High Yield Ethanol Production Research Articles

Key role of K+ and Ca2+ in high-yield ethanol production by S. Cerevisiae from concentrated sugarcane molasses

BackgroundSaccharomyces cerevisiae is an important microorganism in ethanol synthesis, and with sugarcane molasses as the feedstock, ethanol is being synthesized sustainably to meet growing demands. However, high-concentration ethanol fermentation based on high-concentration sugarcane molasses—which is needed for reduced energy consumption of ethanol distillation at industrial scale—is yet to be achieved.ResultsIn the present study, to identify the main limiting factors of this process, adaptive laboratory evolution and high-throughput screening (Py-Fe3+) based on ARTP (atmospheric and room-temperature plasma) mutagenesis were applied. We identified high osmotic pressure, high temperature, high alcohol levels, and high concentrations of K+, Ca2+, K+ and Ca2+ (K+&Ca2+), and sugarcane molasses as the main limiting factors. The robust S. cerevisiae strains of NGT-F1, NGW-F1, NGC-F1, NGK+, NGCa2+ NGK+&Ca2+-F1, and NGTM-F1 exhibited high tolerance to the respective limiting factor and exhibited increased yield. Subsequently, ethanol synthesis, cell morphology, comparative genomics, and gene ontology (GO) enrichment analysis were performed in a molasses broth containing 250 g/L total fermentable sugars (TFS). Additionally, S. cerevisiae NGTM-F1 was used with 250 g/L (TFS) sugarcane molasses to synthesize ethanol in a 5-L fermenter, giving a yield of 111.65 g/L, the conversion of sugar to alcohol reached 95.53%. It is the highest level of physical mutagenesis yield at present.ConclusionOur results showed that K+ and Ca2+ ions primarily limited the efficient production of ethanol. Then, subsequent comparative transcriptomic GO and pathway analyses showed that the co-presence of K+ and Ca2+ exerted the most prominent limitation on efficient ethanol production. The results of this study might prove useful by promoting the development and utilization of green fuel bio-manufactured from molasses.

Enhancement of bioethanol production from Moso bamboo pretreated with biodiesel crude glycerol: Substrate digestibility, cellulase absorption and fermentability

Utilization of sustainable energy is limited by energy requirement for the manufacturing of renewable fuels. Moso bamboo was pretreated with industrially derived crude glycerol obtained from different sources at 150/160 °C for 3 h. This bamboo, pretreated with base biodiesel glycerol with pressure filtration removal method, showed a high glucose yield of 94.95% and an ethanol yield of 73.10% of the theoretical. Major glycerol content was removed by pressure filtration, leaving a small amount of fatty acid soap in the pretreated sample, which formed an emulsion that reduced lignin redisposition onto the biomass surface and effectively blocked lignin absorption of cellulase, allowing greater enzymatic hydrolysis and fermentation system function. The surface was more hydrophilic and a higher lignin removal was achieved: 39.24% with base biodiesel glycerol pretreatment compared to 26.08% with sodium hydroxide glycerol pretreatment. This study provides a useful and cost-effective process, BBGP, for high-yield ethanol production.

Expressing the Thermoanaerobacterium saccharolyticum pforA in engineered Clostridium thermocellum improves ethanol production

