Xylose-fermenting Yeast Research Articles

Isolation, Screening and Characterization of Xylose-Fermenting Yeasts Isolated from Saw Dust

Yeasts have been less frequently reported as xylanase producers compared to bacteria and filamentous fungi. Different cellulosic materials including sawdust are produced on a large scale and these can be used for the production of useful enzymes such as xylanases. Xylanases are a group of enzymes that hydrolyze plant fibers made of xylan hemicellulose. Xylose-fermenting yeasts isolated from soil at a wood processing factory were isolated and qualitatively and quantitatively screened for xylanase production using xylose supplemented medium and congo red as indicator. Xylanase enzyme was produced using different xylose concentrations (0.5%, 1.0%, 1.5% and 2.0%). Pichia chambardii isolate, which was later identified as Wickerhamomyces chambardii by molecular techniques, showed the highest xylanase activity of 199.31U mL-1. Maximum xylanase activity (275.83U mL-1) was achieved at 1.5 %w/v xylose. This study showed that yeasts have a high potential for the production of xylanase enzymes.

Oxygenation influences xylose fermentation and gene expression in the yeast genera Spathaspora and Scheffersomyces

BackgroundCost-effective production of biofuels from lignocellulose requires the fermentation of d-xylose. Many yeast species within and closely related to the genera Spathaspora and Scheffersomyces (both of the order Serinales) natively assimilate and ferment xylose. Other species consume xylose inefficiently, leading to extracellular accumulation of xylitol. Xylitol excretion is thought to be due to the different cofactor requirements of the first two steps of xylose metabolism. Xylose reductase (XR) generally uses NADPH to reduce xylose to xylitol, while xylitol dehydrogenase (XDH) generally uses NAD+ to oxidize xylitol to xylulose, creating an imbalanced redox pathway. This imbalance is thought to be particularly consequential in hypoxic or anoxic environments.ResultsWe screened the growth of xylose-fermenting yeast species in high and moderate aeration and identified both ethanol producers and xylitol producers. Selected species were further characterized for their XR and XDH cofactor preferences by enzyme assays and gene expression patterns by RNA-Seq. Our data revealed that xylose metabolism is more redox balanced in some species, but it is strongly affected by oxygen levels. Under high aeration, most species switched from ethanol production to xylitol accumulation, despite the availability of ample oxygen to accept electrons from NADH. This switch was followed by decreases in enzyme activity and the expression of genes related to xylose metabolism, suggesting that bottlenecks in xylose fermentation are not always due to cofactor preferences. Finally, we expressed XYL genes from multiple Scheffersomyces species in a strain of Saccharomyces cerevisiae. Recombinant S. cerevisiae expressing XYL1 from Scheffersomyces xylosifermentans, which encodes an XR without a cofactor preference, showed improved anaerobic growth on xylose as the primary carbon source compared to S. cerevisiae strain expressing XYL genes from Scheffersomyces stipitis.ConclusionCollectively, our data do not support the hypothesis that xylitol accumulation occurs primarily due to differences in cofactor preferences between xylose reductase and xylitol dehydrogenase; instead, gene expression plays a major role in response to oxygen levels. We have also identified the yeast Sc. xylosifermentans as a potential source for genes that can be engineered into S. cerevisiae to improve xylose fermentation and biofuel production.

Application of natural fungi in bioconversion of lignocellulosic waste to second-generation ethanol

