Mixed Sugar Fermentation Research Articles

Engineering Saccharomyces cerevisiae for Enhanced Production of Protopanaxadiol with Cofermentation of Glucose and Xylose.

Protopanaxadiol (PPD), an active triterpene compound, is the precursor of high-value ginsenosides. In this study, we report a strategy for the enhancement of PPD production in Saccharomyces cerevisiae by cofermentation of glucose and xylose. In mixed sugar fermentation, strain GW6 showed higher PPD titer and yield than that obtained from glucose cultivation. Then, engineering strategies were implemented on GW6 to enhance the PPD yields, such as adjustment of the central carbon metabolism, optimization of the mevalonate pathway, reinforcement of the xylose assimilation pathway, and regulation of cofactor balance, namely, overexpression of xPK/PTA, ERG10/ERG12/ERG13, XYL1/XYL2/TAL1, and POS5, respectively. In particular, the final obtained strain GW10, harboring overexpressed POS5, exhibited the highest PPD yield, which was 2.06 mg of PPD/g of mixed sugar. In a 5-L fermenter, PPD titer reached 152.37 mg/L. These promising results demonstrate the great advantages of mixed sugar over glucose for high-yield production of PPD.

Augmenting Pentose Utilization and Ethanol Production of Native Saccharomyces cerevisiae LN Using Medium Engineering and Response Surface Methodology.

Economics of ethanol production from lignocellulosic biomass depends on complete utilization of constituent carbohydrates and efficient fermentation of mixed sugars present in biomass hydrolysates. Saccharomyces cerevisiae, the commercial strain for ethanol production uses only glucose while pentoses remain unused. Recombinant strains capable of utilizing pentoses have been engineered but with limited success. Recently, presence of endogenous pentose assimilation pathway in S. cerevisiae was reported. On the contrary, evolutionary engineering of native xylose assimilating strains is promising approach. In this study, a native strain S. cerevisiae LN, isolated from fruit juice, was found to be capable of xylose assimilation and mixed sugar fermentation. Upon supplementation with yeast extract and peptone, glucose (10%) fermentation efficiency was 78% with ~90% sugar consumption. Medium engineering augmented mixed sugars (5% glucose + 5% xylose) fermentation efficiency to ~50 and 1.6% ethanol yield was obtained with concomitant sugar consumption ~60%. Statistical optimization of input variables Glucose (5.36%), Xylose (3.30%), YE (0.36%), and peptone (0.25%) with Response surface methodology led to improved sugar consumption (74.33%) and 2.36% ethanol within 84 h. Specific activities of Xylose Reductase and Xylitol Dehydrogenase exhibited by S. cerevisiae LN were relatively low. Their ratio indicated metabolism diverted toward ethanol than xylitol and other byproducts. Strain was tolerant to concentrations of HMF, furfural and acetic acid commonly encountered in biomass hydrolysates. Thus, genetic setup for xylose assimilation in S. cerevisiae LN is not merely artifact of xylose metabolizing pathway and can be augmented by adaptive evolution. This strain showed potential for commercial exploitation.

Co-fermentation of cellobiose and xylose by mixed culture of recombinant Saccharomyces cerevisiae and kinetic modeling.

