Metabolism of novel potential syntrophic acetate-oxidizing bacteria in thermophilic methanogenic chemostats.

Acetate is a major intermediate in the anaerobic digestion of organic waste to produce CH4. In methanogenic systems, acetate degradation is carried out by either acetoclastic methanogenesis or syntrophic degradation by acetate oxidizers and hydrogenotrophic methanogens. Due to challenges in the isolation of syntrophic acetate-oxidizing bacteria (SAOB), the diversity and metabolism of SAOB and the mechanisms of their interactions with methanogenic partners are not fully characterized. In this study, the in situ activity and metabolic characteristics of potential SAOB and their interactions with methanogens were elucidated through metagenomics and metatranscriptomics. In addition to the reported SAOB classified in the genera Tepidanaerobacter, Desulfotomaculum, and Thermodesulfovibrio, we identified a number of potential SAOB that are affiliated with Clostridia, Thermoanaerobacteraceae, Anaerolineae, and Gemmatimonadetes. The potential SAOB possessing the glycine-mediated acetate oxidation pathway dominates SAOB communities. Moreover, formate appeared to be the main product of the acetate degradation by the most active potential SAOB. We identified the methanogen partner of these potential SAOB in the acetate-fed chemostat as Methanosarcina thermophila. The dominated potential SAOB in each chemostat had similar metabolic characteristics, even though they were in different fatty-acid-fed chemostats. These novel syntrophic lineages are prevalent and may play critical roles in thermophilic methanogenic reactors. This study expands our understanding of the phylogenetic diversity and in situ biological functions of uncultured syntrophic acetate degraders and presents novel insights into how they interact with methanogens.IMPORTANCECombining reactor operation with omics provides insights into novel uncultured syntrophic acetate degraders and how they perform in thermophilic anaerobic digesters. This improves our understanding of syntrophic acetate degradation and contributes to the background knowledge necessary to better control and optimize anaerobic digestion processes.

Response mechanisms of different Saccharomyces cerevisiae strains to succinic acid.

BACKGROUND: The production of succinic acid (SA) from biomass has attracted worldwide interest. Saccharomyces cerevisiae is preferred for SA production due to its strong tolerance to low pH conditions, ease of genetic manipulation, and extensive application in industrial processes. However, when compared with bacterial producers, the SA titers and productivities achieved by engineered S. cerevisiae strains were relatively low. To develop efficient SA-producing strains, it's necessary to clearly understand how S. cerevisiae cells respond to SA. RESULTS: In this study, we cultivated five S. cerevisiae strains with different genetic backgrounds under different concentrations of SA. Among them, KF7 and NBRC1958 demonstrated high tolerance to SA, whereas NBRC2018 displayed the least tolerance. Therefore, these three strains were chosen to study how S. cerevisiae responds to SA. Under a concentration of 20 g/L SA, only a few differentially expressed genes were observed in three strains. At the higher concentration of 60 g/L SA, the response mechanisms of the three strains diverged notably. For KF7, genes involved in the glyoxylate cycle were significantly downregulated, whereas genes involved in gluconeogenesis, the pentose phosphate pathway, protein folding, and meiosis were significantly upregulated. For NBRC1958, genes related to the biosynthesis of vitamin B6, thiamin, and purine were significantly downregulated, whereas genes related to protein folding, toxin efflux, and cell wall remodeling were significantly upregulated. For NBRC2018, there was a significant upregulation of genes connected to the pentose phosphate pathway, gluconeogenesis, fatty acid utilization, and protein folding, except for the small heat shock protein gene HSP26. Overexpression of HSP26 and HSP42 notably enhanced the cell growth of NBRC1958 both in the presence and absence of SA. CONCLUSIONS: The inherent activities of small heat shock proteins, the levels of acetyl-CoA and the strains' potential capacity to consume SA all seem to affect the responses and tolerances of S. cerevisiae strains to SA. These factors should be taken into consideration when choosing host strains for SA production. This study provides a theoretical basis and identifies potential host strains for the development of robust and efficient SA-producing strains.

The responses of mesophilic and thermophilic anaerobic digestion of municipal sludge to periodic fluctuation disturbance of organic loading rate.

