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.

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.

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.

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.

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.

Continuous thermophilic composting of distilled grain waste improved organic matter stability and succession of bacterial community.

Continuous thermophilic composting (CTC) is potentially helpful in shortening the composting cycle. However, its universal effectiveness and the microbiological mechanisms involved are unclear. Here, the physicochemical properties and bacterial community dynamics during composting of distilled grain waste in conventional and CTC models were compared. CTC accelerated the organic matter degradation rate (0.2 vs. 0.1 d-1) and shortened the composting cycle (24 vs. 65 d), mainly driven by the synergism of bacterial genera. Microbial analysis revealed that the abundance of Firmicutes was remarkably improved compared to that in conventional composting, and Firmicutes became the primary bacterial phylum (relative abundance >70 %) during the entire CTC process. Moreover, correlation analysis demonstrated that bacterial composition had a remarkable effect on the seed germination index. Therefore, controlling the composting process under continuous thermophilic conditions is beneficial for enhancing composting efficiency and strengthening the cooperation between bacterial genera.

Evaluation of physicochemical properties, bacterial community, and product fertility during rice straw composting supplemented with different nitrogen-rich wastes.

This study evaluated the compostability of rice straw as the main feedstock (75 % in dry weight), supplemented with three different nitrogen-rich wastes, namely food waste (FW), dairy manure (DM), and sewage sludge (SS). Organic matter (OM) degradation, maturity and fertility of the end-product, and bacterial community structure during the composting processes were compared. All composting processes generated mature end-product within 51 days. Notably, FW addition was more effective to accelerate rice straw OM degradation and significantly improved end-product fertility with a high yield of Chinese cabbage. The succession of the bacterial community was accelerated with FW supplementation. Genera Geobacillus, Chryseolinea, and Blastocatella were significantly enriched during the composting of rice straw with FW supplementation. Finally, temperature, total nitrogen, moisture, pH, and total carbon were the key factors affecting microorganisms. This study provides a promising alternative method to enhance the disposal of larger amounts of rice straw in a shorter time.

Different responses of mesophilic and thermophilic anaerobic digestion of waste activated sludge to PVC microplastics.

The effect of microplastics (MPs) retained in waste activated sludge (WAS) on anaerobic digestion (AD) performance has attracted more and more attention. However, their effect on thermophilic AD remains unclear. Here, the influence of polyvinyl chloride (PVC) MPs on methanogenesis and active microbial communities in mesophilic (37 °C) and thermophilic (55 °C) AD was investigated. The results showed that 1, 5, and 10 mg/L PVC MPs significantly promoted the cumulative methane yield in mesophilic AD by 5.62%, 7.36%, and 8.87%, respectively, while PVC MPs reduced that in thermophilic AD by 13.30%, 18.82%, and 19.99%, respectively. Moreover, propionate accumulation was only detected at the end of thermophilic AD with PVC MPs. Microbial community analysis indicated that PVC MPs in mesophilic AD enriched hydrolytic and acidifying bacteria (Candidatus Competibacter, Lentimicrobium, Romboutsia, etc.) together with acetoclastic methanogens (Methanosarcina, Methanosaeta). By contrast, most carbohydrate-hydrolyzing bacteria, propionate-oxidizing bacterium (Pelotomaculum), and Methanosarcina were inhibited by PVC MPs in thermophilic AD. Network analysis further suggested that PVC MPs significantly changed the relationship of key microorganisms in the AD process. A stronger correlation among the above genera occurred in mesophilic AD, which may promote the methanogenic performance. These results suggested that PVC MPs affected mesophilic and thermophilic AD of WAS via changing microbial activities and interaction.

Valorizing kitchen waste to produce value-added fertilizer by thermophilic semi-continuous composting followed by static stacking: Performance and bacterial community succession analysis.