Efficient conversion of cellulosic sugars in cellulosic hydrolysates is important for economically viable production of biofuels from lignocellulosic biomass, but the goal remains a critical challenge. The present study reports a new approach for simultaneous fermentation of cellobiose and xylose by using the co-culture consisting of recombinant Saccharomyces cerevisiae specialist strains. The co-culture system can provide competitive advantage of modularity compared to the single culture system and can be tuned to deal with fluctuations in feedstock composition to achieve robust and cost-effective biofuel production. This study characterized fermentation kinetics of the recombinant cellobiose-consuming S. cerevisiae strain EJ2, xylose-consuming S. cerevisiae strain SR8, and their co-culture. The motivation for kinetic modeling was to provide guidance and prediction of using the co-culture system for simultaneous fermentation of mixed sugars with adjustable biomass of each specialist strain under different substrate concentrations. The kinetic model for the co-culture system was developed based on the pure culture models and incorporated the effects of product inhibition, initial substrate concentration and inoculum size. The model simulations were validated by results from independent fermentation experiments under different substrate conditions, and good agreement was found between model predictions and experimental data from batch fermentation of cellobiose, xylose and their mixtures. Additionally, with the guidance of model prediction, simultaneous co-fermentation of 60 g/L cellobiose and 20 g/L xylose was achieved with the initial cell densities of 0.45 g dry cell weight /L for EJ2 and 0.9 g dry cell weight /L SR8. The results demonstrated that the kinetic modeling could be used to guide the design and optimization of yeast co-culture conditions for achieving simultaneous fermentation of cellobiose and xylose with improved ethanol productivity, which is critically important for robust and efficient renewable biofuel production from lignocellulosic biomass.

Laboratory evolution for forced glucose-xylose co-consumption enables identification of mutations that improve mixed-sugar fermentation by xylose-fermenting Saccharomyces cerevisiae.

Simultaneous fermentation of glucose and xylose can contribute to improved productivity and robustness of yeast-based processes for bioethanol production from lignocellulosic hydrolysates. This study explores a novel laboratory evolution strategy for identifying mutations that contribute to simultaneous utilisation of these sugars in batch cultures of Saccharomyces cerevisiae. To force simultaneous utilisation of xylose and glucose, the genes encoding glucose-6-phosphate isomerase (PGI1) and ribulose-5-phosphate epimerase (RPE1) were deleted in a xylose-isomerase-based xylose-fermenting strain with a modified oxidative pentose-phosphate pathway. Laboratory evolution of this strain in serial batch cultures on glucose–xylose mixtures yielded mutants that rapidly co-consumed the two sugars. Whole-genome sequencing of evolved strains identified mutations in HXK2, RSP5 and GAL83, whose introduction into a non-evolved xylose-fermenting S. cerevisiae strain improved co-consumption of xylose and glucose under aerobic and anaerobic conditions. Combined deletion of HXK2 and introduction of a GAL83G673T allele yielded a strain with a 2.5-fold higher xylose and glucose co-consumption ratio than its xylose-fermenting parental strain. These two modifications decreased the time required for full sugar conversion in anaerobic bioreactor batch cultures, grown on 20 g L−1 glucose and 10 g L−1 xylose, by over 24 h. This study demonstrates that laboratory evolution and genome resequencing of microbial strains engineered for forced co-consumption is a powerful approach for studying and improving simultaneous conversion of mixed substrates.

Biosynthesis of D-glucaric acid from sucrose with routed carbon distribution in metabolically engineered Escherichia coli

D-glucaric acid is a promising platform compound used to synthesize many other value-added or commodity chemicals. The engineering of Escherichia coli for efficiently converting D-glucose to D-glucaric acid has been attempted for several years, with mixed sugar fermentation recently gaining growing interests due to the increased D-glucaric acid yield. Here, we co-expressed cscB, cscA, cscK, ino1, miox, udh, and suhB in E. coli BL21 (DE3), functionally constructing an unreported route from sucrose to D-glucaric acid. Further deletion of chromosomal zwf, pgi, ptsG, uxaC, gudD, over-expression of glk, and use of a D-fructose-dependent translation control system for pgi enabled the strain to use sucrose as the sole carbon source while achieving a high product titer and yield. The titer of D-glucaric acid in M9 medium containing 10 g/L sucrose reached ~1.42 g/L, with a yield of ~0.142 g/g on sucrose.

Engineering cellulolytic bacterium Clostridium thermocellum to co-ferment cellulose- and hemicellulose-derived sugars simultaneously.

