High-temperature Fermentation Research Articles

Competition between biodetoxification fungus and lactic acid bacterium in the biorefinery processing chain for production of cellulosic L-lactic acid

Biodetoxification fungus selectively degrades toxic inhibitors generated from pretreatment of lignocellulose without consuming fermentable sugars. However, one barrier for practical application is the sustained cell viability in the consequent fermentation step to compete the fermentable sugars with fermenting strains, resulting in sugar loss and reduced target product yield. This study investigated the competitive growth property between the biodetoxification fungus Paecilomyces variotii FN89 and the L-lactic acid bacterium Pediococcus acidilactici ZY271 under varying temperature and lactic acid osmatic stress. The results show that the L-lactic acid bacterium Ped. acidilactici ZY271 showed less thermotolerance to Pae. variotii FN89 at high temperature of 45 °C to 50 °C in both synthetic medium and wheat straw hydrolysate. In the higher temperature environment, the growth of the biodetoxification strian failed to compete with the lactic acid fermentation strain and was quickly eliminated from the fermentation system. The high temperature fermentation facilitated a fast transition from the detoxification stage to the fermentation stage for higher production of L-lactic acid.

The impact of microbial inoculants on large-scale composting of straw and manure under natural low-temperature conditions

Understanding large-scale composting under natural conditions is essential for improving waste management and promoting sustainable agriculture. In this study, corn straw (400 tons) and pig manure (200 tons) were composted with microbial inoculants. The thermophilic phase of composting lasted for fourteen weeks, resulting in an alkaline final product. Microbial systems with low-temperature initiation and high-temperature fermentation played a crucial role in enhancing lignocellulose degradation and humic substances (HS) formation. Adding microbes, including Rhodanobacter, Pseudomonas, and Planococcus, showed a positive correlation with degradation rates of cellulose, hemicellulose, and lignin. Bacillus, Planococcus, and Acinetobacter were positively correlated with HS formation. Microorganisms facilitated efficient hydrolysis of lignocelluloses, providing humic precursors to accelerate composting humification through phenolic protein and Maillard pathways. This study provides significant insights into large-scale composting under natural conditions, contributing to the advancement of waste management strategies and the promotion of sustainable agriculture.

餐厨垃圾高温好氧发酵工艺的研究

With the rapid urbanization in China, there has been a gradual increase in the production of kitchen waste, which poses significant environmental challenges. High-temperature aerobic fermentation is an effective method for recycling kitchen waste. This study focuses on utilizing kitchen waste, wood chips, and compound microbial agents as the main raw materials for fermentation. Various parameters such as temperature, moisture content, and amount of compound microbial agent were selected to conduct experiments on the high-temperature stage of the aerobic fermentation process for kitchen waste. Through response surface optimization experiments, it was determined that the optimal conditions for achieving fast high-temperature fermentation of kitchen waste are as follows: temperature at 60℃, moisture content at 60%, and amount of compound microbial agent at 10%. The order of influence on the reduction rate of kitchen waste is found to be temperature followed by the amount of compound microbial agent and moisture content. These findings provide valuable insights into resource utilization strategies for managing kitchen waste.

Pediococcus spp.-mediated competition interaction within Daqu microbiota determines the temperature formation and metabolic profiles.