BackgroundClostridium thermocellum has been the subject of multiple metabolic engineering strategies to improve its ability to ferment cellulose to ethanol, with varying degrees of success. For ethanol production in C. thermocellum, the conversion of pyruvate to acetyl-CoA is catalyzed primarily by the pyruvate ferredoxin oxidoreductase (PFOR) pathway. Thermoanaerobacterium saccharolyticum, which was previously engineered to produce ethanol of high yield (> 80%) and titer (70 g/L), also uses a pyruvate ferredoxin oxidoreductase, pforA, for ethanol production.ResultsHere, we introduced the T. saccharolyticum pforA and ferredoxin into C. thermocellum. The introduction of pforA resulted in significant improvements to ethanol yield and titer in C. thermocellum grown on 50 g/L of cellobiose, but only when four other T. saccharolyticum genes (adhA, nfnA, nfnB, and adhEG544D) were also present. T. saccharolyticum ferredoxin did not have any observable impact on ethanol production. The improvement to ethanol production was sustained even when all annotated native C. thermocellum pfor genes were deleted. On high cellulose concentrations, the maximum ethanol titer achieved by this engineered C. thermocellum strain from 100 g/L Avicel was 25 g/L, compared to 22 g/L for the reference strain, LL1319 (adhA(Tsc)-nfnAB(Tsc)-adhEG544D (Tsc)) under similar conditions. In addition, we also observed that deletion of the C. thermocellum pfor4 results in a significant decrease in isobutanol production.ConclusionsHere, we demonstrate that the pforA gene can improve ethanol production in C. thermocellum as part of the T. saccharolyticum pyruvate-to-ethanol pathway. In our previous strain, high-yield (~ 75% of theoretical) ethanol production could be achieved with at most 20 g/L substrate. In this strain, high-yield ethanol production can be achieved up to 50 g/L substrate. Furthermore, the introduction of pforA increased the maximum titer by 14%.

Fed-batch Saccharomyces cerevisiae fermentation of hydrolysate sugars: A dynamic model-based approach for high yield ethanol production

The efficient fermentation of hydrolyzed sugars from lignocellulosic biomass feedstock to ethanol remains a complex multi-parametric problem. Thus, in the present study, an advanced structured dynamic model for the simulation of the fermentative ethanol production from hydrolysate sugars is developed. The model is combined with a statistical experimental design to determine an optimal operating strategy that maximizes ethanol production and serves for the systematic evaluation of critical process variables. In particular, the effects of various operating conditions and feeding strategies on the dynamic behavior of batch and fed-batch fermentation processes are explored. The deviation from the desired product or the metabolic inhibition of ethanol production are related with the applied environmental conditions and substrate and product inhibition phenomena. The operating strategy, designed with the assistance of the mathematical tools proposed in this study, includes an exponential addition policy of substrate. This strategy is experimentally proved to enhance the final product concentration, raising the ethanol productivity to 2.27 g L−1 h−1 and the ethanol yield to 53.5% of the maximum theoretical value. Moreover, the simulated strategies were in excellent agreement with the experimental results obtained from the real process using low and high glucose initial concentration, under batch and fed-batch conditions, in both flask- and bioreactor-scale cultivations, proving the model's predictive and optimization capabilities. Further improvement of process performance is expected when combining the proposed dynamic model with advanced optimization algorithms to derive the optimal bioprocess operating strategy.

Enhanced characteristics of genetically modified switchgrass (Panicum virgatum L.) for high biofuel production

BackgroundLignocellulosic biomass is one of the most promising renewable and clean energy resources to reduce greenhouse gas emissions and dependence on fossil fuels. However, the resistance to accessibility of sugars embedded in plant cell walls (so-called recalcitrance) is a major barrier to economically viable cellulosic ethanol production. A recent report from the US National Academy of Sciences indicated that, “absent technological breakthroughs”, it was unlikely that the US would meet the congressionally mandated renewable fuel standard of 35 billion gallons of ethanol-equivalent biofuels plus 1 billion gallons of biodiesel by 2022. We here describe the properties of switchgrass (Panicum virgatum) biomass that has been genetically engineered to increase the cellulosic ethanol yield by more than 2-fold.ResultsWe have increased the cellulosic ethanol yield from switchgrass by 2.6-fold through overexpression of the transcription factor PvMYB4. This strategy reduces carbon deposition into lignin and phenolic fermentation inhibitors while maintaining the availability of potentially fermentable soluble sugars and pectic polysaccharides. Detailed biomass characterization analyses revealed that the levels and nature of phenolic acids embedded in the cell-wall, the lignin content and polymer size, lignin internal linkage levels, linkages between lignin and xylans/pectins, and levels of wall-bound fucose are all altered in PvMYB4-OX lines. Genetically engineered PvMYB4-OX switchgrass therefore provides a novel system for further understanding cell wall recalcitrance.ConclusionsOur results have demonstrated that overexpression of PvMYB4, a general transcriptional repressor of the phenylpropanoid/lignin biosynthesis pathway, can lead to very high yield ethanol production through dramatic reduction of recalcitrance. MYB4-OX switchgrass is an excellent model system for understanding recalcitrance, and provides new germplasm for developing switchgrass cultivars as biomass feedstocks for biofuel production.

