Mixture Of Xylose Research Articles

Characterization of Bioethanol Production from Hexoses and Xylose by the White Rot Fungus Trametes versicolor

Bioethanol production by white rot fungus (Trametes versicolor), identified from fungal mixture in naturally decomposing wood samples, from hexoses and xylose was characterized. Results showed that T. versicolor can grow in culture, under hypoxic conditions, with various mixtures of hexoses and xylose and only xylose. Xylose was efficiently fermented to ethanol in media containing mixtures of hexoses and xylose, such as MBMC and G11XY11 media (Table 1), yielding ethanol concentrations of 20.0 and 9.02 g/l, respectively, after 354 h of hypoxic culture. Very strong correlations were found between ethanolic fermentation (alcohol dehydrogenase activity and ethanol production), sugar consumption and xylose catabolism (xylose reductase, xylitol dehydrogenase and xylulokinase activities) after 354 h in culture in MBMC medium. In a medium (G11XY11) containing a 1:1 glucose/xylose ratio, fermentation efficiency of total sugars into ethanol was 80% after 354 h.

Bioethanol production from rice straw by a sequential use of Saccharomyces cerevisiae and Pichia stipitis with heat inactivation of Saccharomyces cerevisiae cells prior to xylose fermentation

In order to establish an efficient bioethanol production system from rice straw, a new strategy to ferment the mixture of glucose and xylose by a sequential application of Saccharomyces cerevisiae and Pichia stipitis was developed, in which heat inactivation of S. cerevisiae cells before addition of P. stipitis was employed. The results showed that heating at 50°C for 6h was sufficient to give high xylose fermentation efficiency. By application of the inactivation process, 85% of the theoretical yield was achieved in the fermentation of the synthetic medium. At the same time, the xylitol production was reduced by 42.4% of the control process. In the simultaneous saccharification and fermentation of the lime-pretreated and CO(2)-neutralized rice straw, the inactivation of S. cerevisiae cells enabled the full conversion of glucose and xylose within 80 h. Finally, 21.1g/l of ethanol was produced from 10% (w/w) of pretreated rice straw and the ethanol yield of rice straw reached 72.5% of the theoretical yield. This process is expected to be useful for the ethanol production from lignocellulosic materials in the regions where large-scale application of recombinant microorganisms was restricted.

Improved NADPH supply for xylitol production by engineered Escherichia coli with glycolytic mutations

Escherichia coli engineered to uptake xylose while metabolizing glucose was previously shown to produce high levels of xylitol from a mixture of glucose and xylose when expressing NADPH-dependent xylose reductase from Candida boidinii (CbXR) (Cirino et al., Biotechnol Bioeng. 2006;95:1167-1176). We then described the effects of deletions of key metabolic pathways (e.g., Embden-Meyerhof-Parnas and pentose phosphate pathway) and reactions (e.g., transhydrogenase and NADH dehydrogenase) on resting-cell xylitol yield (Y RPG: moles of xylitol produced per mole of glucose consumed) (Chin et al., Biotechnol Bioeng. 2009;102:209-220). These prior results demonstrated the importance of direct NADPH supply by NADP+-utilizing enzymes in central metabolism for driving heterologous NADPH-dependent reactions. This study describes strain modifications that improve coupling between glucose catabolism (oxidation) and xylose reduction using two fundamentally different strategies. We first examined the effects of deleting the phosphofructokinase (pfk) gene(s) on growth-uncoupled xylitol production and found that deleting both pfkA and sthA (encoding the E. coli-soluble transhydrogenase) improved the xylitol Y RPG from 3.4 ± 0.6 to 5.4 ± 0.4. The second strategy focused on coupling aerobic growth on glucose to xylitol production by deleting pgi (encoding phosphoglucose isomerase) and sthA. Impaired growth due to imbalanced NADPH metabolism (Sauer et al., J Biol Chem. 2004;279:6613-6619) was alleviated upon expressing CbXR, resulting in xylitol production similar to that of the growth-uncoupled precursor strains but with much less acetate secretion and more efficient utilization of glucose. Intracellular nicotinamide cofactor levels were also quantified, and the magnitude of the change in the NADPH/NADP+ ratio measured from cells consuming glucose in the absence vs. presence of xylose showed a strong correlation to the resulting Y RPG.

