Effects of Weak Electric Fields on the Denitrification Performance of Pseudomonas stutzeri: Insights into Enzymes and Metabolic Pathways.

Enhanced denitrification has been reported under weak electric fields. However, it is difficult to investigate the mechanism of enhanced denitrification due to the complex interspecific interactions of mixed-culture systems. In this study, Pseudomonas stutzeri, capable of denitrification under anaerobic conditions, was selected for treating low COD/N (2.0, ratio between concentration of chemical oxygen demand and NO3--N) artificial wastewater under constant external voltages of 0.2, 0.4, and 0.6 V. The results revealed that P. stutzeri exhibited the highest efficiency in nitrate reduction at 0.2 V. Moreover, the maximum nitrate removal rate was 15.96 mg/(L·h) among the closed-circuit groups, 19.39% higher than that under the open-circuit group. Additionally, a notable reduction in nitrite accumulation was observed under weak electric fields. Enzyme activity analysis showed that the nitrate reductase activities were significantly increased among the closed-circuit groups, while nitrite reductase activities were inhibited. Transcriptomic analysis indicated that amino acid metabolism, carbohydrate metabolism, and energy metabolism were increased, enhancing the resistance of P. stutzeri to environmental stress and the efficiency of carbon source utilization for denitrification. The current study examined the impacts of weak electric fields on enzyme activities and microbial metabolic pathways and offers valuable insights into the mechanism by which denitrification is enhanced by weak electric fields.

Ecological Risk Assessment of Three Pesticide Additives in Soil and Application to the Remediation of Contaminated Soil.

Pesticide additives (PAs) are auxiliary ingredients added to the pesticide manufacturing and use processes, constituting 1% to 99% of the pesticide and often composed of benzene and chlorinated hydrocarbons. We selected three typical PAs, toluene, chloroform, and trichloroethylene, to evaluate their retention function toxicity and ecological risk in soil. Soil immobilization techniques and aquatic model organisms were used to demonstrate the effectiveness of the immobilized soil method to determine the ecological risk of chemicals. The 48-h median lethal concentrations of toluene, chloroform, and trichloroethylene alone in spiked soil on Daphnia magna were 10.5, 2.3, and 1.1 mg/L (medium, high, and high toxicity, respectively). The toxicity of the three-PA mixtures showed an antagonistic effect. The risk levels of toluene, chloroform, and trichloroethylene in the soil were evaluated as moderate to high, low to high, and high risk, respectively. The toxicity of two pesticide-contaminated sites in the Yangtze River Delta before and after remediation was successfully evaluated by immobilized soil technology. The toxicity of two soil sampling points was reduced from medium toxic to low toxic and no toxic, respectively, after remediation. The results of our study give a rationale for and prove the validity of the aquatic model organisms and soil immobilization techniques in assessing the soil retention functions toxicity of PAs. Environ Toxicol Chem 2024;43:1677-1689. © 2024 SETAC.

Unveiling the Occurrence and Non-Negligible Role of Amino Sugars in Waste Activated Sludge Fermentation by an Enriched Chitin-Degradation Consortium.

Polysaccharides in extracellular polymeric substances (EPS) can form a hybrid matrix network with proteins, impeding waste-activated sludge (WAS) fermentation. Amino sugars, such as N-acetyl-d-glucosamine (GlcNAc) polymers and sialic acid, are the non-negligible components in the EPS of aerobic granules or biofilm. However, the occurrence of amino sugars in WAS and their degradation remains unclear. Thus, amino sugars (∼6.0%) in WAS were revealed, and the genera of Lactococcus and Zoogloea were identified for the first time. Chitin was used as the substrate to enrich a chitin-degrading consortium (CDC). The COD balances for methane production ranged from 83.3 and 95.1%. Chitin was gradually converted to oligosaccharides and GlcNAc after dosing with the extracellular enzyme. After doing enriched CDC in WAS, the final methane production markedly increased to 60.4 ± 0.6 mL, reflecting an increase of ∼62%. Four model substrates of amino sugars (GlcNAc and sialic acid) and polysaccharides (cellulose and dextran) could be used by CDC. Treponema (34.3%) was identified as the core bacterium via excreting chitinases (EC 3.2.1.14) and N-acetyl-glucosaminidases (EC 3.2.1.52), especially the genetic abundance of chitinases in CDC was 2.5 times higher than that of WAS. Thus, this study provides an elegant method for the utilization of amino sugar-enriched organics.