Single-step ethanol production from lignocellulose using novel extremely thermophilic bacteria

BackgroundConsolidated bioprocessing (CBP) of lignocellulosic biomass to ethanol using thermophilic bacteria provides a promising solution for efficient lignocellulose conversion without the need for additional cellulolytic enzymes. Most studies on the thermophilic CBP concentrate on co-cultivation of the thermophilic cellulolytic bacterium Clostridium thermocellum with non-cellulolytic thermophilic anaerobes at temperatures of 55°C-60°C.ResultsWe have specifically screened for cellulolytic bacteria growing at temperatures >70°C to enable direct conversion of lignocellulosic materials into ethanol. Seven new strains of extremely thermophilic anaerobic cellulolytic bacteria of the genus Caldicellulosiruptor and eight new strains of extremely thermophilic xylanolytic/saccharolytic bacteria of the genus Thermoanaerobacter isolated from environmental samples exhibited fast growth at 72°C, extensive lignocellulose degradation and high yield ethanol production on cellulose and pretreated lignocellulosic biomass. Monocultures of Caldicellulosiruptor strains degraded up to 89-97% of the cellulose and hemicellulose polymers in pretreated biomass and produced up to 72 mM ethanol on cellulose without addition of exogenous enzymes. In dual co-cultures of Caldicellulosiruptor strains with Thermoanaerobacter strains the ethanol concentrations rose 2- to 8.2-fold compared to cellulolytic monocultures. A co-culture of Caldicellulosiruptor DIB 087C and Thermoanaerobacter DIB 097X was particularly effective in the conversion of cellulose to ethanol, ethanol comprising 34.8 mol% of the total organic products. In contrast, a co-culture of Caldicellulosiruptor saccharolyticus DSM 8903 and Thermoanaerobacter mathranii subsp. mathranii DSM 11426 produced only low amounts of ethanol.ConclusionsThe newly discovered Caldicellulosiruptor sp. strain DIB 004C was capable of producing unexpectedly large amounts of ethanol from lignocellulose in fermentors. The established co-cultures of new Caldicellulosiruptor strains with new Thermoanaerobacter strains underline the importance of using specific strain combinations for high ethanol yields. These co-cultures provide an efficient CBP pathway for ethanol production and represent an ideal starting point for development of a highly integrated commercial ethanol production process.

Cell recycle batch fermentation of high-solid lignocellulose using a recombinant cellulase-displaying yeast strain for high yield ethanol production in consolidated bioprocessing

The aim of this study is to develop a scheme of cell recycle batch fermentation (CRBF) of high-solid lignocellulosic materials. Two-phase separation consisting of rough removal of lignocellulosic residues by low-speed centrifugation and solid–liquid separation enabled effective collection of Saccharomyces cerevisiae cells with decreased lignin and ash. Five consecutive batch fermentation of 200g/L rice straw hydrothermally pretreated led to an average ethanol titer of 34.5g/L. Moreover, the display of cellulases on the recombinant yeast cell surface increased ethanol titer to 42.2g/L. After, five-cycle fermentation, only 3.3g/L sugar was retained in the fermentation medium, because cellulase displayed on the cell surface hydrolyzed cellulose that was not hydrolyzed by commercial cellulases or free secreted cellulases. Fermentation ability of the recombinant strain was successfully kept during a five-cycle repeated batch fermentation with 86.3% of theoretical yield based on starting biomass.

