Metabolic engineering of bacteria for ethanol production

Technologies are available which will allow the conversion of lignocellulose into fuel ethanol using genetically engineered bacteria. Assembling these into a cost-effective process remains a challenge. Our work has focused primarily on the genetic engineering of enteric bacteria using a portable ethanol production pathway. Genes encoding Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase have been integrated into the chromosome of Escherichia coli B to produce strain KO11 for the fermentation of hemicellulose-derived syrups. This organism can efficiently ferment all hexose and pentose sugars present in the polymers of hemicellulose. Klebsiella oxytoca M5A1 has been genetically engineered in a similar manner to produce strain P2 for ethanol production from cellulose. This organism has the native ability to ferment cellobiose and cellotriose, eliminating the need for one class of cellulase enzymes. The optimal pH for cellulose fermentation with this organism (pH 5.0-5.5) is near that of fungal cellulases. The general approach for the genetic engineering of new biocatalysts has been most successful with enteric bacteria thus far. However, this approach may also prove useful with Gram-positive bacteria which have other important traits for lignocellulose conversion. Many opportunities remain for further improvements in the biomass to ethanol processes. These include the development of enzyme-based systems which eliminate the need for dilute acid hydrolysis or other pretreatments, improvements in existing pretreatments for enzymatic hydrolysis, process improvements to increase the effective use of cellulase and hemicellulase enzymes, improvements in rates of ethanol production, decreased nutrient costs, increases in ethanol concentrations achieved in biomass beers, increased resistance of the biocatalysts to lignocellulosic-derived toxins, etc. To be useful, each of these improvements must result in a decrease in the cost for ethanol production. Copyright 1998 John Wiley & Sons, Inc.     

Thermophilic,lignocellulolytic bacteria for ethanol production: current state and perspectives

Lignocellulosic biomass contains a variety of carbohydrates, and their conversion into ethanol by fermentation requires an efficient microbial platform to achieve high yield, productivity, and final titer of ethanol. In recent years, growing attention has been devoted to the development of cellulolytic and saccharolytic thermophilic bacteria for lignocellulosic ethanol production because of their unique properties. First of all, thermophilic bacteria possess unique cellulolytic and hemicellulolytic systems and are considered as potential sources of highly active and thermostable enzymes for efficient biomass hydrolysis. Secondly, thermophilic bacteria ferment a broad range of carbohydrates into ethanol, and some of them display potential for ethanologenic fermentation at high yield. Thirdly, the establishment of the genetic tools for thermophilic bacteria has allowed metabolic engineering, in particular with emphasis on improving ethanol yield, and this facilitates their employment for ethanol production. Finally, different processes for second-generation ethanol production based on thermophilic bacteria have been proposed with the aim to achieve cost-competitive processes. However, thermophilic bacteria exhibit an inherent low tolerance to ethanol and inhibitors in the pretreated biomass, and this is at present the greatest barrier to their industrial application. Further improvement of the properties of thermophilic bacteria, together with the optimization production processes, is equally important for achieving a realistic industrial ethanol production.     

Solvent Production and Morphological Changes in Clostridium acetobutylicum

The morphological and cytological changes which occurred in Clostridium acetobutylicum P262 during the production of acetone, butanol, and ethanol in an industrial fermentation medium were identified and correlated with the growth and physiological changes. The swollen, cigar-shaped clostridial forms were involved in the conversion of acids to neutral solvents, and there was a correlation between the number of clostridial forms and the production of solvents. Sporulation mutants which were unable to form clostridial stages (cls mutants) did not produce solvents. Oligosporogenous mutants which showed reduced clostridial stage formation produced intermediate levels of solvents. Sporulation mutants blocked after the clostridial stage, which were unable to form mature spores (spo mutants), produced normal levels of solvents.     

Solventogenic-cellulolytic clostridia from 4-step-screening process in agricultural waste and cow intestinal tract

