Consolidated bioprocessing and simultaneous saccharification and fermentation of lignocellulose to ethanol with thermotolerant yeast strains

Consolidated bioprocessing (CBP), which integrates enzyme production, saccharification and fermentation into a single process, is a promising strategy for effective ethanol production from lignocellulosic materials because of the resulting reduction in utilities, the substrate and other raw materials and simplification of operation. CBP requires a highly engineered microbial strain capable of hydrolyzing biomass with enzymes produced on its own and producing high-titer ethanol. Recently, heterologous production of cellulolytic enzymes has been pursued with yeast hosts, which has realized direct conversion of cellulose to ethanol. Specifically, the development of cell surface engineering, which provides a display of cellulolytic enzymes on the yeast cell surface, facilitates effective biomass hydrolysis concomitantly with ethanol production. On the other hand, the difference in optimum temperature between saccharification and fermentation is a drawback of efficient ethanol production in the simultaneous saccharification and fermentation (SSF). The application of thermotolerant yeast strains engineered to the SSF process would overcome the drawback by performing hydrolysis and fermentation at elevated temperature. In this review, we focus on the recent advances in the application of thermotolerant yeast to CBP and SSF of lignocellulosic material to ethanol. The development of thermotolerant and ethanologenic yeast strains with the ability to hydrolyze lignocellulosic materials is emphasized for high-temperature CBP.     

兼性厌氧复合菌群H纤维素降解和产乙醇能力及生态组成初探

通过限制性培养条件和连续继代培养,筛选获得了一组具有高效稳定降解纤维素能力的复合菌群H。该菌群在传代30代以上仍能保持各项性状稳定,其工作pH为6～9,3 d可以完全降解置于100 mL PCS缓冲液培养基中的滤纸,发酵液中能够检出1.54 g/L乙醇。通过16S rDNA扩增和DGGE的方法,对菌群在不同阶段的微生物组成进行了研究,确定了琥珀酸嗜热梭菌Clostridium thermo succinogene、产气荚膜梭菌Clostridium straminisolvens和紫色板蓝根梭菌Clostridium isatidis等多种可直接实现纤维素到乙醇转化的菌株。菌群通过菌种之间的协同作用,共同维持了体系的稳定及降解能力的稳定。明确菌系的组成,对于进一步研究菌群降解机理、优化菌群和提高乙醇产率意义重大。     

Engineering microbes for direct fermentation of cellulose to bioethanol

Consolidated bioprocessing (CBP) by micro-organisms is desired for efficient conversion of lignocellulosic biomass to bioethanol fuels. Potential candidates have been discovered, including cellulolytic bacteria and filamentous fungi. Genetic and metabolic manipulation of these organisms further promotes their fermentation capacities and the ethanol tolerance. In addition, Saccharomyces cerevisiae and several other yeasts were genetically modified to express recombinant cellulases in media or display them on the cell surface for CBP of cellulose. To compensate the insufficient capacity of a single strain, various microbial consortia have also been developed. In this article, we reviewed the recent advances in CBP microbes and focused on the efforts in strain improvement employing genetic engineering.     

Consolidated bioprocessing strategy for ethanol production from Jerusalem artichoke tubers by Kluyveromyces marxianus under high gravity conditions

Aims: Developing an innovative process for ethanol fermentation from Jerusalem artichoke tubers under very high gravity (VHG) conditions. Methods and Results: A consolidated bioprocessing (CBP) strategy that integrated inulinase production, saccharification of inulin contained in Jerusalem artichoke tubers and ethanol production from sugars released from inulin by the enzyme was developed with the inulinase‐producing yeast Kluyveromyces marxianus Y179 and fed‐batch operation. The impact of inoculum age, aeration, the supplementation of pectinase and nutrients on the ethanol fermentation performance of the CBP system was studied. Although inulinase activities increased with the extension of the seed incubation time, its contribution to ethanol production was negligible because vigorously growing yeast cells harvested earlier carried out ethanol fermentation more efficiently. Thus, the overnight incubation that has been practised in ethanol production from starch‐based feedstocks is recommended. Aeration facilitated the fermentation process, but compromised ethanol yield because of the negative Crabtree effect of the species, and increases the risk of contamination under industrial conditions. Therefore, nonaeration conditions are preferred for the CBP system. Pectinase supplementation reduced viscosity of the fermentation broth and improved ethanol production performance, particularly under high gravity conditions, but the enzyme cost should be carefully balanced. Medium optimization was performed, and ethanol concentration as high as 94·2 g l^?1 was achieved when 0·15 g l^?1 K₂HPO₄ was supplemented, which presents a significant progress in ethanol production from Jerusalem artichoke tubers. Conclusions: A CBP system using K. marxianus is suitable for efficient ethanol production from Jerusalem artichoke tubers under VHG conditions. Significance and Impact of the Study: Jerusalem artichoke tubers are an alternative to grain‐based feedstocks for ethanol production. The high ethanol concentration achieved using K. marxianus with the CBP system not only saves energy consumption for ethanol distillation, but also significantly reduces the amount of waste distillage discharged from the distillation system.     