Fluctuation disturbance of organic loading rate (OLR) is common in actual anaerobic digestion (AD), but its effects on AD of municipal sludge gets little attention. This study investigated the responses of reactor performance and active microbial community in mesophilic and thermophilic AD of municipal sludge before, during and after OLR periodic fluctuation disturbance. The performance of both reactors were similar before and after disturbance although some parameter values changed during the disturbance, which indicated their enough buffer capacity to OLR periodic fluctuation. Different microbial community at RNA level was observed in the two reactors. When the OLR disturbance commenced, the microbial community changed greatly in thermophilic AD. Error and attack tolerance of the microbial network was analyzed in order to learn the response mechanisms to OLR disturbance. The results assisted that the thermophilic microbial community was more vulnerable, but the reactor performance of which could be maintained using the functional redundancy strategy under OLR fluctuation disturbance.

Comparative transcriptome analysis reveals different adaptation mechanisms for degradation of very long-chain and normal long-chain alkanes in Dietzia sp. DQ12-45-1b.

Hydrocarbon-degrading bacteria typically metabolize a broad range of alkane substrates, but global metabolic characteristics of strains growing on alkane substrates in different chain lengths remain unclear. In this study, we analysed the transcriptional profiles of a hydrocarbon degrading bacterium, Dietzia sp. DQ12-45-1b, during growth on octacosane (C28), hexadecane (C16) and glucose as the sole carbon sources. Our results highlight that C16 and C28 induced common genes of core alkane degradation pathways in DQ12-45-1b, whereas transcriptional patterns of genes related to lipid metabolism, energy metabolism, biomass synthesis, and metal ion transportation were distinct. In addition, the transcriptional differences of genes related to glyoxylate shunt (GS) as well as growth phenotypes of mutant strain with defects in GS demonstrated that GS is essential for C16 degradation, though it is dispensable for C28 degradation in DQ12-45-1b. These results demonstrate that DQ12-45-1b cells exhibited considerable metabolic flexibility by using various mechanisms during growth on alkane substrates in different chain lengths. This study advances our knowledge of microbial hydrocarbon degradation and provides valuable information for the application of alkane-degrading bacteria in bioremediation and microbial enhanced oil recovery.

Saccharomyces cerevisiae employs complex regulation strategies to tolerate low pH stress during ethanol production.

BACKGROUND: Industrial bioethanol production may involve a low pH environment caused by inorganic acids, improving the tolerance of Saccharomyces cerevisiae to a low pH environment is of industrial importance to increase ethanol yield, control bacterial contamination, and reduce production cost. In our previous study, acid tolerance of a diploid industrial Saccharomyces cerevisiae strain KF-7 was chronically acclimatized by continuous ethanol fermentation under gradually increasing low-pH stress conditions. Two haploid strains B3 and C3 having excellent low pH tolerance were derived through the sporulation of an isolated mutant. Diploid strain BC3 was obtained by mating these two haploids. In this study, B3, C3, BC3, and the original strain KF-7 were subjected to comparison transcriptome analysis to investigate the molecular mechanism of the enhanced phenotype. RESULT: The comparison transcriptome analysis results suggested that the upregulated vitamin B1 and B6 biosynthesis contributed to the low pH tolerance. Amino acid metabolism, DNA repairment, and general stress response might also alleviate low pH stress. CONCLUSION: Saccharomyces cerevisiae seems to employ complex regulation strategies to tolerate low pH during ethanol production. The findings provide guides for the construction of low pH-tolerant industrial strains that can be used in industrial fermentation processes.

Regulatory mechanism of Haa1p and Tye7p in Saccharomyces cerevisiae when fermenting mixed glucose and xylose with or without inhibitors.

