Thermotolerant Yeast Research Articles

Optimization Conditions for Ethanol Production from Sweet Sorghum Juice by Thermotolerant Yeast Saccharomyces cerevisiae: Using a Statistical Experimental Design

The optimum fermentation conditions for ethanol production from sweet sorghum juice (SSJ) by the thermotolerant yeast Saccharomyces cerevisiae DBKKUY-53 were determined using a statistical experimental design. Based on the Plackett–Burman design (PBD), yeast cell concentration, sugar concentration, and yeast extract were the significant independent fermentation factors affecting the ethanol production from SSJ at 37 °C by S. cerevisiae DBKKUY-53. These significant factors were optimized using response surface methodology (RSM) based on a central composite design (CCD). The result revealed that the optimum conditions for ethanol fermentation were 7.85 × 107 cells/mL yeast cell concentration, 247 g/L sugar concentration, and 9.99 g/L yeast extract. Verification of the ethanol production using the optimum conditions revealed that the maximum ethanol concentration of 99.75 g/L and the productivity of 2.77 g/L/h were achieved. When the ethanol production was carried out in a 2 L fermentor under optimum conditions, the ethanol concentration was 101.81 g/L and the productivity was 2.83 g/L/h. This finding suggested that the thermotolerant yeast S. cerevisiae DBKKUY-53 has excellent potential for commercial ethanol production at high temperatures.

Metabolomics Study Reveals Biomarker L-Proline as Potential Stress-Protectant Compound for High-Temperature Bioethanol Fermentation by Yeast Pichia kudriavzevii 1P4.

High-temperature ethanol fermentation (> 40°C) can be applied as effective bioprocess technology to increase ethanol production. Thermotolerant yeast Pichia kudriavzevii 1P4 showed the ability to produce ethanol at optimum 37°C. Thus, this study evaluated the ethanol productivity of isolate 1P4 at high-temperature ethanol fermentation (42 and 45°C) and the identification of metabolite biomarkers using untargeted metabolomics with liquid chromatography-tandem mass spectrometry (LC-MS/MS). 1P4 showed tolerance to temperature stress up to 45°C and thus relevant for high-temperature fermentation. As measured by gas chromatography (GC), bioethanol production of 1P4 at 30, 37, 42, and 45°C was 5.8g/l, 7.1g/l, 5.1g/l, and 2.8g/l, respectively. The classification of biomarker compounds was based on orthogonal projection analysis to latent structure discriminant analysis (OPLS-DA), resulting in L-proline being a suspected biomarker compound for isolate 1P4 tolerance against high-temperature stress. Indeed, supplementation of L-proline on fermentation medium supported the growth of 1P4 at high temperatures (> 40°C) than without L-proline. The bioethanol production with the addition of the L-proline resulted in the highest ethanol concentration (7.15g/l) at 42°C. Supplementation of L-proline as a stress-protective compound increased ethanol productivity at high-temperature fermentation of 42 and 45°C by 36.35% and 83.33%, respectively, compared without the addition of L-proline. Preliminary interpretation of these results indicates that bioprocess engineering through supplementation of stress-protective compounds L-proline increases the fermentation efficiency of isolate 1P4 at higher temperatures (42°C and 45°C).

Thermotolerance improvement of engineered Saccharomyces cerevisiae ERG5 Delta ERG4 Delta ERG3 Delta, molecular mechanism, and its application in corn ethanol production

