Enhanced Biofuel Production Research Articles

Enhanced biofuel production through coupled acetic acid and xylose consumption by engineered yeast

The anticipation for substituting conventional fossil fuels with cellulosic biofuels is growing in the face of increasing demand for energy and rising concerns of greenhouse gas emissions. However, commercial production of cellulosic biofuel has been hampered by inefficient fermentation of xylose and the toxicity of acetic acid, which constitute substantial portions of cellulosic biomass. Here we use a redox balancing strategy to enable efficient xylose fermentation and simultaneous in situ detoxification of cellulosic feedstocks. By combining a nicotinamide adenine dinucleotide (NADH)-consuming acetate consumption pathway and an NADH-producing xylose utilization pathway, engineered yeast converts cellulosic sugars and toxic levels of acetate together into ethanol under anaerobic conditions. The results demonstrate a breakthrough in making efficient use of carbon compounds in cellulosic biomass and present an innovative strategy for metabolic engineering whereby an undesirable redox state can be exploited to drive desirable metabolic reactions, even improving productivity and yield.

Enhanced Biofuel Production from High-Concentration Bioethanol Wastewater by a Newly Isolated Heterotrophic Microalga, Chlorella vulgaris LAM-Q

Microalgal biofuel production from wastewater has economic and environmental advantages. This article investigates the lipid production from high chemical oxygen demand (COD) bioethanol wastewater without dilution or additional nutrients, using a newly isolated heterotrophic microalga, Chlorella vulgaris LAM-Q. To enhance lipid accumulation, the combined effects of important operational parameters were studied via response surface methodology. The optimal conditions were found to be temperature of 22.8℃, initial pH of 6.7, and inoculum density of 1.2 × 10(8) cells/ml. Under these conditions, the lipid productivity reached 195.96 mg/l/d, which was markedly higher than previously reported values in similar systems. According to the fatty acid composition, the obtained lipids were suitable feedstock for biodiesel production. Meanwhile, 61.40% of COD, 51.24% of total nitrogen, and 58.76% of total phosphorus were removed from the bioethanol wastewater during microalgal growth. In addition, 19.17% of the energy contained in the wastewater was transferred to the microalgal biomass in the fermentation process. These findings suggest that C. vulgaris LAMQ can efficiently produce lipids from high-concentration bioethanol wastewater, and simultaneously performs wastewater treatment.

Enhancing biofuel production by ultrasonics

This work evaluated the use of high-powered ultrasonics to enhance biofuel production in terms of efficiency and costs. A wide range of feed stocks, including switch grass, corn stover, and soft wood, were studied. The effect of ultrasonic pretreatment on the removal of lignin for hydrolysis of starches and cellulose to fermentable sugars was studied. It was found that many of the pretreatments were very successful in enhancing lignin removal. For example, time of dissolution of lingo-cellulosic biomass in ionic liquids was reduced from hours to minutes accompanied by a significant decrease in energy consumption compared to mechanical stirring. In addition, it was found that hydrolysis of corn starch could be greatly accelerated utilizing ultrasonics. Economic models showed that the technology, once implemented, would have a payback period of less than one year. The work also focused on biodiesel production. It was seen that ultrasonics accelerated the transesterification process so that soy bean oil could be converted to biodiesel in less than a minute, compared to 45 min using traditional methods. It was shown that yeast grown from glycerin, a co-product of biodiesel production, could be extracted and simultaneously converted to biodiesel with ultrasonics in less than a minute, compared to traditional techniques that require multiple processes and relatively long cycle times (+1 h).

Effect of fermentation inhibitors in the presence and absence of activated charcoal on the growth of Saccharomyces cerevisiae

The acidic hydrolysis of biomass generates numerous inhibitors of fermentation, which adversely affect cell growth and metabolism. The goal of the present study was to determine the effects of fermentation inhibitors on growth and glucose consumption by Saccharomyces cerevisiae. We also conducted in situ adsorption during cell cultivation in synthetic broth containing fermentation inhibitors. In order to evaluate the effect of in situ adsorption on cell growth, five inhibitors, namely 5-hydroxymethylfurfural, levulinic acid, furfural, formic acid, and acetic acid, were introduced into synthetic broth. The existence of fermentation inhibitors during cell culture adversely affects cell growth and sugar consumption. Furfural, formic acid, and acetic acid were the most potent inhibitors in our culture system. The in situ adsorption of inhibitors by the addition of activated charcoal to the synthetic broth increased cell growth and sugar consumption. Our results indicate that detoxification of fermentation media by in situ adsorption may be useful for enhancing biofuel production.