Identification and degradation of structural extracellular polymeric substances in waste activated sludge via a polygalacturonate-degrading consortium.

By maintaining the cell integrity of waste activated sludge (WAS), structural extracellular polymeric substances (St-EPS) resist WAS anaerobic fermentation. This study investigates the occurrence of polygalacturonate in WAS St-EPS by combining chemical and metagenomic analyses that identify ∼22% of the bacteria, including Ferruginibacter and Zoogloea, that are associated with polygalacturonate production using the key enzyme EC 5.1.3.6. A highly active polygalacturonate-degrading consortium (GDC) was enriched and the potential of this GDC for degrading St-EPS and promoting methane production from WAS was investigated. The percentage of St-EPS degradation increased from 47.6% to 85.2% after inoculation with the GDC. Methane production was also increased by up to 2.3 times over a control group, with WAS destruction increasing from 11.5% to 28.4%. Zeta potential and rheological behavior confirmed the positive effect which GDC has on WAS fermentation. The major genus in the GDC was identified as Clostridium (17.1%). Extracellular pectate lyases (EC 4.2.2.2 and 4.2.2.9), excluding polygalacturonase (EC 3.2.1.15), were observed in the metagenome of the GDC and most likely play a core role in St-EPS hydrolysis. Dosing with GDC provides a good biological method for St-EPS degradation and thereby enhances the conversion of WAS to methane.

Enhanced photocatalytic degradation of tetracycline hydrochloride over Au-doped BiOBr nanosheets under visible light irradiation.

Bismuth(III) oxybromide (BiOBr) is a typical photocatalyst with a unique layered structure. However, the response of BiOBr to visible light is not strong enough for practical application. Moreover, the charge separation efficiency of BiOBr still needs to be improved. In this study, series of Au-doped BiOBr photocatalysts was prepared through a facile one-step hydrothermal method. The as-prepared Au0.3-BiOBr nanosheets exhibited an excellent electrochemical performance. The charge separation efficiency of Au0.3-BiOBr nanosheets was enhanced by 18.5 times compared with that of BiOBr. The intrinsic photocatalytic activity of Au0.3-BiOBr nanosheets in the degradation of tetracycline hydrochloride was approximately twice higher than that of BiOBr under visible light irradiation. In addition, three pathways were identified for the photocatalytic degradation and mineralization of tetracycline hydrochloride, which involve four reactions: hydroxylation, demethylation, ring opening and mineralization. Accordingly, this study proposes a feasible and effective Au-doped BiOBr photocatalyst, and describes a promising strategy for the design and synthesis of high-performance photocatalysts.

Light-dependent enhancement of sulfadiazine detoxification and mineralization by non-photosynthetic methanotrophs.

Co-metabolism and photodegradation are two approaches for remediating trace organic compounds (TOrCs), however, interactions between the two with regards to TOrCs degradation have not been elucidated. In this study, sulfadiazine (SDZ) was chosen as a representative TOrC and Methylocystis bryophila as a typical strain. Under light conditions, about 80.6% of SDZ was removed by M. bryophila, but only 7.6% or 28.9% of SDZ was eliminated by either individual photodegradation or by co-metabolism. The SDZ stimulated more extracellular organic matter (EOM) production by M. bryophila. The enhanced SDZ degradation was attributed to indirect photolysis caused by the excited triplet state of EOM (3EOM*) and co-metabolism. The UPLC-QTOF-MS analysis showed that due to co-metabolism, the pyrimidine ring was broken and could further be oxidized into smaller molecules under light conditions, such as formic and acetic acids. The SDZ mineralization ratio increased from 9.9% under the co-metabolic condition alone to 36.5% under co-metabolism coupled with photodegradation. The Ames tests confirmed that the SDZ degradation products by co-metabolism were mutagenic, however, their toxicity was ameliorated by light during co-metabolism. In conclusion, light plays a crucial role in co-metabolic processes of TOrCs.