Cellulose and hemicellulose are the most abundant components in plant biomass. A preferred Consolidated Bioprocessing (CBP) system is one which can directly convert both cellulose and hemicellulose into target products without adding the costly hydrolytic enzyme cocktail. In this work, the thermophilic, cellulolytic, and anaerobic bacterium, Clostridium thermocellum DSM 1313, was engineered to grow on xylose in addition to cellulose. Both xylA (encoding for xylose isomerase) and xylB (encoding for xylulokinase) genes from the thermophilic anaerobic bacterium Thermoanaerobacter ethanolicus were introduced to enable xylose utilization while still retaining its inherent ability to grow on 6-carbon substrates. Targeted integration of xylAB into C. thermocellum genome realized simultaneous fermentation of xylose with glucose, with cellobiose (glucose dimer), and with cellulose, respectively, without carbon catabolite repression. We also showed that the respective H2 and ethanol production were twice as much when both xylose and cellulose were consumed simultaneously than when consuming cellulose alone. Moreover, the engineered xylose consumer can also utilize xylo-oligomers (with degree of polymerization of 2-7) in the presence of xylose. Isotopic tracer studies also revealed that the engineered xylose catabolism contributed to the production of ethanol from xylan which is a model hemicellulose in mixed sugar fermentation, demonstrating immense potential of this enhanced CBP strain in co-utilizing both cellulose and hemicellulose for the production of fuels and chemicals.

Assessing the effect of d-xylose on the sugar signaling pathways of Saccharomyces cerevisiae in strains engineered for xylose transport and assimilation.

One of the challenges of establishing an industrially competitive process to ferment lignocellulose to value-added products using Saccharomyces cerevisiae is to get efficient mixed sugar fermentations. Despite successful metabolic engineering strategies, the xylose assimilation rates of recombinant S. cerevisiae remain significantly lower than for the preferred carbon source, glucose. Previously, we established a panel of in vivo biosensor strains (TMB371X) where different promoters (HXT1/2/4p; SUC2p, CAT8p; TPS1p/2p, TEF4p) from the main sugar signaling pathways were coupled with the yEGFP3 gene, and observed that wild-type S. cerevisiae cannot sense extracellular xylose. Here, we expand upon these strains by adding a mutated galactose transporter (GAL2-N376F) with improved xylose affinity (TMB372X), and both the transporter and an oxidoreductase xylose pathway (TMB375X). On xylose, the TMB372X strains displayed population heterogeneities, which disappeared when carbon starvation was relieved by the addition of the xylose assimilation pathway (TMB375X). Furthermore, the signal in the TMB375X strains on high xylose (50g/L) was very similar to the signal recorded on low glucose (≤5g/L). This suggests that intracellular xylose triggers a similar signal to carbon limitation in cells that are actively metabolizing xylose, in turn causing the low assimilation rates.

Single cell oil production by Lipomyces starkeyi: Biphasic fed-batch fermentation strategy providing glucose for growth and xylose for oil production

The aim of this study was to produce single cell oil (SCO) from Lipomyces starkeyi ATCC 56304 using a biphasic fed-batch fermentation strategy. Fed-batch fermentations were performed to compare oil production using glucose, xylose, mixed-sugars (1:1 ratio of glucose:xylose), and a biphasic system supplying glucose for cell growth and xylose for oil production. The fermentation using xylose as the sole carbon source generated an oil yield of 0.16gg−1 and a productivity of 0.12gL−1h−1, two-times the oil yield (0.08gg−1) and productivity (0.06gL−1h−1) achieved with glucose. At 120h, the biphasic fermentation produced a greater oil productivity (0.13gL−1h−1) compared to the mixed-sugar fermentation (0.09gL−1h−1) but similar oil productivities (0.14gL−1h−1) were achieved for both at longer fermentation times. The biphasic fermentation generated a final oil yield of 0.17gg−1 and an oil content of 60.1% (w w−1), but oil productivity showed minor improvement at 0.14gL−1h−1 after 255h. Oleic acid accounted for at least 65% of the fatty acid content, suggesting that oil from L. starkeyi has potential for application in the biofuel and oleochemical industries.

Succinic acid production by Actinobacillus succinogenes from batch fermentation of mixed sugars.