The second generation (2G) or cellulosic ethanol can help with diversification of the use of fossil energy sources. However, as bioconversion of plant waste into 2G bioethanol requires expensive additional steps of pre-treatment/hydrolysis of lignocellulosic materials, and this technology has not yet reached the technological readiness level which would allow it to be scaled-up, this process needs more interdisciplinary and comprehensive studies. This work was aimed at experimental study of a full cycle of successive processes of pre-treatment/saccharification using cellulolytic enzymes of filamentous fungi and fermentation of obtained syrups by xylose-fermenting yeast, using selected natural microorganisms for the fungal-based bioconversion of lignocellulosic agricultural waste to 2G ethanol. Using the Plackett-Burman and Box-Behnken methods of mathematical statistics, the optimal conditions for pre-treatment and enzymatic hydrolysis of wheat straw by a hemi- and cellulolytic multi-enzyme complex of the selected fungal strain Talaromyces funiculosus UCM F-16795 were established: microwave-assisted alkali pre-treatment with sodium hydroxide (NaOH) solutions (concentration range 4.6–4.8%), and saccharification conditions of medium pH 4, temperature 40 °С, hydrolysis duration 18 hours, and dilution of culture liquid with a buffer solution 1:1. The total energy of microwave irradiation 1.2 kJ and the ratio of substrate/enzyme solution 100 mg/1 mL were used. Under optimized conditions, wheat straw hydrolysates contained 5.0–7.5 g/L of reducing sugars, which, according to HPLC assessment, contained 0.7–1.0 g/L of glucose, 2.2–2.9 g/L of xylose and 0.7–0.8 g/L cellobiose. We used the selected strain of xylose-fermenting yeast in fermentation of mixtures of the most important monosaccharides in hydrolysates, xylose and glucose, in the concentration range relevant for syrups obtained by us during the optimized saccharification of lignocellulosic substrates with T. funiculosus enzymes. Based on sequencing and phylogenetic analysis, strain UCM Y-2810 was confirmed as Scheffersomyces stipitis; its nucleotide sequences of ITS region and 28S gene rDNA were deposited in GenBank under the accession numbers OP931914 and OP931915, respectively. The ethanologenic process for S. stipitis UCM Y-2810 was studied according to Box-Behnken design, assessing ethanol concentration by gas chromatography-mass spectrometry. Yeast fermentation under static microaerophilic conditions showed a 1.5 times higher rate of bioethanol production and 1.7 times greater efficiency of ethanologenesis per yeast biomass than for submerged cultivation. Optimization of the process of ethanologenesis resulted in the maximum rate of fermentation mixture of sugars, being 11.30 ± 0.36 g/L of ethanol, with optimal values of factors: 30 g/L of xylose, 5.5 g/L of glucose and cultivation for 5.5 days. It was revealed that the tested glucose concentrations did not significantly affect the process of xylose-fermentation by yeast, and non-competitive inhibition of xylose transport by glucose into yeast cells did not occur. This study demonstrated the potential of a full cycle bioconversion of lignocellulosic waste to 2G ethanol based on use of natural fungal strains and optimization of conditions for all steps.

BLINCAR: a reusable bioluminescent and Cas9-based genetic toolset for repeatedly modifying wild-type Scheffersomyces stipitis.

Scheffersomyces stipitis is a yeast that robustly ferments the 5-carbon sugar xylose, making the yeast a valuable candidate for lignocellulosic ethanol fermentation. However, the non-canonical codon usage of S. stipitis is an obstacle for implementing molecular tools that were developed for other yeast species, thereby limiting the molecular toolset available for S. stipitis. Here, we developed a series of molecular tools for S. stipitis including BLINCAR, a Bio-Luminescent Indicator that is Nullified by Cas9-Actuated Recombination, which can be used repeatedly to add different exogenous DNA payloads to the wild-type S. stipitis genome or used repeatedly to remove multiple native S. stipitis genes from the wild-type genome. Through the use of BLINCAR tools, one first produces antibiotic-resistant, bioluminescent colonies of S. stipitis whose bioluminescence highlights those clones that have been genetically modified; then second, once candidate clones have been confirmed, one uses a transient Cas9-producing plasmid to nullify the antibiotic resistance and bioluminescent markers from the prior introduction, thereby producing non-bioluminescent colonies that highlight those clones which have been re-sensitized to the antibiotic and are therefore susceptible to another round of BLINCAR implementation. IMPORTANCE Cellulose and hemicellulose that comprise a large portion of sawdust, leaves, and grass can be valuable sources of fermentable sugars for ethanol production. However, some of the sugars liberated from hemicellulose (like xylose) are not easily fermented using conventional glucose-fermenting yeast like Saccharomyces cerevisiae, so engineering robust xylose-fermenting yeast that is not inhibited by other components liberated from cellulose/hemicellulose will be important for maximizing yield and making lignocellulosic ethanol fermentation cost efficient. The yeast Scheffersomyces stipitis is one such yeast that can ferment xylose; however, it possesses several barriers to genetic manipulation. It is difficult to transform, has only a few antibiotic resistance markers, and uses an alternative genetic code from most other organisms. We developed a genetic toolset for S. stipitis that lowers these barriers and allows a user to deliver and/or delete multiple genetic elements to/from the wild-type genome, thereby expanding S. stipitis's potential.

Xylose consumption and ethanol production by Pichia guilliermondii and Candida oleophila in the presence of furans, phenolic compounds, and organic acids commonly produced during the pre-treatment of plant biomass.