Influence of low air pressure on combined nitritation and anaerobic ammonium oxidation process.

At high altitude, wastewater aeration efficiency is low, which is detrimental to nitrification in conventional biological nitrogen removal. The combined partial nitritation and anaerobic ammonium oxidation (CPNA) process requires little oxygen and can be appropriate in low-pressure conditions. As such, in this study, we investigated the effect of air pressure on CPNA using a laboratory-scale reactor. We found that low air pressure promoted the removal of total inorganic nitrogen (TIN), achieving a TIN removal rate of 43,000 mg·N/(kg·VSS·d). The secretion of extracellular polymeric substances under low air pressure was not significantly different from that under ordinary air pressure, indicating no adverse effects on microbial aggregation ability, stability, or settleability. The abundance of aerobic ammonia-oxidizing bacteria (AeAOB) increased from 0.2% to 5.6%, and the activity of anaerobic ammonia-oxidizing bacteria (AnAOB) enhanced, giving AeAOB and AnAOB a competitive advantage over nitrite-oxidizing bacteria, thus forming a microbial community structure favorable to the CPNA process. Our further analysis of the results of batch tests in serum bottles confirmed the positive effect of low air pressure on the anaerobic ammonium oxidation (anammox) process, with a 28.5% ± 1.9% improvement in the specific anammox rate at 70 kPa compared with 100 kPa. AnAOB activity increased, which was reflected in the intracellular heme content increasing from 0.56 ± 0.18 µmol/(g·VSS) at 100 kPa to 2.56 ± 0.20 µmol/(g·VSS) at 70 kPa. We clarified the CPNA-process-promoting effect of low air pressure, which shows potential for nitrogen removal in high-altitude regions.

Mixotrophic Cultivation of Microalgae Using Biogas as the Substrate.

Biogas utilization through biotechnology represents a potential and novel technology. We propose the microalgal mixotrophic cultivation to convert biogas to microalgae-based biodiesel, in which methanotroph was co-cultured to convert CH4 to organic intermediate (and CO2) for microalgal mixotrophic growth. This study constructed a co-culture of Methylocystis bryophila (methanotroph) and Scenedesmus obliquus (microalgae) with biogas feeding. Compared with the single culture of S. obliquus, higher microalgal biomass but a lower chlorophyll concentration was observed. The organic metabolism-related genes were upregulated, verifying microalgal mixotrophic growth. The stoichiometric calculation of M. bryophila culture shows that M. bryophila tends to release organic matter rather than grow under a low O2 content. M. bryophila rarely grew under five different light intensities, indicating that M. bryophila acts as a biocatalyst in the co-culture. The organic intermediate released by methanotroph increased the maximum biomass of microalgal culture, accelerated nitrogen absorption, accumulated more monounsaturated fatty acids, and improved the adaptation to light. The co-culture of microalgae and methanotroph may provide new opportunities for microalgae-based biodiesel production using biogas as a substrate.

Simultaneous removal of sulfamethoxazole and enhanced denitrification process from simulated municipal wastewater by a novel 3D-BER system.

In this study, at an electric current intensity at 60 mA, more than 90.50 ± 4.76% of Sulfamethoxazole (SMX) was degraded. The strengthening of bacterial metabolisms and the sustainment of electrical stimulation contributed to the rapid removal of SMX and nitrates from simulated wastewater by a novel 3D-BER system. From the literature, very few studies have been performed to investigate the high risk of nitrates and antibiotics SMX found in wastewater treatment. The highest antibiotic SMX and nitrogen removal efficiency was 96.45 ± 2.4% (nitrate-N), 99.5 ± 1.5% (nitrite-N), 88.45 ± 1.4% (ammonia-N), 78.6 ± 1.0% (total nitrogen), and SMX (90.50 ± 4.76%), respectively. These results were significantly higher as compared to control system (p < 0.05). The highest denitrification efficiency was achieved at the pH level of 7.0 ± 0.20 - 7.5 ± 0.31. Lower or higher pH value can effect on an approach of heterotrophic-autotrophic denitrification. Moreover, low current intensity did not show any significant effect on the degradation, however, enhanced the removal rate of nitrate or nitrite as well as antibiotic SMX. Based on the results of HPLC and LC-MS/MS analysis, the intermediate products were proposed after efficient biodegradation of SMX. Finally, these results is expected to provide some new insights towards the high electric currents, changes the bacterial community structure, and the activated sludge which played an important role in the biodegradation of SMX and nitrates removal more efficiently.

