Xylose Medium Research Articles

Production of poly-γ-glutamic acid (γ-PGA) from xylose-glucose mixtures by Bacillus amyloliquefaciens C1.

Due to the promising applications, the demand to enhance poly-γ-glutamic acid (γ-PGA) production while decreasing the cost has increased in the past decade. Here, xylose/glucose mixture and corncob hydrolysate (CCH) was evaluated as alternatives for γ-PGA production by Bacillus amyloliquefaciens C1. Although both have been validated to support cell growth, glucose and xylose were not simutaneously consumed and exhibited a diauxic growth pattern due to carbon catabolite repression (CCR) in B. amyloliquefaciens C1, while the enhanced transcription of araE alleviated the xylose transport bottleneck across a cellular membrane. Additionally, the xyl operon (xylA and xylB), which was responsible for xylose metabolism, was strongly induced by xylose at the transcriptional level. When cultured in a mixed medium, xylR was sharply induced to 3.39-folds during the first 8-h while reduced to the base level similar to that in xylose medium. Finally, pre-treated CCH mainly contained a mixture of glucose and xylose was employed for γ-PGA fermentation, which obtained a final concentration of 6.56 ± 0.27g/L. Although the glucose utilization rate (84.91 ± 1.81%) was lower than that with chemical substrates, the xylose utilization rate (43.41 ± 2.14%) and the sodium glutamate conversion rate (77.22%) of CCH were acceptable. Our study provided a promising approach for the green production of γ-PGA from lignocellulosic biomass and circumvent excessive non-food usage of glucose.

Optimized Bioconversion of Xylose Derived from Pre-Treated Crop Residues into Xylitol by Using Candida boidinii

Crop residues can serve as low-cost feedstocks for microbial production of xylitol, which offers many advantages over the commonly used chemical process. However, enhancing the efficiency of xylitol fermentation is still a barrier to industrial implementation. In this study, the effects of oxygen transfer rate (OTR) (1.1, 2.1, 3.1 mmol O2/(L × h)) and initial xylose concentration (30, 55, 80 g/L) on xylitol production of Candida boidinii NCAIM Y.01308 on xylose medium were investigated and optimised by response surface methodology, and xylitol fermentations were performed on xylose-rich hydrolysates of wheat bran and rice straw. High values of maximum xylitol yields (58–63%) were achieved at low initial xylose concentration (20–30 g/L) and OTR values (1.1–1.5 mmol O2/(L × h)). The highest value for maximum xylitol productivity (0.96 g/(L × h)) was predicted at 71 g/L initial xylose and 2.7 mmol O2/(L × h) OTR. Maximum xylitol yield and productivity obtained on wheat bran hydrolysate were 60% and 0.58 g/(L × h), respectively. On detoxified and supplemented hydrolysate of rice straw, maximum xylitol yield and productivity of 30% and 0.19 g/(L × h) were achieved. This study revealed the terms affecting the xylitol production by C. boidinii and provided validated models to predict the achievable xylitol yields and productivities under different conditions. Efficient pre-treatments for xylose-rich hydrolysates from rice straw and wheat bran were selected. Fermentation using wheat bran hydrolysate and C. boidinii under optimized condition is proved as a promising method for biotechnological xylitol production.

Optimization with Response Surface Methodology of Microwave-Assisted Conversion of Xylose to Furfural.

The production of furfural from renewable sources, such as lignocellulosic biomass, has gained great interest within the concept of biorefineries. In lignocellulosic materials, xylose is the most abundant pentose, which forms the hemicellulosic part. One of the key steps in the production of furfural from biomass is the dehydration reaction of the pentoses. The objective of this work was to assess the conditions under which the concentration of furfural is maximized from a synthetic, monophasic, and homogeneous xylose medium. The experiments were carried out in a microwave reactor. FeCl3 in different proportions and sulfuric acid were used as catalysts. A two-level, three-factor experimental design was developed for this purpose. The results were further analyzed through a second experimental design and optimization was performed by response surface methodology. The best operational conditions for the highest furfural yield (57%) turned out to be 210 °C, 0.5 min, and 0.05 M FeCl3.