Fermented microbiota is critical to the formation of microenvironment and metabolic profiles in spontaneous fermentation. Microorganisms generate a diverse array of metabolites concurrent with the release of heat energy. In the case of Daqu fermentation, the peak temperature exceeded 60°C, forming a typical high-temperature fermentation system known as high-temperature Daqu. However, microorganisms that cause the quality variation in Daqu and how they affect the functional microbiota and microenvironment in the fermentation process are not yet clear. This study adopted high-throughput sequencing and monitored the dynamic fluctuations of metabolites and environmental factors to identify the pivotal microorganism responsible for the alterations in interaction patterns of functional keystone taxa and quality decline in the fermentation system of different operational areas during the in situ fermentation process that had been mainly attributed to operational taxonomic unit (OTU)_22 (Pediococcus acidilactici). Additionally, we used isothermal microcalorimetry, plate inhibition experiments, and in vitro simulation fermentation experiments to explore the impact of Pediococcus spp. on heat generation, microorganisms, and metabolite profiles. Results showed the heat peak generated by Pediococcus spp. was significantly lower than that of Bacillus spp., filamentous fungi, and yeast. In addition, the preferential growth of P. acidilactici strain AA3 would obviously affect other strains to colonize through competition, and its metabolites made a significant impact on filamentous fungi. The addition of P. acidilactici strain AA3 in simulated fermentation would cause the loss of pyrazines and acids in metabolites. These evidences showed that the overgrowth of Pediococcus spp. greatly influenced the formation of high temperatures and compounds in solid-state fermentation systems. Our work illustrated the vital impact of interaction variability mediated by Pediococcus spp. for microbial assembly and metabolites, as well as in forming temperature. These results emphasized the functional role of Daqu microbiota in metabolites and heat production and the importance of cooperation in improving the fermentation quality.IMPORTANCEThe stable and high-quality saccharifying and fermenting starter in traditional solid-state fermentation was the prerequisite for liquor brewing. An imbalance of microbial homeostasis in fermentation can adversely impact production quality. Identification of such critical microorganisms and verifying their associations with other fermentation parameters pose a challenge in a traditional fermentation environment. To enhance the quality of spontaneous fermented products, strategies such as bioaugmentation or the control of harmful microorganisms would be employed. This work started with the differences in high-temperature Daqu metabolites to explore a series of functional microorganisms that could potentially contribute to product disparities, and found that the differences in interactions facilitated directly or indirectly by Pediococcus spp. seriously affected the development of microbial communities and metabolites, as well as the formation of the microenvironment. This study not only identified functional microbiota in Daqu that affected fermentation quality, but also demonstrated how microorganisms interact to affect the fermentation system, which would provide guidance for microbial supervision in the actual production process. Besides, the application of isothermal microcalorimetry in this study was helpful for us to understand the heat production capacity of microorganisms and their adaptability to the environment. This study presented a commendable framework for improving and controlling the quality of traditional fermentation and inspired further investigations in similar systems.

High-temperature fermentation of corn stover for biodiesel production using Candida tropicalis with enhanced lipid accumulation: A new strategy for breeding thermotolerant biodiesel-production strains

Efficient biodiesel production from biomass is essential for the green-oriented energy transition. The current incongruity between fermentation temperature and microbial thermotolerance is one of the key factors restricting biodiesel production, and it is difficult to solve this problem relying only on conventional microbial characterization and breeding methods. We propose a strategy to solve the corn stover cellulose utilization problem by developing thermotolerant biodiesel-production yeast strains. Candida tropicalis was initially improved by adaptive laboratory evolution and then mutated by atmospheric and room-temperature plasma mutagenesis to obtain a thermotolerant strain capable of growing at temperatures up to 45 °C. Two rounds of fluorescence-activated cell sorting were used to select high lipid content cells, which resulted in a 16.8% increase in lipid accumulation capacity compared to the original cells. The simultaneous saccharification fermentation of the generated strain with pretreated corn stover at 44 °C yielded a lipid generation efficiency of 86.2 ± 2 mg/g. This study provides a new method and an alternative strain for the selection and breeding of high-thermotolerance and high biodiesel-production yeasts and offers a feasible strategy for lignocellulosic fermentation for biodiesel production at elevated temperatures using the new industrial strain.

A thermostable type I-B CRISPR-Cas system for orthogonal and multiplexed genetic engineering

Thermophilic cell factories have remarkably broad potential for industrial applications, but are limited by a lack of genetic manipulation tools and recalcitrance to transformation. Here, we identify a thermophilic type I-B CRISPR-Cas system from Parageobacillus thermoglucosidasius and find it displays highly efficient transcriptional repression or DNA cleavage activity that can be switched by adjusting crRNA length to less than or greater than 26 bp, respectively, without ablating Cas3 nuclease. We then develop an orthogonal tool for genome editing and transcriptional repression using this type I-B system in both thermophile and mesophile hosts. Empowered by this tool, we design a strategy to screen the genome-scale targets involved in transformation efficiency and established dynamically controlled supercompetent P. thermoglucosidasius cells with high efficiency ( ~ 108 CFU/μg DNA) by temporal multiplexed repression. We also demonstrate the construction of thermophilic riboflavin cell factory with hitherto highest titers in high temperature fermentation by genome-scale identification and combinatorial manipulation of multiple targets. This work enables diverse high-efficiency genetic manipulation in P. thermoglucosidasius and facilitates the engineering of thermophilic cell factories.