For 2G ethanol production, pentose fermentation and yeast tolerance to lignocellulosic hydrolyzate components are essential to improve biorefinery yields. Generally, physicochemical pre-treatment methodologies are used to facilitate access to cellulose and hemicellulose in plant material, which consequently can generate microbial growth inhibitory compounds, such as furans, weak acids, and phenolic compounds. Because of the unsatisfactory yield of wild-type Saccharomyces cerevisiae during pentose fermentation, the search for xylose-fermenting yeasts tolerant to microbial growth inhibitors has gained attention. In this study, we investigated the ability of the yeasts Pichia guilliermondii G1.2 and Candida oleophila G10.1 to produce ethanol from xylose and tolerate the inhibitors furfural, 5-hydroxymethylfurfural (HMF), acetic acid, formic acid, ferulic acid, and vanillin. We demonstrated that both yeasts were able to grow and consume xylose in the presence of all single inhibitors, with greater growth limitation in media containing furfural, acetic acid, and vanillin. In saline medium containing a mixture of these inhibitors (2.5-3.5 mM furfural and HMF, 1 mM ferulic acid, 1-1.5 mM vanillin, 10-13 mM acetic acid, and 5-7 mM formic acid), both yeasts were able to produce ethanol from xylose, similar to that detected in the control medium (without inhibitors). In future studies, the proteins involved in the transport of pentose and tolerance to these inhibitors need to be investigated.

Pentose-Fermenting Yeasts in Nature: Ecology, Biodiversity and Applications

The world’s energy sector has undergone drastic changes in the last decades due to the climate change and the turn to renewable energy sources. Biomass is the largest renewable source of carbohydrates on the Earth and is an important substrate for biofuel production. Saccharomyces cerevisiae yeasts are the main producer of first-generation ethanol from corn and sugarcane. However, these yeasts lack the ability to utilize the important components of lignocelluloses such as pentoses D-xylose and L-arabinose. Pentose-fermenting yeasts could become an alternative to S. cerevisiae in ethanol production from lignocelluloses. This review focuses on the ecology, geographical distribution, taxonomy, and potential applications of naturally-occurring pentose-fermenting yeasts. Pentose-fermenting yeasts have been frequently found in the lignocelluloseassociated substrates. Decaying and rotten wood and the gut of wood-boring insects are especially important natural reservoirs of this group of yeasts. Simple sugars xylose and L-arabinose would be present in such habitats as suitable nutrients for pentose-assimilating yeasts. The other natural habitats reported for pentose-fermenting yeasts are soil, plants, and herbivore faeces. Pentose-fermenting yeasts are found in many geographical regions and have been isolated on almost each continent. Dozens of novel pentose-fermenting yeast species have been discovered in the last decade. The previously poorly explored regions, including Brazil, China, and several Asian countries were especially oft en reported as sites of isolation of such yeasts. Most xylose-fermenting yeasts belong to genera Scheffersomyces, Candida, Spathaspora, Sugiyamaella, and Pachysolen, while the most efficient ethanol producers are represented by species Scheffersomyces stipitis and Spathaspora passalidarum. The vast majority of research on the biotechnological application of pentose-fermenting yeasts focuses on their role in the production of bioethanol from lignocellulose. This group of yeasts could be either directly involved in the fermentation stage of ethanol production or serve as a source of genetic material for the genetic manipulation of other industrial yeast strains. Pentose-fermenting yeasts could also be involved in the production of various chemicals from lignocellulosic substrates, mainly polyols, xylitol, and arabitol. Thus, the search for novel pentose-fermenting yeasts that could become new efficient ethanol producers or donors of new genetic material is still ongoing. The previously unexplored or poorly studied geographical regions and natural habitats can hide many novel yeasts with huge biotechnological potential.

Evaluation of Xylose-Utilising Yeasts for Xylitol Production from Second-Generation Ethanol Vinasse and Effect of Agitation Intensity in Flask-Scale Xylitol Production

This study aimed to select a yeast strain that effectively utilises xylose to produce xylitol from the vinasse of ethanol broth obtained from the fermentation of sugarcane bagasse hydrolysate. Eleven strains of xylose-fermenting yeasts were evaluated for their abilities to utilise xylose and produce xylitol. Two strains that showed outstanding performance in the semi-defined xylose medium were selected for further testing with a vinasse medium. Candida guilliermondii TISTR 5068 showed a superior xylitol production of 7.03 ± 0.08 g/L with the xylitol yield of 0.70 g/gxylose when cultured in bagasse-based ethanol vinasse. The strain was further tested for its xylitol production performance when cultured at four different agitation intensities. Excessive agitation resulted in a rapid xylitol production rate but caused xylitol consumption once the xylose was depleted. Moderate agitation resulted in the highest xylitol yield of 0.79 g/gxylose. The results of this study have provided important information for the development of the xylitol production process using waste streams from cellulosic ethanol production.