Metabolic engineering of Corynebacterium glutamicum for production of 1,5‐diaminopentane from hemicellulose

In the present work, the bio-based production of 1,5-diaminopentane (cadaverine), an important building block for bio-polyamides, was extended to hemicellulose a non-food raw material. For this purpose, the metabolism of 1,5-diaminopentane-producing Corynebacterium glutamicum was engineered to the use of the C(5) sugar xylose. This was realized by heterologous expression of the xylA and xylB genes from Escherichia coli, mediating the conversion of xylose into xylulose 5-phosphate (an intermediate of the pentose phosphate pathway), in a defined diaminopentane-producing C. glutamicum strain, recently obtained by systems metabolic engineering. The created mutant, C. glutamicum DAP-Xyl1, exhibited efficient production of the diamine from xylose and from mixtures of xylose and glucose. Subsequently, the novel strain was tested on industrially relevant hemicellulose fractions, mainly containing xylose and glucose as carbon source. A two-step process was developed, comprising (i) enzymatic hydrolysis of hemicellulose from dried oat spelts, and (ii) biotechnological 1,5-diaminopentane production from the obtained hydrolysates with the novel C. glutamicum strain. This now opens a future avenue towards bio-based 1,5-diaminopentane and bio-polyamides thereof from non-food raw materials.

Production of Astaxanthin from Cellulosic Biomass Sugars by Mutants of the Yeast Phaffia rhodozyma

Astaxanthin is a potential high-value coproduct in an ethanol biorefinery. Three mutant strains of the astaxanthin-producing yeast Phaffia rhodozyma, which were derived from the parent strain ATCC 24202 (UCD 67-210) and designated JTM166, JTM185, and SSM19, were tested for their capability of utilizing the major sugars that can be generated from cellulosic biomass, including glucose, xylose, and arabinose, for astaxanthin production. While all three strains were capable of metabolizing these sugars, individually and in mixtures, JTM185 demonstrated the greatest sugar utilization and astaxanthin production. Astaxanthin yield by this strain (milligrams astaxanthin per gram of sugar consumed) was highest for xylose, followed by arabinose and then glucose. The kinetics of sugar utilization by strain JTM185 was studied in fermenters using mixtures of glucose, xylose, and arabinose at varied concentrations. It was found that glucose was utilized preferentially, followed by xylose, and lastly, arabinose. Astaxanthin yield was significantly affected by sugar concentrations. Highest yields were observed with sugar mixtures containing the highest concentrations of xylose and arabinose. Hydrolysates produced from sugarcane bagasse and barley straw pretreated by the soaking in aqueous ammonia method and hydrolyzed with the commercial cellulase preparation, Accellerase™ 1000, were used for astaxanthin production by the mutant strain JTM185. The organism was capable of metabolizing all of the sugars present in the hydrolysates from both biomass sources and produced similar amounts of astaxanthin from both hydrolysates, although these amounts were lower when compared to yields obtained with reagent grade sugars.

Absence of Diauxie during Simultaneous Utilization of Glucose and Xylose bySulfolobus acidocaldarius

Sulfolobus acidocaldarius utilizes glucose and xylose as sole carbon sources, but its ability to metabolize these sugars simultaneously is not known. We report the absence of diauxie during growth of S. acidocaldarius on glucose and xylose as co-carbon sources. The presence of glucose did not repress xylose utilization. The organism utilized a mixture of 1 g/liter of each sugar simultaneously with a specific growth rate of 0.079 h(-1) and showed no preference for the order in which it utilized each sugar. The organism grew faster on 2 g/liter xylose (0.074 h(-1)) as the sole carbon source than on an equal amount of glucose (0.022 h(-1)). When grown on a mixture of the two carbon sources, the growth rate of the organism increased from 0.052 h(-1) to 0.085 h(-1) as the ratio of xylose to glucose increased from 0.25 to 4. S. acidocaldarius appeared to utilize a mixture of glucose and xylose at a rate roughly proportional to their concentrations in the medium, resulting in complete utilization of both sugars at about the same time. Gene expression in cells grown on xylose alone was very similar to that in cells grown on a mixture of xylose and glucose and substantially different from that in cells grown on glucose alone. The mechanism by which the organism utilized a mixture of sugars has yet to be elucidated.