BackgroundThe thermotolerant yeast is beneficial in terms of efficiency improvement of processes and reduction of costs, while Saccharomyces cerevisiae does not efficiently grow and ferment at high-temperature conditions. The sterol composition alteration from ergosterol to fecosterol in the cell membrane of S. cerevisiae affects the thermotolerant capability.ResultsIn this study, S. cerevisiae ERG5, ERG4, and ERG3 were knocked out using the CRISPR–Cas9 approach to impact the gene expression involved in ergosterol synthesis. The highest thermotolerant strain was S. cerevisiae ERG5ΔERG4ΔERG3Δ, which produced 22.1 g/L ethanol at 37 °C using the initial glucose concentration of 50 g/L with an increase by 9.4% compared with the wild type (20.2 g/L). The ethanol concentration of 9.4 g/L was produced at 42 ℃, which was 2.85-fold of the wild-type strain (3.3 g/L). The molecular mechanism of engineered S. cerevisiae at the RNA level was analyzed using the transcriptomics method. The simultaneous deletion of S. cerevisiae ERG5, ERG4, and ERG3 caused 278 up-regulated genes and 1892 down-regulated genes in comparison with the wild-type strain. KEGG pathway analysis indicated that the up-regulated genes relevant to ergosterol metabolism were ERG1, ERG11, and ERG5, while the down-regulated genes were ERG9 and ERG26. S. cerevisiae ERG5ΔERG4ΔERG3Δ produced 41.6 g/L of ethanol at 37 °C with 107.7 g/L of corn liquefied glucose as carbon source.ConclusionSimultaneous deletion of ERG5, ERG4, and ERG3 resulted in the thermotolerance improvement of S. cerevisiae ERG5ΔERG4ΔERG3Δ with cell viability improvement by 1.19-fold at 42 °C via modification of steroid metabolic pathway. S. cerevisiae ERG5ΔERG4ΔERG3Δ could effectively produce ethanol at 37 °C using corn liquefied glucose as carbon source. Therefore, S. cerevisiae ERG5ΔERG4ΔERG3Δ had potential in ethanol production at a large scale under supra-optimal temperature.

Mitochondrial Irc3 helicase of the thermotolerant yeast Ogataea polymorpha displays dual DNA- and RNA-stimulated ATPase activity

Irc3 is one of the six mitochondrial helicases described in Saccharomyces cerevisiae. Physiological functions of Irc3 are not completely understood as both DNA metabolic processes and mRNA translation have been suggested to be direct targets of the helicase. In vitro analysis of Irc3 has been hampered by the modest thermostability of the S. cerevisiae protein. Here, we purified a homologous helicase (Irc3op) of the thermotolerant yeast Ogataea polymorpha that retains structural integrity and catalytic activity at temperatures above 40 °C. Irc3op can complement the respiratory deficiency phenotype of a S. cerevisiae irc3Δ mutant, indicating conservation of biochemical functions. The ATPase activity of Irc3op is best stimulated by branched and double- stranded DNA cofactors. Single-stranded DNA binds Irc3op tightly but is a weak activator of the ATPase activity. We could also detect a lower level stimulation with RNA, especially with molecules possessing a compact three-dimensional structure. These results support the idea that that Irc3 might have dual specificity and remodel both DNA and RNA molecules in vivo. Furthermore, our analysis of kinetic parameters predicts that Irc3 could have a regulatory function via sensing changes of the mitochondrial ATP pool or respond to the accumulation of single-stranded DNA.

Isolation and Characterization of Yeasts from Locally Available Foods

A study was conducted to identify different isolates of yeasts which are prospective to be utilized in various industries from locally available foods. Altogether 24 yeast isolates were obtained from fermented fruits and vegetables (banana, cabbage, grapes, lime and mango), pudding, bee honey, toddy and fermented fish samples using the pour plate method. Characterization of the yeasts using several biochemical tests (urease, catalase, liquid carbon and nitrogen assimilation and sugar fermentation tests) revealed that this pool was composed with yeast strains belong to genera of Saccharomyces, Kluvyeromyces, Candida, Pseudozyma, Cryptococcus, Rhodotorula and Debaryomyces. The most effective lactose fermenters were identified as viable candidates for bioethanol production and for manufacturing fermented dairy products for lactose intolerant people. Yeasts with the highest biomass production were suggested as the best viable candidates for industrial single cell protein production using whey, the major byproduct of the dairy industry. The five thermo-tolerant yeasts (Y55, Y57, Y58, Y59 and Y70) and Y069 which was optimally active under 10C were recognized as suitable for industrial applications. The isolates tolerant for high osmotic pressure conditions were identified as potential isolates to be used in highly concentrated food products. Sugarcane juice was recognized as a possible medium for the cultivation of these yeasts in industrial settings. The beneficial yeasts forecasting in this study are expected to screen using molecular biological methods to utilize them in industrial applications.