Constraining nitrification by intermittent aeration to achieve methane-driven ammonia recovery of the mainstream anaerobic effluent.

Mainstream anaerobic treatment has the potential to capture organic energy, and represents a sustainable development trend, but with the problems of low biogas quality and dissolved methane emissions. In this study, methane-driven ammonia recovery of anaerobic effluent was proposed. A 380-day long-term experiment, which was divided into four phases according to different aeration modes, was conducted. The ammonia conversion and microbial characteristics shows that ammonia oxidizing bacteria (AOB) were constrained during Phases 2 (DO: <0.2 mg L-1) and 4 (DO: 0.1-1.6 mg L-1), and were active during Phase 3 (DO: 2-4 mg L-1). During phase 4, when the intermittent aeration was used, the total nitrogen removal rate was higher than during Phases 2 and 3, and nearly 100% ammonia was removed. Methylomonas, a genus of methane oxidizing bacteria (MOB), was enriched during Phase 4. The serum bottle experiment confirmed that the ammonia removal occurred through the MOB assimilation. The protein content in the CH4-added group was 35.5%, which was higher than in the group without CH4 (23.3%). The powerful ammonia assimilation and protein synthesis capabilities of MOB give a meaning to the anaerobic effluent for ammonia recovery and protein production. Intermittent aeration could be used to constrain AOB and improve ammonia recovery efficiency.

Acute toxicity and ecological risk assessment of 4,4'-dihydroxybenzophenone, 2,4,4'-trihydroxybenzophenone and 4-MBC in ultraviolet (UV)-filters.

Ultraviolet (UV) filters are used in cosmetics, personal care products and packaging materials to provide sun protection for human skin and other substances. Little is known about these substances, but they continue to be released into the environment. The acute toxicity of 4,4'-dihydroxybenzophenone, 2,4,4'-trihydroxybenzophenone and 4-MBC to Chlorella vulgaris and Daphnia magna were analyzed in this study. The 96 h-EC50 values of 4,4'-dihydroxybenzophenone, 2,4,4'-trihydroxybenzophenone and 4-MBC on C. vulgaris were 183.60, 3.50 and 0.16874 mg/L, respectively. The 48 h-LC50 of 4,4'-dihydroxybenzophenone, 2,4,4'-trihydroxybenzophenone and 4-MBC on D. magna were 12.50, 3.74 and 0.54445 mg/L, respectively. The toxicity of a mixture of 4,4'-dihydroxybenzophenone and 4-MBC showed addictive effect on C. vulgaris, while the toxicity of mixtures of 4,4'-dihydroxybenzophenone and 2,4,4'-trihydroxybenzophenone, 2,4,4'-trihydroxybenzophenone and 4-MBC as well as 4,4'-dihydroxybenzophenone, 2,4,4'-trihydroxybenzophenone and 4-MBC all showed antagonistic effect on C. vulgaris. The induced no-effect concentrations of 4,4'-dihydroxybenzophenone, 2,4,4'-trihydroxybenzophenone and 4-MBC by the assessment factor (AF) method were 0.0125, 0.00350 and 0.000169 mg/L, respectively.

Effects of the carbon/nitrogen (C/N) ratio on a system coupling simultaneous nitrification and denitrification (SND) and denitrifying phosphorus removal (DPR).

