The efficiency of biochar in enhancing carbon and nitrogen retention during distilled grain waste (DGW) composting is often limited. Microbial metabolic regulators, like malonic acid (MA), provide a potential strategy to overcome these limitations. This study investigated the effects of MA addition at different composting stages on biochar-assisted DGW composting and elucidated the underlying mechanisms. MA addition reduced carbon and nitrogen losses by 9.3%‒12.8% and 6.3%‒29.0%, respectively, owing to suppressed emissions of carbon- and nitrogen-containing gases and increased formation of NO3-, organic carbon, and nitrogen. Notably, MA addition during the thermophilic stage exhibited excellent efficiency. Bacterial community analysis revealed that MA addition regulated the abundance of genera (such as Bacillus, Ureibacillus, Filomicrobium, Thermobifida, Luteimonas, Nitrosomonas, etc.) involved in carbon and nitrogen conversions and promoted cooperative interactions among bacterial populations. Moreover, MA addition modified the activities of key functional enzymes in carbon and nitrogen metabolic pathways. Particularly, MA addition reduced the early phase of carbon- and nitrogen-containing gases emissions, and facilitated the later phase of carbon and nitrogen fixation, aromatic amino acid synthesis, nitrification, and ammonia assimilation. This study highlights the effectiveness and underlying mechanisms of MA addition in promoting carbon and nitrogen retention in biochar-assisted composting. These findings provided a novel strategy to improve compost quality and support carbon-neutral DGW management.

Polyhydroxyalkanoates (PHA) are microbial biopolyesters with promising potential as biodegradable bioplastics, but their production from waste-derived carboxylates is often limited by fluctuating substrate composition and inconsistent polymer accumulation. This study developed a substrate-specialized mixed microbial culture (MMC) strategy to improve PHA accumulation and regulate polymer monomer composition from variable short-chain carboxylates. Five MMCs were enriched separately with acetate, propionate, butyrate, valerate, or lactate, and then used as functional modules for constructing mixed MMCs. Genome-resolved metagenomic and metatranscriptomic analyses were used to identify putative PHA-storing populations associated with different carboxylates. Azoarcus and Azomonas were mainly associated with acetate- and propionate-enriched cultures. Paracoccus dominated butyrate- and valerate-enriched cultures, and Thauera were active in the lactate-enriched culture. When exposed to mixed-carboxylate substrates, the modularly assembled MMCs achieved 1.3–1.8-fold higher PHA contents than the corresponding individual cultures. The selected mixed MMC further accumulated 65.8 ± 0.3% and 58.5 ± 1.3% PHA from acidogenic broths derived from pretreated corn cobs and corn stover, respectively, with carbon conversion efficiencies of 63.0 ± 0.2% and 56.5 ± 4.2%. These results demonstrate that substrate-specialized cultures can serve as functional modules for constructing mixed-culture bioprocesses adapted to variable carboxylate feedstocks.

This study explores the microbial succession and metabolite changes during solid-state fermentation of Luzhou-flavor liquor in two types of pits: a traditional sealing pit (TSP) and a novel sealing pit (NSP) equipped with a stainless-steel cover, real-time monitoring, and an annular liquid-filled sealing trough. The results revealed that NSP enhanced microbial diversity and facilitated the production of a higher concentration and variety of flavor compounds compared to TSP. The microbial communities in NSP exhibited greater complexity and stronger correlations with flavor compounds. Although TSP contained a greater number of core microbial genera (28) than NSP (17), fewer of these genera were strongly correlated with flavor compounds. This difference may explain the higher fermentation efficiency and the development of more distinctive flavor profiles in NSP. Our findings suggest that the novel sealing method improves fermentation quality by fostering a favorable microbial environment, thus enhancing the overall flavor and aroma characteristics of the liquor.