ЗБРОДЖУВАННЯ КВАСНОГО СУСЛА ТЕРМОТОЛЕРАНТНИМИ ШТАМАМИ МІКРООРГАНІЗМІВ

The regularities of fermentation of yeast wort by thermotolerant strains of microorganisms are studied. For effective fermentation of yeast wort used a culture of thermotolerant yeast Saccharomyces cerevisiae MP-10 and complex lactic acid bacteria in the form of leavens “Kefir”, “Yogurt”, “Symbiotic”, “Bifivit”. The dynamics of changes in dry matter concentration and acidity in different conditions of yeast wort distribution are studied. The optimal content of yeast and lactic acid bacteria is established, namely 4.0 % suspension of yeast and LAB with a yeast content of 80 % of the mass. and lactic acid bacteria 20 % of the mass. It was shown that the drying process can be optimized by using thermotolerant strains of microorganisms and increasing the propagation temperature to 35 °C.

Potato peels waste as a sustainable source for biotechnological production of biofuels: Process optimization

Potato peel waste (PPW) is a starchy by-product generated in great amounts during the industrial processing of potatoes. It can be used as a low cost alternative, and renewable feedstock for the production of second generation bioethanol. In order to intensify this process, Saccharomyces cerevisiae Ethanol Red®, a robust and thermotolerant yeast strain, was selected and two experimental designs and response surfaces assessment were conducted to enable very high gravity fermentations (VHGF) using PPW as feedstock. The first one focused on the optimization of the liquefaction and enzymatic hydrolysis stages, enabling a maximum ethanol concentration of 116.5 g/L and a yield of 80.4 % at 72 h of fermentation; whereas, the second one, focus on the optimization of the pre-saccharification and fermentation stages, which further increased process productivity, leading to a maximum ethanol concentration of 108.8 g/L and a yield of 75.1 % after 54 h of fermentation.These results allowed the definition of an intensified pre-saccharification and simultaneous saccharification and fermentation (PSSF) process for ethanol production from PPW, resorting to short liquefaction and pre-saccharification times, 2 h and 10 h respectively, at an enzyme loading of 80 U/g PPW of Viscozyme and 5 UE/g PPW of SAN Super and a higher fermentation temperature of 34 °C due to the use of a thermotolerant yeast. Overall, with these conditions and solely from PPW without any supplementation, the outlined PSSF process allowed reaching a high ethanol concentration and yield (104.1 g/L and 71.9 %, respectively) standing at high productivities with only 54 h of fermentation.

Higher thermal and ethanol tolerance of a yeast strain isolated from oral cavity

Efficient bioethanol producing microorganisms must be endowed with peculiar physiological and technological traits, such as, higher thermal and ethanol tolerance. This encompasses a strain thermotolerance and an ability to grow at elevated sugar and ethanol concentration or ability to sustain dehydration process such as freeze drying. In this study, we characterized a thermotolerant yeast strain isolated from the human oral cavity regarding the above mentioned parameters. Such an uncommon niche was considered as the great potential reservoir, to isolate strains endowed with metabolic aptitudes requested for ethanol production. In the process, we have defined the YTerm-1strain ability to sustain high sugar and ethanol concentration that are two technological constrains in bioethanol production. Finally, we highlighted that the strain YTerm-1 was able to accumulate, at a high level, trehalose and β-glucan, two compounds conferring the cells an increased resistance to freeze drying process and osmotic stress. Our results suggest that the Yterm-1 strain showed a better growth ability and higher ethanol yield as compared to the industrial strain. Other metabolic traits, such as resistance to dehydration stress, tolerance to ethanol, accumulation of intracellular trehalose or membrane β-glucan confer to that isolate all the characteristics requested in industrial production of ethanol.