The Brazilian Amazonian rainforest harbors a high diversity of yeasts associated with rotting wood, including many candidates for new yeast species.

This study investigated the diversity of yeast species associated with rotting wood in Brazilian Amazonian rainforests. A total of 569 yeast strains were isolated from rotting wood samples collected in three Amazonian areas (Universidade Federal do Amazonas-Universidade Federal do Amazonas [UFAM], Piquiá, and Carú) in the municipality of Itacoatiara, Amazon state. The samples were cultured in yeast nitrogen base (YNB)-d-xylose, YNB-xylan, and sugarcane bagasse and corncob hemicellulosic hydrolysates (undiluted and diluted 1:2 and 1:5). Sugiyamaella was the most prevalent genus identified in this work, followed by Kazachstania. The most frequently isolated yeast species were Schwanniomyces polymorphus, Scheffersomyces amazonensis, and Wickerhamomyces sp., respectively. The alpha diversity analyses showed that the dryland forest of UFAM was the most diverse area, while the floodplain forest of Carú was the least. Additionally, the difference in diversity between UFAM and Carú was the highest among the comparisons. Thirty candidates for new yeast species were obtained, representing 36% of the species identified and totaling 101 isolates. Among them were species belonging to the clades Spathaspora, Scheffersomyces, and Sugiyamaella, which are recognized as genera with natural xylose-fermenting yeasts that are often studied for biotechnological and ecological purposes. The results of this work showed that rotting wood collected from the Amazonian rainforest is a tremendous source of diverse yeasts, including candidates for new species.

Evaluation of Non-Conventional Yeasts Isolated from Rotten Wood for Hydrolytic Activities and Xylose Fermentation

Hydrolysis of lignocellulose to fermentable sugars and their subsequent conversion to ethanol remain great challenges in the biofuel industry. Rotten wood is first colonized by bacteria and molds that possess strong hydrolases. Yeasts are also an important group of microorganisms that may participate in wood hydrolysis. Decaying wood could provide a rich natural reservoir of yeasts possessing promising hydrolytic activities, including xylanases, cellulases, β-glucosidases, or abilities essential for the fermentation of pentose sugars derived from lignocellulose degradation, especially xylose. Therefore, the aim of this work was to screen yeasts isolated from rotten wood samples for the production of hydrolytic enzymes directed at lignocellulose components and the ability to ferment xylose, L-arabinose, and cellobiose. Methods. Yeast strains were isolated from 22 samples of rotten wood and identified by phenotypic characteristics according to Kurtzman et al. Hydrolytic properties and the ability of the isolated strains to ferment xylose, L-arabinose, and cellobiose were determined using conventional methods. Results. 30 strains of yeasts and yeast-like micromycetes were isolated from 22 samples of rotten wood in the Holosiivskyi Forest, Kyiv. Based on phenotypic properties, most of the isolated yeasts belonged to ascomycetous yeasts and were represented by the following genera: Candida (8 strains), Debaryomyces (5 strains), Kluyveromyces (5 strains), Pichia (5 strains), Scheffersomyces (2 strains), Lachancea, Hanseniaspora, Saccharomyces, and Geotrichum/Galactomyces. A strain of yeast-like non-photosynthetic alga Prototheca sp. was also detected. Most of the isolated microfungi (66.6% isolates) exhibited extracellular β-glucosidase activity, two Candida tropicalis strains possessed weak pectinase and xylanase activity. None of the isolates demonstrated extracellular cellulase activity. Two yeast strains preliminarily identified as Scheffersomyces stipitis were able to ferment xylose at a concentration of 20—100 g/L over a wide temperature range up to 37°C. Acetic acid at 0.25—1% (v/v) concentration resulted in the complete inhibition of xylose fermentation. Ethanol production from xylose up to 6 g/L was observed under the microaerobic fermentation conditions for 24 hr at the substrate concentration 40 g/L, but the subsequent fermentation resulted in decreasing ethanol concentration presumably due to ethanol re-assimilation. None of the isolated strains was capable of fermenting cellobiose or L-arabinose under the microaerobic conditions. Conclusions. This work provides the characterization of yeast microbiota of rotten wood that was represented predominantly by ascomycetous yeasts. The dominant extracellular hydrolytic activity of the isolates was β-glucosidase. This is the first report on the isolation of xylose-fermenting yeasts Scheffersomyces stipitis in Ukraine, which comprised 7% of all the microfungi isolated from rotten wood.