Quantitative Secretomic Analysis of Trichoderma reesei Strains Reveals Enzymatic Composition for Lignocellulosic Biomass Degradation

Trichoderma reesei is a mesophilic, filamentous fungus, and it is a major industrial source of cellulases, but its lignocellulolytic protein expressions on lignocellulosic biomass are poorly explored at present. The extracellular proteins secreted by T. reesei QM6a wild-type and hypercellulolytic mutant Rut C30 grown on natural lignocellulosic biomasses were explored using a quantitative proteomic approach with 8-plex high throughput isobaric tags for relative and absolute quantification (iTRAQ) and analyzed by liquid chromatography tandem mass spectrometry. We quantified 230 extracellular proteins, including cellulases, hemicellulases, lignin-degrading enzymes, proteases, protein-translocating transporter, and hypothetical proteins. Quantitative iTRAQ results suggested that the expressions and regulations of these lignocellulolytic proteins in the secretome of T. reesei wild-type and mutant Rut C30 were dependent on both nature and complexity of different lignocellulosic carbon sources. Therefore, we discuss here the essential lignocellulolytic proteins for designing an enzyme mixture for optimal lignocellulosic biomass hydrolysis.

Elucidating and reprogramming Escherichia coli metabolisms for obligate anaerobic n-butanol and isobutanol production

Elementary mode (EM) analysis based on the constraint-based metabolic network modeling was applied to elucidate and compare complex fermentative metabolisms of Escherichia coli for obligate anaerobic production of n-butanol and isobutanol. The result shows that the n-butanol fermentative metabolism was NADH-deficient, while the isobutanol fermentative metabolism was NADH redundant. E. coli could grow and produce n-butanol anaerobically as the sole fermentative product but not achieve the maximum theoretical n-butanol yield. In contrast, for the isobutanol fermentative metabolism, E. coli was required to couple with either ethanol- or succinate-producing pathway to recycle NADH. To overcome these "defective" metabolisms, EM analysis was implemented to reprogram the native fermentative metabolism of E. coli for optimized anaerobic production of n-butanol and isobutanol through multiple gene deletion (~8-9 genes), addition (~6-7 genes), up- and downexpression (~6-7 genes), and cofactor engineering (e.g., NADH, NADPH). The designed strains were forced to couple both growth and anaerobic production of n-butanol and isobutanol, which is a useful characteristic to enhance biofuel production and tolerance through metabolic pathway evolution. Even though the n-butanol and isobutanol fermentative metabolisms were quite different, the designed strains could be engineered to have identical metabolic flux distribution in "core" metabolic pathways mainly supporting cell growth and maintenance. Finally, the model prediction in elucidating and reprogramming the native fermentative metabolism of E. coli for obligate anaerobic production of n-butanol and isobutanol was validated with published experimental data.

ALGAE FOR BIOFUEL: WILL THE EVOLUTION OF WEEDS LIMIT THE ENTERPRISE?

Algae hold promise as a source of biofuel. Yet, the manner in which algae are most efficiently propagated and harvested is different from that used in traditional agriculture. In theory, algae can be grown in continuous culture and harvested frequently to maintain high yields with a short turnaround time. However, the maintenance of the population in a state of continuous growth will likely impose selection for fast growth, possibly opposing the maintenance of lipid stores desirable for fuel. Any harvesting that removes a subset of the population and leaves the survivors to establish the next generation may quickly select traits that escape harvesting. An understanding of these problems should help identify methods for retarding the evolution and enhancing biofuel production.

Engineering Genetic Control to Enhance Biofuel Production

Engineering Genetic Control to Enhance Biofuel Production

Engineering Genetic Control to Enhance Biofuel Production

Using fuels and chemicals derived from fossil sources underpins much of our everyday life and industrialized economy. However, obtaining carbon from oil and gas presents a number of environmental, economic, and political challenges. One solution to these challenges is to derive fuels and chemicals from renewable sources of carbon such as biomass or atmospheric carbon dioxide, thereby closing the carbon loop and moving towards a carbon neutral economy. Biological systems have evolved to harness carbon and energy from the environment for their own survival and can be engineered to produce fuels and chemicals. Although much progress has been made in this area, we present several challenges and areas of opportunity for development. We suggest that the primary barrier to large-scale commercialization of bioderived fuels and chemicals is the availability of feedstocks, which makes the conversion yield (feedstocks to fuels) a critical parameter in determining the impact and sustainability of biofuels processes. Engineering fuel-producing organisms to more efficiently capture carbon and energy, route metabolic fluxes to desired products, and tolerate industrial production conditions is a significant opportunity to increase the yields and economics of biofuels.