Lychee-Derived, Thermotolerant Yeasts for Second-Generation Bioethanol Production

Thermotolerant yeasts are widely considered to be alternative strains to traditional yeasts for bioethanol production at high temperatures. In this study, thirty-two yeasts isolated from lychees were screened for thermotolerance, and seven selected isolates were identified as Candida tropicalis (isolates H8, H19, and H23), Meyerozyma guilliermondii (isolates H1 and H12) and Saccharomyces cerevisiae (isolates H10 and H18). They tolerated up to 45 °C, 12% (v/v) ethanol concentration, 10 g/L acetic acid, and 5 g/L furfural, respectively, and produced 47.96 to 70.18 g/L of ethanol from 160 g/L glucose at 40 °C during 48 h of fermentation. Among the evaluated yeasts, M. guilliermondii H1 showed great potential for second-generation bioethanol fermentation with its ability to ferment xylose and arabinose. Under the optimal conditions resulting from a Plackett Burman design and a Box Behnken design, the highest ethanol concentration of 11.12 g/L was produced from 40 g/L substrate-based sugarcane bagasse hydrolysate (non-detoxified hydrolysate) at 40 °C by M. guilliermondii H1. These findings suggested that the newly isolated thermotolerant yeast M. guilliermondii H1 is a good candidate for ethanol production from agricultural wastes.

RNA polymerase II-driven CRISPR-Cas9 system for efficient non-growth-biased metabolic engineering of Kluyveromyces marxianus

The thermotolerant yeast Kluyveromyces marxianus has gained significant attention in recent years as a promising microbial candidate for industrial biomanufacturing. Despite several contributions to the expanding molecular toolbox for gene expression and metabolic engineering of K. marxianus, there remains a need for a more efficient and versatile genome editing platform. To address this, we developed a CRISPR-based editing system that enables high efficiency marker-less gene disruptions and integrations using only 40 bp homology arms in NHEJ functional and non-functional K. marxianus strains. The use of a strong RNA polymerase II promoter allows efficient expression of gRNAs flanked by the self-cleaving RNA structures, tRNA and HDV ribozyme, from a single plasmid co-expressing a codon optimized Cas9. Implementing this system resulted in nearly 100% efficiency of gene disruptions in both NHEJ-functional and NHEJ-deficient K. marxianus strains, with donor integration efficiencies reaching 50% and 100% in the two strains, respectively. The high gRNA targeting performance also proved instrumental for selection of engineered strains with lower growth rate but improved polyketide biosynthesis by avoiding an extended outgrowth period, a common method used to enrich for edited cells but that fails to recover advantageous mutants with even slightly impaired fitness. Finally, we provide the first demonstration of simultaneous, markerless integrations at multiple loci in K. marxianus using a 2.6 kb and a 7.6 kb donor, achieving a dual integration efficiency of 25.5% in a NHEJ-deficient strain. These results highlight both the ease of use and general robustness of this system for rapid and flexible metabolic engineering in this non-conventional yeast.

High-temperature ethanol fermentation from pineapple waste hydrolysate and gene expression analysis of thermotolerant yeast Saccharomyces cerevisiae

High-temperature ethanol fermentation by thermotolerant yeast is considered a promising technology for ethanol production, especially in tropical and subtropical regions. In this study, optimization conditions for high-temperature ethanol fermentation of pineapple waste hydrolysate (PWH) using a newly isolated thermotolerant yeast, Saccharomyces cerevisiae HG1.1, and the expression of genes during ethanol fermentation at 40 °C were carried out. Three independent variables, including cell concentration, pH, and yeast extract, positively affected ethanol production from PWH at 40 °C. The optimum levels of these significant factors evaluated using response surface methodology (RSM) based on central composite design (CCD) were a cell concentration of 8.0 × 107 cells/mL, a pH of 5.5, and a yeast extract concentration of 4.95 g/L, yielding a maximum ethanol concentration of 36.85 g/L and productivity of 3.07 g/L. Gene expression analysis during high-temperature ethanol fermentation using RT–qPCR revealed that the acquisition of thermotolerance ability and ethanol fermentation efficiency of S. cerevisiae HG1.1 are associated with genes responsible for growth and ethanol stress, oxidative stress, acetic acid stress, DNA repair, the pyruvate-to-tricarboxylic acid (TCA) pathway, and the pyruvate-to-ethanol pathway.

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.