Elucidation and engineering mitochondrial respiratory-related genes for improving bioethanol production at high temperature in Saccharomyces cerevisiae

Industrial manufacturing of bioproducts, especially bioethanol, can benefit from high-temperature fermentation, which requires the use of thermotolerant yeast strains. Mitochondrial activity in yeast is closely related to its overall metabolism. However, the mitochondrial respiratory changes in response to adaptive thermotolerance are still poorly understood and have been rarely utilized for developing thermotolerant yeast cell factories. Here, adaptive evolution and transcriptional sequencing, as well as whole-genome-level gene knockout, were used to obtain a thermotolerant strain of Saccharomyces cerevisiae. Furthermore, thermotolerance and bioethanol production efficiency of the engineered strain were examined. Physiological evaluation showed the boosted fermentation capacity and suppressed mitochondrial respiratory activity in the thermotolerant strain. The improved fermentation produced an increased supply of adenosine triphosphate required for more active energy-consuming pathways. Transcriptome analysis revealed significant changes in the expression of the genes involved in the mitochondrial respiratory chain. Evaluation of mitochondria-associated gene knockout confirmed that ADK1, DOC1, or MET7 were the key factors for the adaptive evolution of thermotolerance in the engineered yeast strain. Intriguingly, overexpression of DOC1 with TEF1 promoter regulation led to a 10.1% increase in ethanol production at 42 °C. The relationships between thermotolerance, mitochondrial activity, and respiration were explored, and a thermotolerant yeast strain was developed by altering the expression of mitochondrial respiration-related genes. This study provides a better understanding on the physiological mechanism of adaptive evolution of thermotolerance in yeast.

Draft genome of the Ogataea polymorpha type strain CBS 4732.

The methylotrophic yeast Ogataea polymorpha is of significant biotechnological interest, particularly in high-temperature fermentations and for recombinant protein production. Here, we present a high-quality genome assembly for the O. polymorpha type strain CBS 4732.

Transcriptomic analysis reveals hub genes and pathways in response to acetic acid stress in Kluyveromyces marxianus during high-temperature ethanol fermentation

The thermotolerant yeast Kluyveromyces marxianus is known for its potential in high-temperature ethanol fermentation, yet it suffers from excess acetic acid production at elevated temperatures, which hinders ethanol production. To better understand how the yeast responds to acetic acid stress during high-temperature ethanol fermentation, this study investigated its transcriptomic changes under this condition. RNA sequencing (RNA-seq) was used to identify differentially expressed genes (DEGs) and enriched gene ontology (GO) terms and pathways under acetic acid stress. The results showed that 611 genes were differentially expressed, and GO and pathway enrichment analysis revealed that acetic acid stress promoted protein catabolism but repressed protein synthesis during high-temperature fermentation. Protein–protein interaction (PPI) networks were also constructed based on the interactions between proteins coded by the DEGs. Hub genes and key modules in the PPI networks were identified, providing insight into the mechanisms of this yeast's response to acetic acid stress. The findings suggest that the decrease in ethanol production is caused by the imbalance between protein catabolism and protein synthesis. Overall, this study provides valuable insights into the mechanisms of K. marxianus's response to acetic acid stress and highlights the importance of maintaining a proper balance between protein catabolism and protein synthesis for high-temperature ethanol fermentation.

Temperature monitoring system of beer fermentation and brewing based on immune fuzzy PID controller

AbstractBeer is one of the popular drinks, the temperature control in the process of beer fermentation plays a crucial role. The current temperature control method mainly uses the traditional PID control, but its control adjustment time is long, the overshoot is large, the control effect still needs to be improved. A beer fermentation and brewing temperature monitoring system based on immune fuzzy PID controller was designed in this experiment. Immune fuzzy PID controller is a nonlinear controller, which combines the advantages of traditional PID controller and fuzzy controller and refers to the regulatory mechanism of biological immune system, and obtains good suitable characteristics by controlling the parameter values of the system. PID converts the rule information into fuzzy information by fuzzy basic theory and stores it in computer database. By referring to the actual situation of PID, the computer uses fuzzy reasoning to adjust the PID parameters. The beer fermentation temperature monitoring system based on the traditional PID controller is compared with the proposed system. Under the control of the designed temperature monitoring system, the temperature has a certain effect on the fermentation speed of beer. The fermentation time of high temperature fermentation (16°C) is 3 days shorter than that of normal temperature fermentation (10°C). The robustness and applicability of the system are verified.

