Improved fermentation of cellobiose,glucose, and xylose to ethanol by Zymomonas anaerobia and a high ethanol tolerant strain of Clostridium saccharolyticum

Summary To improve single step conversion of sugar mixtures containing cellobiose, glucose, and xylose to ethanol by a coculture of Zymomonas anaerobia and Clostridium saccharolyticum, an ethanol tolerant mutant of C. saccharolyticum was obtained. The mutant obtained by the enrichment procedure was able to grow in the presence of 75 g·l^-1 ethanol, with improved ability to utilize cellobiose, and little or no change in its ability to convert xylose to ethanol. This mutant in coculture with Zymomonas anaerobia produced over 50 g·l^-1 ethanol in media containing 130 g·l^-1 total sugars comprising of 60% glucose, 20% cellobiose, and 20% xylose.Issued as NRCC No. 23936     

Mutants of the pentose‐fermenting yeast Pichia stipitis with improved tolerance to inhibitors in hardwood spent sulfite liquor

Mutants of Pichia stipitis NRRL Y‐7124 able to tolerate and produce ethanol from hardwood spent sulfite liquor (HW SSL) were obtained by UV mutagenesis. P. stipitis cells were subjected to three successive rounds of UV mutagenesis, each followed by screening first on HW SSL gradient plates and then in diluted liquid HW SSL. Six third generation mutants with greater tolerance to HW SSL as compared to the wild type (WT) were isolated. The WT strain could not grow in HW SSL unless it was diluted to 65% (v/v). In contrast, the third generation mutants were able to grow in HW SSL diluted to 75% (v/v). Mutants PS301 and PS302 survived even in 80% (v/v) HW SSL, although there was no increase in cell number. All the third generation mutants exhibited higher growth rates but significantly lower growth yields on xylose or glucose compared to the WT. The mutants fermented 4% (w/v) glucose as efficiently as the WT and fermented 4% (w/v) xylose more efficiently with a higher ethanol yield than the WT. In a medium containing 4% (w/v) each of xylose and glucose, all the third generation mutants utilized glucose as efficiently and xylose more efficiently than the WT. This resulted in higher ethanol yield by the mutants. The mutants retained the ability to utilize galactose and mannose and ferment them to ethanol. Arabinose was consumed slowly by both the mutants and WT with no ethanol production. In 60% (v/v) HW SSL, the mutants utilized and fermented glucose, mannose, galactose and xylose while the WT could not ferment any of these sugars. Biotechnol. Bioeng. 2009; 104: 892–900. © 2009 Wiley Periodicals, Inc.     

Ethanol production from xylose with a pyruvate-formate-lyase mutant of Klebsiella planticola carrying a pyruvate-decarboxylase gene from Zymomonas mobilis

Summary Grown anaerobically on d-xylose, Klebsiella planticola ATCC 33531 produced acetate, formate, lactate, CO₂ and ethanol as major end-products. A Mu-insertion mutant which lacked pyruvate-formate-lyase showed among its fermentation products more than 70% d-lactate with residual acetate, 2,3-butanediol, and traces of ethanol, formate, and CO₂. After the introduction of a plasmid carrying the gene for the enzyme pyruvate decarboxylase from Zymomonas mobilis, this Klebsiella mutant became an efficient ethanol producer. The recombinant strain produced 387 mM ethanol from 275 mM xylose in 80 h, about 83% of the theoretical maximal yield. Furthermore, this mutant consumed more than double the amount of xylose (41 g/l) compared to the wild-type, due to reduced production of inhibiting acids during growth.Dedicated to Professor Dr. Zähner on the occasion of his 60th birthday     

Overcoming inhibitors in a hemicellulosic hydrolysate: improving fermentability by feedstock detoxification and adaptation of <Emphasis Type="Italic">Pichia stipitis</Emphasis>