Microbial removal of acetate selectively from sugar mixtures

Acetic acid is an unavoidable constituent of the biomass hydrolysates generated from acetylated hemicellulose and lignin, and acetate affects the performance of microbes used to convert these hydrolysates into biofuels or other biochemicals. In this study, acetate was selectively removed from synthetic mixtures of glucose and xylose using metabolically engineered Escherichia coli strains having mutations in the glucose phosphotransferase system (PTS) genes (ptsG, manZ, crr), glucokinase (glk), and xylose (xylA). In batch culture, ALS1060 (ptsG manZ glk xylA) consumed exclusively acetate to depletion, and then consumed the two sugars only at a very slow rate (a growth rate of about 0.01 h(-1)). We also examined the effects of an additional knockout of either malX, fruA, fruB, bglF, or crr, genes that are involved in other PTSs, and a batch process using KD840 (ptsG manZ glk crr xylA) demonstrated a further reduction in glucose or xylose consumption by E. coli. These results demonstrate the feasibility of using a substrate-selective approach for the pre-treatment of biomass hydrolysate for microbial processes.

Renewable gasoline from aqueous phase hydrodeoxygenation of aqueous sugar solutions prepared by hydrolysis of maple wood

In this paper we demonstrate an integrated process for the production of high octane gasoline from maple wood by hydrolysis of maple wood into aqueous carbohydrate solutions followed by aqueous phase hydrodeoxygenation of the sugar solutions. The aqueous carbohydrate solutions were prepared by both hydrolysis in hot water and hydrolysis with dilute acids (H2SO4, oxalic acid). The aqueous carbohydrate solutions were a mixture of xylose, water soluble hemicellulose oligomers, acetic acid, glucose, glucose oligomers, and probably some lignin polymers. Hydrolysis with hot water produced primarily hemicellulose oligomers whereas hydrolysis with acids produced mainly xylose and acetic acid. The hydrolysis co-product was a solid enriched in cellulose and lignin. The aqueous streams were hydrodeoxygenated by a two step catalytic process in which the first catalyst bed contained a Ru/C catalyst at 393 K and the second catalyst bed contained a Pt/zirconium phosphate (Pt/ZrP) catalyst at 518 K. The Ru/C catalyst was able to selectively hydrogenate xylose into xylitol but could not selectively hydrogenate the xylose oligomers. The two stage process was able to convert the aqueous carbohydrate streams derived from maple wood into gasoline range products with carbon yields of up to 57% and an estimated octane number of 96.5. No significant catalyst deactivation was observed indicating that the catalysts are very stable. The highest gasoline yield from this two stage process was obtained from the stream produced by acid hydrolysis of maple wood with 0.5 wt% oxalic acid at 433 K for 0.5 h. These results suggest that aqueous phase processing of sugars obtained by hydrolysis is a promising option for the production of fuels and chemicals from lignocellulosic biomass.