Display of cellulases on the cell surface of Saccharomyces cerevisiae for high yield ethanol production from high-solid lignocellulosic biomass

Economically feasible processes for industrial cellulosic ethanol production requires increasing the final ethanol titer during fermentation due to the high energy demands of the subsequent ethanol distillation. In the present study, high-yield ethanol production was achieved by short-term liquefaction and fermentation of lignocellulose biomass in a novel drum-type rotary fermentation system using a yeast strain developed for cell-surface display of fungal endoglucanase, cellobiohydrolase, and β-glucosidase. In the presence of 10FPU/g-biomass cellulase added, the recombinant cellulolytic strain produced 1.4-fold higher ethanol (89% of theoretical yield) from high-solid (200g-dry weight/L) rice straw within 72h of fermentation than wild type strain. Cell-surface engineering successfully reduced the amount of commercial enzyme required for the fermentation of cellulose. This study demonstrates that cellulases displayed on the yeast cell surface are capable of hydrolyzing cellulose that was not hydrolyzed by commercial cellulases, leading to increased sugar utilization for improved ethanol production.

Bioenergy II: Comparison of Laboratory and Industrial Saccharomyces cerevisiae Strains for their Stress Tolerance

There are various kinds of stresses during the process of ethanol fermentation and more inhibitory factors are produced when lignocelluloses hydrolysate is used as the substrate. The pretreatment of lignocelluloses biomass causes the increase in the amount of acids and thus the decrease in pH. Low-molecular weight aliphatic acids, furaldehydes and a broad range of aromatic compounds are produced during the pretreatment process. They are the inhibitors for the ethanol producers, e.g. Saccharomyces cerevisiae. Furthermore, besides glucose, lignocellulose hydrolysate contains other sugars, such as xylose, arabinose, galactose and mannose etc., among which xylose is taking the major proportion. Stress tolerance and xylose utilization are therefore essential for Saccharomyces cerevisiae strains to get high-efficiency fermentation and high-yield ethanol production from lignocellulosic materials. In this study, a few laboratory and industrial Saccharomyces cerevisiae strains were selected for the evaluation of their potentials in pH tolerance, inhibitor resistance, temperature and ethanol tolerance, and xylose resistance. The results revealed that strains ATCC 96581 and ATCC 24860 are the most stress tolerant S. cerevisiae strains for ethanol fermentation. Such strains will be improved for inhibitor resistance and xylose utilization in the future by metabolic engineering and directed evolution.

Metabolic engineering of Geobacillus thermoglucosidasius for high yield ethanol production

We describe the metabolic engineering of two strains of Geobacillus thermoglucosidasius to divert their fermentative carbon flux from a mixed acid pathway, to one in which ethanol becomes the major product. This involved elimination of the lactate dehydrogenase and pyruvate formate lyase pathways by disruption of the ldh and pflB genes, respectively, together with upregulation of expression of pyruvate dehydrogenase. Unlike the situation in Escherichia coli, pyruvate dehydrogenase is active under anaerobic conditions in thermophilic bacilli, but expressed sub-optimally for a role as the primary fermentation pathway. Mutants were initially characterised in batch culture using glucose as carbon substrate and strains with all three modifications shown to form ethanol efficiently and rapidly at temperatures in excess of 60 °C in yields in excess of 90% of theoretical. The strain containing the 3 modifications, TM242, was also shown to efficiently ferment cellobiose and a mixed hexose and pentose feed.