In order to improve the fermentative efficiency of sugar maple hemicellulosic hydrolysates for fuel ethanol production, various methods to mitigate the effects of inhibitory compounds were employed. These methods included detoxification treatments utilizing activated charcoal, anion exchange resin, overliming, and ethyl acetate extraction. Results demonstrated the greatest fermentative improvement of 50% wood hydrolysate (v/v) by Pichia stipitis with activated charcoal treatment. Another method employed to reduce inhibition was an adaptation procedure to produce P. stipitis stains more tolerant of inhibitory compounds. This adaptation resulted in yeast variants capable of improved fermentation of 75% untreated wood hydrolysate (v/v), one of which produced 9.8 g/l ± 0.6 ethanol, whereas the parent strain produced 0.0 g/l ± 0.0 within the first 24 h. Adapted strains RS01, RS02, and RS03 were analyzed for glucose and xylose utilization and results demonstrated increased glucose and decreased xylose utilization rates in comparison to the wild type. These changes in carbohydrate utilization may be indicative of detoxification or tolerance activities related to proteins involved in glucose and xylose metabolism.     

Ethanol fermentation of hexose and pentose wood sugars produced by hydrogen-fluoride solvolysis of aspen chips

Summary Hexose and pentose sugars, produced by hydrogen-fluoride solvolysis of aspen wood chips, were totally consumed in a coculture fermentation by Zymomonas mobilis and a mutant of Clostridium saccharolyticum. Z. mobilis converted the glucose to ethanol, while the mutant, which was improved in both ethanol production and tolerance, converted the xylose component to ethanol. A high conversion efficiency of wood sugars to ethanol was obtained, and the cells after the fermentation were successfully used for cell recycle.NRCC no. 23211     

Conversion of cellobiose and xylose to ethanol by immobilized growing cells of Clostridium saccharolyticum on charcoal support

Summary An immobilization technique has been developed for the conversion of both cellobiose and xylose to ethanol, which may be considered as one stage of a process for the conversion of cellulosic biomass to ethanol. Relatively inexpensive charcoal was used as a support material, with 23 mg dry weight of Clostridium saccharolyticum cells per g dry weight of support. Tests were run for 170 h at 0.15 1/h dilution rate. From a 3% (w/v) sugar mixture, 0.7% (w/v) ethanol was obtained with over 97% cellobiose and 62% xylose utilization.     

Metabolic analysis of acetate accumulation during xylose consumption by<Emphasis Type="Italic"> Paenibacillus polymyxa</Emphasis>

Paenibacillus polymyxa ATCC 12321 produced more acetic acid and less butanediol from xylose than from glucose. The product yields from xylose were ethanol (0.72 mol/mol sugar), (R,R)-2,3-butanediol (0.31 mol/mol sugar), and acetate (0.38 mol/mol sugar) while those from glucose were ethanol (0.74 mol/mol sugar), (R,R)-2,3-butanediol (0.46 mol/mol sugar), and acetate (0.05 mol/mol sugar). Higher acetate kinase activity and lower acetate uptake ability were found in xylose-grown cells than in glucose-grown cells. Furthermore, phosphoketolase activity was higher in xylose-grown cells than in glucose-grown cells. In fed-batch culture on xylose, glucose feeding raised the butanediol yield to 0.56 mol/mol sugar and reduced acetate accumulation to 0.04 mol/mol sugar.     

Cloning of <Emphasis Type="SmallCaps">l</Emphasis>-lactate dehydrogenase and elimination of lactic acid production via gene knockout in<Emphasis Type="Italic"> Thermoanaerobacterium saccharolyticum</Emphasis> JW/SL-YS485

The gene encoding l-lactate dehydrogenase from Thermoanaerobacterium saccharolyticum JW/SL-YS485 was cloned, sequenced, and used to obtain an l-ldh deletion mutant strain (TD1) following a site-specific double-crossover event as confirmed by PCR and Southern blot. Growth rates and final cell densities were similar for strain TD1 and the wild-type grown on glucose and xylose. Lactic acid was below the limit of detection (0.3 mM) for strain TD1 on both glucose and xylose at all times tested, but was readily detected for the wild-type strain, with average final concentrations of 8.1and 1.8 mM on glucose and xylose, respectively. Elimination of lactic acid as a fermentation product was accompanied by a proportional increase in the yields of acetic acid and ethanol. The results reported here represent a step toward using metabolic engineering to develop strains of thermophilic anaerobic bacteria that do not produce organic acids, and support the methodological feasibility of this goal.     