Biofuel policies under pressure

The US position on biofuels is criticised but there is still optimism in future potential. Nigel Williams reports. The US position on biofuels is criticised but there is still optimism in future potential. Nigel Williams reports. The US has attracted many critics for its policy on biofuels, particularly the use of crops that otherwise could produce food, and does little to combat the emission of greenhouse gases. The biofuels policy took a hit last month when 17 senators signed a letter calling ethanol ‘fiscally indefensible’ and ‘environmentally unwise’. Led by Democrat Dianne Feinstein and Republican Jon Kyl, the group argued that Congress shouldn't extend certain subsidies that were due to expire at the end of last year, including the 45-cent-per-gallon tax credit for blending ethanol into gasoline and tariffs on cheaper imports. Conservatives dislike the costly industrial policy, while liberals are against the hefty carbon emissions that come with corn fuels, the Wall Street Journal reported. And energy secretary, Steven Chu, told a meeting of the National Press Club last month that “ethanol is not an ideal transportation fuel,” and that the government's focus should be on ways “that we can actually go beyond ethanol”. But efforts are under way worldwide to produce ethanol more cheaply and efficiently, and in ways that minimise the impact of carbon emissions. In Denmark, as elsewhere, companies see a big business potential in helping to curb the world's reliance on petrochemicals. Two companies in the country produce 70 per cent of the world's market for enzymes that go into making second-generation ethanol and are collaborating with another company to manufacture a biofuel to be sold across Denmark. The new fuel blend, which costs about 10 US cents more per gallon than regular fuel, has been sold since last October by around 100 fuel stations. The new fuel is a blend of petroleum and second-generation ethanol. If the experiment succeeds, it could become part of a business worth billions of dollars, say energy analysts. The two companies, Danisco and Novoenzymes, supply enzymes to the producer, Inbicon, which produces ethanol at a plant in western Zealand. Whereas first-generation ethanol is produced from plant products such as corn, wheat and sugar beet that could otherwise be used for food or animal feed, second-generation ethanol is made from agricultural waste materials — straw, husks and leaves — and therefore avoids any food-versus-fuel controversy. Tom Knutzen, Danisco's chief executive, believes developing second-generation ethanol will pay huge business dividends. The two most common forms of biofuels are biodiesel and ethanol. Biodiesel is made by combining alcohol with vegetable oils or animal fats and recycled cooking oils and remains the fuel of choice in Europe, while ethanol is widely used in the US. As a result, Danisco wants to establish a significant presence in the US market. The demand for second-generation ethanol is also driving research into more efficient production. Many companies are investing in research and new facilities. And a new report in the Proceedings of the National Academy of Sciences (published online) describes a genetically modified yeast cell able to simultaneously ferment both cellobiose and xylose into ethanol. Suk-Jin Ha at the University of Illinois, Urbana, and colleagues at the University of California, Berkeley and Seoul National University, Korea, describe the need for an efficient means of producing fuel from the plant waste matter, lignocellulose. The use of plant biomass for fuel production will require efficient utilization of the sugars in this material, they say, primarily glucose and xylose. “However, strains of Saccharomyces cerevisiae presently ferment glucose but not xylose,” they write. But the researchers describe engineered yeasts that are able to co-ferment mixtures of xylose and cellobiose with improved ethanol yield when compared to fermentation with either cellobiose or xylose as sole carbon sources. “The successful integration of cellobiose and xylose fermentation pathways in yeast is a critical step towards enabling economic biofuel production,” they write, as many new approaches to ethanol production are under scrutiny. Such potential is timely but not enough for the disgruntled senators. “Historically our government has helped a product compete in one of three ways: subsidize it, protect it from competition, or require its use. We understand that ethanol may be the only product receiving all three forms of support from the US government at this time.”

Development of Grilled-type Shrimp Flavor by Maillard Reaction and Sensory Evaluation

A grilled-type shrimp flavor was developed through the Maillard reaction to reduce or mask fish odor or off-flavor in seafood. Model systems were created by using enzymatic hydrolysate of shrimp and adding precursors to increase flavor quality and stability. Amino acid precursors such as cysteine and methionine, sugar precursors such as glucose, xylose, ribose, and sucrose, and one particular compound of glucosamine were tried and their flavor qualities were tested by sensory evaluation. Also, the optimum reaction condition was investigated using the pH values of pH 5, 6, 7, and 8 with reaction times of 1 hr, 2 hr and 3 hr after the best precursors were determined. The best condition of the precursors for grilled-type shrimp flavor was the mixtures of methionine, threonine, xylose, and glucosamine. The optimum reaction condition was at pH 8.0 and 2 hr reaction time.

Engineered Saccharomyces cerevisiae capable of simultaneous cellobiose and xylose fermentation

The use of plant biomass for biofuel production will require efficient utilization of the sugars in lignocellulose, primarily glucose and xylose. However, strains of Saccharomyces cerevisiae presently used in bioethanol production ferment glucose but not xylose. Yeasts engineered to ferment xylose do so slowly, and cannot utilize xylose until glucose is completely consumed. To overcome these bottlenecks, we engineered yeasts to coferment mixtures of xylose and cellobiose. In these yeast strains, hydrolysis of cellobiose takes place inside yeast cells through the action of an intracellular β-glucosidase following import by a high-affinity cellodextrin transporter. Intracellular hydrolysis of cellobiose minimizes glucose repression of xylose fermentation allowing coconsumption of cellobiose and xylose. The resulting yeast strains, cofermented cellobiose and xylose simultaneously and exhibited improved ethanol yield when compared to fermentation with either cellobiose or xylose as sole carbon sources. We also observed improved yields and productivities from cofermentation experiments performed with simulated cellulosic hydrolyzates, suggesting this is a promising cofermentation strategy for cellulosic biofuel production. The successful integration of cellobiose and xylose fermentation pathways in yeast is a critical step towards enabling economic biofuel production.