BACKGROUND: Various inhibitors coexist in the hydrolysate derived from lignocellulosic biomass. They inhibit the performance of Saccharomyces cerevisiae and further restrict the development of industrial bioethanol production. Transcription factors are regarded as targets for constructing robust S. cerevisiae by genetic engineering. The tolerance-related transcription factors have been successively reported, while their regulatory mechanisms are not clear. In this study, we revealed the regulation mechanisms of Haa1p and Tye7p that had outstanding contributions to the improvement of the fermentation performance and multiple inhibitor tolerance of S. cerevisiae. RESULTS: Comparative transcriptomic analyses were applied to reveal the regulatory mechanisms of Haa1p and Tye7p under mixed sugar fermentation conditions with mixed inhibitors [acetic acid and furfural (AFur)] or without inhibitor (C) using the original strain s6 (S), the HAA1-overexpressing strain s6H3 (H), and the TYE7-overexpressing strain s6T3 (T). The expression of the pathways related to carbohydrate, amino acid, transcription, translation, cofactors, and vitamins metabolism was enhanced in the strains s6H3 and s6T3. Compared to C_H vs. C_S group, the unique DEGs in AFur_H vs. AFur_S group were further involved in oxidative phosphorylation, purine metabolism, vitamin B6 metabolism, and spliceosome under the regulation of Haa1p. A similar pattern appeared under the regulation of Tye7p, and the unique DEGs in AFur_T vs. AFur_S group were also involved in riboflavin metabolism and spliceosome. The most significant difference between the regulations of Haa1p and Tye7p was the intracellular energy supply. Haa1p preferred to enhance oxidative phosphorylation, while Tye7p tended to upregulate glycolysis/gluconeogenesis. CONCLUSIONS: Global gene expressions could be rewired with the overexpression of HAA1 or TYE7. The positive perturbations of energy and amino acid metabolism were beneficial to the improvement of the fermentation performance of the strain. Furthermore, strengthening of key cofactor metabolism, and transcriptional and translational regulation were helpful in improving the strain tolerance. This work provides a novel and comprehensive understanding of the regulation mechanisms of Haa1p and Tye7p in S. cerevisiae.

Microbial communities in crude oil phase and filter-graded aqueous phase from a Daqing oilfield after polymer flooding.

AIMS: The aim was to characterize indigenous micro-organisms in oil reservoirs after polymer flooding (RAPF). METHODS: The microbial communities in the crude oil phase (Oil) and in the filter-graded aqueous phases Aqu0.22 (>0.22 µm) and Aqu0.1 (0.1-0.22 µm) were investigated by 16S rRNA gene high-throughput sequencing. RESULTS: Indigenous micro-organisms related to hydrocarbon degradation prevailed in the three phases of each well. However, obvious differences in bacterial compositions were observed amongst the three phases of the same well and amongst the same phase of different wells. The crude oil and Aqu0.22 shared many dominant bacteria. Aqu0.1 contained a unique bacterial community in each well. Most bacteria in Aqu0.1 were affiliated to culturable genera, suggesting that they may adapt to the oil reservoir environment by reduction of cell size. Contrary to the bacterial genera, archaeal genera were similar in the three phases but varied in relative abundances. The observed microbial differences may be driven by specific environmental factors in each oil well. CONCLUSIONS: The results suggest an application potential of microbial enhanced oil recovery (MEOR) technology in RAPF. The crude oil and Aqu0.1 contain many different functional micro-organisms related to hydrocarbon degradation. Both should not be overlooked when investing and exploring the indigenous micro-organisms for MEOR. SIGNIFICANCE AND IMPACT OF THE STUDY: This work facilitates the understanding of microbial community structures in RAPF and provides information for microbial control in oil fields.

Improving Acetic Acid and Furfural Resistance of Xylose-Fermenting Saccharomyces cerevisiae Strains by Regulating Novel Transcription Factors Revealed via Comparative Transcriptomic Analysis.

Acetic acid and furfural are the two prevalent inhibitors coexisting with glucose and xylose in lignocellulosic hydrolysate. The transcriptional regulations of Saccharomyces cerevisiae in response to acetic acid (Aa), furfural (Fur), and the mixture of acetic acid and furfural (Aa_Fur) were revealed during mixed glucose and xylose fermentation. Carbohydrate metabolism pathways were significantly enriched in response to Aa, while pathways of xenobiotic biodegradation and metabolism were significantly enriched in response to Fur. In addition to these pathways, other pathways were activated in response to Aa_Fur, i.e., cofactor and vitamin metabolism and lipid metabolism. Overexpression of Haa1p or Tye7p improved xylose consumption rates by nearly 50%, while the ethanol yield was enhanced by nearly 8% under acetic acid and furfural stress conditions. Co-overexpression of Haa1p and Tye7p resulted in a 59% increase in xylose consumption rate and a 12% increase in ethanol yield, revealing the beneficial effects of Haa1p and Tye7p on improving the tolerance of yeast to mixed acetic acid and furfural.IMPORTANCE Inhibitor tolerance is essential for S. cerevisiae when fermenting lignocellulosic hydrolysate with various inhibitors, including weak acids, furans, and phenols. The details regarding how xylose-fermenting S. cerevisiae strains respond to multiple inhibitors during fermenting mixed glucose and xylose are still unknown. This study revealed the transcriptional regulation mechanism of an industrial xylose-fermenting S. cerevisiae strain in response to acetic acid and furfural. The transcription factor Haa1p was found to be involved in both acetic acid and furfural tolerance. In addition to Haa1p, four other transcription factors, Hap4p, Yox1p, Tye7p, and Mga1p, were identified as able to improve the resistance of yeast to these two inhibitors. This study underscores the feasibility of uncovering effective transcription factors for constructing robust strains for lignocellulosic bioethanol production.