Comparative fermentability of enzymatic and acid hydrolysates of steam-pretreated aspenwood hemicellulose by Pichia stipitis CBS 5776

Summary Enzymatic hydrolysates of hemicellulose from steam-pretreated aspenwood were more fermentable than the acid hydrolysate after rotoevaporation or ethyl acetate extraction treatments to remove acetic acid and sugar- and lignin-degradation products prior to fermentation by Pichia stipitis CBS 5776. Total xylose and xylobiose utilization from 5.0% (w/v) ethyl acetate extracted enzymatic hydrolysate was observed with an ethanol yield of 0.47 g ethanol/g total available substrate and an ethanol production rate of 0.20 g·l^-1 per hour in 72 h batch fermentation.     

Enhancement of (R,R)-2,3-butanediol production from xylose by Paenibacillus polymyxa at elevated temperatures

Conversion of xylose to (R,R)-2,3-butanediol by Paenibacillus polymyxa in anaerobic batch and continuous cultures was increased by 39% and 52%, respectively, by increasing the growth temperatures from 30 to 39 °C. There was no effect of temperature when glucose was used as substrate. 39 mM (R,R)-2,3-butanediol, 65 mM ethanol, and 47 mM acetate were obtained from 100 mM xylose after 24 h batch culture at 39 °C. With 100 mM glucose and 100 mM xylose used together in a batch culture at 39 °C, all xylose was consumed after 24 h and 82 mM (R,R)-2,3-butanediol, 124 mM ethanol and 33 mM acetate were produced.     

Single step conversion of cellulose to ethanol by a mesophilic coculture

Summary A coculture consisting of two mesophilic anaerobes, produced about 0.8 mole of ethanol per mole of cellulose from a variety of cellulosic materials. The non-cellulolytic member of this coculture, Clostridium saccharolyticum sp. nov. converted glucose and xylose to ethanol and acetic acid in ratios over 4 to 1.     

Simultaneous fermentation and isomerization of xylose

Summary Ethanol was produced from xylose by converting the sugar to xylulose, using commercial xylose isomerases, and simultaneously converting the xylulose to ethanol by anaerobic fermentation using different yeast strains. The process was optimized with the yeast strain Schizosaccharomyces pombe (Y-164). The data show that the simultaneous fermentation and isomerization of 6% xylose can produce final ethanol concentrations of 2.1% w/v within 2 days at temperatures as high as 39°C.Nomenclature SFIX simultaneous fermentation and isomerization of xylose - V _p volumetric production (g ethanol·l^-1 per hour) - Q _p specific rate (g ethanol·g^-1 cells per hour) - Y _s yield from substrate consumed (g ethanol, g^-1 xylose) - ET ethanol concentration (% wt/vol) - XT xylitol concentration (% wt/vol) - Glu glucose - Xyl xylose - --m maximum - --f final     

Ethanol production during D-xylose,L-arabinose,and D-ribose fermentation by Bacteroides polypragmatus

Summary The specific growth rate () during cultivation of Bacteroides polypragmatus in 2.51 batch cultures in 4–5% (w/v) l-arabinose medium was 0.23 h^-1 while that in either d-xylose or d-ribose medium was lower (=0.19 h^-1). Whereas growth on arabinose or xylose occurred after about 6–8 h lag period, growth on ribose commenced after a 30 h lag phase. The maximum substrate utilization rate for arabinose, ribose and xylose in media with an initial substrate concentration of 4–5% (w/v) was 0.77, 0.76, and 0.60 g/l/h respectively. In medium containing a mixture of glucose, arabinose, and xylose, the utilization of all three substrates occurred concurrently. The maximum amount of ethanol produced after 72 h growth in 4–5% (w/v) of arabinose, xylose, and ribose was 9.4, 6.5, and 5.3 g/l, respectively. The matabolic end products (mol/mol substrate) of growth in 4.4% (w/v) xylose medium were 0.73 ethanol, 0.49 acetate, 1.39 CO₂, 1.05 H₂, and 0.09 butyrate.National Research Council of Canada No. 23406     