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.

Dynamic flux balance modeling of microbial co‐cultures for efficient batch fermentation of glucose and xylose mixtures

Sequential uptake of pentose and hexose sugars that compose lignocellulosic biomass limits the ability of pure microbial cultures to efficiently produce value-added bioproducts. In this work, we used dynamic flux balance modeling to examine the capability of mixed cultures of substrate-selective microbes to improve the utilization of glucose/xylose mixtures and to convert these mixed substrates into products. Co-culture simulations of Escherichia coli strains ALS1008 and ZSC113, engineered for glucose and xylose only uptake respectively, indicated that improvements in batch substrate consumption observed in previous experimental studies resulted primarily from an increase in ZSC113 xylose uptake relative to wild-type E. coli. The E. coli strain ZSC113 engineered for the elimination of glucose uptake was computationally co-cultured with wild-type Saccharomyces cerevisiae, which can only metabolize glucose, to determine if the co-culture was capable of enhanced ethanol production compared to pure cultures of wild-type E. coli and the S. cerevisiae strain RWB218 engineered for combined glucose and xylose uptake. Under the simplifying assumption that both microbes grow optimally under common environmental conditions, optimization of the strain inoculum and the aerobic to anaerobic switching time produced an almost twofold increase in ethanol productivity over the pure cultures. To examine the effect of reduced strain growth rates at non-optimal pH and temperature values, a break even analysis was performed to determine possible reductions in individual strain substrate uptake rates that resulted in the same predicted ethanol productivity as the best pure culture.

Elimination of carbon catabolite repression in Klebsiella oxytoca for efficient 2,3-butanediol production from glucose–xylose mixtures

Microbial preference for glucose implies incomplete and/or slow utilization of lignocellulose hydrolysates, which is caused by the regulatory mechanism named carbon catabolite repression (CCR). In this study, a 2,3-butanediol (2,3-BD) producing Klebsiella oxytoca strain was engineered to eliminate glucose repression of xylose utilization. The crp(in) gene, encoding the mutant cyclic adenosine monophosphate (cAMP) receptor protein CRP(in), which does not require cAMP for functioning, was characterized and overexpressed in K. oxytoca. The engineered recombinant could utilize a mixture of glucose and xylose simultaneously, without CCR. The profiles of sugar consumption and 2,3-BD production by the engineered recombinant, in glucose and xylose mixtures, were examined and showed that glucose and xylose could be consumed simultaneously to produce 2,3-BD. This study offers a metabolic engineering strategy to achieve highly efficient utilization of sugar mixtures derived from the lignocellulosic biomass for the production of bio-based chemicals using enteric bacteria.

Repeated-batch fermentations of xylose and glucose–xylose mixtures using a respiration-deficient Saccharomyces cerevisiae engineered for xylose metabolism

Xylose-fermenting Saccharomyces strains are needed for commercialization of ethanol production from lignocellulosic biomass. Engineered Saccharomyces cerevisiae strains expressing XYL1, XYL2 and XYL3 from Pichia stipitis, however, utilize xylose in an oxidative manner, which results in significantly lower ethanol yields from xylose as compared to glucose. As such, we hypothesized that reconfiguration of xylose metabolism from oxidative into fermentative manner might lead to efficient ethanol production from xylose. To this end, we generated a respiration-deficient (RD) mutant in order to enforce engineered S. cerevisiae to utilize xylose only through fermentative metabolic routes. Three different repeated-batch fermentations were performed to characterize characteristics of the respiration-deficient mutant. When fermenting glucose as a sole carbon source, the RD mutant exhibited near theoretical ethanol yields (0.46 g g −1) during repeated-batch fermentations by recycling the cells. As the repeated-batch fermentation progressed, the volumetric ethanol productivity increased (from 7.5 to 8.3 g L −1 h −1) because of the increased biomass from previous cultures. On the contrary, the mutant showed decreasing volumetric ethanol productivities during the repeated-batch fermentations using xylose as sole carbon source (from 0.4 to 0.3 g L −1 h −1). The mutant did not grow on xylose and lost fermenting ability gradually, indicating that the RD mutant cannot maintain a good fermenting ability on xylose as a sole carbon source. However, the RD mutant was capable of fermenting a mixture of glucose and xylose with stable yields (0.35 g g −1) and productivities (0.52 g L −1 h −1) during the repeated-batch fermentation. In addition, ethanol yields from xylose during the mixed sugar fermentation (0.30 g g −1) were higher than ethanol yields from xylose as a sole carbon source (0.21 g g −1). These results suggest that a strategy for increasing ethanol yield through respiration-deficiency can be applied for the fermentation of lignocellulosic hydrolyzates containing glucose and xylose.

