Overflow metabolism at the thermodynamic limit of life: How carboxydotrophic acetogens mitigate carbon monoxide toxicity

Carboxydotrophic metabolism is gaining interest due to its applications in gas fermentation technology, enabling the conversion of carbon monoxide to fuels and commodities. Acetogenic carboxydotrophs play a central role in current gas fermentation processes. In contrast to other energy-rich microbial substrates, CO is highly toxic, which makes it a challenging substrate to utilize. Instantaneous scavenging of CO upon entering the cell is required to mitigate its toxicity. Experiments conducted with Clostridium autoethanogenum at different biomass-specific growth rates show that elevated ethanol production occurs at increasing growth rates. The increased allocation of electrons towards ethanol at higher growth rates strongly suggests that C. autoethanogenum employs a form of overflow metabolism to cope with high dissolved CO concentrations. We argue that this overflow branch enables acetogens to efficiently use CO at highly variable substrate influxes by increasing the conversion rate almost instantaneously when required to remove toxic substrate and promote growth. In this perspective, we will address the case study of C. autoethanogenum grown solely on CO and syngas mixtures to assess how it employs acetate reduction to ethanol as a form of overflow metabolism.     

Process design and economic studies of alternative fermentation methods for the production of ethanol

Cell recycle and vacuum fermentation processes are described for the continuous production of ethanol. Preliminary process design studies are employed to make an economic comparison of these alternative fermentation schemes with continuous and batch fermentation technologies. Designs are based on a production capacity of 78,000 gal 95% ethanol (EtOH)/day employing molasses as the fermentation substrate. The studies indicate that a 57% reduction in fixed capital investment is realized by continuous rather than batch operation. Further decreases in required capital investment of 68 and 71% over batch fermentation were obtained for cell recycle and vacuum operation, respectively. However, ethanol production costs were dominated by the cost of molasses, representing over 75% of the total manufacturing cost. But, when a reasonable yeast by-product credit was assumed, the net production cost for 95% ethanol was estimated at 82.3 and 80.6 cent/gal, for the cell recycle and vacuum processes, respectively.     

Ethanol and lactic acid production from sugar and starch wastes by anaerobic acidification

Anaerobic conversion of carbohydrates can generate various end‐products. Besides physical parameters such as pH and temperature, the types of carbohydrate being fermented influences the fermentation pattern. Under uncontrolled pH, microbial mixed cultures from activated sludge and anaerobic digester sludge anaerobically produced ethanol from glucose while producing lactic acid from starch conversion. This trend was not only observed in batch trials. Also, continuous chemostat operation of anaerobic digester sludge resulted in the reproducible predominance of ethanol fermentation from glucose solution and lactic acid production from starch. Different feeding regimes and substrate availability (shock load versus continuous feeding) in glucose fermentation under non‐controlled pH did not affect the ethanol production as the major end product. Shifts in feed composition from glucose to starch and vice versa result in an immediate change of fermentation end products formation.     

粗糙脉孢菌(Neurospora crassa)木糖发酵的研究

研究了不同通氧条件和培养基初始pH等对粗糙脉孢菌(Neurospora crassa)AS3．1602木糖发酵的影响。结果表明,粗糙脉孢菌具有较强的发酵木糖产生乙醇及木糖醇的能力。通气量对木糖发酵有较大的影响。乙醇发酵适合在半好氧条件下进行,此时乙醇的转化率达到63．2％。木糖醇发酵适合在微好氧的条件下进行,转化率达到31．8％。木糖醇是在培养基中乙醇达到一定浓度后才开始积累。培养基的初始pH对木糖发酵产物有较大的影响,乙醇产生最适pH5．0,木糖醇产生最适pH4．0。在培养基pH为碱性条件时,木糖发酵受到很大的抑制。初始木糖浓度对产物乙醇及木糖醇的产率有很大的影响。葡萄糖的存在会抑制木糖的利用,对乙醇和木糖醇的产生也有很大的影响。     

Cellulosic alcoholic fermentation using recombinant Saccharomyces cerevisiae engineered for the production of Clostridium cellulovorans endoglucanase and Saccharomycopsis fibuligeraβ-glucosidase

In this study, Saccharomyces cerevisiae was engineered for simultaneous saccharification and fermentation of cellulose by the overexpression of the endoglucanase D (EngD) from Clostridium cellulovorans and the β-glucosidase (Bgl1) from Saccharomycopsis fibuligera . To promote secretion of the two enzymes, the genes were fused to the secretion signal of the S. cerevisiae α mating factor gene. The recombinant developed yeast could produce ethanol through simultaneous production of sufficient extracellular endoglucanase and β-glucosidase. When direct ethanol fermentation from 20 g L⁻¹β-glucan as a substrate was performed with our recombinant strains, the ethanol concentration reached 9.15 g L⁻¹ after 50 h of fermentation. The conversion ratio of ethanol from β-glucan was 80.3% of the theoretical ethanol concentration produced from 20 g L⁻¹β-glucan. In conclusion, we have demonstrated the construction of a yeast strain capable of conversion of a cellulosic substrate to ethanol, representing significant progress towards the realization of processing of cellulosic biomass in a consolidated bioprocessing configuration.     