To explore an effective decentralized kitchen waste (KW) treatment system, the performance and bacterial community succession of thermophilic semi-continuous composting (TSC) of KW followed by static stacking (SS) was studied. A daily feeding ratio of 10% ensured stable performance of TSC using an integrated automatic reactor; the efficiencies of organic matter degradation and seed germination index (GI) reached 80.88% and 78.51%, respectively. SS for seven days further promoted the quality of the compost by improving the GI to 91.58%. Alpha- and beta-diversity analyses revealed significant differences between the bacterial communities of TSC and SS. Firmicutes, Actinobacteria, Chloroflexi, Gemmatimonadetes, and Myxococcota were dominant during the TSC of KW, whereas the members of Proteobacteria and Bacteroidetes responsible for product maturity rapidly proliferated during the subsequent SS and ultimately dominated the compost with Firmicutes and Actinobacteria. These results provide new perspectives for decentralized KW treatment using TSC for practical applications.

Production of volatile fatty acid from fruit waste by anaerobic digestion at high organic loading rates: Performance and microbial community characteristics.

This study examined the performance and microbial community dynamics of an anaerobic volatile fatty acid (VFA) production reactor for treating fruit waste by stepwise increasing organic loading rates (OLRs) from 8 to 24 g volatile total solids (VTS)/(L·d). Results showed that higher VFA concentrations of 52.25-61.90 g chemical oxygen demand (COD)/L can be maintained at each OLR, thereby resulting to a production of 0.70-0.76 g chemical oxygen demand (COD)VFA/g VTS. Notably, an increase in OLR from 8 to 14 g VTS/(L·d) was beneficial for achieving higher VFA concentrations and yields. Moreover, an increase in OLR affected the VFA distribution significantly; acetate and butyrate became dominant in the fermentation liquid at OLRs ≥ 14 g VTS/(L·d). Microbial community dynamics analysis revealed that phyla Firmicutes and Actinobacteriota were predominant at each OLR, and the genera Lactobacillus, Clostridium_sensu_stricto_12, and Caproiciproducens were closely related to anaerobic VFA production.

Screening novel genes by a comprehensive strategy to construct multiple stress-tolerant industrial Saccharomyces cerevisiae with prominent bioethanol production.

BACKGROUND: Strong multiple stress-tolerance is a desirable characteristic for Saccharomyces cerevisiae when different feedstocks are used for economical industrial ethanol production. Random mutagenesis or genome shuffling has been applied for improving multiple stress-tolerance, however, these techniques are generally time-consuming and labor cost-intensive and their molecular mechanisms are unclear. Genetic engineering, as an efficient technology, is poorly applied to construct multiple stress-tolerant industrial S. cerevisiae due to lack of clear genetic targets. Therefore, constructing multiple stress-tolerant industrial S. cerevisiae is challenging. In this study, some target genes were mined by comparative transcriptomics analysis and applied for the construction of multiple stress-tolerant industrial S. cerevisiae strains with prominent bioethanol production. RESULTS: Twenty-eight shared differentially expressed genes (DEGs) were identified by comparative analysis of the transcriptomes of a multiple stress-tolerant strain E-158 and its original strain KF-7 under five stress conditions (high ethanol, high temperature, high glucose, high salt, etc.). Six of the shared DEGs which may have strong relationship with multiple stresses, including functional genes (ASP3, ENA5), genes of unknown function (YOL162W, YOR012W), and transcription factors (Crz1p, Tos8p), were selected by a comprehensive strategy from multiple aspects. Through genetic editing based on the CRISPR/Case9 technology, it was demonstrated that expression regulation of each of these six DEGs improved the multiple stress-tolerance and ethanol production of strain KF-7. In particular, the overexpression of ENA5 significantly enhanced the multiple stress-tolerance of not only KF-7 but also E-158. The resulting engineered strain, E-158-ENA5, achieved higher accumulation of ethanol. The ethanol concentrations were 101.67% and 27.31% higher than those of the E-158 when YPD media and industrial feedstocks (straw, molasses, cassava) were fermented, respectively, under stress conditions. CONCLUSION: Six genes that could be used as the gene targets to improve multiple stress-tolerance and ethanol production capacities of S. cerevisiae were identified for the first time. Compared to the other five DEGs, ENA5 has a more vital function in regulating the multiple stress-tolerance of S. cerevisiae. These findings provide novel insights into the efficient construction of multiple stress-tolerant industrial S. cerevisiae suitable for the fermentation of different raw materials.