Succinic acid production from the monosaccharides xylose, arabinose, glucose, mannose and galactose was studied using the bacterium Actinobacillus succinogenes. In Duran bottle cultures, containing 10g/L of each of sugar, succinic acid was produced from all sugars except for galactose. The highest succinate yield, 0.56g/g, was obtained with glucose, whereas the succinate yield was 0.42, 0.38 and 0.44g/g for xylose, mannose and arabinose, respectively. The specific succinate productivity was 0.7g/gh for glucose, but below 0.2g/gh for the other sugars. Batch bioreactor fermentations were carried out using a sugar mixture of the five sugars giving a total concentration of 50g/L, mimicking the distribution of sugars in spent sulfite liquor (SSL) from Eucalyptus which is rich in xylose. In this mixture, an almost complete conversion of all sugars (except galactose) was achieved resulting in a final succinate concentration of 21.8-26.8g/L and a total yield of 0.59-0.68g/g. There was evidence of co-consumption of glucose and xylose, whereas mannose was consumed after glucose. The main by-products were acetate 0.14-0.20g/g and formate 0.08-0.13g/g. NADH balance calculations suggested that NADH required for succinate production was not met solely from formate and acetate production, but other means of NADH production was necessary. Results from mixed sugar fermentations were verified using SSL as substrate resulting in a succinate yield of 0.60g/g. In addition, it was found that CO2 sparging could replace carbonate supply in the form of MgCO3 without affecting the succinate yield.

Mutation in galP improved fermentation of mixed sugars to succinate using engineered Escherichia coli AS1600a and AM1 mineral salts medium

Escherichia coli KJ122 was engineered to produce succinate from glucose using the wild type GalP for glucose uptake instead of the native phosphotransferase system (ptsI mutation). This strain now ferments 10% xylose poorly. Mutants were selected by serial transfers in AM1 mineral salts medium with 10% xylose. Clones from this population all exhibited a similar improvement, co-fermentation of an equal mixture of xylose and glucose. One of these, AS1600a, produced 84.26±1.37g/L succinate, equivalent to that produced by the parent (KJ122) from 10% glucose (85.46±1.78g/L). AS1600a was sequenced and found to contain a mutation in galactose permease (GalP, G236D). This mutation was shown to be responsible for the improvement in fermentation using KJΔgalP as the host and expression vectors with native galP and with mutant galP∗. Strain AS1600a and KJΔgalP(pLOI5746; galP∗) also co-fermented a mixture of glucose, xylose, arabinose, and galactose in sugarcane bagasse hydrolysate using mineral salts medium.

Deletion of D-ribulose-5-phosphate 3-epimerase (RPE1) induces simultaneous utilization of xylose and glucose in xylose-utilizing Saccharomyces cerevisiae.

Simultaneous co-utilization of xylose and glucose is a key issue in engineering microbes for cellulosic ethanol production. We coupled xylose utilization with glucose metabolism by deletion of D-ribulose-5-phosphate 3-epimerase (RPE1) through pentose phosphate pathway flux. Simultaneous utilization of xylose and glucose then occurred in the engineered Saccharomyces cerevisiae strain with the xylose utilization pathway. Xylose consumption occurred at the beginning of glucose consumption by the engineered yeast without RPE1 in a mixed sugar fermentation. About 3.2 g xylose l(-1) was utilized simultaneously with consumption of 40.2 g glucose l(-1) under O2-limited conditions. In addition, an approximate ratio (~1:10) for xylose and glucose consumption was observed in the fermentation with different sugar concentration by the engineered strain without RPE1. Simultaneous utilization of xylose is realized by the coupling of glucose metabolism and xylose utilization through RPE1 deletion in xylose-utilizing S. cerevisiae.

Overexpression of the Aspergillus niger GatA transporter leads to preferential use of D-galacturonic acid over D-xylose.