Furfural, 5-hydroxymethyl furfural, and acetoin act as external electron acceptors during anaerobic fermentation of xylose in recombinant Saccharomyces cerevisiae

The electron acceptors acetoin, acetaldehyde, furfural, and 5-hydroxymethylfurfural (HMF) were added to anaerobic batch fermentation of xylose by recombinant, xylose utilising Saccharomyces cerevisiae TMB 3001. The intracellular fluxes during xylose fermentation before and after acetoin addition were calculated with metabolic flux analysis. Acetoin halted xylitol excretion and decreased the flux through the oxidative pentose phosphate pathway. The yield of ethanol increased from 0.62 mol ethanol/mol xylose to 1.35 mol ethanol/mol xylose, and the cell more than doubled its specific ATP production after acetoin addition compared to fermentation of xylose only. This did, however, not result in biomass growth. The xylitol excretion was also decreased by furfural and acetaldehyde but was unchanged by HMF. Thus, furfural present in lignocellulosic hydrolysate can be beneficial for ethanolic fermentation of xylose. Enzymatic analyses showed that the reduction of acetoin and furfural required NADH, whereas the reduction of HMF required NADPH. The enzymatic activity responsible for furfural reduction was considerably higher than for HMF reduction and also in situ furfural conversion was higher than HMF conversion.     

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.     

一步法发酵菊芋生产乙醇

利用马克斯克鲁维酵母(Kluyveromyces marxianus)YX01具有菊粉酶生产能力且乙醇发酵性能良好的特点,直接发酵菊粉生成乙醇.在摇瓶中考察了该菌株最适发酵温度,进而在2.5L发酵罐中考察了通气量和底物浓度的影响.实验结果表明:该菌株最适发酵温度为35℃;在通气量为50 mL/min和100 mL/min时菌体生长加快,发酵时间缩短,但在不通气条件下糖醇转化率明显提高;在菊粉浓度235 g/L时,发酵终点乙醇浓度达到92.2 g/L,乙醇对糖的得率为0.436,为理论值的85.5%.在此基础上,使用近海滩涂种植海水灌溉收获的菊芋为底物,以批式补料方式直接发酵菊芋干粉浓度为280 g/L的底物,发酵终点乙醇浓度为84.0 g/L,乙醇对糖的得率为0.405,为理论值的80.0%.这些研究工作,为以菊芋为原料的燃料乙醇技术开发奠定了基础.     

Comparison of solid-state and submerged-state fermentation for the bioprocessing of switchgrass to ethanol and acetate by Clostridium phytofermentans

The conversion of sustainable energy crops using microbiological fermentation to biofuels and bioproducts typically uses submerged-state processes. Alternatively, solid-state fermentation processes have several advantages when compared to the typical submerged-state processes. This study compares the use of solid-state versus submerged-state fermentation using the mesophilic anaerobic bacterium Clostridium phytofermentans in the conversion of switchgrass to the end products of ethanol, acetate, and hydrogen. A shift in the ratio of metabolic products towards more acetate and hydrogen production than ethanol production was observed when C. phytofermentans was grown under solid-state conditions as compared to submerged-state conditions. Results indicated that the end product concentrations (in millimolar) obtained using solid-state fermentation were higher than using submerged-state fermentation. In contrast, the total fermentation products (in weight of product per weight of carbohydrates consumed) and switchgrass conversion were higher for submerged-state fermentation. The conversion of xylan was greater than glucan conversion under both fermentation conditions. An initial pH of 7 and moisture content of 80 % resulted in maximum end products formation. Scanning electron microscopy study showed the presence of biofilm formed by C. phytofermentans growing on switchgrass under submerged-state fermentation whereas bacterial cells attached to surface and no apparent biofilm was observed when grown under solid-state fermentation. To our knowledge, this is the first study reporting consolidated bioprocessing of a lignocellulosic substrate by a mesophilic anaerobic bacterium under solid-state fermentation conditions.     