Bioaugmentation with syntrophic volatile fatty acids-oxidizing consortia to alleviate the ammonia inhibition in continuously anaerobic digestion of municipal sludge.

Ammonia inhibition easily affects the performance of anaerobic digestion (AD) for municipal sludge and the oxidization of volatile fatty acids (VFAs) is the rate-limiting step of this process. Bioaugmentation is considered to be an effective method to alleviate ammonia inhibition of AD, but most study used the hydrogenotrophic methanogens as the bioaugmentation culture. In this study, bioaugmentation of mesophilic AD (MAD) and thermophilic AD (TAD) under ammonia inhibition with syntrophic acetate and propionate oxidizing consortia was investigated. The results showed that the bioaugmented reactors recovered earlier than control reactors with 20 (MAD) and 8 (TAD) days, respectively. The high-throughput 16S rRNA gene sequencing indicated that the relative abundance of carbohydrates fermenter (Lentimicrobium), syntrophic VFAs-oxidizing bacteria (Rikenellaceae_DMER64, Smithella and Syntrophobacter) and acetoclastic and hydrogenotrophic methanogens (Methanosaeta, Methanolinea and Methanospirillum) increased in MAD after bioaugmentation. However, part of the bioaugmentation culture could not adapt to the high free ammonia (FAN) concentration in MAD and the effect was weakened. In TAD, proteolytic bacteria (Keratinibaculum and Tepidimicrobium), syntrophic VFAs-oxidizing bacteria (Syntrophomonas) and hydrogenotrophic methanogen (Methanosarcina) were strengthened. The effect of bioaugmentation in TAD was durable even at higher organic loading rate (OLR), due to its positive influence on microbial community. These results suggested that the different bioaugmentation mechanism occurred in MAD and TAD, which are derived from the synergetic effects of ammonia tolerance and microbial interactions. Our study revealed the VFAs-oxidizing consortia as bioaugmented culture could be the potential strategy to alleviate the ammonia stress of AD.

Bacterial Community Structure and Metabolic Function Succession During the Composting of Distilled Grain Waste.

Distilled grain waste (DGW) can be converted to organic fertilizer via aerobic composting process without inoculating exogenous microorganisms. To illustrate the material conversion mechanism, this study investigated the dynamic changes of bacterial community structure and metabolic function involved in DGW composting. Results showed that a significant increase in microbial community alpha diversity was observed during DGW composting. Moreover, unique community structures occurred at each composting stage. The dominant phyla were Firmicutes, Proteobacteria, Actinobacteriota, Bacteroidota, Myxococcota, and Chloroflexi, whose abundance varied according to different composting stages. Keystone microbes can be selected as biomarkers for each stage, and Microbispora, Chryseolinea, Steroidobacter, Truepera, and Luteimonas indicating compost maturity. Co-occurrence network analysis revealed a significant relationship between keystone microbes and environmental factors. The carbohydrate and amino acid metabolism were confirmed as the primary metabolic pathways by metabolic function profiles. Furthermore, nitrogen metabolism pathway analysis indicated that denitrification and NH3 volatilization induced higher nitrogen loss during DGW composting. This study can provide new understanding of the microbiota for organic matter and nitrogen conversion in the composting process of DGW.

A comparative study of mesophilic and thermophilic anaerobic digestion of municipal sludge with high-solids content: Reactor performance and microbial community.