Identification of traits to improve co-assimilation of glucose and xylose by adaptive evolution of Spathaspora passalidarum and Scheffersomyces stipitis yeasts.

Lignocellulosic biomass is a renewable raw material for producing several high-value-added chemicals and fuels. In general, xylose and glucose are the major sugars in biomass hydrolysates, and their efficient utilization by microorganisms is critical for an economical production process. Yeasts capable of co-consuming mixed sugars might lead to higher yields and productivities in industrial fermentation processes. Herein, we performed adaptive evolution assays with two xylose-fermenting yeasts, Spathaspora passalidarum and Scheffersomyces stipitis, to obtain derived clones with improved capabilities of glucose and xylose co-consumption. Adapted strains were obtained after successive growth selection using xylose and the non-metabolized glucose analog 2-deoxy-D-glucose as a selective pressure. The co-fermentation capacity of evolved and parental strains was evaluated on xylose-glucose mixtures. Our results revealed an improved co-assimilation capability by the evolved strains; however, xylose and glucose consumption were observed at slower rates than the parental yeasts. Genome resequencing of the evolved strains revealed genes affected by non-synonymous variants that might be involved with the co-consumption phenotype, including the HXT2.4 gene that encodes a putative glucose transporter in Sp. passalidarum. Expression of this mutant HXT2.4 in Saccharomyces cerevisiae improved the cells' co-assimilation of glucose and xylose. Therefore, our results demonstrated the successful improvement of co-fermentation through evolutionary engineering and the identification of potential targets for further genetic engineering of different yeast strains. KEY POINTS: • Laboratory evolution assay was used to obtain improved sugar co-consumption of non-Saccharomyces strains. • Evolved Sp. passalidarum and Sc. stipitis were able to more efficiently co-ferment glucose and xylose. • A mutant Hxt2.4 permease, which co-transports xylose and glucose, was identified.

RNA-seq based transcriptomic analysis of the non-conventional yeast Spathaspora passalidarum during Melle-boinot cell recycle in xylose-glucose mixtures

The Melle-Boinot is a promising process for second-generation ethanol production by xylose-fermenting yeasts. However, the impact of this process on the physiology of the non-conventional yeast Spathaspora passalidarum during second-generation ethanol production remains unclear. Therefore, we performed a transcriptomic analysis of S. passalidarum to determine the differences and global responses of differentially expressed genes (DEGs) during five fed-batch fermentations with cell recycle. A cycle-to-cycle metabolic reprogramming was observed resulting in an increase in ethanol yield (32%), volumetric productivity (33%), and titer (33%); and an expressive decrease of 94% of xylitol production. A broad set of pathways operated synergically such as fatty acid metabolism, N-glycan biosynthesis, glyoxylate and dicarboxylate metabolism, oxidative phosphorylation, glutathione metabolism and sulfur metabolism, indicating those as important mechanisms for cell recycling adaptations due to increased ethanol concentration, long-term ethanol exposure and osmotic stress. Together these results suggest that cellular energy was redirected towards the production of cell wall components enabling S. passalidarum cells to thrive on consecutive recycles. Furthermore, these results are instrumental to guide genetic-engineering efforts of xylose-fermenting yeasts to improve the productiveness and feasibility of renewable energy production from lignocellulosic biomass and hemicellulosic hydrolysates.

The role of hexose transporter-like sensor hxs1 and transcription activator involved in carbohydrate sensing azf1 in xylose and glucose fermentation in the thermotolerant yeast Ogataea polymorpha