A clostridial bacterium is accepted to be one of the important and efficient microorganisms for the application in fuel fermentation process. However, the lack of cellulolytic activity of cellulosome in this organism appears to be one of the main important problems for efficient production of the fuel. It is therefore interesting to search for the genetic resource of natural clostridial bacteria for the application in bioengineering. Presently, Clostridium species selection and identification are based on various physiological properties tests. This article developed the way for a 4-step screening process via mainly three criteria and 16S rDNA identification. In this study, solvent-producing clostridial bacteria were successfully isolated from decomposed sources, cow feaces, and dry grass in Thailand. Anaerobes were screened by cellulolytic activity and butanol tolerance in selective media that composed of basal media supplemented with 2% cellulose and 5% butanol. Thirty isolates of cellulolytic and butanol-tolerant anaerobic bacteria were obtained from screening in this medium. Fifteen isolates were rapidly classified as in the class Clostridia by three selected criteria (endospore formation, sulfite-reducing ability, and metabolic products). Secondary metabolites of the bacteria such as acetone, butanol and ethanol were varied depending on the process. Clostridial differential medium was used as a genus identification tool. Finally, PCR-amplified gene fragments coding for 16S rDNA were analyzed as a key to identify bacteria species. This process can be used to screen and identify Clostridium species in short period. Cellulosome and non-cellulosome cellulases productivity were analyzed. The results revealed that the selected cellulolytic strains (such as Fea-PA) exhibited EngD non-cellulosome cellulase activity especially endoglucanase activity on carboxymethyl cellulose. The selective system in this research was appropriate for the screening of Clostridiaceae in a similarity range between 83% and 100%.     

Microbial production of ethanol from carbon monoxide

Production of ethanol from fermentation of CO has received much attention in the last few years with several companies proposing to use CO fermentation in their ethanol production processes. The genomes of two CO fermenters, Clostridium ljungdahlii and Clostridium carboxidivorans, have recently been sequenced. The genetic information obtained from this sequencing is aiding molecular biologists who are enhancing ethanol and butanol production by genetic manipulation. Several studies have optimized media for CO fermentation, which has resulted in enhanced ethanol production. Also, new reactor designs involving the use of hollow fiber membranes have reduced mass transfer barriers that have hampered previous CO fermentation efforts.     

Yeasts in sustainable bioethanol production: A review

Bioethanol has been identified as the mostly used biofuel worldwide since it significantly contributes to the reduction of crude oil consumption and environmental pollution. It can be produced from various types of feedstocks such as sucrose, starch, lignocellulosic and algal biomass through fermentation process by microorganisms. Compared to other types of microoganisms, yeasts especially Saccharomyces cerevisiae is the common microbes employed in ethanol production due to its high ethanol productivity, high ethanol tolerance and ability of fermenting wide range of sugars. However, there are some challenges in yeast fermentation which inhibit ethanol production such as high temperature, high ethanol concentration and the ability to ferment pentose sugars. Various types of yeast strains have been used in fermentation for ethanol production including hybrid, recombinant and wild-type yeasts. Yeasts can directly ferment simple sugars into ethanol while other type of feedstocks must be converted to fermentable sugars before it can be fermented to ethanol. The common processes involves in ethanol production are pretreatment, hydrolysis and fermentation. Production of bioethanol during fermentation depends on several factors such as temperature, sugar concentration, pH, fermentation time, agitation rate, and inoculum size. The efficiency and productivity of ethanol can be enhanced by immobilizing the yeast cells. This review highlights the different types of yeast strains, fermentation process, factors affecting bioethanol production and immobilization of yeasts for better bioethanol production.     

Engineering and Two-Stage Evolution of a Lignocellulosic Hydrolysate-Tolerant Saccharomyces cerevisiae Strain for Anaerobic Fermentation of Xylose from AFEX Pretreated Corn Stover

The inability of the yeast Saccharomyces cerevisiae to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of S. cerevisiae to convert xylose efficiently into ethanol in defined lab medium, few strains are able to ferment xylose from lignocellulosic hydrolysates in the absence of oxygen. This limited xylose conversion is believed to result from small molecules generated during biomass pretreatment and hydrolysis, which induce cellular stress and impair metabolism. Here, we describe the development of a xylose-fermenting S. cerevisiae strain with tolerance to a range of pretreated and hydrolyzed lignocellulose, including Ammonia Fiber Expansion (AFEX)-pretreated corn stover hydrolysate (ACSH). We genetically engineered a hydrolysate-resistant yeast strain with bacterial xylose isomerase and then applied two separate stages of aerobic and anaerobic directed evolution. The emergent S. cerevisiae strain rapidly converted xylose from lab medium and ACSH to ethanol under strict anaerobic conditions. Metabolomic, genetic and biochemical analyses suggested that a missense mutation in GRE3, which was acquired during the anaerobic evolution, contributed toward improved xylose conversion by reducing intracellular production of xylitol, an inhibitor of xylose isomerase. These results validate our combinatorial approach, which utilized phenotypic strain selection, rational engineering and directed evolution for the generation of a robust S. cerevisiae strain with the ability to ferment xylose anaerobically from ACSH.     

Enteric bacterial catalysts for fuel ethanol production.