The combined effects of microplastics (MPs) and antibiotics on anaerobic digestion (AD) remain unclear. This study investigated the responses of AD performance, the microbial communities, and antibiotic resistance genes (ARGs) to the co-exposure of individual MP types (PE, PVC, or PS) with tetracycline (TC) in mesophilic and thermophilic sludge reactors. Results indicated that all thermophilic reactors could maintain methane production levels. However, methane production significantly decreased in mesophilic reactors containing MPs, reaching 53.52% to 66.48% of control levels under a TC concentration of 2 mg L-1 d-1 and 30.37% to 41.83% under 10 mg L-1 d-1, potentially due to the suppression of hydrolysis-related bacteria. Metagenomic analysis revealed that thermophilic AD continued to reduce ARGs and mobile genetic elements (MGEs) even when TC and MPs were present together. Conversely, the interaction between MPs and TC appeared to enhance the transfer and potential spread of ARGs within mesophilic AD systems. Notably, six out of nine dominant ARGs significantly increased in the PVC reactor under a TC concentration of 10 mg L-1 d-1, with tetracycline resistance genes being enriched up to 3.76-fold. Network analysis further suggested that horizontal gene transfer (HGT) of ARGs among low-abundance bacteria might be the primary cause of ARG enrichment. These findings highlight that the combined impact of MPs and TC on mesophilic AD warrants greater attention.

Hexanoic acid (HA), a vital platform chemical, can be produced by various mixed cultures using ethanol or ethanol-containing waste. However, systematic research remains lacking on its ecological succession, community assembly, and core synthetic pathways. In this study, an ethanol-based HA-producing synthetic community (SynCom) was constructed from the pit mud using a top-down strategy, and dominated by Clostridium sensu stricto 12 (primarily Clostridium kluyveri, 86%), Clostridium sensu stricto 18 and Pseudoclavibacter. A highly significant positive correlation (P < 0.01) was observed between Clostridium sensu stricto 12 and HA biosynthesis. Ecological analysis revealed that community assembly exhibited significant diversity dynamics during enrichment. While overall assembly was predominantly governed by stochastic processes, colonization of the key HA-producing lineage Clostridium sensu stricto 12 was distinctly driven by deterministic processes. Functional predictions and fermentation profiling suggested a potential associationbetween HA production by SynCom (G4) and the reverse β-oxidation pathway. Under optimized conditions (pH 7.9, 37.6°C, 8.2% inoculum, 77% loading volume), SynCom (G4) achieved a HA yield of 4.69 g/L. This study demonstrated that a deterministically assembled Clostridium sensu stricto 12-dominated in this synthetic community may effectively promote HA production, providing mechanistic insights into microbial community assembly and offering a simplified yet functional model in Baijiu fermentation.

A substantial proportion of carbon (> 50%) is lost as CO2 emissions during composting of distilled grain waste (DGW). The porous structure and alkaline surface properties of biochar facilitate in-situ CO2 capture within the composting environment, thereby potentially reducing carbon loss and promoting humification. However, the efficacy of biochar is influenced by its feedstock and pyrolysis conditions, underscoring the need for targeted modifications to achieve consistent and enhanced performance. This study systematically evaluated pristine biochar (T1) and KOH-modified biochar (T2) on maturity, carbon loss, and humification during DGW composting, using a no-biochar control (CK). Self-organizing map (SOM) analysis showed that T2 reached in 8 d the maturity achieved by CK at 16 d and T1 at 12 d. T2 reduced cumulative CO2 emissions by 31.28% and 30.75% compared with CK and T1, respectively. Carbon loss decreased by 13.24% (T1) and 20.66% (T2) versus CK. Moreover, compared with CK, the Fmax of the humic acid (HA)-like substances increased by 16.7% (T1) and 18.3% (T2). Mechanistically, KOH-modified biochar initially creates a porous, alkaline microenvironment that accelerates mineralization of labile organic matter while adsorbing and chemically fixing in situ CO2 as carbonates. As succession proceeds, autotrophic microorganisms assimilate inorganic carbon into organic carbon. Subsequently, key bacteria convert this fixed carbon into HA precursors via the shikimate pathway. These precursors then polymerize via the polyphenol pathway and the Maillard reaction, yielding stable, highly aromatic HA. These results offer mechanistic evidence for KOH-modified biochar in DGW composting, reducing carbon loss and improving HA quality.

