not benefit from ethanol production or microbial growth. The possibility of noncompetitive inhibition within such a system was deduced from the calculation of the kinetic constants K(m) and V(max). Continuous fermentations were carried out at 60 degrees C and pH 7.0 using 1.5 and 3% pure cellulose as a limiting substrate. The maximum ethanol concentration reached during the 1.5% cellulose fermentation was 0.3%. and 0.9% during the 3% cellulose fermentation. The yield of ethanol was about 0.3 grams per gram of consumed cellulose. The overall yield in both schemes was around 0.45 and 0.75 grams per gram of cellulose degraded. It was concluded that cellulose could be degraded continuously in a system with C. thermocellum for production of ethanol. While the continuous system like the batch method is feasible, it may not be promising as yet because of the slow generation time of this microorganism.     

Eastern gamagrass as an alternative cellulosic feedstock for bioethanol production

Eastern gamagrass (Trypsacum dactyloides) is a C₄ perennial grass, native to the USA with desirable characteristics that warrants further investigation as a new lignocellulosic crop for bioethanol production. Chemical composition assays showed that eastern gamagrass had comparable cellulose, hemicellulose and lignin compositions to those of switchgrass (Panicum virgatum). With the cellulose solvent-based lignocellulose fractionation (CSLF) pretreatment and subsequent enzymatic saccharification, 80.5–99.8% of cellulosic glucose was released from the gamagrass biomass, which was 10–17% greater than the glucose release efficiency from switchgrass (73.5–87.1%). Furthermore, the hydrolysate of gamagrass supported greater ethanol fermentation yield (up to 0.496 g/g glucose) than the hydrolysates of switchgrass. As such, in the whole process of biomass-to-ethanol conversion, gamagrass could yield 13–35% more ethanol per gram of biomass than switchgrass, indicating that gamagrass has high potential as an alternative energy feedstock for lignocellulosic ethanol production.     

Fermentation of d-Xylose to Ethanol by Genetically Modified Klebsiella planticola

d-Xylose is a plentiful pentose sugar derived from agricultural or forest residues. Enteric bacteria such as Klebsiella spp. ferment d-xylose to form mixed acids and butanediol in addition to ethanol. Thus the ethanol yield is normally low. Zymomonas spp. and most yeasts are unable to ferment xylose, but they do ferment hexose sugars to ethanol in high yield because they contain pyruvate decarboxylase (EC 4.1.1.1), a key enzyme that is absent from enteric bacteria. This report describes the fermentation of d-xylose by Klebsiella planticola ATCC 33531 bearing multicopy plasmids containing the pdc gene inserted from Zymomonas mobilis. Expression of the gene markedly increased the yield of ethanol to 1.3 mol/mol of xylose, or 25.1 g/liter. Concurrently, there were significant decreases in the yields of formate, acetate, lactate, and butanediol. Transconjugant Klebsiella spp. grew almost as fast as the wild type and tolerated up to 4% ethanol. The plasmid was retained by the cells during at least one batch culture, even in the absence of selective pressure by antibiotics to maintain the plasmid. Ethanol production was 31.6 g/liter from 79.6 g of mixed substrate per liter chosen to simulate hydrolyzed hemicellulose. The physiology of the wild-type of K. planticola is described in more detail than in the original report of its isolation.     

Effects of environmental conditions on xylose fermentation by recombinant Escherichia coli

In batch fermentations, optimal conversion of xylose to ethanol by recombinant Escherichia coli was obtained under the following conditions: 30 to 37 degrees C, pH 6.4 to 6.8, 0.1 to 0.2 M potassium phosphate buffer, and xylose concentrations of 8% or less. A yield of 39.2 g of ethanol per liter (4.9% ethanol by volume) was observed with 80 g of xylose per liter, equivalent to 96% of the maximum theoretical yield. Maximal volumetric productivity was 0.7 g of ethanol per liter per h in batch fermentations and 30 g of ethanol per liter per h in concentrated cell suspensions (analogous to cell recycling).     

Effects of environmental conditions on xylose fermentation by recombinant Escherichia coli.

In batch fermentations, optimal conversion of xylose to ethanol by recombinant Escherichia coli was obtained under the following conditions: 30 to 37 degrees C, pH 6.4 to 6.8, 0.1 to 0.2 M potassium phosphate buffer, and xylose concentrations of 8% or less. A yield of 39.2 g of ethanol per liter (4.9% ethanol by volume) was observed with 80 g of xylose per liter, equivalent to 96% of the maximum theoretical yield. Maximal volumetric productivity was 0.7 g of ethanol per liter per h in batch fermentations and 30 g of ethanol per liter per h in concentrated cell suspensions (analogous to cell recycling).     

Metabolism and Solubilization of Cellulose by Clostridium cellulolyticum H10

When Clostridium cellulolyticum was grown with cellulose MN300 as the substrate, the rates of growth and metabolite production were found to be lower than those observed with soluble sugars as the substrate. At low cellulose concentrations, the growth yields were equal to those obtained with cellobiose. The main fermentation products from cellulose and soluble sugars were the same. Up to 15 mM of consumed hexose, a change in the metabolic pathway favoring lactate production similar to that observed with soluble sugars was found to occur concomitantly with a decrease in molar growth yield. With cellulose concentrations above 5 g/liter, accumulation of soluble sugars occurred once growth had ceased. Glucose accounted for 30% of these sugars. A kinetic analysis of cellulose solubilization revealed that cellulolysis by C. cellulolyticum involved three stages whatever cellulose concentration was used. Analysis of these kinetics showed three consecutive enzymatic activity levels having the same K_m (0.8 g of cellulose per liter, i.e., 5 mM hexose equivalent) but decreasing values of V_max. The hypothesis is suggested that each step corresponds to differences in cellulose structure.     