Efficient xylose utilization leads to highest lipid productivity in Candida tropicalis SY005 among six yeast strains grown in mixed sugar medium

Six local isolates of yeasts were screened for cell mass and lipid production in mixed glucose and xylose medium. Candida tropicalis SY005 and Trichosporon (Apiotrichum) loubieri SY006 showed significant lipid accumulation of 24.6% and 32% (dry cell weight), respectively when grown in medium containing equal mass of both the sugars. SY005 produced relatively higher cell mass of 9.66gL-1 due to higher rate of sugar consumption, which raised the lipid productivity of the organism to 0.792gL-1day-1 as compared to 0.446gL-1day-1 in SY006. When grown with each sugar separately, the xylose consumption rate of SY005 was found to be 0.55gL-1h-1 after 4days as compared to 0.52gL-1h-1 for SY006. Transcript expression of the high affinity xylose transporter (Cthaxt), xylose reductase (Ctxyl1), and xylitol dehydrogenase (Ctxyl2) of SY005 was monitored to unravel such high rate of sugar consumption. Expression of all the three genes was observed to vary in mixed sugars with Cthaxt exhibiting the highest expression in presence of only xylose. Expression levels of both Ctxyl1 and Ctxyl2, involved in xylose catabolism, were maximum during 24-48h of growth, indicating that xylose utilization started in the presence of glucose, which was depleted in the medium after 96h. Together, the present study documents that C. tropicalis SY005 consumes xylose concomitant to glucose during early period of growth, and it is a promising yeast strain for viable production of storage lipid or other high-value oleochemicals utilizing lignocellulose hydrolysate.

Enhancement of Gluconobacter oxydans Resistance to Lignocellulosic-Derived Inhibitors in Xylonic Acid Production by Overexpressing Thioredoxin.

Efficient utilization of lignocellulose is an economically relevant practice for improving the financial prospects of biorefineries. Lignocellulose contains significant levels of xylose that can be converted into valuable xylonic acid. However, some inhibitors of bioconversion processes are produced after pretreatment. Xylonic acid production in bacteria, such as Gluconobacter oxydans, is hindered by poor bacterial tolerance to contaminants. Therefore, in order to enhance bacterial resistance to inhibitors, a recombinant strain of G. oxydans was created by the introduction of the thioredoxin gene. Thioredoxin is a key protein responsible for maintaining cellular redox potential and is critical to the conversion of xylose to xylonate. Overexpression of thioredoxin was confirmed at the enzymatic level, while the recombinant strain showed increased catalytic activity when inhibitors, such as formic acid or p-hydroxybenzaldehyde (PHBA), were added to the synthetic xylose medium (17% and 7% improvement in xylonic acid yield, respectively). To probe the molecular mechanism behind the recombinant strain response to inhibitors, the expression levels of various genes were analyzed by qRT-PCR, which revealed five differentially expressed genes (DEGs) upon exposure to formic acid or PHBA.

Deletion of JEN1 and ADY2 reduces lactic acid yield from an engineered Saccharomyces cerevisiae, in xylose medium, expressing a heterologous lactate dehydrogenase.

Microorganisms have evolved to produce specific end products for many reasons, including maintaining redox balance between NAD+ and NADH. The yeast Saccharomyces cerevisiae, for example, produces ethanol as a primary end product from glucose for the regeneration of NAD+. Engineered S. cerevisiae strains have been developed to ferment lignocellulosic sugars, such as xylose, to produce lactic acid by expression of a heterologous lactate dehydrogenase (ldhA from Rhizopus oryzae) without genetic perturbation to the native ethanol pathway. Surprisingly, the engineered yeast strains predominantly produce ethanol from glucose, but produce lactic acid as the major product from xylose. Here, we provide initial evidence that the shift in product formation from ethanol to lactic acid during xylose fermentation is at least partially dependent on the presence of functioning monocarboxylate transporter genes/proteins, including JEN1 and ADY2, which are downregulated and unstable in the presence of glucose, but upregulated/stable on xylose. Future yeast metabolic engineering studies may find the feedstock/carbon selection, such as xylose, an important step toward improving the yield of target end products.