High-salinity conditions frequently impair the fermentation performance of Saccharomyces cerevisiae in industrial processes involving high-osmolarity substrates. Identifying genetic determinants that enhance salt tolerance is therefore essential for the development of robust yeast cell factories. In this study, a comparative transcriptomic analysis was performed to investigate the transcriptional responses of a salt-tolerant strain, E-158, and its parental strain, KF-7, under 1.25 M NaCl stress, with the aim of identifying potential targets for strain engineering. Comparative transcriptomic analysis revealed extensive transcriptional differences between E-158 and KF-7 under high-salt conditions, involving central carbon and nitrogen metabolism, peroxisome-associated oxidative stress responses, ion transport, cell wall-related processes, and sporulation-related pathways. Based on these profiles, two transcription factors (CUP9 and ZNF1) and three functional genes (DAL1, IDP2, and CTA1) were selected for functional validation. Overexpression or deletion of the transcription factors, as well as overexpression of the functional genes, was carried out in KF-7. Fermentation experiments under 1.25 M NaCl demonstrated that all engineered strains outperformed the parental strain. Among them, overexpression of CTA1 resulted in the greatest improvement, with glucose consumption and ethanol production increased by 35.04

Although biochar has been widely used to regulate carbon-nitrogen transformations and promote compost humification, its performance is highly variable across feedstocks and pyrolysis conditions, and current studies largely emphasize emission mitigation or nitrogen conversion alone. This study introduces Mg-modified biochar (MMB) into distilled grain waste (DGW) composting to quantify carbon-nitrogen synergistic retention and humification. Three treatments were established (CK, pristine biochar (T1), and MMB (T2)) to systematically evaluate their impacts on compost maturity, coupled C/N transformations and losses, and humification dynamics. Results indicate that T2 significantly accelerated the composting process, reducing maturation time by 7 days compared to T1. Self-Organizing Map (SOM) analysis visually confirmed its faster succession trajectory. Compared with the CK treatment, T1 reduced nitrogen and carbon losses by 26.42 % and 13.24 %, respectively. Notably, T2 exhibited superior performance in carbon-nitrogen synergistic retention, achieving reductions of 30.15 % and 19.61 % in nitrogen and carbon losses, while increasing humic acid carbon (HA-C) content by 97.72 %. Furthermore, T2 significantly reshaped the topological structure of microbial communities through increased network complexity and modularity, while directionally enriching lignocellulose-degrading and nitrogenretaining functional communities represented by Thermobifida and Luteimonas. The XGBoostSHAP machine learning model further elucidated that MMB drives ecological functions by optimizing physicochemical conditions (pH, EC) and differentially regulating key species. This primarily involves inhibiting Alcaligenaceae to control nitrogen loss and promoting Saccharomonospora proliferation to accelerate humification. This study provides clear microbiological mechanisms and practical evidence for developing efficient, low-emission technologies for DGW resource utilization.