Recent process improvements for the ammonia fiber expansion (AFEX) process and resulting reductions in minimum ethanol selling price

The ammonia fiber expansion (AFEX) process has been shown to be an effective pretreatment for lignocellulosic biomass. Technological advances in AFEX have been made since previous cost estimates were developed for this process. Recent research has enabled lower overall ammonia requirements, reduced ammonia concentrations, and reduced enzyme loadings while still maintaining high conversions of glucan and xylan to monomeric sugars. A new ammonia recovery approach has also been developed. Capital and operating costs for the AFEX process, as part of an overall biorefining system producing fuel ethanol from biomass have been developed based on these new research results. These new cost estimates are presented and compared to previous estimates. Two biological processing options within the overall biorefinery are also compared, namely consolidated bioprocessing (CBP) and enzymatic hydrolysis followed by fermentation. Using updated parameters and ammonia recovery configurations, the cost of ethanol production utilizing AFEX is calculated. These calculations indicate that the minimum ethanol selling price (MESP) has been reduced from $1.41/gal to $0.81/gal.     

Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status

Fuel ethanol production from plant biomass hydrolysates by Saccharomyces cerevisiae is of great economic and environmental significance. This paper reviews the current status with respect to alcoholic fermentation of the main plant biomass-derived monosaccharides by this yeast. Wild-type S. cerevisiae strains readily ferment glucose, mannose and fructose via the Embden–Meyerhof pathway of glycolysis, while galactose is fermented via the Leloir pathway. Construction of yeast strains that efficiently convert other potentially fermentable substrates in plant biomass hydrolysates into ethanol is a major challenge in metabolic engineering. The most abundant of these compounds is xylose. Recent metabolic and evolutionary engineering studies on S. cerevisiae strains that express a fungal xylose isomerase have enabled the rapid and efficient␣anaerobic fermentation of this pentose. l-Arabinose fermentation, based on the expression of a prokaryotic pathway in S. cerevisiae, has also been established, but needs further optimization before it can be considered for industrial implementation. In addition to these already investigated strategies, possible approaches for metabolic engineering of galacturonic acid and rhamnose fermentation by S. cerevisiae are discussed. An emerging and major challenge is to achieve the rapid transition from proof-of-principle experiments under ‘academic’ conditions (synthetic media, single substrates or simple substrate mixtures, absence of toxic inhibitors) towards efficient conversion of complex industrial substrate mixtures that contain synergistically acting inhibitors.     

Engineering new metabolic capabilities in bacteria: lessons from recombinant cellulolytic strategies

Cellulose waste biomass is the most attractive substrate for 'biorefinery strategies' producing high-value products (e.g. fuels or plastics) by fermentation. However, traditional biomass bioconversions are economically inefficient multistep processes. Thus far, no microorganisms able to perform single-step fermentation into products (consolidated bioprocessing; CBP) have been isolated. Metabolic engineering is currently employed to develop recombinant microorganisms suitable for CBP. The heterologous expression of extracellular proteins (e.g. cellulases or hemicellulases) is the key feature of recombinant cellulolytic strategies, conferring cellulolytic ability to microorganisms exhibiting high product yields and titers. Although more molecular tools are becoming available, efficient heterologous expression of secreted proteins is still a challenge. The present review summarizes both bottlenecks and solutions of organism engineering for biomass biorefinery strategies.     