Thermosynergistes pyruvativorans gen. nov., sp. nov., an anaerobic, pyruvate-degrading bacterium from Shengli oilfield, and proposal of Thermosynergistaceae fam. nov. in the phylum Synergistetes.

A strictly anaerobic, thermophilic, Gram-stain-negative bacterium, named as strain S15T, was isolated from oily sludge of Shengli oilfield in PR China. Cells of strain S15T were straight or slightly curved rods with 0.4-0.8 µm width × 1.4-3 µm length and occurred mostly in pairs or short chains. Endospore-formation was not observed. The strain grew optimally at 55 °C (range from 30-65 °C), pH 6.5 (pH 6.0-8.5) and 0-30 g l-1 NaCl (optimum with 10 g l-1 NaCl). Yeast extract was an essential growth factor for strain S15T. The major cellular fatty acid was iso-C15 : 0 (58.2 %), and the main polar lipids were amino phospholipid (APL), glycolipids (GLs) and phosphatidylethanolamine (PE). The G+C content of DNA of strain S15T was 52.2 mol%. Strain S15T shared 89.8 % 16S rRNA gene similarity with the most related organism Acetomicrobium hydrogeniformans DSM 22491T in the phylum Synergistetes. The paired genomic average amino acid identity (AAI) and percentage of conserved proteins (POCP) values showed relatedness of less than 58.0 and 39.7 % with type strains of the species in the phylum Synergistetes. On the basis of phenotypic, phylogenetic and phylogenomic evidences, strain S15T constitutes a novel species in a novel genus, for the name Thermosynergistes pyruvativorans gen. nov., sp. nov. is proposed. The type strain is S15T (=CCAM 583T=JCM 33159T). Thermosynergistaceae fam. nov. is also proposed.

Metabolic Exchange with Non-Alkane-Consuming Pseudomonas stutzeri SLG510A3-8 Improves n-Alkane Biodegradation by the Alkane Degrader Dietzia sp. Strain DQ12-45-1b.

Biodegradation of alkanes by microbial communities is ubiquitous in nature. Interestingly, the microbial communities with high hydrocarbon-degrading performances are sometimes composed of not only hydrocarbon degraders but also nonconsumers, but the synergistic mechanisms remain unknown. Here, we found that two bacterial strains isolated from Chinese oil fields, Dietzia sp. strain DQ12-45-1b and Pseudomonas stutzeri SLG510A3-8, had a synergistic effect on hexadecane (C16 compound) biodegradation, even though P. stutzeri could not utilize C16 individually. To gain a better understanding of the roles of the alkane nonconsumer P. stutzeri in the C16-degrading consortium, we reconstructed a two-species stoichiometric metabolic model, iBH1908, and integrated in silico prediction with the following in vitro validation, a comparative proteomics analysis, and extracellular metabolomic detection. Metabolic interactions between P. stutzeri and Dietzia sp. were successfully revealed to have importance in efficient C16 degradation. In the process, P. stutzeri survived on C16 metabolic intermediates from Dietzia sp., including hexadecanoate, 3-hydroxybutanoate, and α-ketoglutarate. In return, P. stutzeri reorganized its metabolic flux distribution to fed back acetate and glutamate to Dietzia sp. to enhance its C16 degradation efficiency by improving Dietzia cell accumulation and by regulating the expression of Dietzia succinate dehydrogenase. By using the synergistic microbial consortium of Dietzia sp. and P. stutzeri with the addition of the in silico-predicted key exchanged metabolites, diesel oil was effectively disposed of in 15 days with a removal fraction of 85.54% ± 6.42%, leaving small amounts of C15 to C20 isomers. Our finding provides a novel microbial assembling mode for efficient bioremediation or chemical production in the future.IMPORTANCE Many natural and synthetic microbial communities are composed of not only species whose biological properties are consistent with their corresponding communities but also ones whose chemophysical characteristics do not directly contribute to the performance of their communities. Even though the latter species are often essential to the microbial communities, their roles are unclear. Here, by investigation of an artificial two-member microbial consortium in n-alkane biodegradation, we showed that the microbial member without the n-alkane-degrading capability had a cross-feeding interaction with and metabolic regulation to the leading member for the synergistic n-alkane biodegradation. Our study improves the current understanding of microbial interactions. Because "assistant" microbes showed importance in communities in addition to the functional microbes, our findings also suggest a useful "assistant-microbe" principle in the design of microbial communities for either bioremediation or chemical production.