Blastobotrys adeninivorans and B. raffinosifermentans, two sibling yeast species which accumulate lipids at elevated temperatures and from diverse sugars

BackgroundIn the context of sustainable development, yeast are one class of microorganisms foreseen for the production of oil from diverse renewable feedstocks, in particular those that do not compete with the food supply. However, their use in bulk production, such as for the production of biodiesel, is still not cost effective, partly due to the possible poor use of desired substrates or poor robustness in the practical bioconversion process. We investigated the natural capacity of Blastobotrys adeninivorans, a yeast already used in biotechnology, to store lipids under different conditions.ResultsThe genotyping of seven strains showed the species to actually be composed of two different groups, one that (including the well-known strain LS3) could be reassigned to Blastobotrys raffinosifermentans. We showed that, under nitrogen limitation, strains of both species can synthesize lipids to over 20% of their dry-cell weight during shake-flask cultivation in glucose or xylose medium for 96 h. In addition, organic acids were excreted into the medium. LS3, our best lipid-producing strain, could also accumulate lipids from exogenous oleic acid, up to 38.1 ± 1.6% of its dry-cell weight, and synthesize lipids from various sugar substrates, up to 36.6 ± 0.5% when growing in cellobiose. Both species, represented by LS3 and CBS 8244T, could grow with little filamentation in the lipogenic medium from 28 to 45 °C and reached lipid titers ranging from 1.76 ± 0.28 to 3.08 ± 0.49 g/L in flasks. Under these conditions, the maximum bioconversion yield (YFA/S = 0.093 ± 0.017) was obtained with LS3 at 37 °C. The presence of genes for predicted subunits of an ATP citrate lyase in the genome of LS3 reinforces its oleaginous character.ConclusionsBlastobotrys adeninivorans and B. raffinosifermentans, which are known to be xerotolerant and genetically-tractable, are promising biotechnological yeasts of the Saccharomycotina that could be further developed through genetic engineering for the production of microbial oil. To our knowledge, this is the first report of efficient lipid storage in yeast when cultivated at a temperature above 40 °C. This paves the way to help reducing costs through consolidated bioprocessing.

Proteome analysis of xylose metabolism in Rhodotorula toruloides during lipid production

BackgroundRhodotorula toruloides is a promising platform organism for production of lipids from lignocellulosic substrates. Little is known about the metabolic aspects of lipid production from the lignocellolosic sugar xylose by oleaginous yeasts in general and R. toruloides in particular. This study presents the first proteome analysis of the metabolism of R. toruloides during conversion of xylose to lipids.ResultsRhodotorula toruloides cultivated on either glucose or xylose was subjected to comparative analysis of its growth dynamics, lipid composition, fatty acid profiles and proteome. The maximum growth and sugar uptake rate of glucose-grown R. toruloides cells were almost twice that of xylose-grown cells. Cultivation on xylose medium resulted in a lower final biomass yield although final cellular lipid content was similar between glucose- and xylose-grown cells. Analysis of lipid classes revealed the presence of monoacylglycerol in the early exponential growth phase as well as a high proportion of free fatty acids. Carbon source-specific changes in lipid profiles were only observed at early exponential growth phase, where C18 fatty acids were more saturated in xylose-grown cells. Proteins involved in sugar transport, initial steps of xylose assimilation and NADPH regeneration were among the proteins whose levels increased the most in xylose-grown cells across all time points. The levels of enzymes involved in the mevalonate pathway, phospholipid biosynthesis and amino acids biosynthesis differed in response to carbon source. In addition, xylose-grown cells contained higher levels of enzymes involved in peroxisomal beta-oxidation and oxidative stress response compared to cells cultivated on glucose.ConclusionsThe results obtained in the present study suggest that sugar import is the limiting step during xylose conversion by R. toruloides into lipids. NADPH appeared to be regenerated primarily through pentose phosphate pathway although it may also involve malic enzyme as well as alcohol and aldehyde dehydrogenases. Increases in enzyme levels of both fatty acid biosynthesis and beta-oxidation in xylose-grown cells was predicted to result in a futile cycle. The results presented here are valuable for the development of lipid production processes employing R. toruloides on xylose-containing substrates.

Synthesis of polyketides from low cost substrates by the thermotolerant yeast Kluyveromyces marxianus.

Kluyveromyces marxianus is a promising nonconventional yeast for biobased chemical production due to its rapid growth rate, high TCA cycle flux, and tolerance to low pH and high temperature. Unlike Saccharomyces cerevisiae, K. marxianus grows on low-cost substrates to cell densities that equal or surpass densities in glucose, which can be beneficial for utilization of lignocellulosic biomass (xylose), biofuel production waste (glycerol), and whey (lactose). We have evaluated K. marxianus for the synthesis of polyketides, using triacetic acid lactone (TAL) as the product. The 2-pyrone synthase (2-PS) was expressed on a CEN/ARS plasmid in three different strains, and the effects of temperature, carbon source, and cultivation strategy on TAL levels were determined. The highest titer was obtained in defined 1% xylose medium at 37°C, with substantial titers at 41 and 43°C. The introduction of a high-stability 2-PS mutant and a promoter substitution increased titer four-fold. 2-PS expression from a multi-copy pKD1-based plasmid improved TAL titers a further five-fold. Combining the best plasmid, promoter, and strain resulted in a TAL titer of 1.24 g/L and a yield of 0.0295 mol TAL/molcarbon for this otherwise unengineered strain in 3 ml tube culture. This is an excellent titer and yield (on xylose) before metabolic engineering or fed-batch culture relative to other hosts (on glucose), and demonstrates the promise of this rapidly growing and thermotolerant yeast species for polyketide production.