Modeling and static optimization of the ethanol production in a cascade reactor. I. Modeling

Let us consider the modeling of a cascade reactor for the production of ethanol. The rates of reaction in alcoholic fermentation are modeled so that it can assume both ethanol and substrate inhibition, in relation to the observed results.A nonstructured model, based on biomass, substrate, and ethanol concentrations, is developed and identified. It is a complex model, this being due to the nonlinearity between the specific rate of ethanol production and the growth rate and, on the other hand, the study of the static optimization of ethanol fermentation is performed.     

d-Xylulose Fermentation to Ethanol by Saccharomyces cerevisiae

We used commercial bakers' yeast (Saccharomyces cerevisiae) to study the conversion of d-xylulose to ethanol in the presence of d-xylose. The rate of ethanol production increased with an increase in yeast cell density. The optimal temperature for d-xylulose fermentation was 35 degrees C, and the optimal pH range was 4 to 6. The fermentation of d-xylulose by yeast resulted in the production of ethanol as the major product; small amounts of xylitol and glycerol were also produced. The production of xylitol was influenced by pH as well as temperature. High pH values and low temperatures enhanced xylitol production. The rate of d-xylulose fermentation decreased when the production of ethanol yielded concentrations of 4% or more. The slow conversion rate of d-xylulose to ethanol was increased by increasing the yeast cell density. The overall production of ethanol from d-xylulose by yeast cells under optimal conditions was 90% of the theoretical yield.     

Simultaneous saccharification and fermentation of steam-pretreated bagasse using Saccharomyces cerevisiae TMB3400 and Pichia stipitis CBS6054

Sugarcane bagasse--a residue from sugar and ethanol production from sugar cane--is a potential raw material for lignocellulosic ethanol production. This material is high in xylan content. A prerequisite for bioethanol production from bagasse is therefore that xylose is efficiently fermented to ethanol. In the current study, ethanolic fermentation of steam-pretreated sugarcane bagasse was assessed in a simultaneous saccharification and fermentation (SSF) set-up using either Saccharomyces cerevisiae TMB3400, a recombinant xylose utilizing yeast strain, or Pichia stipitis CBS6054, a naturally xylose utilizing yeast strain. Commercial cellulolytic enzymes were used and the content of water insoluble solids (WIS) was 5% or 7.5%. S. cerevisiae TMB3400 consumed all glucose and large fraction of the xylose in SSF. Almost complete xylose conversion could be achieved at 5% WIS and 32 degrees C. Fermentation did not occur with P. stipitis CBS6054 at pH 5.0. However, at pH 6.0, complete glucose conversion and high xylose conversion (>70%) was obtained. Microaeration was required for P. stipitis CBS6054. This was not necessary for S. cerevisiae TMB3400.     

The use of plant cell vesicles for immobilization of yeast cells producing ethanol

The preparation of immobilized living yeast cells adsorbed into or onto delipided specimens of the dwarf duckweed Wolffia arrhiza (Fam. Lemnaceae) is reported. These yeast cell-plant cell conjugates were used for the repeated batch production of ethanol from glucose (143 to 246 g/l) or saccharose (150 g/l). Up to 25 fermentation cycles at 30°C were performed. The cycle time for complete substrate conversion to ethanol was reduced 10-fold by a 5-fold increase of the yeast cell Wolffia conjugate concentration (ε = 0.08 to ε =0.4) ε = volume of cell conjugate/totnl reaction volume. The corresponding ethanol production was 11.5 to 13.5 vol% and 9 vol% respectively. The reported results on the discontinuous ethanol fermentation with Wolffia-immobilized yeast cells open the field for their application in continuous ethanol production processes.     

Ethanol extraction by supported liquid membrane during fermentation

A supported liquid membrane system was developed for the extraction of ethanol during semicontinuous fermentation of Saccharomyces bayanus. it consisted of a porous Teflon sheet as support, soaked with isotridecanol. This assembly permitted combining biocompatibility, permeation efficiency, and stability. The removal of ethanol from the cultures led to decreased inhibition and, thus, to a gain in conversion of 452 g/L glucose versus 293 g/L glucose without extraction. At the same time, the ethanol volumetric productivity was enhanced 2.5 times, due to an improvement of yeast viability, while the substrate conversion yield was maintained above 95% of its theoretical value. Besides these improvements in fermentation performances, the process resulted in ethanol purification, since the separation was selective towards microbial cells and carbon substrate, and likely selective to mineral ions present in the fermentation broth. For pervaporation, a concentration of ethanol four times greater was obtained in the collected permeate.     