Pectin is a structural heteropolysaccharide of the primary cell walls of plants and as such is a significant fraction of agricultural waste residues that is currently insufficiently used. Its main component, D-galacturonic acid, is an attractive substrate for bioconversion. The complete metabolic pathway is present in the genome of Aspergillus niger, that is used in this study. The objective was to identify the D-galacturonic acid transporter in A. niger and to use this transporter to study D-galacturonic acid metabolism.We have functionally characterized the gene An14g04280 that encodes the D-galacturonic acid transporter in A. niger. In a mixed sugar fermentation it was found that the An14g04280 overexpression strain, in contrast to the parent control strain, has a preference for D-galacturonic acid over D-xylose as substrate. Overexpression of this transporter in A. niger resulted in a strong increase of D-galacturonic acid uptake and induction of the D-galacturonic acid reductase activity, suggesting a metabolite controlled regulation of the endogenous D-galacturonic acid catabolic pathway.

Cofermentation of glucose and galactose by a newly isolated Saccharomyces cerevisiae strain in free and immobilized forms

A newly isolated yeast strain from the soil of grape fruit farm was identified as Saccharomyces cerevisiae Wu-Y2 through molecular characterization. This strain, whether used in free or immobilized form, showed a good performance on co-fermentation of reagent grade mixed sugars comprising of glucose and galactose in production of bioethanol in batch and fed batch culture. The yield coefficient (YE/S; 0.47g/g) for ethanol from monosaccharide and yield efficiency (YE; 92.0%) after 48h fermentation of mixed sugars shown by this wild-type S. cerevisiae Wu-Y2 were much higher than those by engineered S. cerevisiae (D452-2BT) strain (0.34g/g, 67%) and engineered industrial distillers’ yeast (0.28g/g and 55%). The immobilized S. cerevisiae Wu-Y2 cells exhibited a high degree of operational stability for several weeks without significant loss of viability or ethanologenicity in fed batch culture and it can tolerate up to 7–10% of ethanol for more than a month. This strain also showed good ability to utilize glucose and galactose present in red seaweed hydrolysate with high ethanol conversion efficiency; it showed significant potential to be an efficient bioethanol producer using seaweed as a feedstock.

Comparative engineering of Escherichia coli for cellobiose utilization: Hydrolysis versus phosphorolysis

Microbial biocatalysts capable of cellobiose assimilation are of interest in bioconversion of cellulosic materials. This study provides a careful comparison in the two mechanisms of cellobiose assimilation, hydrolysis versus phosphorolysis, between two otherwise isogenic E. coli strains. Relative to cells assimilating cellobiose hydrolytically, phosphorolysis cells tolerated common inhibitors better under both anaerobic and aerobic conditions. Additionally, phosphorolysis cells were able to direct the favorable energy metabolism to recombinant protein production, resulting in up to five fold more recombinant proteins. In a mixed sugar fermentation (5% (w/v) cellobiose+5.0% (w/v) xylose), however, xylose utilization in phosphorolysis cells came to a complete halt after only about 60% consumption whereas the hydrolysis cells were able to ferment both sugars to near completion. These results provide insights into the new metabolic engineering strategy. To our best knowledge, this is the first comparison study in E. coli on the two cellobiose assimilation mechanisms.

Deletion of the HXK2 gene in Saccharomyces cerevisiae enables mixed sugar fermentation of glucose and galactose in oxygen-limited conditions