The recalcitrance of lignocellulosic structures in crop straw constrains compost humification. Reactive oxygen species (ROS) can oxidatively deconstruct lignocellulose and release humification precursors, whereas iron redox cycling may sustain ROS generation. Here, rapeseed straw composting was conducted under three treatments: continuous aeration without FeSO4 addition (CK), continuous aeration with FeSO4 addition (T1), and intermittent aeration with FeSO4 addition (T2). The effects on compost maturation, Fe-ROS dynamics, lignocellulose degradation, humification, and microbial succession were evaluated. Self-organizing map (SOM) analysis showed that T2 reached comparable maturity at 18 d, whereas CK and T1 reached this level at 35 d and 25 d, respectively. During the mid-to-late stages, T2 maintained higher Fe(II) levels and ROS activity, with H2O2 and •OH levels exceeding CK by 20.69–107.63% and 22.57–65.56%, respectively, and T1 by 7.97–33.80% and 13.38–58.14%, respectively, accompanied by enhanced lignocellulose degradation. Compared with CK, the Fmax values of humic-like C1 and humic-acid-like C4 in T2 increased by 16.19% and 35.57%, respectively. FT-ICR MS showed that T2 promoted DOM transformation toward higher unsaturation and aromaticity, with DBEw and AImodw reaching 7.57 and 0.19, respectively, accompanied by lignin-like and tannin-like humification products. In T2, intermittent aeration established a cyclic redox environment, sustaining Fe(II)/Fe(III) cycling via Fe(II)-mediated ROS generation under aerobic conditions and microbial Fe(III) reduction under oxygen-limited conditions. This cyclic environment further maintained ROS activity, enriching lignocellulose-degrading taxa (Thermobifida) and potential Fe-cycling taxa (Lysinibacillus and Geobacillus). Ultimately, the sustained ROS activity and the microbial activity accelerated lignocellulose deconstruction and promoted humification.

The molecular mechanisms underlying the influence of nitrogen sources on xanthan gum production remain poorly understood. This study compared the effects of two nitrogen sources (NH4Cl and glutamate) on xanthan gum production in Xanthomonas campestris. Additionally, the transcriptomic responses to different glutamate concentrations during fermentation were investigated. The findings revealed that glutamate, particularly at a lower concentration of 1 g/L, significantly enhanced xanthan gum yield and viscosity. Comparative transcriptomic analysis across different fermentation stages revealed that nitrogen limitation, observed on day 4 under 1 g/L glutamate and day 6 under 2 g/L glutamate, favored xanthan gum biosynthesis. Furthermore, low glutamate concentration improved xanthan gum production primarily by altering nitrogen assimilation and redirecting carbon metabolic fluxes through modulation in the expression of genes (rpoN, rpfC, and rpfG) associated within two-component regulatory system. These results may provide valuable guidance for xanthan gum production using genetically engineered bacteria.

In this study, thermophilic high-solids anaerobic co-digestion of dewatered sludge (DS) and rice straw (RS) was conducted at a constant C/N ratio of 10 (hereafter called C/N ratio-adjusted DS); the long-term reactor performance and microbial community dynamics were compared with DS mono-digestion. The co-digestion allowed stable performance for 952 days, and biogas evolution was 402 ml/g-VTS with methane content of approximate 50 % at a higher VTS loading rate of 8.0 g/kg-sludge/d. In contrast, the performance of DS mono-digestion was negatively influenced by the varied properties in DS even at a low VTS loading rate of 1-2 g/kg-sludge/d. Interestingly, the deteriorated DS mono-digestion performance was recovered and promoted by adding RS. Beta-diversity analyses revealed significant differences in the microbial community between co-and mono-digestion. The stable performance of co-digestion was attributed to more balanced function microbes in protein and cellulosic matter degradation, as a result, leading to alleviation of ammonia inhibition.

In light of the growing severity of plastic pollution and the fossil resource crisis, replacing conventional fossil-based plastics with bio-based and biodegradable "green plastics" is imperative. Polyhydroxyalkanoates (PHAs), which are synthesized by microorganisms, are considered to be the most promising candidates due to their good biodegradability and material properties. However, their widespread commercial adoption is hindered by their high production costs. To develop low-cost PHA synthesis processes, it is necessary to fully evaluate the successes and the limitations of current technologies. This review summarizes recent advancements in reducing PHA production costs, focusing on the development of low-cost carbon sources, strain engineering, extremophilic PHA fermentation, and mixed microbial cultures (MMCs) fermentation. Among low-cost carbon sources, lignocellulosic biomass, owing to its abundant availability, shows greater potential for driving economically viable, large-scale PHA production. However, current pure-culture fermentation processes face challenges such as low carbon conversion efficiency and susceptibility to inhibitors when utilizing lignocellulosic feedstocks. Strain engineering offers promising solutions to these challenges through strategies such as ribosome-binding site optimization, promoter optimization, metabolic engineering, and cell morphology engineering. In contrast to pure-culture fermentation, MMCs-based fermentation, which operates in open environments, demonstrates superior economic feasibility. Future research should focus on refining protocols for MMCs enrichment and establishing continuous, high-efficiency PHA production systems tailored to industrial requirements. Additionally, the PHA recovery protocols need to be optimized to minimize the chemical usage for economic viability and environmental sustainability.