Development and application of co-culture for ethanol production by co-fermentation of glucose and xylose: a systematic review

This article reviews current co-culture systems for fermenting mixtures of glucose and xylose to ethanol. Thirty-five co-culture systems that ferment either synthetic glucose and xylose mixture or various biomass hydrolysates are examined. Strain combinations, fermentation modes and conditions, and fermentation performance for these co-culture systems are compared and discussed. It is noted that the combination of Pichia stipitis with Saccharomyces cerevisiae or its respiratory-deficient mutant is most commonly used. One of the best results for fermentation of glucose and xylose mixture is achieved by using co-culture of immobilized Zymomonas mobilis and free cells of P. stipitis, giving volumetric ethanol production of 1.277 g/l/h and ethanol yield of 0.49–0.50 g/g. The review discloses that, as a strategy for efficient conversion of glucose and xylose, co-culture fermentation for ethanol production from lignocellulosic biomass can increase ethanol yield and production rate, shorten fermentation time, and reduce process costs, and it is a promising technology although immature.     

A C6/C5 co‐fermenting Saccharomyces cerevisiae strain with the alleviation of antagonism between xylose utilization and robustness

During second‐generation bioethanol production from lignocellulosic biomass, the desired traits for fermenting microorganisms, such as Saccharomyces cerevisiae, are high xylose utilization and high robustness to inhibitors in lignocellulosic hydrolysates. However, as observed previously, these two traits easily showed the antagonism, one rising and the other falling, in the C6/C5 co‐fermenting S. cerevisiae strain. In this study, LF1 obtained in our previous study is an engineered budding yeast strain with a superior co‐fermentation capacity of glucose and xylose, and was then mutated by atmospheric and room temperature plasma (ARTP) mutagenesis to improve its robustness. The ARTP‐treated cells were grown in 50% (v/v) leachate from lignocellulose pretreatment with high inhibitors content for adaptive evolution. After 30 days, the generated mutant LF1‐6 showed significantly enhanced tolerance, with a six‐fold increase in cell density in the above leachate. Unfortunately, its xylose utilization dropped markedly, indicating the recurrence of the negative correlation between xylose utilization and robustness. To alleviate this antagonism, LF1‐6 cells were iteratively mutated with ARTP mutagenesis and then anaerobically grown using xylose as the sole carbon source, and xylose utilization was restored in the resulting strain 6M‐15. 6M‐15 also exhibited increased co‐fermentation performance of xylose and glucose with the highest ethanol productivity reported to date (0.525 g g^?1 h^?1) in high‐level mixed sugars (80 g L^?1 glucose and 40 g L^?1 xylose) with no inhibitors. Meanwhile, its fermentation time was shortened by 8 h compared to that of LF1. During the fermentation of non‐detoxified lignocellulosic hydrolysate with high inhibitor concentrations at pH ~3.5, 6M‐15 can efficiently convert glucose and xylose with an ethanol yield of 0.43 g g^?1. 6M‐15 is also regarded as a potential chassis cell for further design of a customized strain suitable for production of second‐generation bioethanol or other high value‐added products from lignocellulosic biomass.     

A genome shuffling-generated <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> isolate that ferments xylose and glucose to produce high levels of ethanol

Genome shuffling is an efficient approach for the rapid improvement of industrially important microbial phenotypes. This report describes optimized conditions for protoplast preparation, regeneration, inactivation, and fusion using the Saccharomyces cerevisiae W5 strain. Ethanol production was confirmed by TTC (triphenyl tetrazolium chloride) screening and high-performance liquid chromatography (HPLC). A genetically stable, high ethanol-producing strain that fermented xylose and glucose was obtained following three rounds of genome shuffling. After fermentation for 84 h, the high ethanol-producing S. cerevisiae GS3-10 strain (which utilized 69.48 and 100% of the xylose and glucose stores, respectively) produced 26.65 g/L ethanol, i.e., 47.08% higher than ethanol production by S. cerevisiae W5 (18.12 g/L). The utilization ratios of xylose and glucose were 69.48 and 100%, compared to 14.83 and 100% for W5, respectively. The ethanol yield was 0.40 g/g (ethanol/consumed glucose and xylose), i.e., 17.65% higher than the yield by S. cerevisiae W5 (0.34 g/g).     