Evaluation of pretreatment methods for lignocellulosic ethanol production from energy cane variety L 79-1002

Approximately half of the 80 billion tons of crop produced annually around the world remains as residue that could serve as a renewable resource to produce valuable products such as ethanol and butanol. Ethanol produced from lignocellulosic biomass is a promising renewable alternative to diminishing oil and gas liquid fuels. Sugarcane is an important industry in Louisiana. The recently released variety of “energy cane” has great potential to sustain a competitive sugarcane industry. It has been demonstrated that fuel-grade ethanol can be produced from post harvest sugarcane residue in the past, but optimized ethanol production was not achieved. Optimization of the fermentation process requires efficient pretreatment to release cellulose and hemicellulose from lignocellulosic complex of plant fiber. Determining optimal pretreatment techniques for fermentation is essential for the success of lignocellulosic ethanol production process. The purpose of this study was to evaluate three pretreatment methods for the energy cane variety L 79-1002 for maximum lignocellulosic ethanol production. The pretreatments include alkaline pretreatment, dilute acid hydrolysis, and solid-state fungal pretreatment process using brown rot and white rot fungi. Pretreated biomass was enzymatically saccharified and subjected to fermentation using a recombinant Escherichia coli FBR5. The results revealed that all pretreatment processes produced ethanol. However, the best result was observed in dilute acid hydrolysis followed by alkaline pretreatment and solid-state fungal pretreatment.     

Consolidated bioprocessing (CBP) of AFEX™-pretreated corn stover for ethanol production using Clostridium phytofermentans at a high solids loading

Consolidated bioprocessing (CBP) using Clostridium phytofermentans (ATCC 700394) on ammonia fiber expansion (AFEX?)‐treated corn stover (AFEX?‐CS) at a low solids loading showed promising results [Jin et al. (2011) Biotechnol Bioeng 108(6): 1290–1297]. However, industrial relevant process requires high solids loading. Therefore, we studied high solids loading CBP performance on AFEX?‐CS. The factors potentially affecting the performance including solids loading, CBP products acetate and ethanol, and degradation products resulting from pretreatment were investigated. At 4% (w/w) glucan loading, C. phytofermentans performed well on AFEX?‐CS with no nutrients supplementation and reached similar sugar conversions as a fermentation with nutrients supplementation. A glucan conversion of 48.9% and a xylan conversion of 77.9% were achieved after 264 h with 7.0 g/L ethanol and 8.8 g/L acetate produced. Relatively high concentrations of acetate produced at high solids loading was found to be the major factor limiting the CBP performance. Degradation products in AFEX?‐CS helped enhance ethanol production. Biotechnol. Bioeng. 2012; 109:1929–1936. © 2012 Wiley Periodicals, Inc.     

The potential of cellulases and cellulosomes for cellulosic waste management

Lignocellulose is the most abundant plant cell wall component of the biosphere and the most voluminous waste produced by our society. Fortunately, it is not toxic or directly harmful, but our major waste disposal facilities--the landfills--are rapidly filling up with few realistic alternatives. Because cellulose is pure glucose, its conversion to fine products or fuels has remained a romantic and popular notion; however, the heterogeneous and recalcitrant nature of cellulosic waste presents a major obstacle for conventional conversion processes. One paradigm for the conversion of biomass to products in nature relies on a multienzyme complex, the cellulosome. Microbes that produce cellulosomes convert lignocelluose to microbial cell mass and products (e.g. ethanol) simultaneously. The combination of designer cellulosomes with novel production concepts could in the future provide the breakthroughs necessary for economical conversion of cellulosic biomass to biofuels.     

纤维素乙醇统合加工过程中的酵母多酶共展示体系研究进展

利用统合生物加工过程(Consolidated bioprocessing,CBP)生产纤维素乙醇是目前国内外的研究热点。CBP需要一种“集成化”微生物,既能生产水解木质纤维素的多种酶类又能利用水解木质纤维素产生的糖类发酵产乙醇。以酿酒酵母表面展示技术为依托,建立CBP菌株多酶共展示体系的研究主要分为以下两个方向：一是直接将纤维素酶展示在细胞表面,即非复合型纤维素酶体系;另一种是通过表面展示纤维小体(Cellulosome)将纤维素酶间接地锚定在细胞表面,即复合型纤维素酶体系,本文主要从以上两个方向阐述了近几年对于纤维素乙醇生物统合加工过程的研究进展。因纤维小体对纤维素的降解能力比非复合型纤维素酶体系更强,所以其在酿酒酵母细胞表面的组装研究受到越来越多的关注,为了更深入透彻地了解纤维小体的酵母展示技术,文中对纤维小体的结构与功能及其在纤维素乙醇发酵中的应用研究进行重点论述,并对该领域的发展方向进行展望。     