The technology is available to produce fuel ethanol from renewable lignocellulosic biomass. The current challenge is to assemble the various process options into a commercial venture and begin the task of incremental improvement. Current process designs for lignocellulose are far more complex than grain to ethanol processes. This complexity results in part from the complexity of the substrate and the biological limitations of the catalyst. Our work at the University of Florida has focused primarily on the genetic engineering of Enteric bacteria using genes encoding Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase. These two genes have been assembled into a portable ethanol production cassette, the PET operon, and integrated into the chromosome of Escherichia coli B for use with hemicellulose-derived syrups. The resulting strain, KO11, produces ethanol efficiently from all hexose and pentose sugars present in the polymers of hemicellulose. By using the same approach, we integrated the PET operon into the chromosome of Klebsiella oxytoca to produce strain P2 for use in the simultaneous saccharification and fermentation (SSF) process for cellulose. Strain P2 has the native ability to ferment cellobiose and cellotriose, eliminating the need for one class of cellulase enzymes. Recently, the ability to produce and secrete high levels of endoglucanase has also been added to strain P2, further reducing the requirement for fungal cellulase. The general approach for the genetic engineering of new biocatalysts using the PET operon has been most successful with Enteric bacteria but was also extended to Gram positive bacteria, which have other useful traits for lignocellulose conversion. Many opportunities remain for further improvements in these biocatalysts as we proceed toward the development of single organisms that can be used for the efficient fermentation of both hemicellulosic and cellulosic substrates.     

Ethanol and acetate production by <Emphasis Type="Italic">Clostridium ljungdahlii</Emphasis> and <Emphasis Type="Italic">Clostridium autoethanogenum</Emphasis> using resting cells

Combined gasification and fermentation technologies can potentially produce biofuels from renewable biomass. Gasification generates synthesis gas consisting primarily of CO, CO₂, H₂, N₂, with smaller amounts of CH₄, NO_x, O₂, C₂ compounds, ash and tars. Several anaerobic bacteria species can ferment bottled mixtures of pure synthesis gas constituents. However, there are challenges to maintaining culture viability of synthesis gas exposed cells. This study was designed to enhance culture stability and improve ethanol-to-acetate ratios using resting (non-growing) cells in synthesis gas fermentation. Resting cell states were induced in autotrophic Clostridium ljungdahlii cultures with minimal ethanol and acetate production due to low metabolic activity compared to growing cell production levels of 5.2 and 40.1 mM of ethanol and acetate. Clostridium autoethanogenum cultures were not induced into true resting states but did show improvement in total ethanol production (from 5.1 mM in growing cultures to 9.4 in one nitrogen-limited medium) as well as increased shifts in ethanol-to-acetate production ratios.     

Efficient xylose fermentation by the brown rot fungus Neolentinus lepideus

The efficient production of bioethanol on an industrial scale requires the use of renewable lignocellulosic biomass as a starting material. A limiting factor in developing efficient processes is identifying microorganisms that are able to effectively ferment xylose, the major pentose sugar found in hemicellulose, and break down carbohydrate polymers without pre-treatment steps. Here, a basidiomycete brown rot fungus was isolated as a new biocatalyst with unprecedented fermentability, as it was capable of converting not only the 6-carbon sugars constituting cellulose, but also the major 5-carbon sugar xylose in hemicelluloses, to ethanol. The fungus was identified as Neolentinus lepideus and was capable of assimilating and fermenting xylose to ethanol in yields of 0.30, 0.33, and 0.34 g of ethanol per g of xylose consumed under aerobic, oxygen-limited, and anaerobic conditions, respectively. A small amount of xylitol was detected as the major by-product of xylose metabolism. N. lepideus produced ethanol from glucose, mannose, galactose, cellobiose, maltose, and lactose with yields ranging from 0.34 to 0.38 g ethanol per g sugar consumed, and also exhibited relatively favorable conversion of non-pretreated starch, xylan, and wheat bran. These results suggest that N. lepideus is a promising candidate for cost-effective and environmentally friendly ethanol production from lignocellulosic biomass. To our knowledge, this is the first report on efficient ethanol fermentation from various carbohydrates, including xylose, by a naturally occurring brown rot fungus.     