High efficiency hydrogen production from glucose/xylose by the ldh-deleted Thermoanaerobacterium strain

A strictly anaerobic, thermoacidophilic, H(2)-producing bacterium was isolated and designated as Thermoanaerobacterium aotearoense. The optimized cultivation conditions for H(2) production are 55 degrees C, pH 6.5 and 10gl(-1) of glucose or xylose. A metabolic pathway analysis showed that lactate occupied most of the liquid metabolites and consumed a large amount of NADH. To increase the efficiency of hydrogen production, the gene encoding the l-lactate dehydrogenase was knocked out to redirect the NADH flow. Genetic manipulation resulted in the 2 and 2.5 folds increase of the H(2) yield and production rate, respectively. The maximum H(2) yields using the Deltaldh mutant were 2.71, 1.45 and 2.28molH(2)mol(-1) sugar under glucose, xylose and glucose/xylose mixture tests, respectively. The recombinant Deltaldh strain could ferment the mixture of glucose and xylose to produce H(2) effectively, indicating that the performance of Thermoanaerobacterium in H(2) production can be significantly improved by metabolic engineering technique.

Co-fermentation of cellobiose and xylose using beta-glucosidase displaying diploid industrial yeast strain OC-2

The co-utilization of sugars, particularly xylose and glucose, during industrial fermentation is essential for economically feasible processes with high ethanol productivity. However, the major problem encountered during xylose/glucose co-fermentation is the lower consumption rate of xylose compared with that of glucose fermentation. Here, we therefore attempted to construct high xylose assimilation yeast by using industrial yeast strain with high beta-glucosidase activity on the cell surface. We first constructed the triple auxotrophic industrial strain OC2-HUT and introduced four copies of the cell-surface-displaying beta-glucosidase (BGL) gene and two copies of a xylose-assimilating gene into its genome to generate strain OC2-ABGL4Xyl2. It was confirmed that the introduction of multiple copies of the BGL gene increased the cell-surface BGL activity, which was also correlated to the observed increase in xylose-assimilating ability. The strain OC2-ABGL4Xyl2 was able to consume xylose during cellobiose/xylose co-fermentation (0.38 g/h/g-DW) more rapidly than during glucose/xylose co-fermentation (0.18 g/h/g-DW). After 48 h, 5.77% of the xylose was consumed despite the co-fermentation conditions, and the observed ethanol yield was 0.39 g-ethanol/g-total sugar. Our results demonstrate that a BGL-displaying and xylose-assimilating industrial yeast strain is capable of efficient xylose consumption during the co-fermentation with cellobiose. Due to its high performance for fermentation of mixtures of cellobiose and xylose, OC2-ABGL4Xyl2 does not require the addition of beta-glucosidase and is therefore a promising yeast strain for cost-effective ethanol production from lignocellulosic biomass.

Effect of primary degradation–reaction products from Ammonia Fiber Expansion (AFEX)-treated corn stover on the growth and fermentation of Escherichia coli KO11

The primary degradation–reaction products (DRP) identified in Ammonia Fiber Expansion (AFEX)-pretreated corn stover are acetate, lactate, 4-hydroxybenzaldehyde (4HBD) and acetamide. The effects of these products at a broad concentration range were tested on Escherichia coli KO11, a strain engineered for cellulosic ethanol production. Fermentations using glucose or xylose as the sole carbohydrate source and a sugar mixture of glucose and xylose were conducted to determine how these products and sugar selection affected fermentation performance. Co-fermentation of the sugar mixture exhibited the lowest overall ethanol productivity compared to single-sugar fermentations and was more susceptible to inhibition. Metabolic ethanol yield increased with the increasing initial concentration of acetate. Although these degradation–reaction products (with exception of acetamide) are generally perceived to be inhibitory, organic acids and 4-hydroxybenzaldehyde at low levels stimulated fermentation. Adaptation of cells to these products prior to fermentation increased overall fermentation rate.