Bioethanol from Macroalgal Biomass: Utilization of Marine Yeast for Production of the Same

The present study deals with the first systematic study on the isolation, characterization, and utilization of marine yeast for bioethanol production using seaweed biomass. The ability and efficiency of isolated marine yeast to grow and ferment sugar to ethanol in the presence of 2.5 % to 15 % salt concentration was validated by fermenting galactose in the presence of different salts at varied concentrations. Successively, this yeast was employed for fermentation of seaweed hydrolysate, containing high salt concentration, to ethanol. The hydrolysate having varying sugar as well as salt contents, from 2.7 % to 5.5 % and from 6.25 to 11.25 %, respectively, yielded 1.23–1.76 % ethanol. Through biochemical, fatty acid methyl ester analysis, and BioLog, the yeast was identified as Candida sp. The ability of this yeast to function at high salinity can be commercialized for its use to convert seaweed polysaccharide based hydrolysate, rich in salt, to ethanol without desalting process, ultimately making the process more efficient and economically viable. This is the first organized study for the utilization of marine yeast for converting Kappaphycus alvarezii, a red algal biomass, into ethanol as a byproduct, under highly saline condition.     

Immobilized anaerobic fermentation for bio-fuel production by Clostridium co-culture

Clostridium thermocellum/Clostridium thermolacticum co-culture fermentation has been shown to be a promising way of producing ethanol from several carbohydrates. In this research, immobilization techniques using sodium alginate and alkali pretreatment were successfully applied on this co-culture to improve the bio-ethanol fermentation performance during consolidated bio-processing (CBP). The ethanol yield obtained increased by over 60 % (as a percentage of the theoretical maximum) as compared to free cell fermentation. For cellobiose under optimized conditions, the ethanol yields were approaching about 85 % of the theoretical efficiency. To examine the feasibility of this immobilization co-culture on lignocellulosic biomass conversion, untreated and pretreated aspen biomasses were also used for fermentation experiments. The immobilized co-culture shows clear benefits in bio-ethanol production in the CBP process using pretreated aspen. With a 3-h, 9 % NaOH pretreatment, the aspen powder fermentation yields approached 78 % of the maximum theoretical efficiency, which is almost twice the yield of the untreated aspen fermentation.     

燃料乙醇发酵技术研究进展

在分析木质纤维素类生物质制备燃料乙醇原理基础上,重点对燃料乙醇转化过程的发酵工艺进行了论述。目前乙醇发酵工艺主要包括直接发酵、分步糖化发酵、同步糖化发酵、同步糖化共发酵和联合生物加工技术等,对这几种技术的研究现状进行了分析并对其发展趋势进行了展望,通过基因工程构建高效发酵菌种的联合生物加工技术将是未来高效发酵工艺的发展趋势,旨在为有效提高发酵菌株的底物代谢能力,获得高的乙醇产量提供重要参考。     

Isolation and characterization of a Trichodermastrain capable of fermenting cellulose to ethanol

The direct fermentation of cellulosic biomass to ethanol has long been a desired goal. To this end, we screened the environment for fungal strains capable of this conversion when grown on minimal medium. One strain, identified as a member of the genus Trichoderma and designated strain A10, was isolated from cow dung and initially produced about 0.4 g ethanol l(-1). This strain cannot grow on any substrate under anaerobic conditions, but can ferment microcrystalline cellulose or several sugars to ethanol. Ethanol accumulation was eventually increased, by selection and the use of a vented fermentation flask, to 2 g l(-1) when the fermentation was carried out in submerged culture in minimal medium. The highest levels of ethanol, >5.0 g l(-1), were obtained by the fermentation of glucose. Little ethanol was produced by the fermentation of xylose, although other fermentation products such as succinate and acetate were observed. Strain A10 was also found to utilize (aerobically) a wide range of carbon sources. In addition, auxotrophic mutants were generated and used to demonstrate parasexuality by complementation between auxotrophs and between morphological mutants. The ability of this strain to use a wide variety of carbohydrates (including crystalline cellulose) combined with its minimal nutrient requirements and the availability of a genetic system suggests that the strain merits further investigation of its ability to convert biomass to ethanol.     

