The uncultured WOR-3 phylum is widely distributed in anaerobic environments, including hot springs, marine ecosystems, and hydrothermal vents, yet its ecological roles and metabolic capabilities remain poorly understood. In this study, we analyzed 180 medium- to high-quality metagenome-assembled genomes (MAGs), including 59 newly reconstructed from environmental samples and 121 retrieved from the Genome Taxonomy Database (GTDB). Phylogenetic analyses resolved the WOR-3 lineage into four subgroups (subgroup 1-4). Metabolic reconstruction revealed significant divergence of the carbon, sulfur, nitrogen and hydrogen metabolism pathways among the different subgroups. Subgroup 1 was characterized by fermentative metabolism involving formate and ethanol, and uniquely exhibited potential for carbon fixation via Calvin cycle, as indicated by the presence of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) gene. Notably, WOR-3 RuBisCO is phylogenetically affiliated with archaeal form III, although the carbon fixation pathway follows the canonical bacterial Calvin cycle—a feature of potential evolutionary significance. Subgroup 3 exhibits metabolic versatility, including genes for dissimilatory sulfate reduction, sulfur oxidation, partial denitrification, and fatty acid degradation. In addition, all subgroups harbored key components of hydrogen metabolism, including widespread NiFe hydrogenases and Rnf complexes, supporting H₂-dependent electron transfer and energy conservation under anaerobic conditions. Featuring near-universal presence of complexes I and II and frequent occurrence of terminal oxidases ( cyd AB and cox ABC)—suggests a facultative anaerobic lifestyle. Collectively, this study expands the genomic framework for the WOR-3 phylum and provides novel insights into the metabolic versatility and ecological functions of this previously uncharacterized lineage in biogeochemical cycles of carbon, nitrogen, and sulfur. IMPORTANCE The WOR-3 phylum represents a widespread but poorly understood bacterial lineage inhabiting diverse anaerobic/microaerobic environments. By integrating 180 metagenome-assembled genomes, including 59 newly reconstructed, this study provides the most comprehensive genomic framework to date for WOR-3. Phylogenomic and metabolic analyses revealed four distinct subgroups with divergent capacities for carbon, sulfur, and nitrogen metabolism. Notably, subgroup 1 encodes a complete Calvin-Benson-Bassham (CBB) cycle featuring an archaeal-type form III RuBisCO, suggesting an unusual evolutionary trajectory for carbon fixation in this lineage. Subgroup 3 exhibits versatile metabolic potential, including dissimilatory sulfur metabolism, partial denitrification, and fatty acid degradation, highlighting its possible roles in multiple biogeochemical processes. The widespread presence of hydrogenases and respiratory complexes across all subgroups supports energy conservation under anaerobic or microaerobic conditions. These findings not only expand the taxonomic and functional landscape of the WOR-3 phylum but also offer key insights into its ecological roles in global element cycling.

Acidic CO 2 electrolysis, enhanced by the introduction of alkali cations, presents a strategic approach for improving carbon efficiency compared to processes conducted in neutral and alkaline environments. However, a significant challenge arises from the dissolution of both organic acids and alkali cations in a strongly acidic feed stream, resulting in a considerable energy penalty for downstream separation. In this study, we investigate the feasibility of using flow-electrode capacitive deionization (FCDI) technology to separate organic acids and recover alkali cations from a strongly acidic feed stream (pH ~ 1). We show that organic acids, such as formic acid and acetic acid, are retained in molecular form in the separation chamber, achieving a rejection rate of over 90% under all conditions. Alkali cations, such as K + and Cs + , migrate to the cathode chamber in ionic form, with their removal and recovery significantly influenced by their concentration and the pH of the feed stream, but responding differently to the types and concentrations of organic acids. The energy consumption for the removal and recovery of K + is 4 to 8 times higher than for Cs + , and the charge efficiency is significantly influenced by the types of organic acid products and alkali cations. We conduct a series of electrochemical measurements and analyze the impedance spectroscopy, identifying that hindered mass transfer governed the electrode process. Our findings underscore the potential of FCDI as an advanced downstream separation technology for acidic electrocatalysis processes.