BackgroundFuel ethanol from lignocellulose could be important source of renewable energy. However, to make the process feasible, more efficient microbial fermentation of pentose sugars, mainly xylose, should be achieved. The native xylose-fermenting thermotolerant yeast Ogataea polymorpha is a promising organism for further development. Efficacy of xylose alcoholic fermentation by O. polymorpha was significantly improved by metabolic engineering. Still, genes involved in regulation of xylose fermentation are insufficiently studied.ResultsWe isolated an insertional mutant of O.polymorpha with impaired ethanol production from xylose. The insertion occurred in the gene HXS1 that encodes hexose transporter-like sensor, a close homolog of Saccharomyces cerevisiae sensors Snf3 and Rgt2. The role of this gene in xylose utilization and fermentation was not previously elucidated. We additionally analyzed O.polymorpha strains with the deletion and overexpression of the corresponding gene. Strains with deletion of the HXS1 gene had slower rate of glucose and xylose consumption and produced 4 times less ethanol than the wild-type strain, whereas overexpression of HXS1 led to 10% increase of ethanol production from glucose and more than 2 times increase of ethanol production from xylose. We also constructed strains of O.polymorpha with overexpression of the gene AZF1 homologous to S. cerevisiae AZF1 gene which encodes transcription activator involved in carbohydrate sensing. Such transformants produced 10% more ethanol in glucose medium and 2.4 times more ethanol in xylose medium. Besides, we deleted the AZF1 gene in O. polymorpha. Ethanol accumulation in xylose and glucose media in such deletion strains dropped 1.5 and 1.8 times respectively. Overexpression of the HXS1 and AZF1 genes was also obtained in the advanced ethanol producer from xylose. The corresponding strains were characterized by 20–40% elevated ethanol accumulation in xylose medium. To understand underlying mechanisms of the observed phenotypes, specific enzymatic activities were evaluated in the isolated recombinant strains.ConclusionsThis paper shows the important role of hexose sensor Hxs1 and transcription factor Azf1 in xylose and glucose alcoholic fermentation in the native xylose-fermenting yeast O. polymorpha and suggests potential importance of the corresponding genes for construction of the advanced ethanol producers from the major sugars of lignocellulose.

An integrated biorefinery approach for bioethanol production from sugarcane tops

Bioethanol from lignocellulosic biomass is a promising alternative to petroleum-based fuels to alleviate greenhouse gas emissions and reduce the dependency on fossil fuels. The lignocellulosic biomass is exploited for ethanol production due to its sustainability and abundance. Sugarcane tops, an agricultural residue, was employed in the present investigation to assess its potential as a feedstock for bioethanol production by adopting different fermentation approaches, namely separate fermentation, simultaneous saccharification and fermentation (SSF) and partially consolidated bioprocessing (PCBP). The present study demonstrated the potential of mono and co-fermentation for ethanol production. Comparison between separate fermentation and SSF using S.cerevisiae showed the latter to be more efficient with ethanol production of 5.69% (v/v) in 30.67 h of fermentation time than separate fermentation with 3.76% (v/v) ethanol in 48 h. An integrated fermentation strategy stated as partially consolidated bioprocessing (PCBP) was investigated to improve fermentation efficiency. This process integrates simultaneous pretreatment and saccharification (SPS), conducted by enzyme blends of laccase and cellulase followed by co-fermentation using S. cerevisiae and xylose-fermenting yeast AKBR 212. This approach resulted in a 7.57% (v/v) maximum ethanol concentration in 24.30 h. The different fermentation strategies involved enzymes and yeasts only, thus offering a green biotechnology approach towards converting sugarcane tops into bioethanol.

Physiological comparisons among Spathaspora passalidarum, Spathaspora arborariae, and Scheffersomyces stipitis reveal the bottlenecks for their use in the production of second-generation ethanol.

The microbial conversion of pentoses to ethanol is one of the major drawbacks that limits the complete use of lignocellulosic sugars. In this study, we compared the yeast species Spathaspora arborariae, Spathaspora passalidarum, and Sheffersomyces stipitis regarding their potential use for xylose fermentation. Herein, we evaluated the effects of xylose concentration, presence of glucose, and temperature on ethanol production. The inhibitory effects of furfural, hydroxymethylfurfural (HMF), acetic acid, and ethanol were also determined. The highest ethanol yield (0.44g/g) and productivity (1.02g/L.h) were obtained using Sp. passalidarum grown in 100g/L xylose at 32°C. The rate of xylose consumption was reduced in the presence of glucose for the species tested. Hydroxymethylfurfural did not inhibit the growth of yeasts, whereas furfural extended their lag phase. Acetic acid inhibited the growth and fermentation of all yeasts. Furthermore, we showed that these xylose-fermenting yeasts do not produce ethanol concentrations greater than 4% (v/v), probably due to the inhibitory effects of ethanol on yeast physiology. Our data confirm that among the studied yeasts, Sp. passalidarum is the most promising for xylose fermentation, and the low tolerance to ethanol is an important aspect to be improved to increase its performance for second-generation (2G) ethanol production. Our molecular data showed that this yeast failed to induce the expression of some classical genes involved in ethanol tolerance. These findings suggest that Sp. passalidarum may have not activated a proper response to the stress, impacting its ability to overcome the negative effects of ethanol on the cells.