Effects of Formic and Levulinic Acids on Butyric Acid Synthesis by Clostridium tyrobutyricum in Xylose Media

Abstract. Weak acids released during hydrolysis of lignocellulosic biomass are potential inhibitors of microorganism fermentation. In this study, the effects of formic and levulinic acids on butyric acid synthesis by were investigated. With the addition of 1.2 to 4.8 g L-1 of formic acid, increased lag time, decreased cell density, and lower butyric acid productivity were observed. Up to 15% and 56% reduction in peak cell density and butyric acid productivity, respectively, were caused by formic acid addition, whereas there was no significant difference in butyric acid yield between the control and formic acid treated groups (except for the 2.4 g formic acid L-1 treatment). Levulinic acid did not show any notable effects on within the investigated concentration range (0 to 4.8 g L-1). Overall, showed strong tolerance of both formic and levulinic acids, but neither of these acids could be metabolized by the microbe.HighlightsFormic acid had dosage-dependent inhibitory effects on C. tyrobutyricum.Levulinic acid had no effects on cell growth or butyrate synthesis.Neither formic acid nor levulinic acid was metabolized by C. tyrobutyricum.C. tyrobutyricum showed strong tolerance to formic acid and levulinic acid. Keywords: Butyric acid, Clostridium tyrobutyricum, Formic acid, Levulinic acid, Lignocellulosic hydrolysate, Xylose.

Evolutionary engineered Candida intermedia exhibits improved xylose utilization and robustness to lignocellulose-derived inhibitors and ethanol

The development of robust microorganisms that can efficiently ferment both glucose and xylose represents one of the major challenges in achieving a cost-effective lignocellulosic bioethanol production. Candida intermedia is a non-conventional, xylose-utilizing yeast species with a high-capacity xylose transport system. The natural ability of C. intermedia to produce ethanol from xylose makes it attractive as a non-GMO alternative for lignocellulosic biomass conversion in biorefineries. We have evaluated the fermentation capacity and the tolerance to lignocellulose-derived inhibitors and the end product, ethanol, of the C. intermedia strain CBS 141442 isolated from steam-exploded wheat straw hydrolysate. In a mixed sugar fermentation medium, C. intermedia CBS 141442 co-fermented glucose and xylose, although with a preference for glucose over xylose. The strain was clearly more sensitive to inhibitors and ethanol when consuming xylose than glucose. C. intermedia CBS 141442 was also subjected to evolutionary engineering with the aim of increasing its tolerance to inhibitors and ethanol, and thus improving its fermentation capacity under harsh conditions. The resulting evolved population was able to ferment a 50% (v/v) steam-exploded wheat straw hydrolysate (which was completely inhibitory to the parental strain), improving the sugar consumption and the final ethanol concentration. The evolved population also exhibited a better tolerance to ethanol when growing in a xylose medium supplemented with 35.5 g/L ethanol. These results highlight the potential of C. intermedia CBS 141442 to become a robust yeast for the conversion of lignocellulose to ethanol.