Controlling accumulation of fermentation inhibitors in biorefinery recycle water using microbial fuel cells

Background  

Microbial fuel cells (MFC) and microbial electrolysis cells are electrical devices that treat water using microorganisms and convert soluble organic matter into electricity and hydrogen, respectively. Emerging cellulosic biorefineries are expected to use large amounts of water during production of ethanol. Pretreatment of cellulosic biomass results in production of fermentation inhibitors which accumulate in process water and make the water recycle process difficult. Use of MFCs to remove the inhibitory sugar and lignin degradation products from recycle water is investigated in this study.     

Influence of the ethanol and glucose supply rate on the rate and enantioselectivity of 3-oxo ester reduction by baker's yeast

Baker's-yeast-mediated reductions of ketones hold great potential for the industrial production of enantiopure alcohols. In this article we describe the stoichiometry and kinetics of asymmetric ketone reduction by cell suspensions of bakers' yeast (Saccharomyces cerevisiae). A system for quantitative analysis of 3-oxo ester reduction was developed and allowed construction of full mass and redox balances as well as determination of the influence of different process parameters on aerobic ketone reduction. The nature of the electron donor (ethanol or glucose) and its specific consumption rate by the biomass (0-1 mol.kg dw(-1).h(-1)) affected the overall stoichiometry and rate of the process and the final enantiomeric excess of the product. Excess glucose as the electron donor, i.e. a very high consumption rate of glucose, resulted in a high rate of alcoholic fermentation, oxygen consumption, and biomass formation and therefore causing low efficiency of glucose utilization. Controlled supply of the electron donor at the highest rates applied prevented alcoholic fermentation but still resulted in biomass formation and a high oxygen requirement, while low rates resulted in a more efficient use of the electron donor. Low supply rates of ethanol resulted in biomass decrease while low supply rates of glucose provided the most efficient strategy for electron donor provision and yielded a high enantiomeric excess of ethyl (S)-3-hydroxybutanoate. In contrast to batchwise conversions with excess glucose as the electron donor, this strategy prevented by-product formation and biomass increase, and resulted in a low oxygen requirement.     

Simultaneous saccharification and fermentation of steam-exploded corn stover at high glucan loading and high temperature

Background

Simultaneous saccharification and fermentation (SSF) is a promising process for bioconversion of lignocellulosic biomass. High glucan loading for hydrolysis and fermentation is an efficient approach to reduce the capital costs for bio-based products production. The SSF of steam-exploded corn stover (SECS) for ethanol production at high glucan loading and high temperature was investigated in this study.

Results

Glucan conversion of corn stover biomass pretreated by steam explosion was maintained at approximately 71 to 79% at an enzyme loading of 30 filter paper units (FPU)/g glucan, and 74 to 82% at an enzyme loading of 60 FPU/g glucan, with glucan loading varying from 3 to 12%. Glucan conversion decreased obviously with glucan loading beyond 15%. The results indicated that the mixture was most efficient in enzymatic hydrolysis of SECS at 3 to 12% glucan loading. The optimal SSF conditions of SECS using a novel Saccharomyces cerevisiae were inoculation optical density (OD)₆₀₀?=?4.0, initial pH 4.8, 50% nutrients added, 36 hours pre-hydrolysis time, 39°C, and 12% glucan loading (20% solid loading). With the addition of 2% Tween 20, glucan conversion, ethanol yield, final ethanol concentration reached 78.6%, 77.2%, and 59.8 g/L, respectively, under the optimal conditions. The results suggested that the solid and degradation products’ inhibitory effect on the hydrolysis and fermentation of SECS were also not obvious at high glucan loading. Additionally, glucan conversion and final ethanol concentration in SSF of SECS increased by 13.6% and 18.7%, respectively, compared with separate hydrolysis and fermentation (SHF).

Conclusions

Our research suggested that high glucan loading (6 to 12% glucan loading) and high temperature (39°C) significantly improved the SSF performance of SECS using a thermal- and ethanol-tolerant strain of S. cerevisiae due to the removal of degradation products, sugar feedback, and solid’s inhibitory effects. Furthermore, the surfactant addition obviously increased ethanol yield in SSF process of SECS.