Adaptive Laboratory Evolution for Multistress Tolerance, including Fermentability at High Glucose Concentrations in Thermotolerant Candida tropicalis

Candida tropicalis, a xylose-fermenting yeast, has the potential for converting cellulosic biomass to ethanol. Thermotolerant C. tropicalis X-17, which was isolated in Laos, was subjected to repetitive long-term cultivation with a gradual increase in temperature (RLCGT) in the presence of a high concentration of glucose, which exposed cells to various stresses in addition to the high concentration of glucose and high temperatures. The resultant adapted strain demonstrated increased tolerance to ethanol, furfural and hydroxymethylfurfural at high temperatures and displayed improvement in fermentation ability at high glucose concentrations and xylose-fermenting ability. Transcriptome analysis revealed the up-regulation of a gene for a glucose transporter of the major facilitator superfamily and genes for stress response and cell wall proteins. Additionally, hydropathy analysis revealed that three genes for putative membrane proteins with multiple membrane-spanning segments were also up-regulated. From these findings, it can be inferred that the up-regulation of genes, including the gene for a glucose transporter, is responsible for the phenotype of the adaptive strain. This study revealed part of the mechanisms of fermentability at high glucose concentrations in C. tropicalis and the results of this study suggest that RLCGT is an effective procedure for improving multistress tolerance.

Performance of xylose-fermenting yeasts in oat and soybean hulls hydrolysate and improvement of ethanol production using immobilized cell systems.

We investigated the fermentation of a mixture of oat and soybean hulls (1:1) subjected to acid (AH) or enzymatic (EH) hydrolyses, with both showing high osmotic pressures (> 1200Osmkg-1) for the production of ethanol. Yeasts of genera Spathaspora, Scheffersomyces, Sugiymaella, and Candida, most of them biodiverse Brazilian isolates and previously untested in bioprocesses, were cultivated in these hydrolysates. Spathaspora passalidarum UFMG-CM-469 showed the best ethanol production kinetics in suspended cells cultures in acid hydrolysate, under microaerobic and anaerobic conditions. This strain was immobilized in LentiKats® (polyvinyl alcohol) and cultured in AH and EH. Supplementation of hydrolysates with crude yeast extract and peptone was also performed. The highest ethanol production was obtained using hydrolysates supplemented with crude yeast extract (AH-CYE and EH-CYE) showing yields of 0.40 and 0.44gg-1, and productivities of 0.39 and 0.29g(Lh)-1, respectively. The reuse of the immobilized cells was tested in sequential fermentations of AH-CYE, EH-CYE, and a mixture of acid and enzymatic hydrolysates (AEH-CYE) operated under batch fluidized bed, with ethanol yields ranging from 0.31 to 0.40gg-1 and productivities from 0.14 to 0.23g(Lh)-1. These results warrant further research using Spathaspora yeasts for second-generation ethanol production.

The role of Mig1, Mig2, Tup1 and Hap4 transcription factors in regulation of xylose and glucose fermentation in the thermotolerant yeast Ogataea polymorpha.

Glucose is a preferred carbon source for most living organisms. The metabolism and regulation of glucose utilization are well studied mostly for Saccharomyces cerevisiae. Xylose is the main pentose sugar released from the lignocellulosic biomass, which has a high potential as a renewable feedstock for bioethanol production. The thermotolerant yeast Ogataea (Hansenula) polymorpha, in contrast to S. cerevisiae, is able to metabolize and ferment not only glucose but also xylose. However, in non-conventional yeasts, the regulation of glucose and xylose metabolism remains poorly understood. In this study, we characterize the role of transcriptional factors Mig1, Mig2, Tup1 and Hap4 in the natural xylose-fermenting yeast O. polymorpha. The deletion of MIG1 had no significant influence on ethanol production either from xylose or glucose, however the deletion of both MIG1 and MIG2 reduced the amount of ethanol produced from these sugars. The deletion of HAP4-A and TUP1 genes resulted in increased ethanol production from xylose. Inversely, the overexpression of HAP4-A and TUP1 genes reduced ethanol production during xylose alcoholic fermentation. Thus, HAP4-A and TUP1 are involved in repression of xylose metabolism and fermentation in yeast O. polymorpha and their deletion could be a viable strategy to improve ethanol production from this pentose.

