| Bioenergy from lignocellulose offers a great clean sustainable alternative toconventional petroleum-based energy sources which can dramatically impactnational economic growth, national energy security and environmental quality.However, the development of lignocellulose-derived bioenergy is still in its infancy.One of the great challenges is to depolymerize plant materials and release highquality, high quantity sugars for biofuel production. New microbial catalysts foreffectively disrupting plant polymers and efficiently releasing sugars are urgentlyneeded. Therefore, the objective of this study is to obtain and characterize novelfungal strains capable of effectively degrading plant polymers and efficientlyproducing sugars for biofuel production.A novel Shigella strain （Shigella flexneri str. G3） showing high cellulolyticactivity under mesophilic, anaerobic conditions was characterized. The bacteriumdisplays effective production of glucose, cellobiose and other oligosaccharides fromcellulose (Avicel PH-101) at the optimal conditions of40oC and pH6.5.Approximately75%of cellulose was hydrolyzed in the modified ATCC1191medium containing0.3%cellulose, and the oligosaccharide production yield andspecific production rate reached375mg g-1Avicel and6.25mg g-1Avicel h-1after a60-hour incubation, respectively. To our knowledge, this represents the highestoligosaccharide yield and specific rate from cellulose for mesophilic bacterialmonocultures reported so far. The results demonstrate that S. flexneri G3iscapable of rapid conversion of cellulose to oligosaccharides, with potential biofuelapplications under mesophilic conditions.We have obtained a novel fungal strain, Penicillium sp. YT02, which is capableof effectively depolymerizing plant materials such as alfalfa，switchgrass, cornstover and wheat straw. The isolated strain can efficiently produce sugars from plantmaterials with the highest realized theoretical sugar yields reported, and is able toproduce30%more sugars than the control fungus, Trichoderma reesei. The averageefficient of saccharificaiton from varied lignocellulosic substrates was amount to0.665g g-1substrates, the highest oligosaccharides producity from raw fungalenzyme. An novel bio-delignin approach was proposed that in the presence of low C: Nbiomass, such as alfalfa, an essential nutrient source for high-lignin biomass （high C:N）, may improve the decomposition of the high-lignin materials for furtherbioenergy process. In this study, microbial pretreatment with alfalfa woodybiomass mixture system by white-rot P. chrysosprium was able to degrade lignin inhard and soft wood materials and has the potential to be an energy-saving, low cost,simple, two lignocellulosic biomass process simultaneously, and environmentfriendly approach which can reduce the severity of chemical pretreatments. Abalance between lignin degradation and availability of carbohydrates indicates that50g L-1of raw oak biomass and10g L-1of alfalfa and50g L-1of raw pine and15gL-1of alfalfa were the most promising pretreatment, which improve thesaccharification ratio to30%compared with traditional pretreatment methods.Bioaugmented fermentation of cellulosic substrates to produce biohydrogenvia co-culture of isolated strains Shigella flexneri str. G3&Clostridiumacetobutylicum X9was investigated. The ability of the selected strains to effectivelyconvert different cellulosic substrates to hydrogen was tested on carboxymethylcellulose （AVICEL）, as well as pretreated lignocellulosic material such as Bermudagrass, corn stover, rice straw, and corn cob. Results showed that co-culture ofShigella flexneri str G3and Clostridium acetobutylicum X9efficiently improvedcellulose hydrolysis and subsequent hydrogen production from carboxymethylcellulose. Hydrogen production yield approximately reached1.3mol H2（mol glucose）1, compared to0.32mol H2（mol glucose）1of the X9single culture,while the cellulose degradation efficiency increased by50%. Co-culture alsoefficiently improved hydrogen production from natural lignocellulosic materials（which was even70%-80%higher than mono-culture with X9）, and the highestperformance of24.8mmol L-1was obtained on Bermuda grass. The resultsdemonstrate that co-culture of S. flexneri&G3&C. acetobutylicum X9was capableto efficiently enhance cellulose conversion to hydrogen, thus fostering potentialbiofuel applications under mesophilic conditions.In addition, the produced sugars were further examined for producing ethanolcoupled with a yeast strain. In this study, with alfalfa and other lignocellulosicsubstrates （except oak tree）, the ethanol yields ranged from0.11to0.21g ethanol gbiomass-1after24hrs of fermentation, which corresponds to88.2%of the maximal theoretical value. To the best of our knowledge, this could be the highest efficiencyof ethanol production reported for monocultures of yeast with lignocellulosichydrolysate. These results suggested that the novel fungal isolate, YT02, can serveas an effective microbial catalyst for cellulosic ethanol production, and could be agood source of new enzymes for various industrial applications.In order to understand the mechanism that why YT02could produce highactivity enzymes, we sequenced its whole genome sequence and researched itsgenes expression profile of YT02. Consistent with the high saccharificationefficiency, the genome of YT02encodes more carbohydrate-active enzymes（CAZymes） than T. reesei. Less gene numbers encoding HK, PFK-1and PK in EMPpathway and The absence of endocing genes XK in predicted xylose utilizationpathway of YT02was the main reaon why oligosaccharides were “accumulated”during lignocellulosic saccharificaiton. RNA-seq data revealed the abundance ofxylanase and their active expression might attribute to the significantly higherxylanase activity of YT02than T. reesei. The expression of CAZymes genes wassignificantly induced by lingocellulose. Our analysis, coupled with the genomesequence data and RNA-seq data, provides a roadmap for further developingenhanced P. expansum strains for industrial applications such as biofuel production. |
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