Efficient perchlorate reduction in microaerobic environment facilitated by partner methane oxidizers.

The conventional perchlorate (ClO4-) reduction typically necessitates anaerobic conditions. However, in this study, we observed efficient ClO4- reduction using CH4 as the electron donor in a microaerobic environment. The maximum ClO4- removal flux of 2.18 g/m2·d was achieved in CH4-based biofilm. The kinetics of ClO4- reduction showed significant differences, with trace oxygen increasing the reduction rate of ClO4-, whereas oxygen levels exceeding 2 mg/L decelerated the ClO4- reduction. In the absence of exogenous oxygen, anaerobic methanotrophic (ANME) archaea contribute more than 80% electrons through the reverse methanogenesis pathway for ClO4- reduction. Simultaneously, microorganisms activate CH4 by utilizing oxygen generated from chlorite (ClO2-) disproportionation. In the presence of exogenous oxygen, methane oxidizers predominantly consume oxygen to drive the aerobic oxidation of methane. It is indicated that methane oxidizers and perchlorate reducing bacteria can form aggregates to resist external oxygen shocks and achieve efficient ClO4- reduction under microaerobic condition. These findings provide new insights into biological CH4 mitigation and ClO4- removal in hypoxic environment.

Methane-dependent selenate reduction by a bacterial consortium.

Methanotrophic microorganisms play a critical role in controlling the flux of methane from natural sediments into the atmosphere. Methanotrophs have been shown to couple the oxidation of methane to the reduction of diverse electron acceptors (e.g., oxygen, sulfate, nitrate, and metal oxides), either independently or in consortia with other microbial partners. Although several studies have reported the phenomenon of methane oxidation linked to selenate reduction, neither the microorganisms involved nor the underlying trophic interaction has been clearly identified. Here, we provide the first detailed evidence for interspecies electron transfer between bacterial populations in a bioreactor community where the reduction of selenate is linked to methane oxidation. Metagenomic and metaproteomic analyses of the community revealed a novel species of Methylocystis as the most abundant methanotroph, which actively expressed proteins for oxygen-dependent methane oxidation and fermentation pathways, but lacked the genetic potential for selenate reduction. Pseudoxanthomonas, Piscinibacter, and Rhodocyclaceae populations appeared to be responsible for the observed selenate reduction using proteins initially annotated as periplasmic nitrate reductases, with fermentation by-products released by the methanotrophs as electron donors. The ability for the annotated nitrate reductases to reduce selenate was confirmed by gene knockout studies in an isolate of Pseudoxanthomonas. Overall, this study provides novel insights into the metabolic flexibility of the aerobic methanotrophs that likely allows them to thrive across natural oxygen gradients, and highlights the potential role for similar microbial consortia in linking methane and other biogeochemical cycles in environments where oxygen is limited.

NC10 bacteria promoted methane oxidation coupled to chlorate reduction.

The strictly anaerobic serum bottles were applied to investigate methane oxidation coupled to chlorate (ClO3-) reduction (MO-CR) without exogenous oxygen. 0.35 mM ClO3- was consumed within 20 days at the reduction rate of 17.50 µM/d, over three times than that of ClO4-. Chlorite (ClO2-) was not detected throughout the experiment and the mass recovery of Cl- was over 89%. Isotope tracing results showed most of 13CH4 was oxided to CO2, and the electrons recovery reached to 77.6%. Small amounts of 13CH4 was consumed for DOC production probably through aerobic methane oxidation process, with oxygen generated from disproportionation reaction. In pMMO (key enzyme in aerobic oxidation of methane) inhibition tests, ClO3- reduction rate was slowed to 7. 0 µmol/d by 2 mM C2H2, real-time quantitative PCR also showed the transcript abundance of pMMO and Cld were significantly dropped at the later period of experiment, indicating that the O2 disproportionated from ClO2- was utilized to active CH4. NC10 bacteria Candidatus Methylomirabilis, related closely to oxygenic denitrifiers M. oxyfera, was detected in the system, and got enriched along with chlorate reduction. Several pieces of evidence supported that NC10 bacteria promoted CH4 oxidation coupled to ClO3- reduction, these oxygenic denitrifiers may perform ClO2- disproportionation to produce O2, and then oxidized methane intracellularly.

A mixed consortium of methanotrophic archaea and bacteria boosts methane-dependent selenate reduction.