This study involved a comparison between mesophilic (MAD) and thermophilic anaerobic digestion (TAD) of municipal sludge with high (10%) solids content; the reactor performance and the response of total and active microbial communities to changes in sludge properties were monitored. Both TAD and MAD were stably maintained. TAD performed better than MAD in biogas production and volatile total solids reduction upon feeding sludge 1. TAD was slightly inhibited by ammonia, whereas the performance of MAD was improved when sludge 2 was used as the feedstock. Alpha- and beta-diversity analyses revealed significant differences in the microbial community based on DNA and RNA datasets, indicating that not all microbes function in AD. The active microbial community diversity and composition in MAD and TAD were also driven by sludge properties. Moreover, MAD showed significantly higher richness and diversity of the active microbial community compared with TAD, regardless of changes in sludge properties.

Acclimation Improves Methane Production from Molasses Wastewater with High Salinity in an Upflow Anaerobic Filter Reactor: Performance and Microbial Community Dynamics.

This study evaluated the performance of an upflow anaerobic filter (UAF) reactor in the thermophilic methane fermentation of hypersaline molasses wastewater. The high salinity (~ 45 mS/cm) of the undiluted wastewater completely inhibited the biogas production. An acclimation strategy involving gradient dilution of the molasses wastewater was implemented to gradually increase the salt stress. Consequently, the biogas production was recovered, inhibited only slightly by the high salinity of the undiluted wastewater. The reactor steadily achieved a high total organic carbon (TOC) loading rate of 5 g/L/day, with approximately 60% TOC removal efficiency. Acclimation to the gradually increased salt stress leads to a relative abundance of some halotolerant microbes, such as bacteria from Arcobacter, Tissierella, and Ruminococcaceae, which increased as their hydrolytic and acidogenic abilities adjusted to the incremental increase in salinity. Additionally, hydrogenotrophic methanogens, especially Methanoculleus, showed greater resistance to hypersalinity than aceticlastic methanogens. These results suggest that acclimation of the fermentation microbial community to hypersalinity is an effective strategy to improve methane production from hypersaline molasses wastewater in thermophilic UAF reactors.

Response of Propionate-Degrading Methanogenic Microbial Communities to Inhibitory Conditions.

Propionate is a crucial intermediate during methane fermentation. Investigating the effects of different kinds of inhibitors on the propionate-degrading microbial community is necessary to develop countermeasures for improving process stability. In the present study, under inhibitory conditions (acetate, propionate, sulfide, and ammonium addition), the dynamic changes of the propionate-degrading microbial community from a mesophilic chemostat fed with propionate as the sole carbon source were investigated using high-throughput sequencing of 16S rRNA. Sulfide and/or ammonia inhibited specific species in the microbial community. Compared with Syntrophobacter, Smithella was more resistant to inhibition by sulfide and/or ammonia. However, Syntrophobacter demonstrated greater tolerance than Smithella under acid inhibition conditions. Some genera that had close phylogenetic relationships and similar functions showed similar responses to different inhibitors.

Comparative Transcriptome Analysis of Recombinant Industrial Saccharomyces cerevisiae Strains with Different Xylose Utilization Pathways.

A heterologous xylose utilization pathway, either xylose reductase-xylitol dehydrogenase (XR-XDH) or xylose isomerase (XI), is usually introduced into Saccharomyces cerevisiae to construct a xylose-fermenting strain for lignocellulosic ethanol production. To investigate the molecular basis underlying the effect of different xylose utilization pathways on the xylose metabolism and ethanol fermentation, transcriptomes of flocculating industrial strains with the same genetic background harboring different xylose utilization pathways were studied. A different source of xylA did not obviously affect the change of the strains transcriptome, but compared with the XR-XDH strain, several key genes in the central carbon pathway were downregulated in the XI strains, suggesting a lower carbon flow to ethanol. The carbon starvation caused by lower xylose metabolism in XI strains further influenced the stress response and cell metabolism of amino acid, nucleobase, and vitamin. Besides, the downregulated genes mostly included those involved in mitotic cell cycle and the cell division-related process. Moreover, the transcriptomes analysis indicated that the after integrate xylA in the δ region, the DNA and chromosome stability and cell wall integrity of the strains were affected to some extent. The aim of this was to provide some reference for constructing efficient xylose-fermenting strains.