Exploring Natural Fermented Foods as a Source for New Efficient Thermotolerant Yeasts for the Production of Second-Generation Bioethanol

Considering the cost-effectiveness of bioethanol production at high temperatures, there is an enduring need to find new thermotolerant ethanologenic yeasts. In this study, a total of eighteen thermotolerant yeasts were isolated from various natural fermented products in Morocco. Ethanol production using 50 g/L glucose or 50 g/L xylose as the sole carbon source revealed potential yeasts with high productivities and volumetric ethanol productivities at high temperatures. Based on molecular identification, the selected thermotolerant fermentative isolates were affiliated with Pichia kudriavzevii, Kluyveromyces marxianus, and Kluyveromyces sp. During the simultaneous saccharification and fermentation of lignocellulosic biomass at a high temperature (42 °C), the designated yeast P. kudriavzevii YSR7 produced an ethanol concentration of 22.36 g/L, 18.2 g/L and 6.34 g/L from 100 g/L barley straw (BS), chickpea straw (CS), and olive tree pruning (OTP), respectively. It also exhibited multi-stress tolerance, such as ethanol, acetic acid, and osmotic tolerance. Therefore, the yeast P. kudriavzevii YSR7 showed promising attributes for biorefinery-scale ethanol production in the future.

The ability of Pichia kudriavzevii to tolerate multiple stresses makes it promising for developing improved bioethanol production processes.

Thermotolerant ethanol fermenting yeasts have been extensively used in industrial bioethanol production. However, little is known about yeast physiology under stress during bioethanol processing. This study investigated the physiological characteristics of the thermotolerant yeast Pichia kudriavzevii, strains NUNS-4, NUNS-5 and NUNS-6, under the multiple stresses of heat, ethanol and sodium chloride. Results showed that NUNS-4, NUNS-5 and NUNS-6 displayed higher growth rates under each stress condition than the reference strain, Saccharomyces cerevisiae TISTR5606. Maximum specific growth rates under stresses of heat (45°C), 15% v/v ethanol and 1·0 M sodium chloride were 0·23 ± 0·04 (NUNS-4), 0·11 ± 0·01 (NUNS-5) and 0·15 ± 0·01 h-1 (NUNS-5), respectively. Morphological features of all yeast studied changed distinctly with the production of granules and vacuoles when exposed to ethanol, and cells were elongated under increased sodium chloride concentration. This study suggests that the three P. kudriavzevii strains are potential candidates to use in industrial-scale fermentation due to a high specific growth rate under multiple stress conditions. Multiple stress-tolerant P. kudriavzevii NUNS strains have received much attention not only for improving large-scale fuel ethanol production, but also for utilizing these strains in other biotechnological industries.

Production of ethanol, lipid and lactic acid from mixed agrowastes hydrolysate

To combat the shortage of single agro-residue and overcome the problem of seasonal availability, it is beneficial to use mixture of lignocellulosic biomasses. In the present study, efforts were made to use mixed lignocellulosic biomass for production of bioethanol, along with microbial lipids and lactic acid. Upon enzymatic hydrolysis of mixed biomass at varied proportions it was observed that mixture of paddy straw and jute in the ratio 3:1 resulted in best sugar yield (41.50 g/L) at 10% substrate loading. Ethanolic fermentation of mixed substrate hydrolysate by thermotolerant yeast, Saccharomyces cerevisiae JRC6 resulted in 8.39 g/L of ethanol. To maintain sustainability and economic impact, oleaginous yeast (Trichosporon mycotoxinivorans S2) and lactic acid bacteria (Lactobacillus plantarum LP-9) were used for lipid production (14.5 g/L) and lactic acid production (11.08 g/L), respectively. Therefore, this study explored the potential of mixed lignocellulosic biomass to be exploited for production of various value-added products.

Identification of a novel gene required for competitive growth at high temperature in the thermotolerant yeast Kluyveromyces marxianus.