Simultaneous nitrification and denitrification (SND) were coupled with a denitrifying phosphorus removal (DPR) to achieve simultaneous nutrient and carbon removal. With influent chemical oxygen demand (COD), ammonia-N (NH4+-N), and total phosphorus (TP) concentrations of 250, 50, and 8 mg/L, the SND-DPR coupled system achieved stable nutrient removal efficiency of COD, NH4+-N, TN and TP were 91.8 ± 1.7%, 88.4 ± 1.8%, 64 ± 3.3% and 99.2 ± 0.6%, respectively. Enhancing the C/N ratio strengthened the storage of intracellular polymers and provided sufficient intracellular carbon sources for phosphorus uptake. The nutrient removal efficiency reached the highest level at a C/N ratio of 5, and no advantage was observed after increasing the C/N ratio to 7. Nutrients were mainly removed during the aerobic stage at a low DO concentration as well during the anoxic stage, which helped achieve concurrent nitrification and denitrification by ordinary heterotrophic organisms (OHOs), promote denitrifying and aerobic phosphorus removal, and conserve organic carbon demand and energy consumption for aeration. The system was limited for DO in the aerobic stage at a low DO concentration, resulting in a deficiency in electron acceptors (O2 and NO3-N) and limiting the subsequent promotion of phosphorus uptake and TN removal. The limited DO content in the low DO stage was the key factor involved in enhancing the nutrient removal efficiency along with the increasing influent C/N ratio.

Simultaneous nitrification and denitrification in the bio-cathode of a multi-anode microbial fuel cell.

A multi-anode microbial fuel cell (MA-MFC) was developed to investigate simultaneous nitrification and denitrification (SND) in the bio-cathode. As the chemical oxygen demand to nitrogen (COD/N) ratio of the cathode was increased from 0 to 4.5, the electricity-producing quantity ranged between 498 and 543 C and the attained total nitrogen (TN) removal rate reached 12.07 g TN·m-3·d-1, resulting in a TN removal efficiency of 78.8% under the target COD/N ratio of 3.5. The removal of pollutants in series and parallel, open-circuit and closed-circuit were compared, respectively. The removal rates of TN, NH4+-N, and cathode and anode COD were all higher in the parallel connection configuration than in the series configuration. In parallel connection, the TN removal rate reached 14.4 g TN·m-3·d-1, which was 1.9 times that in series connection. Compared with the open-circuit system, the removal rate of TN in the closed-circuit system was improved by 17.8%, which could be ascribed to electrochemical denitrification. The results of high-throughput sequencing confirmed and clarified the presence of autotrophic denitrification and heterotrophic denitrification, including aerobic denitrification, when the MA-MFC had been operated for 18 months.

The performance and microbial communities of an anaerobic membrane bioreactor for treating fluctuating 2-chlorophenol wastewater.

An anaerobic membrane bioreactor (AnMBR) was used to treat low to high (5-200 mg/L) concentrations of 2-chlorophenol (2-CP) wastewater. The AnMBR achieved high and stable chemical oxygen demand removal and 2-CP removal with an average value of 93.2% and 94.2% under long hydraulic retention times (HRTs, 48-96 h), respectively. 2-CP removal efficiency of 98.6 mg/L/d was achieved with 2-CP concentration of 200 mg/L, which was much higher than that of other anaerobic bioreactors. Furthermore, volatile fatty acids didn't accumulate under high 2-CP loading. Long HRTs significantly reduced the membrane fouling as the fouling rate (0.90 × 109-5.44 × 109 m-1h-1) was low. Spirochaetaceae and Methanosaeta were the dominant microbes responsible for dechlorination, methanogenesis, and shock resistance. All these results demonstrate that this AnMBR operated under long HRTs is good and robust for fluctuating chlorophenols wastewater treatment, which has high potential for treating fluctuating refractory organics wastewater with the low membrane fouling rate.

The indispensable role of assimilation in methane driven nitrate removal.