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).

Effect of Different Excipients on Fungal Flora during Aerobic Fermentation of Thermal Hydrolyzed Kitchen Waste

In this paper, a comparative study was conducted on the aerobic fermentation of kitchen waste pretreated by thermal hydrolysis and three different excipients, the dynamic changes of temperature and organic matter of kitchen waste after thermal hydrolysis were measured during aerobic fermentation with peanut shell, corn stalk and corn cob as additives, and high-throughput sequencing was performed on the samples of the three excipients in the high temperature fermentation stage to identify the dominant bacterial community in the high temperature region of different excipients. The results showed that the dominant bacterial communities of peanut shell, corn stalk and corn cob were similar at the phylum classification level. Among them, Ascomycota accounted for 97.68%, 99.55% and 99.98% in HS, YM and YX treatment groups, respectively, which means it was the absolute dominant fungal phyla in high temperature fermentation stage. At the level of genus classification, there were differences among the dominant bacterial genera in each group. The main bacterial genera in peanut shell treatment group were Aspergillus, unclassified_f__Dipodascaceae, Penicillium, unclassified_o__Sordariales, Candida and Diutina. The main bacterial genera in corn stalk treatment group were Candida, Aspergillus and Penicillium. The main bacteria in corn cob treatment group was unclassified_o__Sordariales. The percentage of saprophytic fungi in HS, YM and YX high-temperature treatment groups was 96.40%, 99.25% and 99.97%, respectively. Saprophytic fungi played a major role in the high-temperature fermentation stage.

Activating a dormant metabolic pathway for high-temperature l-alanine production in Bacillus licheniformis.

l-Alanine is an important amino acid widely used in food, medicine, materials, and other fields. Here, we develop Bacillus licheniformis as an efficient l-alanine microbial cell factory capable of realizing high-temperature fermentation. By enhancing the glycolytic pathway, knocking out the by-product pathways andoverexpressing the thermostable alanine dehydrogenase, the engineered B. licheniformis strain BLA3 produced 93.7g/L optically pure l-alanine at 50°C. Subsequently, d-alanine dependence of an alanine racemase-deficient strain is relieved by adaptive laboratory evolution, implying that a dormant alternative pathway for d-alanine synthesis is activated in the evolved strain. The d-amino acid aminotransferase Dat1 is shown to be a key enzyme in the dormant alternative pathway. Molecular mechanism of the d-alanine dependence is revealed via mutational analysis. This study demonstrates a novel technology for high-temperature l-alanine production and shows that activating dormant metabolic pathway(s) is an effective strategy of metabolic engineering.

Peptidomics analysis of Jiang-Flavor Daqu from high-temperature fermentation to mature and in different preparation season

Jiang-Flavor Daqu (JFDQ) is a grain-type fermented starter for brewing Chinese liquor. Peptides, the metabolites of proteins in JFDQ, are important for the quality and flavor of JFDQ or even the liquor. The peptide variations in the progress of JFDQ preparation were investigated using RPLC-MS/MS. The JFDQ after high-temperature fermenting (HTF_SU) and after ripening (M_SU), as well as the mature JFDQ prepared in spring (M_SP) and in summer (M_SU), were compared respectively. These two groups were investigated from peptides, precursor proteins, abundance, interactions, and potential antimicrobial peptides (pAMPs). A total of 177, 158, and 262 peptides from HTF_SU, M_SP, and M_SU were identified, respectively. Significant differences (P < 0.01) in the abundance of shared peptides were found in different fermentation stage group (HTF_M), and stronger positive correlations were observed in different preparation season group (MSP_MSU). The interactions of the shared peptides in HTF_M and in MSP_MSU were investigated respectively. In addition, 8 pAMPs in HTF_SU, 5 in M_SP, and 22 in M_SU were predicted using CAMPR3, and their core functional regions were analyzed. This systematic study demonstrated the influences of fermentation stage and preparation season on the peptide profiles in JFDQ, which would provide theoretical guidance and be helpful for JFDQ production. SignificancePeptidomics analysis showed that the peptide profiles of JFDQ varied in different fermentation stages and different preparation seasons, which mainly resulted from the peptides with high abundance, high interaction degrees, and potential antimicrobial activity, as well as the important precursor proteins such as glutens. This systematic study would benefit for the insufficiency of peptide research of JFDQ till now, and provide theoretical guidance for JFDQ production.