Genomics of clostridial pathogens: implication of extrachromosomal elements in pathogenicity

The recently decoded genomes of the major clostridial toxin-producing pathogens Clostridium perfringens, Clostridium tetani, Clostridium botulinum and Clostridium difficile have provided a huge amount of new sequence data. Recent studies have focused on the identification and investigation of pathogenic determinants and the regulatory events governing their expression. The sequence data revealed also the genomic background of virulence genes, as well as the contribution of extrachromosomal elements to a pathogenic phenotype. This has generated new insights in clostridial pathogenesis - and will continue to do so in the future - and has deepened our understanding of the anaerobic lifestyle of clostridial species.     

Citrate, a specific substrate for the isolation of Clostridium sphenoides.

With a medium containing citrate as the carbon and energy source, 10 clostridial strains were isolated from various mud samples. Characterization of these strains revealed that they all belonged to the same species, Clostridium sphenoides. Strains of this organism obtained from culture collections were also able to grow citrate, whereas 15 other clostridial species tested were not. Citrate was fermented by C. sphenoides to acetate, ethanol, carbon dioxide, and hydrogen. Experiments with stereospecifically 14C-labeled citrate indicated that citrate lyase was involved in citrate degradation.     

The enzymatic hydrolysis and fermentation of pretreated wood substrates

Aspenwood chips were pretreated by steam explosion. The various wood fractions obtained were assayed for their ability to act as substrates for growth and cellulase production of different Trichoderma and Clostridium thermocellum species. Steam exploded aspenwood was as efficiently utilized as solka floc and correspondingly high cellulase activities were detected in the various culture filtrates. When T. harzianum E58 was grown on increasing concentrations of solka floc, highest cellulase and xylanase activities were detected at 1% substrate concentrations while high substrate concentrations (10-20%) inhibited growth and enzyme production. When the cellulosic substrates were supplemented with increasing amounts of glucose, cellulase and xylanase production were inhibited when the glucose concentration exceeded 0.1%. Highest xylanase activities were detected after growth of T. reesei C30 and T. harianum E58 on xylan and solka floc respectively. All of the steam exploded fractions were at least partially hydrolyzed by the T. harzianum E58 cellulase system. The extent of the pretreatment also influenced the ability of Zymomonas mobilis and Saccharomyces cerevisiae to ferment the liberated sugars to ethanol. About 85% of the theoretical yield of ethanol from cellulose could be obtained from the combined hydrolysis and fermentation of pretreated aspenwood.     

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.     

The impacts of deacetylation prior to dilute acid pretreatment on the bioethanol process

Background

There is currently considerable interest in developing renewable sources of energy. One strategy is the biological conversion of plant biomass to liquid transportation fuel. Several technical hurdles impinge upon the economic feasibility of this strategy, including the development of energy crops amenable to facile deconstruction. Reliable assays to characterize feedstock quality are needed to measure the effects of pre-treatment and processing and of the plant and microbial genetic diversity that influence bioconversion efficiency.

Results

We used the anaerobic bacterium Clostridium phytofermentans to develop a robust assay for biomass digestibility and conversion to biofuels. The assay utilizes the ability of the microbe to convert biomass directly into ethanol with little or no pre-treatment. Plant samples were added to an anaerobic minimal medium and inoculated with C. phytofermentans, incubated for 3 days, after which the culture supernatant was analyzed for ethanol concentration. The assay detected significant differences in the supernatant ethanol from wild-type sorghum compared with brown midrib sorghum mutants previously shown to be highly digestible. Compositional analysis of the biomass before and after inoculation suggested that differences in xylan metabolism were partly responsible for the differences in ethanol yields. Additionally, we characterized the natural genetic variation for conversion efficiency in Brachypodium distachyon and shrub willow (Salix spp.).

Conclusion

Our results agree with those from previous studies of lignin mutants using enzymatic saccharification-based approaches. However, the use of C. phytofermentans takes into consideration specific organismal interactions, which will be crucial for simultaneous saccharification fermentation or consolidated bioprocessing. The ability to detect such phenotypic variation facilitates the genetic analysis of mechanisms underlying plant feedstock quality.     

Bioethanol production from steam-exploded rice husk by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> KO11

Rice husk is one of the most abundant types of lignocellulosic biomass. Because of its significant amount of sugars, such as cellulose and hemicellulose, it can be used for the production of biofuels such as bioethanol. However, the complex structure of lignocellulosic biomass, consisting of cellulose, hemicellulose and lignin, is resistant to degradation, which limits biomass utilization for ethanol production. The protection of cellulose by lignin contributes to the recalcitrance of lignocelluloses to hydrolysis. Therefore, we conducted steam-explosion treatment as pretreatment of rice husk. However, recombinant Escherichia coli KO11 did not ferment the reducing sugar solution obtained by enzymatic saccharification of steam-exploded rice husk. When the steam-exploded rice husk was washed with hot water to remove inhibitory substances and M9 medium (without glucose) was used as a fermentation medium, E. coli KO11 completely fermented the reducing sugar solution obtained by enzymatic saccharification of hot water washing-treated steam-exploded rice husk to ethanol. We report here the efficient production of bioethanol using steam-exploded rice husk.     