Modeling Butanol Synthesis in Xylose by Clostridium saccharoperbutylacetonicum

To examine the effect of xylose concentration on butanol synthesis by Clostridium saccharoperbutylacetonicum, a kinetic model of acetone-butanol-ethanol fermentation in the media with various xylose concentrations (40 g/L to 60 g/L) was developed and implemented in COPASI. Batch fermentation experiments were conducted to feed and validate the model, and the highest butanol production was achieved in 45 g/L xylose medium. Strong correlations (R2 > 0.91) between model simulation and experimental results were obtained. The modeling results suggested that the reaction rates in R6 (from acetate to acetyl-CoA), R8 (from acetyl-CoA to acetoacetyl-CoA), R9 (from acetoacetyl-CoA to butyryl-CoA), R10 (from butyryl-CoA to butanol), R14 (from butyrate to butyryl-CoA), and R20 (xylose consumption) were higher in groups with an initial xylose of 45 g/L, 50 g/L, or 55 g/L than those in groups with 40 g/L or 60 g/L xylose. In contrast, the reaction rates in R13 (from butyryl-CoA to butyrate) and R16 (from biomass to inactive cells) were lower in groups with initial xylose of 45 g/L, 50 g/L, or 55 g/L than those in groups with 40 g/L or 60 g/L xylose, which indicated that when initial xylose concentration changed, those reactions were affected, which resulted in different butanol syntheses.

Highly efficient conversion of xylose to ethanol without glucose repression by newly isolated thermotolerant Spathaspora passalidarum CMUWF1\u20132

BackgroundEfficient bioconversion of lignocellulosic biomass to bioethanol is one of key challenges in the situation of increasing bioethanol demand. The ethanologenic microbes for such conversion are required to possess abilities of utilization of various sugars including xylose and arabinose in lignocellulosic biomass. As required additional characteristics, there are a weak or no glucose repression that allows cells to simultaneously utilize various sugars together with glucose and thermotolerance for fermentation at high temperatures, which has several advantages including reduction of cooling cost. Spathaspora passalidarum ATCC MYA-4345, a type strains, isolated previously have mainly of these abilities or characteristics but its thermotolerance is not so strong and its glucose repression on xylose utilization is revealed.ResultsNewly isolated S. passalidarum CMUWF1–2 was found to have a high ability to produce ethanol from various sugars included in lignocellulosic biomass at high temperatures. The strain achieved ethanol yields of 0.43 g, 0.40 g and 0.20 g ethanol/g xylose at 30 °C, 37 °C and 40 °C, respectively. Interestingly, no significant glucose repression was observed in experiments with mixed sugars, being consistent with the strong resistance to 2-deoxyglucose, and antimycin A showed no effect on its growth in xylose medium. Moreover, the strain was tolerant to glucose and ethanol at concentrations up to 35.0% (w/v) and 8.0% (v/v), respectively.ConclusionsS. passalidarum CMUWF1–2 was shown to achieve efficient production of ethanol from various sugars and a high ethanol yield from xylose with little accumulation of xylitol. The strain also exhibited stress-resistance including thermotolerance and no detectable glucose repression as beneficial characteristics. Therefore, S. passalidarum CMUWF1–2 has remarkable potential for conversion of lignocellulosic biomass to bioethanol.

Improving xylose utilization and ethanol production from dry dilute acid pretreated corn stover by two-step and fed-batch fermentation

It is a great challenge to efficiently utilize xylose in fermentation with high solid loadings for the economically feasible production of lignocellulosic ethanol. However, 20% solid loading of dry dilute acid pretreated corn stover (DDAPCS) decreased 66.3% of the xylose consumption compared to yeast extract peptone dextrose and xylose (YPDX) medium, which was significantly larger than 16% solid loading (9.4%). To overcome those problems, several two-step processes for simultaneous saccharification and co-fermentation (SSCF) were developed by inoculating a water extract of 16% solid loading DDAPCS with microorganisms in the first fermentation step. During the first step, the relatively lower inhibitor concentrations (compared to 20% solid loading), decreased glucose concentration, and the absence of enzymatic hydrolysis residues (EHR) were beneficial for the use of xylose and enabled up to 75.6% of the xylose to be converted to ethanol. With an optimized fed-batch strategy of two-step process, the residual xylose concentration reduced by 55.6% with 20% solid loading, and the maximum ethanol concentration and yield increased by 30% and 16.6%, respectively.

Genetic and physiological basis for antibody production by Kluyveromyces marxianus

Kluyveromyces marxianus is a thermotolerant, crabtree-negative yeast, which preferentially directs metabolism (e.g., from the tricarboxylic acid cycle) to aerobic alcoholic fermentation. Thus K. marxianus has great potential for engineering to produce various materials under aerobic cultivation conditions. In this study, we engineered K. marxianus to produce and secrete a single-chain antibody (scFv), a product that is highly valuable but has historically proven difficult to generate at large scale. scFv production was obtained with strains carrying either plasmid-borne or genomically integrated constructs using various combinations of promoters (PMDH1 or PACO1) and secretion signal peptides (KmINUss or Scα-MFss). As the wild-type K. marxianus secretes endogenous inulinase predominantly, the corresponding INU1 gene was disrupted using a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)—associated protein (CRISPR–Cas9) system to re-direct resources to scFv production. Genomic integration was used to replace INU1 with sequences encoding a fusion of the INU1 signal peptide to scFv; the resulting construct yielded the highest scFv production among the strains tested. Optimization of growth conditions revealed that scFv production by this strain was enhanced by incubation at 30 °C in xylose medium containing 200 mM MgSO4. These results together demonstrate that K. marxianus has the potential to serve as a host strain for antibody production.