Microbial electrosynthesis (MES) is a promising platform for renewable energy storage, carbon fixing, and chemicals production. However, the generation of longer chain carboxylates of which their values are higher than acetate remains a grand challenge in traditional autotrophic MES feeding CO2 as the sole carbon source. The present study describes the employment of mixotrophic MES as an important strategy for the production of chemicals with longer carbon chain. With pre-enriched acetogens, the caproate (C6) specificity was increased from 0 % (autotrophic MES) to 12.8 +/- 1.5 % (mixotrophic MES). Without pre-enriched acetogens, no caproate was detected, while the propionate (C3) specificity was increased from 17.1 +/- 7.8 % (autotrophic MES) to 40.1 +/- 0.1 % (mixotrophic MES). Metagenomic sequencing indicated that this strategy increased the abundance of functional microbes capable of chain elongation via the fatty acid biosynthesis (FAB), although the mixotrophic condition slightly decreased the carbon metabolic pathways via Wood-Ljungdahl (WL) pathways and reverse beta-oxidation (RBO). Genome-centric analysis located 60 functional microbes who possessed WL and/or FAB pathways, and 27 of them were significantly enriched in mixotrophic conditions (p < 0.001). In addition, compared with the abundance of these functional microbes, it was proposed that the pre-enriched acetogens were more critical than that of mixotrophic conditions for a shift in product spectrum. These results demonstrate the great potential of mixotrophic MES, as a novel renewable-electricity-driven technology, to produce long -chain compounds from the simultaneous valorization of CO2 and organic waste.

The article provides a pipeline for comparing microbial co-occurrence networks based on the R microeco package and meconetcomp package. It has high flexibility and expansibility and can help users efficiently compare networks built from different groups of samples or different construction approaches.

Microbial conversion of methane to electricity, fuels, and liquid chemicals has attracted much attention. However, due to the low solubility of methane, it is not considered a suitable substrate for microbial fuel cells (MFCs). In this study, a conductive fiber membrane (CFM) module was constructed as the bioanode of methane-driven MFCs, directly delivering methane. After biofilm formation on the CFM surface, a steady voltage output of 0.6 to 0.7 V was recorded, and the CFM-MFCs obtained a maximum power density of 64 ± 2 mW/m2. Moreover, methane oxidation produced a high concentration of intermediate acetate (up to 7.1 mM). High-throughput 16S rRNA gene sequencing suggests that the microbial community was significantly changed after electricity generation. Methane-related archaea formed a symbiotic consortium with characterized electroactive bacteria and fermentative bacteria, suggesting a combination of three types of microorganisms for methane conversion into acetate and electricity.

Microbial species have different niche breadth, leading to distinct habitat ranges. Specialists have narrow niche breadth and high habitat specificity in comparison with the generalists, which possess extensive habitat ranges. Since specialists have low probabilities to occur in various habitats at a specific spatio-temporal scale, communities with different ratios of specialists and generalists may exhibit different species co-occurrence patterns and functional characteristics. However, it is still not clear how the assemblages of specialists and generalists can affect microbial co-occurrence and functional potential at a large biogeographical scale. In this study, we explored the relationship between microbial habitat specificity and the co-occurrence patterns of prokaryotic communities in Chinese wetland soils and the contribution of functional profiles to this relationship. We first identified some specialists and generalists which hold significant roles in the co-occurrence network. Then, we found that communities with more specialists have more simple co-occurrence patterns (fewer linkages in network) than those with more generalists. Soil electrical conductivity was the most significant abiotic factor in structuring this relationship. The biological mechanisms that contribute to this relationship were closely associated with functional differences of taxa. With the increase of specialists in communities, aerobic ammonia oxidation and cellulolysis guilds decreased, and photoautotrophy and sulfate/sulfide respiration populations increased. Furthermore, the abundances of metabolic pathways altered along with the abiotic gradients (e.g., conductivity and pH), including carbohydrate degradation, fermentation, inorganic nutrient metabolism and stress resistance-related pathways. Especially, there were higher abundances of metabolism-related pathways in communities with more specialists compared to those with more generalists. Functional differences at traits, genes, and metabolic pathways reflected the characteristics of microbial co-occurrence patterns and biogeochemical cycles of habitats. In summary, microbial habitat-specificity can modulate the co-occurrence patterns of prokaryotic communities along the environmental gradients in wetland soils. Our findings provide a mechanistic understanding on how microbial niche breadth can affect microbial co-occurrence in wetland soils.