Sessions 2 and 7: Advances in Microbial Science and Technology

The Microbial Science and Technology Sessions emphasized a variety of approaches used for development of superior recombinant microorganisms for fermentation of multiple sugars typically present in any lignocellulosic hydrolyzate to fuels and chemicals. Efficient conversion of lignocellulosic biomass to liquid transportation fuels is a major goal for many in these sessions. The Session 2 contained six oral presentations and 62 poster presentations and Session 7 contained an additional six oral presentations and 49 posters. A brief overview of each oral Session and a summary of topics presented during the poster sessions are provided below. Session 2 opened with a comparison of different pretreatments for corn stover, followed by fermentation using different recombinant organisms. Next, the metabolic engineering of a novel thermophilic Geobacillus sp. capable of high-yield ethanol production was discussed. The pathway for D-galacturonic acid catabolism in fungi was elucidated in the third presentation and determined to be different from any previously described pathway. Butanol production from engineered Escherichia coli and improvements in solventogenic Clostridium butanol production were the topics for another presentation. Metabolic pathway engineering of Thermoanaerobacterium saccharolyticum for efficient ethanol production and reduction of cellulase loading emphasized a consolidated bioprocessing approach. New methods for isolating novel biocatalysts and enzymes from lignocellulosedegrading insects rounded out this first session. Appl Biochem Biotechnol (2009) 153:11–12 DOI 10.1007/s12010-009-8609-9

The fermentability of concentrated softwood-derived hemicellulose fractions with and without supplemental cellulose hydrolysates

The hemicellulose-rich component recovered following the steam explosion of softwood residues typically contain very dilute sugar concentrations, which can impact the overall economics of the bioconversion process. In an attempt to address this concern, the water-soluble fractions derived from steam exploded Douglas-fir whitewood and whitewood/bark chips were subjected to a concentration step (rotary-evaporation or freeze-drying) prior to fermentation. The fermentability of the resultant streams was largely dependent on the accumulation of inhibitory material, with poor fermentation of the whitewood concentrates at as low as a twofold increase in concentration. The bark-containing concentrates were slightly easier to ferment, but above a 2.5-fold concentration, a significant “lag” in fermentation was observed. Concurrently, the water-soluble fractions were supplemented with the glucose-rich cellulose hydrolysate obtained after enzymatic hydrolysis of the water-insoluble, cellulose component. Supplementation of the original water-soluble fraction with the readily-fermentable cellulose hydrolysate resulted in significantly improved fermentation. Using this approach, it was possible to increase the hexose sugar concentration in the water-soluble fraction by more than 50%, while still achieving high yield ethanol production.

High-yield ethanol production from Jerusalem artichoke tubers

Fermentation conditions were optimized for the production of ethanol from Jerusalem artichoke with a strain of Saccharomyces cerevisiae able to use high-concentration juice and undiluted pulp. Yields (95 to 125 g ethanol/l=85 to 98% of the theoretical value) exceeded those obtained with strain of Kluyveromyces used classically.

SO2 prehydrolysis for high yield ethanol production from biomass

The advantages of the use of SO2 in steam pretreatment are described. Two different large scale continuous reactors, the Stake and the Wenger, have been used for this purpose. Pine, aspen and corn stover were prehydrolysed by SO2 in these reactors and hydrolysed by enzymes. The solution of hexoses and pentoses so obtained were fermented byPichia stipitis R, yielding 372, 346 and 388 L ethanol/tonne for the 3 feedstocks, respectively. When a mixed culture ofP. stipitis R, which is an excellent pentose fermenter, andBrettanomyces clausenii which is an excellent cellobiose fermenter, was used in a simultaneous saccharification-fermentation made with SO2-prehydrolysed aspen, the yield rose to 384 L/tonne. These are higher yields than have been reported in the literature to date.

Ethanol production from pineapple juice in Côte d'Ivoire with preselected yeast strains

Fresh pineapple juice was inoculated with 8 preselected yeast strains and incubated for high yield ethanol production at between 25°C and 35°C. The natural pH of the juice did not affect the growth of the strains or the alcohol yields. The higher ethanol concentrations were obtained at 30°C and 35°C in the ranges of 27.17 to 46.50 g/ l and 31.38 to 54.65 g/ l, respectively, with 82% and 89% as the highest yields when referring to the theoretical yield. The study indicated that strain CMI is the best performing strain at 30°C and 35°C according to yield and productivity.