In Silico Structural Determination of GPAT Enzyme from Ostreococcus Lucimarinus for Biotechnological Application of Microalgal Biofuel Production

ABSTRACTGlycerol-3-phosphate acyltransferase (GPAT) is an enzyme in the triacylglycerol (TAG) biosynthetic pathway that catalyses the conversion of glycerol-3-phosphate to lysophosphatidic acid. Targeting key enzymes involved in TAG pathway is considered to be a powerful strategy for augmented lipid accumulation in microorganisms. In the present study three-dimensional structure of the marine microalgae, Ostreococcus lucimarinus GPAT protein was developed based on the crystal structure of Cucurbita moschata GPAT protein. Besides, several structure validation tools were employed to confirm the reliability of the developed model. The predicted and validated model reveals the tertiary structure of GPAT monomer comprising of two domains, the smaller domain I, which folds into a four helix bundle, and the larger domain II, which is constructed from alternating α/β secondary structural elements that give rise to 9-stranded β sheet flanked by 11α helices. Critical structural analysis of the developed model reveals the presence of H(X) 4D motif; the latter being, a consensus sequence conserved amongst many glycerolipid acyltransferase. The detected cluster of positively charged residues H189, K243, H244, R285 and R287 in the model could be conjectured to be important in glycerol-3-phosphate recognition. The structural insight obtained from this in silico study may provide useful clues to further advanced biotechnological studies of strategic site-specific genetic and metabolic engineering of microalgae for enhanced biofuel production.

Comparative genomics of xylose-fermenting fungi for enhanced biofuel production

Cellulosic biomass is an abundant and underused substrate for biofuel production. The inability of many microbes to metabolize the pentose sugars abundant within hemicellulose creates specific challenges for microbial biofuel production from cellulosic material. Although engineered strains of Saccharomyces cerevisiae can use the pentose xylose, the fermentative capacity pales in comparison with glucose, limiting the economic feasibility of industrial fermentations. To better understand xylose utilization for subsequent microbial engineering, we sequenced the genomes of two xylose-fermenting, beetle-associated fungi, Spathaspora passalidarum and Candida tenuis. To identify genes involved in xylose metabolism, we applied a comparative genomic approach across 14 Ascomycete genomes, mapping phenotypes and genotypes onto the fungal phylogeny, and measured genomic expression across five Hemiascomycete species with different xylose-consumption phenotypes. This approach implicated many genes and processes involved in xylose assimilation. Several of these genes significantly improved xylose utilization when engineered into S. cerevisiae, demonstrating the power of comparative methods in rapidly identifying genes for biomass conversion while reflecting on fungal ecology.

Genetic engineering of algae for enhanced biofuel production.

There are currently intensive global research efforts aimed at increasing and modifying the accumulation of lipids, alcohols, hydrocarbons, polysaccharides, and other energy storage compounds in photosynthetic organisms, yeast, and bacteria through genetic engineering. Many improvements have been realized, including increased lipid and carbohydrate production, improved H(2) yields, and the diversion of central metabolic intermediates into fungible biofuels. Photosynthetic microorganisms are attracting considerable interest within these efforts due to their relatively high photosynthetic conversion efficiencies, diverse metabolic capabilities, superior growth rates, and ability to store or secrete energy-rich hydrocarbons. Relative to cyanobacteria, eukaryotic microalgae possess several unique metabolic attributes of relevance to biofuel production, including the accumulation of significant quantities of triacylglycerol; the synthesis of storage starch (amylopectin and amylose), which is similar to that found in higher plants; and the ability to efficiently couple photosynthetic electron transport to H(2) production. Although the application of genetic engineering to improve energy production phenotypes in eukaryotic microalgae is in its infancy, significant advances in the development of genetic manipulation tools have recently been achieved with microalgal model systems and are being used to manipulate central carbon metabolism in these organisms. It is likely that many of these advances can be extended to industrially relevant organisms. This review is focused on potential avenues of genetic engineering that may be undertaken in order to improve microalgae as a biofuel platform for the production of biohydrogen, starch-derived alcohols, diesel fuel surrogates, and/or alkanes.