Rice straw saccharification using cellulolytic cocktail from Aspergillus tubingensis and structure alterations studies of the wall polymer

Optimization of saccharification of rice straw plays a crucial role in harnessing maximum fermentable sugars through enzymatic hydrolysis of this complex polymer. The present study was aimed to evaluate the applicability of A. tubingensis NKBP-55 cellulolytic cocktail for generation of fermentable sugars and to carry out parametric optimization of the saccharification process followed by batch ethanolic fermentation of the rice straw hydrolysate. Also, evaluation of the alterations (composition, crystallinity and structural morphology) occurring in the wall polymer structure after sequential treatments was studied. The cellulolytic cocktail, under the statistically optimized conditions (rice straw 4% w/v and pH 5.0) liberated 578 mg fermentable sugars (glucose 463 mg, xylose 114.35 and cellobiose 0.71 mg) per gram of pretreated rice straw showing 2.17-fold enhancement. Candida shehatae NCIM 3501 utilized both glucose and xylose and produced 16.15 g/L bioethanol along with yield (YP/S) 0.50 after 24 h of fermentation. Its bioethanol production was observed to be better than Saccharomyces cerevisiae (13.39 g/L) and their co-culture (15.57 g/L). Sequential changes brought about by the enzymatic treatment of the solid substrate revealed by X-ray diffraction and FTIR analyses showed decrease in the absolute crystallinity (CrI) (54.80 to 48.31%) and total crystallinity index (TCI) (1.002 to 0.991) and confirmed the degradation of the crystalline domain of cellulose by enzyme. The study demonstrated the potential of cellulolytic cocktail produced by A. tubingensis NKBP-55 in rice straw saccharification and its efficient fermentation by conventional and xylose-fermenting yeast in batch ethanolic fermentation.

Ecofriendly biodegradation of Reactive Black 5 by newly isolated Sterigmatomyces halophilus SSA1575, valued for textile azo dye wastewater processing and detoxification

A total of seven yeast strains from 18 xylanolytic and/or xylose-fermenting yeast species isolated from the wood-feeding termite Reticulitermes chinenesis could efficiently decolorize various azo dyes under high-salt conditions. Of these strains, a novel and unique azo-degrading and halotolerant yeast, Sterigmatomyces halophilus SSA1575, has been investigated in this study. This strain could significantly decolorize four combinations of a mixture of dyes. It showed a high capability for decolorizing Reactive Black 5 (RB5) even at 1,500 mg L−1. The strain SSA1575 still showed a high capability for decolorizing a 50 mg L−1 RB5 with a salt mixing at a NaCl concentration of up to 80 g L−1. It also exhibited significant ability to decolorize repeated additions of dye aliquots, with a reduction in time of up to 18 h. Most of the tested carbon and nitrogen sources could significantly enhance a RB5 decolorization. However, this process was inhibited by the addition of sucrose and sodium nitrate. NADH-dichlorophenol indophenol (NADH-DCIP) reductase and lignin peroxidase were determined as the key reductase and oxidase of S. halophilus SSA1575. Finally, strain SSA1575, can effectively detoxify RB5 into non-toxic products. Overall, S. halophilus SSA1575, might be a promising halotolerant yeast valued for the treatment of various textile effluents with high salinity.

Combining Xylose Reductase from Spathaspora arborariae with Xylitol Dehydrogenase from Spathaspora passalidarum to Promote Xylose Consumption and Fermentation into Xylitol by Saccharomyces cerevisiae

In recent years, many novel xylose-fermenting yeasts belonging to the new genus Spathaspora have been isolated from the gut of wood-feeding insects and/or wood-decaying substrates. We have cloned and expressed, in Saccharomyces cerevisiae, a Spathaspora arborariae xylose reductase gene (SaXYL1) that accepts both NADH and NADPH as co-substrates, as well as a Spathaspora passalidarum NADPH-dependent xylose reductase (SpXYL1.1 gene) and the SpXYL2.2 gene encoding for a NAD+-dependent xylitol dehydrogenase. These enzymes were co-expressed in a S. cerevisiae strain over-expressing the native XKS1 gene encoding xylulokinase, as well as being deleted in the alkaline phosphatase encoded by the PHO13 gene. The S. cerevisiae strains expressing the Spathaspora enzymes consumed xylose, and xylitol was the major fermentation product. Higher specific growth rates, xylose consumption and xylitol volumetric productivities were obtained by the co-expression of the SaXYL1 and SpXYL2.2 genes, when compared with the co-expression of the NADPH-dependent SpXYL1.1 xylose reductase. During glucose-xylose co-fermentation by the strain with co-expression of the SaXYL1 and SpXYL2.2 genes, both ethanol and xylitol were produced efficiently. Our results open up the possibility of using the advantageous Saccharomyces yeasts for xylitol production, a commodity with wide commercial applications in pharmaceuticals, nutraceuticals, food and beverage industries.