Methane is a greenhouse gas that can be released from sludge anaerobic fermentation in wastewater treatment plants. Methane is also an alternative additional carbon source for deep nitrate removal of secondary effluent. A sequencing experiment was conducted to study the efficacy of nitrate removal with methane as the sole carbon source. The maximum nitrate removal rate was 17.2 mg-N·L-1·d-1. Nitrate removal was confirmed to arise via two pathways: aerobic methane oxidation coupled to denitrification (AME-D) contributed to 55% of the nitrate removal with the rest stemming from assimilation by methanotrophs. Additional study revealed that nitrate assimilated by methanotrophs was used for the synthesis of proteins, resulting in a protein content of 52.2% dry weight. Metagenomic sequencing revealed a high abundance of nitrate assimilation and glutamine synthetase genes, which were primarily provided by methanotrophs (mainly Methylomonas). Assimilatory nitrate removal by methanotrophs has a high potential for advanced nitrogen removal and for alleviating methane emissions. The nitrogen-rich biomass produced by nitrate absorption could also be used as a biofertilizer for nitrogen recycling.

Tertiary denitrification and organic matter variations of secondary effluent from wastewater treatment plant by the 3D-BER system.

A three-dimensional biofilm-electrode reactor (3D-BER) was constructed to facilitate the tertiary denitrification of the secondary effluent of wastewater treatment plants (SEWTP) under 12 mA and in the absence of a carbon source. The TN removal efficiency was 63.8%. The path of the formation and transformation of nitrogen, the relationship between the TN and COD removal rate and the relative concentration and composition of organic matter in the influent and effluent were analyzed to clarify the possible pathways of N and C transformation in the 3D-BER system. Under the action of an electric current, 4.4 mg NH4+-N·L-1 and 17.7 mg COD·L-1 accumulated in the 3D-BER system, and the removal rates of TN and COD were strongly and positively correlated (R2 = 0.9353). The microorganisms in the 3D-BER system under the action of electric current secreted organic matter, some of which (humic acid and microbial metabolites) could be further electrolyzed by microorganisms into bioavailable organic matter for heterotrophic denitrification. Partially dissolved organic matter (DOM, tryptophan aromatic protein, humic acid and microbial metabolites) in the SEWTP could be hydrolyzed under the action of the electric current in the 3D-BER system and consisted of bioavailable organic matter for heterotrophic denitrification. The contribution of heterotrophic denitrification to TN removal was greater than 11.7%. Therefore, the 3D-BER system removed a portion of DOM through microbial electrohydrolysis and promoted the coupling of hydrogen autotrophic denitrification and heterotrophic denitrification to enhance the effectiveness of nitrogen removal in SEWTP. Overall, this technique is effective for enhancing tertiary denitrification in SEWTP.

Electron transfer pathways and kinetic analysis of cathodic simultaneous nitrification and denitrification process in microbial fuel cell system.

Microbial fuel cell (MFC) is an innovative bioconversion technology for wastewater treatment accompanied with electricity recovery. In this study, a kinetic model was developed base on Activated Sludge Model No.1 (ASM1) to describe electron transfer pathways during the simultaneous nitrification and denitrification (SND) process in the biocathode system of a dual-chamber MFC. The batch running of the dual-chamber MFC system showed that it produced a power density up to 2.96 W m-3 within 48 h, the achieved SND efficiency and autotrophic denitrification ratio in the cathodic denitrification process were up to 87.3 ± 0.8% and 69.5 ± 6.6%, respectively. Meanwhile, by integrating nitrification, autotrophic denitrification, heterotrophic denitrification, organic carbon oxidation, and oxygen reduction in the cathode, the model was able to precisely fit the concentration variations of NH3-N, dissolved oxygen (DO) and chemical oxygen demand (COD) during the cathodic SND process (R2 ≥ 0.9876). The cathode electrons tended to be completely utilized with the increase of autotrophic denitrification ratio in the cathodic denitrification process. When the nitrification rate was enhanced, the autotrophic denitrification would prevail in the competition with the heterotrophic denitrification. In summary, the developed model was confirmed to be effective and reliable for describing the electron transfer pathways and predicting the performance of the nitrogen removal reactions during the cathodic SND process in a double-chamber MFC.

High-rate anaerobic decolorization of methyl orange from synthetic azo dye wastewater in a methane-based hollow fiber membrane bioreactor.

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.

Humic substances as electron acceptors for anaerobic oxidation of methane driven by ANME-2d.

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.

Microbial selenite reduction coupled to anaerobic oxidation of methane.

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.