Cell growth and hydrogen production on the mixture of xylose and glucose using a novel strain of Clostridium sp. HR-1 isolated from cow dung compost

A novel mesophilic hydrogen-producing bacterium was isolated from cow dung compost and designated as Clostridium sp. HR-1 by 16S rRNA gene sequence. The optimum condition for hydrogen production by strain HR-1 was pH of 6.5, temperature of 37 °C and yeast extract as nitrogen sources. The strain HR-1 has the ability to utilize kinds of hexose and pentose as carbon sources for growth and H 2 production. Cell growth and hydrogen productivity were investigated for batch fermentation on media containing different ratios of xylose and glucose. Glucose was the preferred substrate in the glucose and xylose mixtures. The high glucose fraction had higher cell biomass production rate. The rate of glucose consumption was higher than xylose consumption, and remained essentially constant independent of xylose content of the mixture. The rate of xylose utilization was decreased with increasing of the glucose fraction. The average H 2 yield and specific H 2 production rates with xylose and glucose are 1.63 mol-H 2/mol xylose and 11.14-H 2 mmol/h g-cdw, and 2.02 mol-H 2/mol-glucose and 9.37 mmol-H 2/h g-cdw, respectively. Using the same initial substrate concentration, the maximum average H 2 yield and specific H 2 production rates with the mixtures of 9 g/l xylose and 3 g/l glucose was 2.01 mol-H 2/mol-mixed sugar and 12.56 mmol-H 2/h g-cdw, respectively. During the fermentation, the main soluble microbial products were ethanol and acetate which showed trends with the different ratios of xylose and glucose.

An evolved xylose transporter from Zymomonas mobilis enhances sugar transport in Escherichia coli

BackgroundXylose is a second most abundant sugar component of lignocellulose besides glucose. Efficient fermentation of xylose is important for the economics of biomass-based biorefineries. However, sugar mixtures are sequentially consumed in xylose co-fermentation with glucose due to carbon catabolite repression (CCR) in microorganisms. As xylose transmembrance transport is one of the steps repressed by CCR, it is therefore of interest to develop a transporter that is less sensitive to the glucose inhibition or CCR.ResultsThe glucose facilitator protein Glf transporter from Zymomonas mobilis, also an efficient transporter for xylose, was chosen as the target transporter for engineering to eliminate glucose inhibition on xylose uptake. The evolution of Glf transporter was carried out with a mixture of glucose and xylose in E. coli. Error-prone PCR and random deletion were employed respectively in two rounds of evolution. Aided by a high-throughput screening assay using xylose analog p-nitrophenyl-β-D-xylopyranoside (pNPX) in 96-well plates, a best mutant 2-RD5 was obtained that contains several mutations, and a deletion of 134 residues (about 28% of total residues), or three fewer transmembrane sections (TMSs). It showed a 10.8-fold improvement in terms of pNPX transport activity in the presence of glucose. The fermentation performance results showed that this mutant improved xylose consumption by 42% with M9 minimal medium containing 20 g L-1 xylose only, while with the mixture sugar of xylose and glucose, 28% more glucose was consumed, but no obvious co-utilization of xylose was observed. Further glucose fed-batch experiments suggested that the intracellular metabolism of xylose was repressed by glucose.ConclusionsThrough random mutagenesis and partial deletion coupled with high-throughput screening, a mutant of the Glf transporter (2-RD5) was obtained that relieved the inhibition of xylose transport by glucose. The fermentation tests revealed that 2-RD5 was advantageous in xylose and glucose uptakes, while no obvious advantage was seen for xylose co-consumption when co-fermented with glucose. Further efforts could focus on reducing CCR-mediated repression of intracellular metabolism of xylose. Glf should also serve as a useful model to further exploit the molecular mechanism of xylose transport and the CCR-mediated inhibition.