Anaerobic digestion (AD) of organic wastes relies on the interaction and cooperation of various microorganisms. Phages are crucial components of the microbial community in AD systems, but their diversity and interactions with the prokaryotic populations are still inadequately comprehended. In this study, 2121 viral operational taxonomic units (vOTUs) were recovered from 12 anaerobic fatty acid-fed reactors. Notably, 63.1% of these vOTUs could not be assigned to any known family, revealing a substantial presence of uncharted phages specifically associated with AD environments. Over half of the vOTUs associated with hosts had the capability to infect multiple hosts, ranging from 2 to 49, with a prevalent tendency to infect 2-5 hosts. In silico predictions of phage-host linkages uncovered that only a small fraction of vOTUs were shared across different functional groups, including fermentative bacteria, syntrophic fatty acid-oxidising bacteria (SFOB) and methanogens. Phages linked to hosts in all three groups primarily consisted of generalists and temperate species, especially those linked to SFOB. Additionally, metabolic reconstruction identified auxiliary metabolic genes participating in fatty acid degradation, methanogenesis and energy conservation. The present study provides insights into phage characteristics and their in situ interactions with prokaryotic hosts, highlighting their ecological role in AD systems.

Pristine biochar (DB)-assisted composting can enhance product maturity and mitigate nitrogen loss, but its efficacy varies and is limited by feedstock variability and preparation conditions, highlighting the need for surface modifications to optimize performance. This study systematically investigated the effects of DB and KOH-modified biochar (DBK) on compost maturity, nitrogen loss, and the related microbial mechanisms during the composting of distilled grain waste, using a group without biochar addition (D) as the control. Results indicated that DBK exhibited a specific surface area of 644.33 m2/g and was rich in pore structures and functional groups. DBK significantly promoted compost maturity, with the seed germination index (GI) reaching 70 % by 23 d and biological nitrification occurring earlier (16 d). Additionally, compared to D and DB, DBK reduced nitrogen loss by 34.13 % and 10.47 %, respectively. DBK accelerates critical nitrogen transformation processes by increasing the abundance of nitrogen-fixing bacteria and associated functional genes. Neutral community modeling and symbiotic networks indicated higher microbial community complexity and stochasticity, thereby promoting functional redundancy and improving nitrogen retention. Furthermore, Actinomadura and Chryseolinea were identified as key microbial drivers of nitrogen transformation, with their nxrABC and hao genes playing crucial roles in establishing efficient 'microbe-gene' synergistic mechanisms. Finally, economic analysis indicated that DBK generated a net profit of up to 63.63 RMB/t. These findings provide a theoretical basis for using modified biochar to promote maturity and control nitrogen loss during composting.