Galactose is one of the major sugar components of red seaweeds along with glucose. In Saccharomyces cerevisiae, synthesis of galactose-metabolizing enzymes (the Leloir proteins) is under tight and complex regulation in the presence of glucose by a mechanism called catabolite repression. As a result, when both sugars are concurrently present in the medium under oxygen-limited conditions, the yeast cannot utilize galactose even after glucose consumption. Research efforts on mixed sugar fermentation of glucose/galactose, therefore, were made in the presence of oxygen, thereby resulting in sequential substrate consumption, and low ethanol yield and productivity. In this study, mixed sugar fermentation of glucose and galactose in oxygen-limited conditions was achieved by deleting the HXK2 gene, a moonlighting protein acting as hexokinase or repressor involved in catabolite repression of S. cerevisiae. Remarkably, the S. cerevisiae D452-2Δhxk2 strain utilized galactose after glucose depletion without a diauxic lag period, whereas the parental strain could not use galactose at all under oxygen-limited conditions. The Δhxk2 strain fermented galactose at the consumption rate of 3.02±0.10g/L/h to produce ethanol with a yield of 0.44±0.01g ethanol/g galactose. Complementation of the Δhxk2 strain with the plasmid-harbored HXK2 gene under the control of various promoters clearly showed that the galactose consumption rates were inversely related to the mRNA levels of the HXK2 gene. The transcription of the GAL genes was dramatically elevated by deleting the HXK2 gene, whereas the expressions of hexokinase genes were not significantly affected. We concluded that the HXK2-deleted strain is able to efficiently utilize both glucose and galactose in the oxygen-limited conditions by alleviating catabolite-repression.

Bioconversion of Non-Detoxified Hemicellulose Hydrolysates to Xylitol by Halotolerant Yeast Debaryomyces nepalensis NCYC 3413

Lignocellulosic materials are one of the most abundant renewable resources whose exploitation for the production of biochemicals and biofuels is the major challenge in the area of industrial biotechnology due to inhibition of growth and product formation by the toxic compounds released upon their hydrolysis. Indeed the bioprocess that can produce industrial products from hemicellulose hydrolysates in the presence of toxic compounds is economical than the process which involves detoxification. In this study, the ability of halotolerant strain Debaryomyces nepalensis NCYC 3413 to convert non-detoxified xylose enriched hemicellulose hydrolysates from corn cobs, rice straw, sugarcane bagasse and wheat straw to xylitol was evaluated. It was found that this strain has the capability to grow in all hemicellulose hydrolysates and convert xylose to xylitol without detoxification of hydrolysates. The maximum xylitol concentration of 14.6 g L-1 was obtained from corn cobs and wheat straw with productivities of 0.16 and 0.20 g L-1 h-1 respectively at a yield of 0.30 g g-1. Whereas sugarcane bagasse and rice straw gave xylitol yields of 0.31 and 0.32 g g-1 respectively with 14.2 g L-1 maximum xylitol and productivities were calculated to be 0.20 and 0.15 g L-1 h-1 respectively. The presence of high glucose hindered xylitol production by producing ethanol. Based on our findings, we suggest that (i) D. nepalensis is a promising strain for ecofriendly xylitol production as it exhibits broad specificity to lignocellulose substrates, fermentation of mixed sugars and (ii) tolerance towards lignocellulosic inhibitors making the process more economical.

Kinetics of Bioethanol Production Employing Mono‐ and Co‐Cultures of Saccharomyces cerevisiae and Pichia stipitis

AbstractThe kinetics of bioethanol production using mono‐ and co‐cultures of Saccharomyces cerevisiae and Pichia stipitis with glucose, xylose, and glucose‐xylose sugar mixtures were investigated. A MATLAB® program was formulated for simulation of experimental results in order to get predicted values of ethanol production and sugar consumption and for kinetic parameter estimation. Kinetic parameters implied less extent of substrate and/or product inhibition when the co‐culture scheme of immobilized S. cerevisiae and free P. stipitis was employed for fermentation of mixed sugars. In addition, a high ethanol yield was achieved by applying this co‐culture strategy to wheat straw hydrolysates.