Enhanced production of extracellular inulinase by the yeast Kluyveromyces marxianus in xylose catabolic state

The production of extracellular proteins by the thermotolerant yeast Kluyveromyces marxianus, which utilizes various sugars, was investigated using media containing sugars such as glucose, galactose, and xylose. SDS-PAGE analysis of culture supernatants revealed abundant production of an extracellular protein when cells were grown in xylose medium. The N-terminal sequence of the extracellular protein was identical to a part of the inulinase encoded by INU1 in the genome. Inulinase is an enzyme hydrolyzing β-2,1-fructosyl bond in inulin and sucrose and is not required for xylose assimilation. Disruption of INU1 in the strain DMKU 3-1042 lost the production of the extracellular protein and resulted in growth defect in sucrose and inulin media, indicating that the extracellular protein was inulinase (sucrase). In addition, six K.marxianus strains among the 16 strains that were analyzed produced more inulinase in xylose medium than in glucose medium. However, expression analysis indicated that the INU1 promoter activity was lower in the xylose medium than in the glucose medium, suggesting that enhanced production of inulinase is controlled in a post-transcriptional manner. The production of inulinase was also higher in cultures with more agitation, suggesting that oxygen supply affects the production of inulinase. Taken together, these results suggest that both xylose and oxygen supply shift cellular metabolism to enhance the production of extracellular inulinase.

Selection of yeast Saccharomyces cerevisiae promoters available for xylose cultivation and fermentation

To efficiently utilize xylose, a major sugar component of hemicelluloses, in Saccharomyces cerevisiae requires the proper expression of varied exogenous and endogenous genes. To expand the repertoire of promoters in engineered xylose-utilizing yeast strains, we selected promoters in S.cerevisiae during cultivation and fermentation using xylose as a carbon source. To select candidate promoters that function in the presence of xylose, we performed comprehensive gene expression analyses using xylose-utilizing yeast strains both during xylose and glucose fermentation. Based on microarray data, we chose 29 genes that showed strong, moderate, and weak expression in xylose rather than glucose fermentation. The activities of these promoters in a xylose-utilizing yeast strain were measured by lacZ reporter gene assays over time during aerobic cultivation and microaerobic fermentation, both in xylose and glucose media. In xylose media, PTDH3, PFBA1, and PTDH1 were favorable for high expression, and PSED1, PHXT7, PPDC1, PTEF1, PTPI1, and PPGK1 were acceptable for medium-high expression in aerobic cultivation, and moderate expression in microaerobic fermentation. PTEF2 allowed moderate expression in aerobic culture and weak expression in microaerobic fermentation, although it showed medium-high expression in glucose media. PZWF1 and PSOL4 allowed moderate expression in aerobic cultivation, while showing weak but clear expression in microaerobic fermentation. PALD3 and PTKL2 showed moderate promoter activity in aerobic cultivation, but showed almost no activity in microaerobic fermentation. The knowledge of promoter activities in xylose cultivation obtained in this study will permit the control of gene expression in engineered xylose-utilizing yeast strains that are used for hemicellulose fermentation.

Xylan-hydrolyzing thermotolerant Candida tropicalis HNMA-1 for bioethanol production from sugarcane bagasse hydrolysate