The dynamics of the structure of polyhydroxyalkanoate-producing mixed microbial cultures (PHA-MMCs) during enrichment and maintenance is an unsolved problem. The effect of phages has been proposed as a cause of dynamic changes in community structure, but evidence is lacking. To address this question, five PHA-MMCs were enriched, and biological samples were sampled temporally to study the interactions between phage and bacterial members by combining metagenomics and metatranscriptomics. A total of 963 metagenome-assembled genomes (MAGs) and 4,294 phage operational taxonomic units (pOTUs) were assembled from bulk metagenomic data. The dynamic changes in the structure of phage and bacterial communities were remarkably consistent. Structural equation modeling analysis showed that phages could infect and lyse dominant species to vacate ecological niches for other species, resulting in a community succession state in which dominant species alternated. Seven key auxiliary metabolic genes (AMGs), phaC, fadJ, acs, ackA, phbB, acdAB, and fadD, potentially contributing to PHA synthesis were identified from phage sequences. Importantly, these AMGs were transcribed, indicating that they were in an active expression state. The meta-analysis provides the first catalog of phages in PHA-MMCs and the AMGs they carry, as well as how they affect the dynamic changes in bacterial communities. This study provides a reference for subsequent studies on understanding and regulating the microbial community structure of open microbial systems.IMPORTANCEThe synthesis of biodegradable plastic PHA from organic waste through mixed microbial cultures (PHA-MMCs), at extremely low cost, has the potential for expanded production. However, the dynamics of dominant species in PHA-MMCs are poorly understood. Our results demonstrate for the first time the impact of phages on the structure of bacterial communities in the PHA-MMCs. There are complex interactions between the PHA producers (e.g., Azomonas, Paracoccus, and Thauera) and phages (e.g., Casadabanvirus and unclassified Hendrixvirinae). Phage communities can regulate the activity and structure of bacterial communities. In addition, the AMGs related to PHA synthesis may hitchhike during phage-host infection cycles, enabling their dissemination across bacterial communities, and phages may act as a critical genetic reservoir for bacterial members, facilitating access to PHA synthesis-related functional traits. This study highlights the impact of phages on bacterial community structure, suggesting that phages have the potential to be used as a tool for better controlling the microbial community structure of PHA-MMCs.

Atmospheric and room temperature plasma (ARTP) mutagenesis represents a promising approach for enhancing biosynthesis in various microbes. However, its application to rhamnolipid (RL) production has been limited, with the underlying enhancement mechanisms remaining unclear. In this study, a mutant strain P-41 from Pseudomonas aeruginosa was obtained through ARTP mutagenesis, which exhibited a 2.76-fold increase in the produced RL concentration compared to the original strain. Through comparative genomics and transcriptomics analysis, ARTP-induced mutations in multiple genes, involved in branched-chain amino acid metabolism (bkdA1 and bkdA2), quorum sensing (QS) system (pqsH), transcription regulation (sspA and rpoB), and signaling and cellular processes (tonB, exbB, and tolR), may contribute to the enhanced RL concentration in strain P-41 when cultured under optimal nitrogen source conditions (NaNO3). These genetic modifications appear to have altered nitrogen metabolism, carbohydrate metabolism, motility, sugar precursor allocation, and signaling molecule synthesis, thereby promoting RL synthesis in strain P-41 on both day 2 and day 4 of fermentation. The findings from multi-omics analysis provide a theoretical foundation for understanding the mechanisms that enhance RL production.

IntroductionThe cooperation among members of microbial communities based on the exchange of public goods such as 20 protein amino acids (AAs) has attracted widespread attention. However, little is known about how AAs availability affects interactions among members of complex microbial communities and the structure and function of a community.MethodsTo investigate this question, trace amounts of AAs combinations with different synthetic costs (low-cost, medium-cost, high-cost, and all 20 AAs) were supplemented separately to acetate-degrading thermophilic methanogenic reactors, and the differences in microbial community structure and co-occurring networks of main members were compared to a control reactor without AA supplementation.ResultsThe structure of the microbial community and the interaction of community members were influenced by AAs supplementation and the AAs with different synthetic costs had different impacts. The number of nodes, links, positive links, and the average degree of nodes in the co-occurrence network of the microbial communities with AAs supplementation was significantly lower than that of the control without AAs supplementation, especially for all 20 AAs supplementation followed by the medium- and high-cost AAs supplementation. The average proportion of positive interactions of microbial members in the systems supplemented with low-cost, medium-cost, high-cost, all AAs, and the control group were 0.42, 0.38, 0.15, 0.4, and 0.45, respectively. In addition, the ecological functions of community members possibly changed with the supplementation of different cost AAs.DiscussionThese findings highlight the effects of AAs availability on the interactions among members of complex microbial communities, as well as on community function.