Improving xylitol yield by deletion of endogenous xylitol-assimilating genes: a study of industrial Saccharomyces cerevisiae in fermentation of glucose and xylose.

Engineered Saccharomyces cerevisiae can reduce xylose to xylitol. However, in S.cerevisiae, there are several endogenous enzymes including xylitol dehydrogenase encoded by XYL2, sorbitol dehydrogenases encoded by SOR1/SOR2 and xylulokinase encoded by XKS1 may lead to the assimilation of xylitol. In this study, to increase xylitol accumulation, these genes were separately deleted through CRISPR/Cas9 system. Their effects on xylitol yield of an industrial S. cerevisiae CK17 overexpressing Candida tropicalis XYL1 (encoding xylose reductase) were investigated. Deletion of SOR1/SOR2 or XKS1 increased the xylitol yield in both batch and fed-batch fermentation with different concentrations of glucose and xylose. The analysis of the transcription level of key genes in the mutants during fed-batch fermentation suggests that SOR1/SOR2 are more crucially responsible for xylitol oxidation than XYL2 under the genetic background of S.cerevisiae CK17. The deletion of XKS1 gene could also weaken SOR1/SOR2 expression, thereby increasing the xylitol accumulation. The XKS1-deleted strain CK17ΔXKS1 produced 46.17 g/L of xylitol and reached a xylitol yield of 0.92 g/g during simultaneous saccharification and fermentation (SSF) of pretreated corn stover slurry. Therefore, the deletion of XKS1 gene provides a promising strategy to meet the industrial demands for xylitol production from lignocellulosic biomass.

Zhaonella formicivorans gen. nov., sp. nov., an anaerobic formate-utilizing bacterium isolated from Shengli oilfield, and proposal of four novel families and Moorellales ord. nov. in the phylum Firmicutes.

A novel obligately anaerobic, thermophilic and formate-utilizing bacterium K32T was isolated from Shengli oilfield of China. Cells were straight rods (0.4-0.8 µm × 2.5-8.0 µm), Gram-stain-positive, non-spore-forming and slightly motile. Optimum growth occurred with pH of 7 and 0.5 g l-1 NaCl under temperature of 55-60 °C. Nitrate could be reduced into nitrite, syntrophic formate oxidation to methane and carbon dioxide occurred when co-culturing strain K32T and Methanothermobacter thermautotrophicus ΔH. The main cellular fatty acids were iso-C15 : 0 (24.0 %), anteiso-C15 : 0 (21.7 %), C16 : 0 (12.7 %) and C14 : 0 (10.8 %), and the main polar lipid was phosphatidylglycerol. The G+C content of the genomic DNA was 46.3 mol%. The 16S rRNA gene sequence of K32T shared ≤90.4 % of sequence similarity to closest type strains of Desulfitibacter alkalitolerans, Calderihabitans maritimus and members of the genus Moorella. Based on the phenotypic, biochemical and genotypic characterization, Zhaonella formicivorans gen. nov., sp. nov. is proposed with K32T (=CCAM 584T =DSM 107278T=CGMCC1.5297T) as the type strain, which is the first representative of Zhaonellaceae fam. nov. In addition, the order Thermoanaerobacterales and family Peptococcaceae were reclassified, and three novel families in the novel order of Moorellales ord. nov. were also proposed.