The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass

Lignocellulosic biomass is a renewable and abundant resource with great potential for bioconversion to value-added bioproducts. However, the biorefining process remains economically unfeasible due to a lack of biocatalysts that can overcome costly hurdles such as cooling from high temperature, pumping of oxygen/stirring, and, neutralization from acidic or basic pH. The extreme environmental resistance of bacteria permits screening and isolation of novel cellulases to help overcome these challenges. Rapid, efficient cellulase screening techniques, using cellulase assays and metagenomic libraries, are a must. Rare cellulases with activities on soluble and crystalline cellulose have been isolated from strains of Paenibacillus and Bacillus and shown to have high thermostability and/or activity over a wide pH spectrum. While novel cellulases from strains like Cellulomonas flavigena and Terendinibacter turnerae, produce multifunctional cellulases with broader substrate utilization. These enzymes offer a framework for enhancement of cellulases including: specific activity, thermalstability, or end-product inhibition. In addition, anaerobic bacteria like the clostridia offer potential due to species capable of producing compound multienzyme complexes called cellulosomes. Cellulosomes provide synergy and close proximity of enzymes to substrate, increasing activity towards crystalline cellulose. This has lead to the construction of designer cellulosomes enhanced for specific substrate activity. Furthermore, cellulosome-producing Clostridium thermocellum and its ability to ferment sugars to ethanol; its amenability to co-culture and, recent advances in genetic engineering, offer a promising future in biofuels. The exploitation of bacteria in the search for improved enzymes or strategies provides a means to upgrade feasibility for lignocellulosic biomass conversion, ultimately providing means to a ''greener'' technology.     

Development and application of co-culture for ethanol production by co-fermentation of glucose and xylose: a systematic review

This article reviews current co-culture systems for fermenting mixtures of glucose and xylose to ethanol. Thirty-five co-culture systems that ferment either synthetic glucose and xylose mixture or various biomass hydrolysates are examined. Strain combinations, fermentation modes and conditions, and fermentation performance for these co-culture systems are compared and discussed. It is noted that the combination of Pichia stipitis with Saccharomyces cerevisiae or its respiratory-deficient mutant is most commonly used. One of the best results for fermentation of glucose and xylose mixture is achieved by using co-culture of immobilized Zymomonas mobilis and free cells of P. stipitis, giving volumetric ethanol production of 1.277 g/l/h and ethanol yield of 0.49–0.50 g/g. The review discloses that, as a strategy for efficient conversion of glucose and xylose, co-culture fermentation for ethanol production from lignocellulosic biomass can increase ethanol yield and production rate, shorten fermentation time, and reduce process costs, and it is a promising technology although immature.     

Comparative Analysis of Carbohydrate Active Enzymes in Clostridium termitidis CT1112 Reveals Complex Carbohydrate Degradation Ability

Clostridium termitidis strain CT1112 is an anaerobic, gram positive, mesophilic, cellulolytic bacillus isolated from the gut of the wood-feeding termite, Nasutitermes lujae. It produces biofuels such as hydrogen and ethanol from cellulose, cellobiose, xylan, xylose, glucose, and other sugars, and therefore could be used for biofuel production from biomass through consolidated bioprocessing. The first step in the production of biofuel from biomass by microorganisms is the hydrolysis of complex carbohydrates present in biomass. This is achieved through the presence of a repertoire of secreted or complexed carbohydrate active enzymes (CAZymes), sometimes organized in an extracellular organelle called cellulosome. To assess the ability and understand the mechanism of polysaccharide hydrolysis in C. termitidis, the recently sequenced strain CT1112 of C. termitidis was analyzed for both CAZymes and cellulosomal components, and compared to other cellulolytic bacteria. A total of 355 CAZyme sequences were identified in C. termitidis, significantly higher than other Clostridial species. Of these, high numbers of glycoside hydrolases (199) and carbohydrate binding modules (95) were identified. The presence of a variety of CAZymes involved with polysaccharide utilization/degradation ability suggests hydrolysis potential for a wide range of polysaccharides. In addition, dockerin-bearing enzymes, cohesion domains and a cellulosomal gene cluster were identified, indicating the presence of potential cellulosome assembly.