It is important to understand the basis of thermotolerance in yeasts to broaden their application in industrial biotechnology. The capacity to run bioprocesses at temperatures above 40 °C is of great interest but this is beyond the growth range of most of the commonly used yeast species. In contrast, some industrial yeasts such as Kluyveromyces marxianus can grow at temperatures of 45 °C or higher. Such species are valuable for direct use in industrial biotechnology and as a vehicle to study the genetic and physiological basis of yeast thermotolerance. In previous work, we reported that evolutionarily young genes disproportionately changed expression when yeast were growing under stressful conditions and postulated that such genes could be important for long-term adaptation to stress. Here, we tested this hypothesis in K. marxianus by identifying and studying species-specific genes that showed increased expression during high-temperature growth. Twelve such genes were identified and 11 were successfully inactivated using CRISPR-mediated mutagenesis. One gene, KLMX_70384, is required for competitive growth at high temperature, supporting the hypothesis that evolutionary young genes could play roles in adaptation to harsh environments. KLMX_70384 is predicted to encode an 83 aa peptide, and RNA sequencing and ribo-sequencing were used to confirm transcription and translation of the gene. The precise function of KLMX_70384 remains unknown but some features are suggestive of RNA-binding activity. The gene is located in what was previously considered an intergenic region of the genome, which lacks homologues in other yeasts or in databases. Overall, the data support the hypothesis that genes that arose de novo in K. marxianus after the speciation event that separated K. marxianus and K. lactis contribute to some of its unique traits.

Valorization of Pinus taeda hemicellulosic hydrolysate for the production of value-added compounds in an ethanol biorefinery

Production of cellulosic ethanol from lignocellulosic biomass leads to the generation of a hemicellulosic hydrolysate during the feedstock pretreatment. This hydrolysate is rich in sugars, but also contains inhibitory compounds (mainly acetic acid and phenolic compounds) in concentrations that may be toxic to microbial growth. Currently, this side-stream of the cellulosic ethanol production process is processed as a waste, due to the lack of feasible alternatives for tackling the complexity of wood hemicellulosic hydrolysate. Thus, this work evaluated the ability of six microorganisms to metabolize the raw and detoxified hemicellulosic hydrolysate produced from Pinus taeda for the production of lactic acid, ethanol, xylitol, single-cell protein, lipids and carotenoids, with the aim of selecting a potential alternative for valorization of this side stream generated during the production of cellulosic ethanol contributing to the implementation of a sustainable advanced biorefinery. The tested microorganisms included a lactic acid bacterium, Bacillus coagulans; a probiotic bacterium, Lactobacillus salivarius; two oleaginous yeasts, Rhodosporidium toruloides and Saitoella coloradoensis; a thermotolerant yeast, Kluyveromyces marxianus; and a methylotrophic yeast, Hansenula polymorpha. L. salivarius, K. marxianus, and H. polymorpha showed promising ability to metabolize the partially detoxified hydrolysate (composed of (g/L): mannose, 29.27; glucose, 17.25; galactose, 6.18; xylose, 4.94; arabinose, 1.23; acetic acid, 7.99; formic acid, 4.86; levulinic acid, 4.04; 5-hydroxymethylfurfural, 0.74; total phenolic compounds, 0.40). On the other hand, the oleaginous yeasts and B. coagulans presented high sensitivity to the inhibitory compounds. L. salivarius produced lactic acid with high yield (1.1 g/g), which was limited by product inhibition. K. marxianus produced xylitol at 0.37 g/g xylose and ethanol at 0.19 g/g hexoses. Finally, H. polymorpha converted hexoses and acetic acid into single-cell protein with yield of 0.27 g/g. The production of lactic acid by L. salivarius proved to be a promising alternative for valorization of Pinus hemicellulosic hydrolysate in an ethanol biorefinery.

Viability of probiotics, physicochemical and microbiological characterization of beverage (smoothie) with symbiotic yogurt and berries pulp