Different transcriptional responses of haploid and diploid S. cerevisiae strains to changes in cofactor preference of XR.

BACKGROUND: Xylitol accumulation is a major barrier for efficient ethanol production through heterologous xylose reductase-xylitol dehydrogenase (XR-XDH) pathway in recombinant Saccharomyces cerevisiae. Mutated NADH-preferring XR is usually employed to alleviate xylitol accumulation. However, it remains unclear how mutated XR affects the metabolic network for xylose metabolism. In this study, haploid and diploid strains were employed to investigate the transcriptional responses to changes in cofactor preference of XR through RNA-seq analysis during xylose fermentation. RESULTS: For the haploid strains, genes involved in xylose-assimilation (XYL1, XYL2, XKS1), glycolysis, and alcohol fermentation had higher transcript levels in response to mutated XR, which was consistent with the improved xylose consumption rate and ethanol yield. For the diploid strains, genes related to protein biosynthesis were upregulated while genes involved in glyoxylate shunt were downregulated in response to mutated XR, which might contribute to the improved yields of biomass and ethanol. When comparing the diploids with the haploids, genes involved in glycolysis and MAPK signaling pathway were significantly downregulated, while oxidative stress related transcription factors (TFs) were significantly upregulated, irrespective of the cofactor preference of XR. CONCLUSIONS: Our results not only revealed the differences in transcriptional responses of the diploid and haploid strains to mutated XR, but also provided underlying basis for better understanding the differences in xylose metabolism between the diploid and haploid strains.

Different Interspecies Electron Transfer Patterns during Mesophilic and Thermophilic Syntrophic Propionate Degradation in Chemostats.

Propionate is one of the major intermediates in anaerobic digestion of organic waste to CO2 and CH4. In methanogenic environments, propionate is degraded through a mutualistic interaction between symbiotic propionate oxidizers and methanogens. Although temperature heavily influences the microbial ecology and performance of methanogenic processes, its effect on syntrophic interaction during propionate degradation remains poorly understood. In this study, metagenomics and metatranscriptomics were employed to compare mesophilic and thermophilic propionate degradation communities. Mesophilic propionate degradation involved multiple syntrophic organisms (Syntrophobacter, Smithella, and Syntrophomonas), pathways, interactions, and preference toward formate-based electron transfer to methanogenic partners (i.e., Methanoculleus). In thermophilic propionate degradation, one syntrophic organism predominated (Pelotomaculum), interspecies H2 transfer played a major role, and phylogenetically and metabolically diverse H2-oxidizing methanogens were present (i.e., Methanoculleus, Methanothermobacter, and Methanomassiliicoccus). This study showed that microbial interactions, metabolic pathways, and niche diversity are distinct between mesophilic and thermophilic microbial communities responsible for syntrophic propionate degradation.

Novel Syntrophic Isovalerate-Degrading Bacteria and Their Energetic Cooperation with Methanogens in Methanogenic Chemostats.

Isovalerate is an important intermediate in anaerobic degradation of proteins/amino acids. Little is known about how this compound is degraded due to challenges in cultivation and characterization of isovalerate-degrading bacteria, which are thought to symbiotically depend on methanogenic archaea. In this study, we successfully enriched novel syntrophic isovalerate degraders (uncultivated Clostridiales and Syntrophaceae members) through operation of mesophilic and thermophilic isovalerate-fed anaerobic reactors. Metagenomics- and metatranscriptomics-based metabolic reconstruction of novel putative syntrophic isovalerate metabolizers uncovered the catabolic pathway and byproducts (i.e., acetate, H2, and formate) of isovalerate degradation, mechanisms for electron transduction from isovalerate degradation to H2 and formate generation (via electron transfer flavoprotein; ETF), and biosynthetic metabolism. The identified organisms tended to prefer formate-based interspecies electron transfer with methanogenic partners. The byproduct acetate was further converted to CH4 and CO2 by either Methanothrix (mesophilic) and Methanosarcina (thermophilic), which employed different approaches for acetate degradation. This study presents insights into novel mesophilic and thermophilic isovalerate degraders and their interactions with methanogens.