Xanthan gum, produced by the aerobic fermentation of carbohydrates by Xanthomonas campestris, is a significant natural and industrial biopolymer known for its exceptional properties. Enhancing the yield of xanthan gum production remains a critical challenge. This study employed atmospheric and room temperature plasma (ARTP) technology to induce mutagenesis in X. campestris, resulting in a high-yielding strain, X20. The X20 mutant exhibited a substantial increase in xanthan gum yield, ranging from 13.3 % to 30.0 % over the starting strain across NaCl concentrations of 0, 6.0, and 8.0 g/L, along with improved viscosity and molecular weight. In the whole genome of X20 mutant, a total of 80 variant sites differing from the reference genome were identified, involving 76 mutated genes. Among these, 19 were missense mutations primarily associated with the twocomponent system. Transcriptome analysis highlighted their role in enhancing flagellar movement, biofilm formation, and metabolic synthesis, thereby elevating the capability of the mutant strain in xanthan gum production. This study demonstrates the potential of ARTP as an effective tool for microbial mutagenesis breeding, providing theoretical guidance for future studies on the synthesis regulation of xanthan gum and the engineering modification of X. campestris.

Background Acetate is the major intermediate of anaerobic digestion of organic waste to CH4. In anaerobic methanogenic systems, acetate degradation is carried out by either acetoclastic methanogenesis or a syntrophic degradation by a syntrophy of acetate oxidizers and hydrogenotrophic methanogens. Due to challenges in isolation of syntrophic acetate-oxidizing bacteria (SAOB), the diversity and metabolism of SAOB, as well as the mechanisms of their interactions with methanogenic partners remain poorly understood. Results In this study, we successfully enriched previously unknown SAOB by operating continuous thermophilic anaerobic chemostats fed with acetate, propionate, butyrate, or isovalerate as the sole carbon and energy source. They represent novel clades belonging to Clostridia, Thermoanaerobacteraceae, Anaerolineae, and Gemmatimonadetes. In these SAOB, acetate is degraded through reverse Wood-Ljungdahl pathway or an alternative pathway mediated by the glycine cleavage system, while the SAOB possessing the latter pathway dominated the bacterial community. Moreover, H2 is the major product of the acetate degradation by these SAOB, which is mediated by [FeFe]-type electron-confurcating hydrogenases, formate dehydrogenases, and NADPH reoxidation complexes. We also identified the methanogen partner of these SAOB in acetate-fed chemostat, Methanosarcina thermophila, which highly expressed genes for CO2-reducing methanogenesis and hydrogenases to supportively consuming H2 at transcriptional level. Finally, our bioinformatical analyses further suggested that these previously unknown syntrophic lineages were prevalent and might play critical roles in thermophilic methanogenic reactors. Conclusion This study expands our understanding on the phylogenetic diversity and in situ biological functions of uncultured syntrophic acetate degraders, and presents novel insights on how they interact with their methanogens partner. These knowledges strengthen our awareness on the important role of SAO in thermophilic methanogenesis and may be applied to manage microbial community to improve the performance and efficiency of anaerobic digestion.

Haloarchaea with the capacity to degrade alkanes is promising to deal with petroleum pollution in hypersaline environments. However, only a limited number of haloarchaeal species are investigated, and their pathway and mechanism for alkane degradation remain unclear. In this study, Halogranum rubrum RO2-11, a haloarchaeal strain, verified the ability to degrade kerosene and hexadecane in 184 g/L NaCl, with 53% and 52% degradation rates after 9 and 4 days, respectively. Genome sequencing and gene annotation indicated that strain RO2-11 possesses a complete potential alkane-degrading pathway, of which alkane hydroxylases may include CYP450, AlmA, and LadA. Transcriptome and metabolome analyses revealed that the upregulation of related genes in TCA cycle, lysine biosynthesis, and acetylation may help improve hexadecane degradation. Additionally, an alternative degrading pathway of hexadecane based on dual-terminal β-oxidation may occur in strain RO2-11. It is likely to be the first report of alkane degradation by the genus Halogranum, which may be helpful for applications of oil-pollution bioremediation under high-salt conditions.