Continuous SSCF of AFEX™ pretreated corn stover for enhanced ethanol productivity using commercial enzymes and Saccharomyces cerevisiae 424A (LNH‐ST)

High productivity processes are critical for commercial production of cellulosic ethanol. One high productivity process—continuous hydrolysis and fermentation—has been applied in corn ethanol industry. However, little research related to this process has been conducted on cellulosic ethanol production. Here, we report and compare the kinetics of both batch SHF (separate hydrolysis and co‐fermentation) and SSCF (simultaneous saccharification and co‐fermentation) of AFEX? (Ammonia Fiber Expansion) pretreated corn stover (AFEX?‐CS). Subsequently, we designed a SSCF process to evaluate continuous hydrolysis and fermentation performance on AFEX?‐CS in a series of continuous stirred tank reactors (CSTRs). Based on similar sugar to ethanol conversions (around 80% glucose‐to‐ethanol conversion and 47% xylose‐to‐ethanol conversion), the overall process ethanol productivity for continuous SSCF was 2.3‐ and 1.8‐fold higher than batch SHF and SSCF, respectively. Slow xylose fermentation and high concentrations of xylose oligomers were the major factors limiting further enhancement of productivity. Biotechnol. Bioeng. 2013; 110: 1302–1311. © 2012 Wiley Periodicals, Inc.     

Micro and macroalgal biomass: A renewable source for bioethanol

Population outburst together with increased motorization has led to an overwhelming increase in the demand for fuel. In the milieu of economical and environmental concern, algae capable of accumulating high starch/cellulose can serve as an excellent alternative to food crops for bioethanol production, a green fuel for sustainable future. Certain species of algae can produce ethanol during dark-anaerobic fermentation and thus serve as a direct source for ethanol production. Of late, oleaginous microalgae generate high starch/cellulose biomass waste after oil extraction, which can be hydrolyzed to generate sugary syrup to be used as substrate for ethanol production. Macroalgae are also harnessed as renewable source of biomass intended for ethanol production. Currently there are very few studies on this issue, and intense research is required in future in this area for efficient utilization of algal biomass and their industrial wastes to produce environmentally friendly fuel bioethanol.     

Engineering of Saccharomyces cerevisiae for efficient anaerobic alcoholic fermentation of L-arabinose

For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as l-arabinose, is required. Wild-type strains of Saccharomyces cerevisiae, the organism used in industrial ethanol production, cannot ferment xylose and arabinose. Although metabolic and evolutionary engineering has enabled the efficient alcoholic fermentation of xylose under anaerobic conditions, the conversion of l-arabinose into ethanol by engineered S. cerevisiae strains has previously been demonstrated only under oxygen-limited conditions. This study reports the first case of fast and efficient anaerobic alcoholic fermentation of l-arabinose by an engineered S. cerevisiae strain. This fermentation was achieved by combining the expression of the structural genes for the l-arabinose utilization pathway of Lactobacillus plantarum, the overexpression of the S. cerevisiae genes encoding the enzymes of the nonoxidative pentose phosphate pathway, and extensive evolutionary engineering. The resulting S. cerevisiae strain exhibited high rates of arabinose consumption (0.70 g h(-1) g [dry weight](-1)) and ethanol production (0.29 g h(-1) g [dry weight](-1)) and a high ethanol yield (0.43 g g(-1)) during anaerobic growth on l-arabinose as the sole carbon source. In addition, efficient ethanol production from sugar mixtures containing glucose and arabinose, which is crucial for application in industrial ethanol production, was achieved.     

Optimization of alkaline pretreatment of coffee pulp for production of bioethanol

The use of lignocellulosic raw materials in bioethanol production has been intensively investigated in recent years. However, for efficient conversion to ethanol, many pretreatment steps are required prior to hydrolysis and fermentation. Coffee stands out as the most important agricultural product in Brazil and wastes such as pulp and coffee husk are generated during the wet and dry processing to obtain green grains, respectively. This work focused on the optimization of alkaline pretreatment of coffee pulp with the aim of making its use in the alcoholic fermentation. A central composite rotatable design was used with three independent variables: sodium hydroxide and calcium hydroxide concentrations and alkaline pretreatment time, totaling 17 experiments. After alkaline pretreatment the concentration of cellulose, hemicellulose, and lignin remaining in the material, the subsequent hydrolysis of the cellulose component and its fermentation of substrate were evaluated. The results indicated that pretreatment using 4% (w/v) sodium hydroxide solution, with no calcium hydroxide, and 25 min treatment time gave the best results (69.18% cellulose remaining, 44.15% hemicelluloses remaining, 25.19% lignin remaining, 38.13 g/L of reducing sugars, and 27.02 g/L of glucose) and produced 13.66 g/L of ethanol with a yield of 0.4 g ethanol/g glucose. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:451–462, 2014     

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。     

湿氧化爆破稻秆的同步糖化发酵条件研究

探讨了木质纤维素经过湿氧化爆破后在同步糖化发酵过程中酵母产乙醇的基本规律.采用单因素方法对湿氧化爆破条件、酶系组成和添加量以及预酶解时间和温度进行了优化.不同湿氧化爆破预处理条件下的稻秆对同步糖化发酵工艺的影响较大,在预处理温度160 ℃,进氧压力为4×105 Pa,碱用量为6%(w/w),反应时间为20 min的条件...