Improved Sugar Conversion and Ethanol Yield for Forage Sorghum (Sorghum bicolor L. Moench) Lines with Reduced Lignin Contents

Lignin is known to impede conversion of lignocellulose into ethanol. In this study, forage sorghum plants carrying brown midrib (bmr) mutations, which reduce lignin contents, were evaluated as bioenergy feedstocks. The near-isogenic lines evaluated were: wild type, bmr-6, bmr-12, and bmr-6 bmr-12 double mutant. The bmr-6 and bmr-12 mutations were equally efficient at reducing lignin contents (by 13% and 15%, respectively), and the effects were additive (27%) for the double mutant. Reducing lignin content was highly beneficial for improving biomass conversion yields. Sorghum biomass samples were pretreated with dilute acid and recovered solids washed and hydrolyzed with cellulase to liberate glucose. Glucose yields for the sorghum biomass were improved by 27%, 23%, and 34% for bmr-6, bmr-12, and the double mutant, respectively, compared to wild type. Sorghum biomass was also pretreated with dilute acid followed by co-treatment with cellulases and Saccharomyces cerevisiae for simultaneous saccharification and fermentation (SSF) into ethanol. Conversion of cellulose to ethanol for dilute-acid pretreated sorghum biomass was improved by 22%, 21%, and 43% for bmr-6, bmr-12, and the double mutant compared to wild type, respectively. Electron microscopy of dilute-acid treated samples showed an increased number of lignin globules in double-mutant tissues as compared to the wild-type, suggesting the lignin had become more pliable. The mutations were also effective for improving ethanol yields when the (degrained) sorghum was pretreated with dilute alkali instead of dilute acid. Following pretreatment with dilute ammonium hydroxide and SSF, ethanol conversion yields were 116 and 130 mg ethanol/g dry biomass for the double-mutant samples and 98 and 113 mg/g for the wild-type samples.     

Heterotrophic Growth and Production of Xanthophylls by Chlorella pyrenoidosa

The growth and level of xanthophylls of several representative species of green algae were investigated as a possible source of pigmentation for the egg yolk and broiler markets. Chlorella pyrenoidosa 7-11-05 was selected for fermentation studies because of its high level of xanthophylls and wide temperature range for growth. The heterotrophic metabolism was preferred because of the ease of adaptability to present fermentation equipment. When used as the sole carbon source, glucose was the only sugar, among many tested, that gave appreciable growth in illuminated shaken flasks. A dry cell weight of 90 g per liter and total xanthophylls of 450 mg per liter were obtained from 190 g per liter of glucose monohydrate in 168-hr illuminated shaken flasks. Higher levels of glucose decreased yields. In combination with glucose, monosaccharides, such as fructose and galactose, were readily assimilated. The 7-11-05 strain was adapted to galactose as the sole carbon source after six vegetative passages. Light of the proper intensity and duration stimulated total xanthophylls approximately 35%. The effect on dry cell weight and total xanthophylls of seven antibiotics added at various levels in shaken flasks was studied. Erythromycin was essentially stable throughout the fermentation and nontoxic up to 25 μg/ml, with only slight toxicity at higher levels. Both erythromycin and ristocetin were effective in controlling a high incidence of bacterial contamination in 30-liter fermentors. With the higher agitation and aeration rates possible in 30-liter fermentors, dry cell weights in excess of 100 g per liter and total xanthophylls of 467 to 512 mg per liter were readily obtained from 230 to 260 g per liter of glucose in 162-hr illuminated batch-type fermentations. Continuous-feed runs yielded a dry cell weight of 302 g per liter and total xanthophylls of 650 mg per liter from 520 g per liter of glucose. The type of Chlorella cell produced was an important consideration with respect to the availability of the xanthophylls in pigmenting egg yolks and broilers.     

Cellulose catabolism by Clostridium cellulolyticum growing in batch culture on defined medium

A reinvestigation of cellulose degradation by Clostridium cellulolyticum in a bioreactor with pH control of the batch culture and using a defined medium was performed. Depending on cellulose concentration, the carbon flow distribution was affected, showing the high flexibility of the metabolism. With less than 6.7 g of cellulose liter(-1), acetate, ethanol, H(2), and CO(2) were the main end products of the fermentation and cellulose degradation reached more than 85% in 5 days. The electron flow from the glycolysis was balanced by the production of H(2) and ethanol, the latter increasing with increasing initial cellulose concentration. From 6.7 to 29.1 g of cellulose liter(-1), the percentage of cellulose degradation declined; most of the cellulase activity remained on the cellulose fibers, the maximum cell density leveled off, and the carbon flow was reoriented from ethanol to acetate. In addition to that of previously indicated end products, lactate production rose, and, surprisingly enough, pyruvate overflow occurred. Concomitantly the molar growth yield and the energetic yield of the biomass decreased. Growth arrest may be linked to sufficiently high carbon flow, leading to the accumulation of an intracellular inhibitory compound(s), as observed on cellobiose (E. Guedon, M. Desvaux, S. Payot, and H. Petitdemange, Microbiology 145:1831-1838, 1999). These results indicated that bacterial metabolism exhibited on cellobiose was distorted compared to that exhibited on a substrate more closely related to the natural ecosystem of C. cellulolyticum. To overcome growth arrest and to improve degradation at high cellulose concentrations (29.1 g liter(-1)), a reinoculation mode was evaluated. This procedure resulted in an increase in the maximum dry weight of cells (2,175 mg liter(-1)), cellulose solubilization (95%), and end product concentrations compared to a classical batch fermentation with a final dry weight of cells of 580 mg liter(-1) and 45% cellulose degradation within 18 days.