Fe(III) has been recognized as a potential electron sink for the anaerobic oxidation of methane (Fe-AOM) in diverse environments. However, most of previous Fe-AOM processes are limited to ANME archaea and the Fe-AOM mechanism remains unclear. Here we investigate, for the first time, the Fe-AOM performance and mechanisms by a single methanogen Methanosarcina barkeri. The results showed that M. barkeri was capable of oxidizing methane to CO2 and reducing ferrihydrite to siderite simultaneously. The presence of methane enhanced both the abundances of redox-active species (such as cytochromes) and electrochemical activity of M. barkeri. The proteomic analyses revealed that M. barkeri up-regulated the expressions of a number of methanogenic enzymes during Fe-AOM, and significantly enriched metabolic pathways of amino acid synthesis and nitrogen fixation. Metabolic inhibition experiments indicated that membrane-bound redox-active components (cytochromes, methanophenazine and F420H2:quinone oxidoreductase) were probably involved in extracellular electron transfer (EET) from cells to ferrihydrite. Overall, these results provide a deep insight into the single‑carbon metabolism and survival strategy for methanogens and suggest that methanogens may play an important role in linking methane and iron cycling in the substrate-limited environments.

Enhanced biological phosphorus removal (EBPR) process is known to mainly rely on the ability of phosphorus-accumulating organisms to take up, transform and store excess amount of phosphorus (P) inside the cells. However, recent studies have revealed considerable accumulation of P also in the extracellular polymeric substances (EPS) of sludge, implying a non-negligible role of EPS in P removal by EBPR sludge. However, the contribution of EPS to P uptake and the forms of accumulated extracellular P vary substantially in different studies, and the underlying mechanism of P transformation and transportation in EPS remains poorly understood. This review provides a new recognition into the P removal process in EBPR system by incorporating the role of EPS. It overviews on the characteristics of P accumulation in EPS, explores the mechanism of P transformation and transportation in EBPR sludge and EPS, summarizes the main influential factors for the P-accumulation properties of EPS, and discusses the remaining knowledge gaps and needed future efforts that may lead to better understanding and use of such an EPS role for maximizing P recovery from wastewater.

Much research has been conducted to improve sludge dewaterability and reveal its dewatering process. However, to date, two key issues remain unsolved: (1) the difficulty of dewatering sludge and (2) why suitable chemical conditioning can improve sludge dewaterability. This study is focused on addressing these two problems using a rheological analysis. Flow and dynamic measurements were performed to investigate the rheological properties of three types of sludge samples (raw sludge, acid-conditioned sludge, and Fenton-conditioned sludge) with different dewatering performances and a total solid content (TS) range of 20-62 g/L (2-6.2 wt %). The measured data were fitted to predict the rheological behaviors of the sludge at moderate and high TS values (15-50 wt %). A horizontal analysis and comparison of the rheological characteristics of the sludge samples indicated that rheological properties of the sludge exhibited a strong dependence on the solids content of the raw and treated sludge. An increase in the solids content significantly strengthened the solid-like properties and increased the deformation resistance, causing the sludge to become more resistant to flow and resulting in lower flowability of the sludge system; this provided an explanation for the difficulty in sludge dewatering. A vertical comparison and analysis of the rheological behavior of the sludge samples addressed the second key issue and indicated that a suitable conditioning method such as acid and Fenton oxidation conditioning had positive effects on the evolution of the rheological parameters of the sludge; namely, the conditioning resulted in weaker viscoelastic properties and better flowability. These results provide insights into the fundamental mechanism of sludge dewatering through rheological analysis.