The aim of this study was to evaluate the viability of commercial probiotic bacteria (bifidobacteria) in symbiotic beverages made with symbiotic yoghurts and berries pulp, after manufacturing and refrigerated storage. Six beverage formulations were prepared using symbiotic yogurt (60%) containing inulin or fructooligosaccharides (FOS) and commercial probiotic cultures (Bifidobacterium spp.) and berries pulp (40%), as follows: F1 (Howaru HN 019, inulin); F2 (Howaru HN 019, FOS); F3 (Lafti B 94, inulin); F4 (Lafti B 94, FOS); F5 (Kit Bifi, inulin); F6 (Kit Bifi, FOS). The beverages were evaluated for microbiological quality (total and thermotolerant coliforms, molds and yeasts), viability of probiotics, pH and titratable acidity. The pH, acidity and probiotic counts were investigated during 30 days of storage. The physicochemical characterization of the formulations with better performance regarding the maintenance of probiotic counts was carried out. The beverages elaborated showed appropriate sanitary hygienic quality, decreased pH and increased acidity, which is common in fermented dairy products. The probiotic Howaru HN019 exhibited better stability in the beverage than Lafti B4 and Kit Bifi. The highest level of bifidobacteria was found in F1 and F2 beverages containing B. animalis (Howaru HN019) and inulin or FOS, remained around 8 log CFU.mL-1 for up to 30 days, whereas levels of 6 to 7 log CFU.mL-1 were maintained in the other beverages. The beverages made with symbiotic yogurt and berries pulp can be considered an appropriate vehicle for the incorporation of probiotics and fibers, whereas, the probiotics remain at a satisfactory level throughout storage.

Respiratory reoxidation of NADH is a key contributor to high oxygen requirements of oxygen-limited cultures of Ogataea parapolymorpha.

While thermotolerance is an attractive trait for yeasts used in industrial ethanol production, oxygen requirements of known thermotolerant species are incompatible with process requirements. Analysis of oxygen-sufficient and oxygen-limited chemostat cultures of the facultatively fermentative, thermotolerant species Ogataea parapolymorpha showed its minimum oxygen requirements to be an order of magnitude larger than those reported for the thermotolerant yeast Kluyveromyces marxianus. High oxygen requirements of O. parapolymorpha coincided with a near absence of glycerol, a key NADH/NAD+ redox-cofactor-balancing product in many other yeasts, in oxygen-limited cultures. Genome analysis indicated absence of orthologs of the Saccharomyces cerevisiae glycerol-3-phosphate-phosphatase genes GPP1 and GPP2. Co-feeding of acetoin, whose conversion to 2,3-butanediol enables reoxidation of cytosolic NADH, supported a 2.5-fold increase of the biomass concentration in oxygen-limited cultures. An O. parapolymorpha strain in which key genes involved in mitochondrial reoxidation of NADH were inactivated did produce glycerol, but transcriptome analysis did not reveal a clear candidate for a responsible phosphatase. Expression of S. cerevisiae GPD2, which encodes NAD+-dependent glycerol-3-phosphate dehydrogenase, and GPP1 supported increased glycerol production by oxygen-limited chemostat cultures of O. parapolymorpha. These results identify dependence on respiration for NADH reoxidation as a key contributor to unexpectedly high oxygen requirements of O. parapolymorpha.

Distinct Metabolic Flow in Response to Temperature in Thermotolerant Kluyveromyces marxianus.

The intrinsic mechanism of the thermotolerance of Kluyveromyces marxianus was investigated by comparison of its physiological and metabolic properties at high and low temperatures. After glucose consumption, the conversion of ethanol to acetic acid became gradually prominent only at a high temperature (45°C) and eventually caused a decline in viability, which was prevented by exogenous glutathione. Distinct levels of reactive oxygen species (ROS), glutathione, and NADPH suggest a greater accumulation of ROS and enhanced ROS-scavenging activity at a high temperature. Fusion and fission forms of mitochondria were dominantly observed at 30°C and 45°C, respectively. Consistent results were obtained by temperature upshift experiments, including transcriptomic and enzymatic analyses, suggesting a change of metabolic flow from glycolysis to the pentose phosphate pathway. The results of this study suggest that K. marxianus survives at a high temperature by scavenging ROS via metabolic change for a period until a critical concentration of acetate is reached. IMPORTANCE Kluyveromyces marxianus, a thermotolerant yeast, can grow well at temperatures over 45°C, unlike Kluyveromyces lactis, which belongs to the same genus, or Saccharomyces cerevisiae, which is a closely related yeast. K. marxianus may thus bear an intrinsic mechanism to survive at high temperatures. This study revealed the thermotolerant mechanism of the yeast, including ROS scavenging with NADPH, which is generated by changes in metabolic flow.