Sugarcane bagasse is one of the low-cost substrates used for bioethanol production. In order to solubilize sugars in hemicelluloses like xylan, a new thermotolerant isolate of Candida tropicalis HNMA-1 with xylan-hydrolyzing ability was identified and characterized. The strain showed relative tolerance to high temperature. Our results demonstrated 0.211 IU ml−1 xylanase activity at 40 °C compared to 0.236 IU ml−1 at 30 °C. The effect of high temperature on the growth and fermentation of xylose and sugarcane bagasse hydrolysate were also investigated. In both xylose or hydrolysate medium, increased growth was recorded at 40 °C. Meanwhile, the efficiency of ethanol fermentation was adversely affected by temperature since yields of 0.088 g g−1 and 0.076 g g−1 in the xylose medium, in addition to 0.090 g g−1 and 0.078 g g−1 in the hydrolysate medium were noticed at 30 °C and 40 °C, respectively. Inhibitory compounds in the hydrolysate medium demonstrated negative effects on fermentation and productivity, with maximum ethanol concentration attained after 48 h in the hydrolysate, as opposed to 24 h in the xylose medium. Our data show that the newly thermotolerant isolate, C. tropicalis HNMA-1, is able to efficiently ferment xylose and hydrolysate, and also has the capacity for application in ethanol production from hemicellulosic sources.

Transcriptional activator Cat8 is involved in regulation of xylose alcoholic fermentation in the thermotolerant yeast Ogataea (Hansenula) polymorpha

BackgroundEfficient xylose alcoholic fermentation is one of the key to a successful lignocellulosic ethanol production. However, regulation of this process in the native xylose-fermenting yeasts is poorly understood. In this work, we paid attention to the transcriptional factor Cat8 and its possible role in xylose alcoholic fermentation in Ogataea (Hansenula) polymorpha. In Saccharomyces cerevisiae, organism, which does not metabolize xylose, gene CAT8 encodes a Zn-cluster transcriptional activator necessary for expression of genes involved in gluconeogenesis, respiration, glyoxylic cycle and ethanol utilization. Xylose is a carbon source that could be fermented to ethanol and simultaneously could be used in gluconeogenesis for hexose synthesis. This potentially suggests involvement of CAT8 in xylose metabolism.ResultsHere, the role of CAT8 homolog in the natural xylose-fermenting thermotolerant yeast O. polymorpha was characterized. The CAT8 ortholog was identified in O. polymorpha genome and deleted both in the wild-type strain and in advanced ethanol producer from xylose. Constructed cat8Δ strain isolated from wild strain showed diminished growth on glycerol, ethanol and xylose as well as diminished respiration on the last substrate. At the same time, cat8Δ mutant isolated from the best available O. polymorpha ethanol producer showed only visible defect in growth on ethanol. CAT8 deletant was characterized by activated transcription of genes XYL3, DAS1 and RPE1 and slight increase in the activity of several enzymes involved in xylose metabolism and alcoholic fermentation. Ethanol production from xylose in cat8Δ mutants in the background of wild-type strain and the best available ethanol producer from xylose increased for 50 and 30%, respectively. The maximal titer of ethanol during xylose fermentation was 12.5 g ethanol/L at 45 °C. Deletion of CAT8 did not change ethanol production from glucose. Gene CAT8 was also overexpressed under control of the strong constitutive promoter GAP of glyceraldehyde-3-phosphate dehydrogenase. Corresponding strains showed drop in ethanol production in xylose medium whereas glucose alcoholic fermentation remained unchanged. Available data suggest on specific role of Cat8 in xylose alcoholic fermentation.ConclusionsThe CAT8 gene is one of the first identified genes specifically involved in regulation of xylose alcoholic fermentation in the natural xylose-fermenting yeast O. polymorpha.

Butyric Acid Fermentation in Xylose and Glucose by Clostridium tyrobutyricum

The objective of this study was to understand the effect of different concentrations of xylose and glucose on butyric acid production by Clostridium tyrobutyricum. C. tyrobutyricum was cultured in a medium containing xylose, glucose, or mixtures of xylose and glucose as the main carbon source. The butyric acid concentration increased from 3.5 to 16.3 g/L in the xylose media, and from 2.6 to 27.0 g/L in the glucose media when the initial sugar concentration increased from 5 to 75 g/L. The yield from xylose to butyric acid started to decrease as the sugar concentration was above 35 g/L, while for glucose media higher glucose concentration resulted in higher yield. At low sugar concentrations (5 g/L or 15 g/L), xylose was more efficient than glucose for butyric acid generation, but at high concentrations (55 or 75 g/L), glucose was more efficient. In mixtures containing both sugars, glucose was the preferred sugar for bacteria growth and xylose was rapidly consumed only after the glucose was exhausted. The xylose to glucose ratio affected bacterial growth and butyric acid production. High xylose to glucose ratios (4:1 or 3:2) showed better butyric acid production than low ratios (1:1, 2:3, or 1:4) when the total initial sugar content in the media was kept at 30 g/L.