Waste C1 gases as alternatives to pure CO2 can potentially facilitate the practical application of microbial electrosynthesis (MES) for chemical production and further decrease the total cost. This work evaluated the impact of the CO fraction on MES of C2-C6 carboxylates. Using the CO/CO2 mixture as the substrate in MES favors simultaneous acetogenesis, solventogenesis, and chain elongation. The test of CO-50% achieved the best performance of production of C4 and C6 carboxylates, of which the specificity based on the electron recovery was 43.11 +/- 1.31% for acetate, 34.11 +/- 0.11% for butyrate, and 15.41 +/- 1.48% for caproate, while the lag phase was 32 days for butyrate and 44 days for caproate. However, the specificity observed with pure CO2 (CO-0% test) was 85.37 +/- 3.24% for acetate, 10.68 +/- 2.54% for butyrate, and 0% for caproate. The increase of CO fraction decreased the electron recovery to methane. Microbial diversity analysis demonstrated that the elevated CO fraction increased the relative abundance of Acetobacterium as well as Clostridium. This study proved that waste C1 gases as alternatives to pure CO2 could stimulate the generation of C4 and C6 carboNylates in MES.

Anaerobic biological techniques are widely used in the reductive decolorization of textile wastewater. However, the decolorization efficiency of textile wastewater by conventional anaerobic biological techniques is generally limited due to the low biomass retention capacity and short hydraulic retention time (HRT). In this study, a methane-based hollow fiber membrane bioreactor (HfMBR) was initially inoculated with an enriched anaerobic methane oxidation (AOM) culture to rapidly form an anaerobic biofilm. Then, synthetic azo dye wastewater containing methyl orange (MO) was fed into the HfMBR. MO decolorization efficiency of - 100 % (HRT = 2 to 1.5 days) and maximum decolorization rate of 883 mg/L/day (HRT = 0.5 day) were obtained by the stepwise increase of the MO loading rate into the methane-based HfMBR. Scanning electron microscopy (SEM) and fluorescence in situ hybridization (FISH) analysis visually revealed that archaea clusters formed synergistic consortia with adjacent bacteria. Quantitative PCR (qPCR), phylogenetic and high-throughput sequencing analysis results further confirmed the biological consortia formation of methane-related archaea and partner bacteria, which played a synergistic role in MO decolorization. The high removal efficiency and stable microbial structure in HfMBR suggest it is a potentially effective technique for high-toxic azo dyes removal from textile wastewater.

Denitrifying anaerobic methane oxidation (DAMO) is the process of coupling the anaerobic oxidation of methane (AOM) with denitrification, which plays an important part in controlling the flow of methane in anoxic niches. In this study, we explored the feasibility of microbial selenite reduction using methane by DAMO culture. Isotopic 13CH4 and long-term experiments showed that selenite reduction was coupled to methane oxidation, and selenite was ultimately reduced to Se (0) by the analyses of scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The introduction of nitrate, the original electron acceptor in the DAMO culture, inhibited selenite reduction. Meanwhile, the microbial community of DAMO culture was significantly changed when the electron acceptor was changed from nitrate to selenite after long-term selenite reduction. High-throughput 16S rRNA gene sequencing indicated that Methylococcus (26%) became the predominant microbe performing selenite reduction and methane oxidation and the possible pathways of AOM accompanied with selenite reduction were proposed. This study revealed more potential relation during the biogeochemical cycle of carbon, nitrogen, and selenium.

The nontemplating preparation of porous carbon materials by using specially designed polymer precursors for supercapacitor is attracting considerable research attention because of the more controllable frame structure and easier processes than templating methods. Herein, a deliberately designed cross-linking polyphosphamide resin with defined N and P structure is synthesized and then carbonized to obtain porous carbon material. The as-obtained porous carbon material has a specific surface area of 2,620 m(2) g(-1), high porosity of 1.49 cm(3) g(-1), and well-distributed micro/mesoporous carbon structure. Different from activation by post-added NH4H2PO4, the confined N and P in the polymer frame are confirmed to play an important role in pore structure development by forming in situ highly dispersed NH4H2PO4 during carbonization. When evaluated as the electrode material for supercapacitors, the polyphosphamide-resin-based porous carbon material demonstrates excellent capacitance (440 F g(-1) under 0.5 A g(-1)) and high stability (retention of 93% over 10,000 cycles).