Insights into organic matter evolution during food waste stabilization induced by 14-hour high-temperature fermentation.

Large amounts of secondary pollutants are released during traditional composting, and rapid fermentation is desirable for the stabilization of food wastes. Food wastes were mixed with rice husk, placed in a bioreactor, and stirred continuously to achieve high-temperature fermentation for 14h. The transformations of the mixtures were analyzed using elemental and spectral analysis combined with kinetic equations and two-dimensional correlation spectroscopy. The carbohydrates, proteins, and aliphatic compounds of food waste were degraded after 4h of fermentation. Transformations of dissolved organic and sulfur- and nitrogen-containing substances followed first-order kinetic equations with reaction rate constants of 0.142h-1, 0.098h-1, and 0.016h-1, respectively. Organic matter conversion was in the following order: aliphatic → protein → carbohydrate and followed the order, acrylamide C → O-alkyl C → anomeric C at the molecular level. The fermentation process was characterized by the increase in protein- and fulvic-like compounds. Fulvic acid substances gradually accumulated during the late fermentation period. Thus, dissolved organic matter components were gradually transformed into humic substances with increasing fermentation time. The sequence of transformation during the fermentation process was, tyrosine-like → tryptophan-like → fulvic-like substances. Humification mainly occurred in the mature stage of composting; therefore, it was verified that the food waste was stabilized by a 14-h fermentation.

MICRO-POSITIVE PRESSURE AERATION FOR WINDROW COMPOSTING OF MUNICIPAL SOLID WASTE IN CHANDIGARH: AN OPPORTUNITY IN A CARBON CONSCIOUS WORLD FOR COMBATING CLIMATE CHANGE

The present study was a pilot project initiated in Chandigarh to discuss the feasibility andeffectiveness of adept schemes for successful co-composting of green waste and kitchen waste(food and fruit) waste in 64 m2 open covered windrows with bulking grass layer bottom upventilation system generating micro-positive pressure, making windrow piles aerobic, organolepticwith reduced green -house gases (GHGs) emission. The compost temperature profile shows twothermophilic peaks; first in bioreactor (74 oC) and second in charging pit (76.2 oC) after 40 days oninoculation and the high temperature fermentation was maintained for 80days. The high qualityfinal compost has 40oC temperature, 7.6pH, 42% moisture content, 3.36ds/m electrical conductivityand 25 C/N ratio. The main objective of the present study is to analyze and overview theconfiguration technology and analytical parameters to limit the global temperature rise to 1.5 oC,to meet the goals of the Paris agreement to avoid catastrophic impacts of climate change. Theinference of the study is the mitigation of carbon leakage, generating 346.7 carbon credits and 564quintals organic compost worth 25 lakhs to achieve sustainable zero waste future.

Combined effects of fermentation temperature and Saccharomyces cerevisiae strains on free amino acids, flavor substances, and undesirable secondary metabolites in huangjiu fermentation

Fermentation temperature (FT) is one of the most critical factors, which can be used to control the growth conditions of S. cerevisiae to obtain excellent final products in winemaking. In this study, we analyzed the responses of six S. cerevisiae strains with different temperature preferences to FT (20 °C, 30 °C, and 35 °C) in huangjiu fermentation. The flavor substances, free amino acids and undesirable secondary metabolites related to huangjiu quality were determined. Results indicated that both S. cerevisiae strains and FT had significant effects on huangjiu fermentation, while the effects were strain-independent and differentiated temperature preferences for different fermentation characteristics. We found that the effects of FT were greater than that of S. cerevisiae strains under the premise of satisfying fermentation completion. Low temperature (20 °C) and high temperature (35 °C) fermentation were possible in the production of differentiated industrial huangjiu styles, while the effects of FT on undesirable secondary metabolites needed to be considered before industrial application. The results showed that a combination of FT with one or more S. cerevisiae strains could be used as a fermentation starter in huangjiu production for different types of products.