Engineering of ethanolic E. coli with the Vitreoscilla hemoglobin gene enhances ethanol production from both glucose and xylose

Escherichia coli strain FBR5, which has been engineered to direct fermentation of sugars to ethanol, was further engineered, using three different constructs, to contain and express the Vitreoscilla hemoglobin gene (vgb). The three resulting strains expressed Vitreoscilla hemoglobin (VHb) at various levels, and the production of ethanol was inversely proportional to the VHb level. High levels of VHb were correlated with an inhibition of ethanol production; however, the strain (TS3) with the lowest VHb expression (approximately the normal induced level in Vitreoscilla) produced, under microaerobic conditions in shake flasks, more ethanol than the parental strain (FBR5) with glucose, xylose, or corn stover hydrolysate as the predominant carbon source. Ethanol production was dependent on growth conditions, but increases were as high as 30%, 119%, and 59% for glucose, xylose, and corn stover hydrolysate, respectively. Only in the case of glucose, however, was the theoretical yield of ethanol by TS3 greater than that achieved by others with FBR5 grown under more closely controlled conditions. TS3 had no advantage over FBR5 regarding ethanol production from arabinose. In 2 L fermentors, TS3 produced about 10% and 15% more ethanol than FBR5 for growth on glucose and xylose, respectively. The results suggest that engineering of microorganisms with vgb/VHb could be of significant use in enhancing biological production of ethanol.     

Characterization of the impact of acetate and lactate on ethanolic fermentation by Thermoanaerobacter ethanolicus

Ethanolic fermentation of simple sugars is an important step in the production of bioethanol as a renewable fuel. Significant levels of organic acids, which are generally considered inhibitory to microbial metabolism, could be accumulated during ethanolic fermentation, either as a fermentation product or as a by-product generated from pre-treatment steps. To study the impact of elevated concentrations of organic acids on ethanol production, varying levels of exogenous acetate or lactate were added into cultures of Thermoanaerobacter ethanolicus strain 39E with glucose, xylose or cellobiose as the sole fermentation substrate. Our results found that lactate was in general inhibitory to ethanolic fermentation by strain 39E. However, the addition of acetate showed an unexpected stimulatory effect on ethanolic fermentation of sugars by strain 39E, enhancing ethanol production by up to 394%. Similar stimulatory effects of acetate were also evident in two other ethanologens tested, T. ethanolicus X514, and Clostridium thermocellum ATCC 27405, suggesting the potentially broad occurrence of acetate stimulation of ethanolic fermentation. Analysis of fermentation end product profiles further indicated that the uptake of exogenous acetate as a carbon source might contribute to the improved ethanol yield when 0.1% (w/v) yeast extract was added as a nutrient supplement. In contrast, when yeast extract was omitted, increases in sugar utilization appeared to be the likely cause of higher ethanol yields, suggesting that the characteristics of acetate stimulation were growth condition-dependent. Further understanding of the physiological and metabolic basis of the acetate stimulation effect is warranted for its potential application in improving bioethanol fermentation processes.     

Isolation and preliminary characterization of a Zymomonas mobilis mutant with an altered preference for xylose and glucose utilization

The narrow substrate range of Zymomonas mobilis CP4 has been extended previously to include metabolism of the pentose sugar, xylose, by Zhang et al. (Science 267: 240–243). The strain CP4(pZB5) co-ferments both glucose and xylose in mixed sugar fermentations, however glucose is utilized preferentially. The present work reports the isolation of a new mutant from CP4(pZB5) which displays an altered carbon substrate preference. The mutant, CP4(pZB5) M1-2, metabolizes xylose more rapidly than glucose in mixed glucose/xylose media. Sequence data analysis revealed mutations in both the glucose facilitator (glf) and glucokinase (glk) genes.