The extracellular electron transfer (EET) efficiency in bioelectrochemical systems has been proven to be dependent on anode potentials. To explore the underlying mechanism, previous studies have mainly focused on EET conduit and bacterial biomass but rarely concerned with the role of extracellular polymeric substances (EPS) surrounding electroactive cells. In this study, the response of Geobacter biofilms to anode potentials was investigated with a special emphasis on the mechanistic role of EPS. The electrochemical activities and cell viabilities of Geobacter soli biofilms were simultaneously attenuated at 0.4 and 0.6 V compared to -0.2 and 0 V. It was found that the biofilms (especially the biofilm region closer to electrode surface) grown at -0.2 and 0 V produced relatively more extracellular redox-active proteins and less extracellular polysaccharides, which conferred higher electron accepting/donating capacities to EPS and consequently facilitated EET. Meanwhile, electrically nonconductive extracellular polysaccharide-dominated interior layers were formed in the biofilms grown at 0.4 and 0.6 V, which limited direct EET but might serve as physical barriers for protecting cells in these biofilms from the increasing stress by poised electrodes. These results demonstrated that the production of EPS under different anode potentials might be finely regulated by cells to keep balance between EET efficiency and cell-protection. This study provides a new insight to investigate the Geobacter biofilms coping with various environments, and is useful for optimizing electrochemical activity of anode biofilms. (C) 2019 Elsevier Ltd. All rights reserved.

ABSTRACTBACKGROUNDThe selection of hydrodynamic properties of microalgae such as agitation speed is usually done according to empirical values without a technical basis, and the selected agitation speeds are not consistent with each other in many references. In this study, we aimed to provide a possible technical basis for the setup of the agitation speed of microalgae suspensions based on basic rheological analysis, in terms of viscosity of microalgae suspensions at different agitation speeds, using the red microalga Porphyridium cruentum as a model.RESULTSThe agitation speed of 150 rpm was optimal for P. cruentum cultivation, with its biomass concentration increased by about 19–57% compared with the microalgae cultured at other agitation speeds. Meanwhile, the content of linoleic acid, arachidonic acid and total fatty acids increased by maximum 103%, 58% and 48%, respectively, compared with those at other agitation speeds. Furthermore, a low rotational speed was beneficial for eicosapentaenoic acid formation (which increased by 72–106%).CONCLUSIONBasic rheological analysis of microalgal suspensions can be used as a guide in setting a reasonable and suitable agitation speed. Meanwhile, the agitation speed could affect both the biomass growth and the formation of certain high‐value products of microalgae. © 2019 Society of Chemical Industry

Humic substances (humics) are ubiquitous in terrestrial and aquatic environments where they can serve as electron acceptors for anaerobic oxidation of organic compounds. Methane is a powerful greenhouse gas, as well as the least reactive organic molecule. Anaerobic oxidation of methane (AOM) coupled to microbial reduction of various electron acceptors plays a crucial role in mitigating methane emissions. Here, we reported that humics could serve as terminal electron acceptors for AOM using enriched nitrate-reducing AOM microorganisms. AOM coupled to the reduction of humics was demonstrated based on the production of 13C-labelled carbon dioxide, and AOM activity was evaluated with different methane partial pressures and electron acceptor concentrations. After three-cycle reduction, both AOM activity and copy numbers of the archaea 16S rRNA and mcrA genes were the highest when anthraquinone-2,6-disulfonic acid and anthraquinone-2-sulfonic acid were electron acceptors. The high-throughput sequencing results suggested that ANME-2d were the dominant methane oxidation archaea after humics reduction, although the partner bacteria NC10 trended downward, other reported humics reduction bacteria (Geobactor and Anammox) appeared. The potential electron transfer models from ANME-2d to humics were proposed. These results enable a better understanding of available electron acceptors for AOM in natural environments and broaden our insight into the significant role of ANME-2d.

Although a large amount of textile wastewater is discharged at high temperatures, azo dye reduction under extreme-thermophilic conditions by mixed cultures has gained little attention. In this study, Acid Orange 7 (AO7) was used as the model azo dye to demonstrate the decolorization ability of an extreme-thermophilic mixed culture. The results showed that a decolorization efficiency of over 90% was achieved for AO7. The neutral red (NR, 0.1 mM) could promote AO7 decolorization, in which the group of Cell+NR offered the highest decolorization rate of 1.568 1/h and t(1/2) was only 0.44 h, whereas after CuCl2 addition, the decolorization rate (0.141 1/h) was lower and t(1/2) (4.92 h) was much longer. Thus, CuCl2 notably inhibited this process. Caldanaerobacter (64.0%) and Pseudomonas (25.4%) were the main enriched bacteria, which were not reported to have the ability for dye decolorization. Therefore, this study extends the application of extreme-thermophilic biotechnology.