Description of Biomaibacter acetigenes gen. nov., sp. nov., and proposal of Thermosediminibacterales ord. nov. containing two novel families of Tepidanaerobacteraceae fam. nov. and Thermosediminibacteraceae fam. nov.

A Gram-stain-negative, rod-shaped, chemoorganotrophic and anaerobic bacterium, strain SK-G1T, was isolated from oily sludge sampled at the Shengli oilfield in PR China. Growth occurred with 0-30 g l-1 NaCl, at 40-65 °C and at pH 6.0-8.5. The predominant fatty acids were C14 : 0 and C13 : 0, and the major cellular polar lipids were phosphatidylglycerol and phosphatidylethanolamine. No respiratory quinone was detected. The genomic G+C content was 43.9 mol%. The strain had highest 16S rRNA gene sequence similarity (93.2 % identity) to Tepidanaerobacter syntrophicus DSM 15584T. The phylogenetic, phenotypic and chemotaxonomic data showed that strain SK-G1T (=CCAM 530T=KCTC 15783T=JCM 33158T) represents a novel species of a new genus Biomaibacteracetigenes gen. nov., sp. nov. The results of phylogenetic and phylogenomic analyses indicated that the genera Biomaibacter, Caldanaerovirga, Fervidicola, Tepidanaerobacter, Thermosediminibacter, Thermovenabulum and Thermovoraxformed a clade with high bootstrap support distinguishing to other taxon within the order Thermoanaerobacterales. This clade is proposed as Thermosediminibacterales ord. nov. and includes Tepidanaerobacteraceae fam. nov. and Thermosediminibacteraceae fam. nov. Emended descriptions of the order Thermoanaerobacterales and family Syntrophomonadaceae are also provided.

Identification of novel potential acetate-oxidizing bacteria in thermophilic methanogenic chemostats by DNA stable isotope probing.

Syntrophic oxidization of acetate and propionate are both critical steps of methanogenesis during thermophilic anaerobic digestion. However, knowledge on syntrophic acetate-oxidizing bacteria (SAOB) and syntrophic propionate-oxidizing bacteria (SPOB) is limited because of the difficulty in pure culture isolation due to symbiotic relationship. In this study, two thermophilic acetate-fed anaerobic chemostats, ATL (dilution rate of 0.025 day-1) and ATH (0.05 day-1) and one thermophilic propionate-fed anaerobic chemostat PTL (0.025 day-1) were constructed, AOB and POB in these chemostats were studied via microbial community analysis and DNA stable-isotope probing (SIP). The results showed that, in addition to Tepidanaerobacter, a known SAOB, species of Thauera, Thermodesulfovibrio, Anaerobaculum, Ruminiclostridium, Comamonas, and uncultured bacteria belonging to Lentimicrobiaceae, o_MBA03, Thermoanaerobacteraceae, Anaerolineaceae, Clostridiales, and Ruminococcaceae were determined to be potential AOB in chemostats. Pelotomaculum was the key SPOB detected in the propionate-fed chemostat. Based on the intense fluorescence of coenzyme F420, majority of Methanosarcina cells in acetate-fed chemostats were involved in hydrogenotrophic methanogenesis, suggesting the existence of highly active SAOB among the detected AOB. In the propionate-fed chemostat, most of the species detected as AOB were similar to those detected in the acetate-fed chemostats, suggesting the contribution of the syntrophic acetate oxidization pathway for methane generation. These results revealed the existence of previously unknown AOB with high diversity in thermophilic chemostats and suggested that methanogenesis from acetate via the syntrophic oxidization pathway is relevant for thermophilic anaerobic digestion.

Transcriptome changes in adaptive evolution of xylose-fermenting industrial Saccharomyces cerevisiae strains with δ-integration of different xylA genes.