Highly efficient production of 2,3-butanediol from xylose and glucose by newly isolated thermotolerant Cronobacter sakazakii

Background2,3-Butanediol (2,3-BD), a valuable compound used for chemicals, cosmetics, pesticides and pharmaceuticals, has been produced by various microbes. However, no high-temperature fermentation of the compound at high productivity has been reported.MethodsThermotolerant xylose-utilizing microbes were isolated from 6 different districts in Laos and screened for a low accumulation of xylitol in a xylose medium at 37 ˚C. One isolate was found to produce 2,3-BD and identified by 16S rDNA sequencing. The 2,3-BD fermentation capacity was investigated at different temperatures using xylose and glucose as carbon sources, and the fermentation parameters were determined by a high-performance liquid chromatography system.ResultsBy screening for a low accumulation of xylitol in a xylose medium, one isolate that accumulated almost no xylitol was obtained. Further analyses revealed that the isolate is Cronobacter sakazakii and that it has the ability to produce 2,3-BD at high temperatures. When xylose and glucose were used, this strain, named C. sakazakii OX-25, accumulated 2,3-BD in a short period before the complete consumption of these sugars and then appeared to convert 2,3-BD to acetoin. The optimum temperature of the 2,3-BD fermentation was 42 ˚C to 45 ˚C, and the maximum yield of 2,3-BD was 0.3 g/g at 12 h in 20 g/l xylose medium and 0.4 g/g at 6 h in 20 g/l glucose medium at 42 ˚C. The 2,3-BD productivity of the strain was higher than the 2,3-BD productivities of other non-genetically engineered microorganisms reported previously, and the highest productivity was 0.6 g/l·h and 1.2 g/l·h for xylose and glucose, respectively.ConclusionsAmong thermotolerant microbes isolated in Laos, we discovered a strain, C. sakazakii OX-25, that can convert xylose and glucose to 2,3-BD with high efficiency and high productivity at high temperatures, suggesting that C. sakazakii OX-25 has the potential for industrial application to produce 2,3-BD as an important platform chemical.

Mutants with Enhanced Cellobiose-Fermenting Ability from Thermotolerant Kluyveromyces marxianus DMKU 3-1042, Which Are Beneficial for Fermentation with Cellulosic Biomass

Several cellulose-hydrolysis enzymes are required for eco-friendly utilization of cellulose as renewable biomass, and it would therefore be beneficial if fermenting microbes can provide such enzymes without genetic engineering. Thermotolerant and multisugar-fermenting Kluyveromyces marxianus is one of the promising yeasts for high-temperature fermentation and has genes for putative oligosaccharide-degradation enzymes. Mutants obtained after multiple mutagenesis showed significantly higher activity than that of the parental strain for cellobiose fermentation. The efficient strains were found to have amino acid substitutions and frame-shift mutations in 26-28 genes including 3 genes for glucose transporters. These strains grown in a cellobiose medium showed higher β-glucosidase than that of the parental strain and greatly reduced glucose utilization. The introduction of KTH2 for a glucose transporter into one of the efficient mutants reduced the cellobiose fermentation activity of the mutant. The results suggest that release from glucose repression significantly promotes the uptake of cellobiose. Co-culture of one efficient strain and the parental strain allowed good fermentation of both glucose and cellobiose, suggesting that the efficient strains are useful for conversion of cellulosic biomass to ethanol.

Transcriptomes analysis of Pichia kudriavzevii UniMAP 3–1 in response to acetic acid supplementation in glucose and xylose medium at elevated fermentation temperature

The yeast Pichia kudriavzevii is gaining attention as a potential lignocellulosic bioethanol producer due to its multiple stress tolerance characteristics. RNA sequencing was performed to analyze the transcriptomics profiling of an isolated strain, Pichia kudriavzevii UniMAP 3–1 in response to acetic acid supplementation at 40ºC in individual glucose and xylose medium. Acetic acid was found to be consumed simultaneously with glucose when grown in glucose media. Transcriptomics profiling revealed that the response of P. kudriavzevii UniMAP 3–1 towards acetic acid includes activation of differentially expressed genes (DEGs) involved in tricarboxylic acid (TCA) cycle, ATP-binding cassette (ABC) transporters, and folding, sorting and degradation, both in glucose and xylose media. Conversely, DEGs involved in energy metabolism were only apparent in xylose media. DEGs categorized under glycolysis, lipid metabolism, amino acid metabolism and hexose transporters are mainly repressed in response to acetic acid supplementation. These results provide new insights on P. kudriavzevii response to acetic acid supplementation in glucose and xylose media during high temperature fermentation, and serve as an important basis to uncover the regulatory mechanisms involved. This study will also provide reference for future studies in developing engineered strains possessing high tolerance to inhibitors at high temperature bioethanol production.