Unmineable coal accounts for over 90% of the world's fossil fuel resources. Fortunately, many coal seams contain indigenous microorganisms capable of utilizing the coal as a carbon source to produce secondary biogenic coalbed natural gas. However, coal bioavailability has been shown to be a significant factor that limits the extent of bioconversion. In this study, we have analyzed the rate and yield of the biogas production and assessed the gas potential, carbon balance, stable carbon isotopes, microbial communities, and microbial pathways changes resulting from the hydrogen peroxide pretreatment with the Wyoming's Powder River Basin subbituminous coal. The results showed that coal pretreated with hydrogen peroxide can significantly enhance the bioavailability of coal for enhancing the biogas production, with a peak yield of 552.6 mu mol/g coal (437.1 Scf/ton coal) at the day 184. The stable carbon isotopic analysis indicated that the delta C-13 values of the methane and carbon dioxide were much less negative than the published field data. This suggested that the enrichment or depletion of the precursor C-13 could contribute to the shift of the carbon isotopic composition in the subsequence processes. Therefore, the data should be used with cautions for interpreting genesis of the thermogenic/biogenic methane and the methanogenic pathways. The methanogenic pathways were also investigated with re-fed experiment and microbial community analysis. The results indicated that the hydrogenotrophic pathway was not active in the original inoculum became activated after the gas production. The microbial community analysis demonstrated that the obligate hydrogenotrophic methanobacterium was the most dominant methanogens in the microcosms after the gas production. This suggested that the chemical treatment of coal has impacts on the microbial structure during the subsequent methane production phase.

Subsequent application of the obtained algae by chemical coagulation (e.g. Fe(III) addition) presents a challenge because of various iron compounds in algae. In this study, algae obtained by chemical coagulation were carbonized to yield an algae-based Fe/C nano-composite with a high capacity for hexavalent chromium (Cr(VI)) removal (236.9 mg/g), which is attributed to the high reductive Fe content (e.g., FeS, Fe(0), and FeO) and specific surface area. The optimal conditions-that is, 100 mg/L Fe(III) addition and 800 °C-were determined. Moreover, the role of extracellular polymeric substances (EPS) in carbonization was examined as it affected the product composition and efficiency of Cr(VI) removal, owing to the stabilizing property effect of EPS in algae. Algal EPS induced the homogeneous distribution of Fe compounds on the surface of the algae, and the generated α-FeOOH nanoparticles were wrapped in organic carbon matrix, resulting in a sufficient reaction between Fe compounds and organic carbon during carbonization. X-ray photoelectron spectroscopy showed that reduction and adsorption contributed 83.44% and 16.56% to Cr(VI) removal, respectively. This study provides a new insight into the role of EPS in the efficient Cr(VI) removal by algae-based Fe/C nano-composite and presents a promising application of this Fe/C nano-composite in environmental remediation.

Microbial fuel cells (MFCs) are a promising technology that converts chemical energy into electricity. However, up to now only few MFCs have been powered by gas fuels, such as methane, and their limited performance is still challenged by the low solubility and bioavailability of gases. Here, we developed a gas diffusion cloth (GDC) anode to significantly enhance the performance of methane-powered MFCs. The GDC anode was constructed by simply coating waterproof GORE-TEX cloth with conductive carbon cloth in one step. After biofilm enrichment, the GDC anodes obtained a methane-dependent current up to 1130.2 mA m-2, which was 165.2 times higher than conventional carbon cloth (CC) anodes. Moreover, MFCs equipped with GDC anodes generated a maximum power density of 419.5 mW m-2. Illumina high-throughput sequencing revealed that the GDC anode biofilm was dominated mainly by Geobacter, in contrast with the most abundant Methanobacterium in planktonic cells. It is hypothesized that Methanobacterium reversed the methanogenesis process by transferring electrons to the anodes, and Geobacter generated electricity via the intermediates (e.g., acetate) of anaerobic methane oxidation. Overall, this work provides an effective route in preparing facile and cost-effective anodes for high-performance methane MFCs.