Modeling alcoholic fermentation of glucose/xylose mixtures by ethanologenic Escherichia coli as a function of pH

In order to understand the effect of pH on growth and ethanol production in ethanologenic Escherichia coli, we investigated the kinetic behavior of ethanologenic E. coli during alcoholic fermentation of glucose or xylose in a controlled pH environment and the fermentation of glucose, xylose, or their mixtures without pH control. Based on the Monod equation, an unstructured and unsegregated kinetic model was proposed as a function of the pH of the fermentation medium. The pH effects on cell growth, sugar consumption, and ethanol production were taken into account in the proposed model. Both cell growth and ethanol production were found to be significantly influenced by the pH of the fermentation medium. The optimal pH range for ethanol production by ethanologenic E. coli on either glucose or xylose was 6.0–6.5. The highest value of the maximum specific growth rate (μm) was obtained at pH 7.0. In the kinetic model of the fermentations of the sugar mixture, two inhibition terms related to glucose concentrations were included in both the cell growth and ethanol production equations because of the strong inhibitions of glucose and glucose metabolites on xylose metabolism. A good fit was found between model predictions and experimental data for both single-sugar and mixed-sugar fermentations without pH control within the experimental domain.

Effect of salts on the Co-fermentation of glucose and xylose by a genetically engineered strain of Saccharomyces cerevisiae

BackgroundA challenge currently facing the cellulosic biofuel industry is the efficient fermentation of both C5 and C6 sugars in the presence of inhibitors. To overcome this challenge, microorganisms that are capable of mixed-sugar fermentation need to be further developed for increased inhibitor tolerance. However, this requires an understanding of the physiological impact of inhibitors on the microorganism. This paper investigates the effect of salts on Saccharomyces cerevisiae 424A(LNH-ST), a yeast strain capable of effectively co-fermenting glucose and xylose.ResultsIn this study, we show that salts can be significant inhibitors of S. cerevisiae. All 6 pairs of anions (chloride and sulfate) and cations (sodium, potassium, and ammonium) tested resulted in reduced cell growth rate, glucose consumption rate, and ethanol production rate. In addition, the data showed that the xylose consumption is more strongly affected by salts than glucose consumption at all concentrations. At a NaCl concentration of 0.5M, the xylose consumption rate was reduced by 64.5% compared to the control. A metabolomics study found a shift in metabolism to increased glycerol production during xylose fermentation when salt was present, which was confirmed by an increase in extracellular glycerol titers by 4 fold. There were significant differences between the different cations. The salts with potassium cations were the least inhibitory. Surprisingly, although salts of sulfate produced twice the concentration of cations as compared to salts of chloride, the degree of inhibition was the same with one exception. Potassium salts of sulfate were less inhibitory than potassium paired with chloride, suggesting that chloride is more inhibitory than sulfate.ConclusionsWhen developing microorganisms and processes for cellulosic ethanol production, it is important to consider salt concentrations as it has a significant negative impact on yeast performance, especially with regards to xylose fermentation.

Production of butanol from glucose and xylose with immobilized cells of Clostridium acetobutylicum

Pretreated cotton towels were used as carriers to immobilize Clostridium acetobutylicum CGMCC 5234 cells for butanol or ABE production from glucose and xylose. Results showed that cell immobilization was a promising method to increase butanol concentration, yield and productivity regardless of the sugar sources compared with cell suspension. In this study, a high butanol concentration of 10.02 g/L with a yield of 0.20 g/g was obtained from 60 g/L xylose with 9.9 g/L residual xylose using immobilized cells compared with 8.48 g/L butanol and a yield of 0.141 g/g with 20.2 g/L residual xylose from 60 g/L xylose using suspended cells. In mixed-sugar fermentation (30 g/L glucose plus 30 g/L xylose), the immobilized cultures produced 11.1 g/L butanol with a yield of 0.190 g/g, which were 28.3% higher than with suspended cells (8.65 g/L) during which 30 g/L glucose was utilized completely using both immobilized and suspended cells while 3.46 and 13.1 g/L xylose maintained untilized for immobilized and suspended cells, respectively. Based on the results, we speculated that immobilized cells showed enhanced tolerance to butanol toxicity and the cultures preferred glucose to xylose during ABE fermentation. Moreover, the cultures showed obvious difference when grown between high initial concentrations of glucose and those of xylose. Repeated-batch fermentations from glucose with immobilized cells showed better long-term stability than from xylose. At last, the morphologies of free and immobilized cells adsorbed on pretreated cotton towels during the growth cycle were examined by SEM.