It is of utmost importance to construct industrial xylose-fermenting Saccharomyces cerevisiae strains for lignocellulosic bioethanol production. In this study, two xylose isomerase-based industrial S. cerevisiae strains, O7 and P5, were constructed by δ-integration of the xylose isomerase (XI) gene xylA from the fungus Orpinomyces sp. and from the bacterium Prevotella ruminicola, respectively. The xylose consumption of the strains O7 and P5 at 48-h fermentation was 17.71 and 26.10 g/L, respectively, in synthetic medium with xylose as the sole sugar source. Adaptive evolution further improved the xylose fermentation capacity of the two strains to 51.0 and 28.9% in average, respectively. The transcriptomes of these two strains before and after evolution were analyzed using RNA-Seq. The expression levels of the genes involved in cell integrity, non-optimal sugar utilization, and stress response to environment were significantly up-regulated after evolution and did not depend on the origin of xylA; the expression levels of the genes involved in transmembrane transport, rRNA processing, cytoplasmic translation, and other processes were down-regulated. The expression of genes involved in central carbon metabolism was fine-tuned after the evolution. The analysis of transcription factors (TFs) indicated that most of the genes with significant differential expression were regulated by the TFs related to cell division, DNA damage response, or non-optimal carbon source utilization. The results of this study could provide valuable references for the construction of efficient xylose-fermenting XI strains.

Comparative transcriptomes reveal novel evolutionary strategies adopted by Saccharomyces cerevisiae with improved xylose utilization capability.

Production of ethanol from xylose by recombinant Saccharomyces cerevisiae is suboptimal with slow fermentation rate, compared with that from glucose. In this study, a strain-expressing Scheffersomyces stipitis xylose reductase-xylitol dehydrogenase (XR-XDH) pathway was subjected to adaptive evolution on xylose; this approach generated populations with the significantly improved cell growth and ethanol production rate. Mutants were isolated, and the best one was used for sporulation to generate eight stable mutant strains with improved xylose fermentation ability. They were used in a microarray assay to study the molecular basis of the enhanced phenotype. The enriched transcriptional differences among the eight mutant strains and the native strain revealed novel responses to xylose, which likely contributes to the improved xylose utilization. The upregulated vitamin B1 and B6 biosynthesis indicated that thiamine served as an important cofactor in xylose metabolism and may alleviate the redox stress. The increased expression of genes involved in sulfur amino acid biosynthesis and the decreased expression of genes related to Fe(II) transport may alleviate redox stress as well. Meanwhile, it was remarkable that several glucose-repressible genes, including genes of the galactose metabolism, gluconeogenesis, and ethanol catabolism, had a lower expression level after adaptive evolution. Concomitantly, the expression levels of two regulators of the glucose signaling pathway, Rgs2 and Sip4, decreased, indicating a reshaped signaling pathway to xylose after adaptive evolution. Our findings provide new targets for construction of a superior bioethanol producing strain through inverse metabolic engineering.

Xylose fermentation efficiency and inhibitor tolerance of the recombinant industrial Saccharomyces cerevisiae strain NAPX37.

Industrial yeast strains with good xylose fermentation ability and inhibitor tolerance are important for economical lignocellulosic bioethanol production. The flocculating industrial Saccharomyces cerevisiae strain NAPX37, harboring the xylose reductase-xylitol dehydrogenase (XR-XDH)-based xylose metabolic pathway, displayed efficient xylose fermentation during batch and continuous fermentation. During batch fermentation, the xylose consumption rates at the first 36 h were similar (1.37 g/L/h) when the initial xylose concentrations were 50 and 75 g/L, indicating that xylose fermentation was not inhibited even when the xylose concentration was as high as 75 g/L. The presence of glucose, at concentrations of up to 25 g/L, did not affect xylose consumption rate at the first 36 h. Strain NAPX37 showed stable xylose fermentation capacity during continuous ethanol fermentation using xylose as the sole sugar, for almost 1 year. Fermentation remained stable at a dilution rate of 0.05/h, even though the xylose concentration in the feed was as high as 100 g/L. Aeration rate, xylose concentration, and MgSO4 concentration were found to affect xylose consumption and ethanol yield. When the xylose concentration in the feed was 75 g/L, a high xylose consumption rate of 6.62 g/L/h and an ethanol yield of 0.394 were achieved under an aeration rate of 0.1 vvm, dilution rate of 0.1/h, and 5 mM MgSO4. In addition, strain NAPX37 exhibited good tolerance to inhibitors such as weak acids, furans, and phenolics during xylose fermentation. These findings indicate that strain NAPX37 is a promising candidate for application in the industrial production of lignocellulosic bioethanol.