Though methane-based selenate reduction has been reported, neither the selenate load nor the removal rate could satisfy practical applications, thus limiting this technique to bio-remediate selenate pollution. In the present study, using a membrane biofilm batch reactor (MBBR), we successfully enriched a consortium performing methane-dependent selenate reduction, with enhanced reduction rates from 16.1 to 28.9 µM-day-1 under a comparable Se concentration to industrial wastewaters (i.e., ~500 µM). During active reduction, 16S rRNA gene copies of Archaea and Bacteria were both increased more than one order of magnitude. Clone library construction and high-throughput sequencing indicated that Methanosarcina and Methylocystis were the only methane-oxidizing microorganisms. The presence of 20 mM bromoethanesulphonate or 0.15 mM acetylene both significantly, but not completely, inhibited methane-dependent selenate reduction, indicating the concurrent contributions of methanotrophic archaea and bacteria. Fluorescence in situ hybridization (FISH) revealed that archaea directly adhered to the surface of the membrane while bacteria were in the outer layer, together forming the mature biofilm. This study highlights the crucial role of both methanotrophic archaea and bacteria in methane-dependent selenate reduction, and lays foundations in applying methane to bio-remediate practical selenate pollution.

Why does sulfate inhibit selenate reduction: Molybdenum deprivation from Mo-dependent selenate reductase.

Selenium pollution has become an increasingly serious global concern. Methane-fed selenate reduction has proven to be of great interest for the bioremediation of selenate-contaminated waters even with the coexistence of nitrate and dissolved oxygen. However, it is unclear if the common concurrent sulfate anion affects selenate removal. To address this question, we first introduced selenate (SeO42-) as the sole influent electron acceptor in a CH4-fed membrane biofilm reactor (CH4-MBfR); then we added different concentrations of sulfate (SO42-). The initial selenate removal efficiency (∼90%) was decreased by 50% in the presence of 15.6 µM of sulfate and completely inhibited after loading with 171.9 µM of sulfate. 16S rRNA gene sequencing showed that the selenate-reducing bacteria decreased after the addition of sulfate. Metagenomic sequencing showed that the abundance of genes encoding molybdenum (Mo)-dependent selenate reductase reduced by >50% when exposed to high concentrations of sulfate. Furthermore, the decrease in the total genes encoding all Mo-oxidoreductases was much greater than that of the genes encoding molybdate transporters, suggesting that the inhibition of selenate reduction by sulfate was most likely via the direct competition with molybdate for the transport system, leading to a lack of available Mo for Mo-dependent selenate reductases and thus reducing their activities. This result was confirmed by a batch test wherein the supplementation of molybdate mitigated the sulfate effect. Overall, this study shed light on the underlying mechanism of sulfate inhibition on selenate reduction and laid the foundation for applying the technology to practical wastewaters.

How nitrate affects perchlorate reduction in a methane-based biofilm batch reactor.

Nitrate (NO3-) affected perchlorate (ClO4-) reduction in a membrane batch biofilm reactor (MBBR), even though the electron donor, CH4, was available well in excess of its demand. For example, the perchlorate-reduction rate was 1.7 mmol/m2-d when perchlorate was the sole electron acceptor, but it dropped to 0.64 mmol/m2-d when nitrate also was present. The perchlorate-reduction rate returned to 1.60 mmol/m2-d after all nitrate was consumed. Denitratisoma and Azospirillum were main genera involved in perchlorate and nitrate reduction, and both could utilize NO3- and ClO4- as electron acceptors. Results of the reverse transcription-polymerase chain reaction (RT-PCR) showed that transcript abundances of nitrate reductase (narG), nitrite reductase (nirS), and perchlorate reductase (pcrA) increased when the perchlorate and nitrate concentrations were higher. Specifically, pcrA transcripts correlated to the sum of perchlorate and nitrate, rather than perchlorate individually. Analysis based on Density Functional Theory (DFT) suggests that bacteria able to utilize both acceptors, preferred NO3- over ClO4- due to nitrate reduction having lower energy barriers for proton and electron transfers.

Methane oxidation coupled to perchlorate reduction in a membrane biofilm batch reactor.

A specially designed CH4-based membrane biofilm batch reactor (MBBR) was applied to investigate anaerobic methane oxidation coupled to perchlorate reduction (AnMO-PR). The 0.21 mM ClO4- added in the first stage of operation was completely reduced in 28 days, 0.40 mM ClO4- was reduced within 23 days in stage 2, and 0.56 mM of ClO4- was reduced within 30 days in stage 3. Although some chlorate (ClO3-) accumulated, the recovery of Cl- was over 92%. Illumina sequencing of the 16S rRNA gene documented that the bacterial community was mainly composed by perchlorate-reducing bacteria (PRB), methanotrophic bacteria, and archaea. Real-time quantitative PCR showed the archaeal 16S rRNA and mcrA genes increased as more ClO4- was reduced, and the predominant archaea belonged to Methanosarcina mazei, which is related to ANME-3, an archaeon able to perform reverse methanogenesis. Several pieces of evidence support that ClO4- reduction by the MBBR biofilm occurred via a synergism between Methanosarcina and PRB: Methanosarcina oxidized methane through reverse methanogesis and provided electron donor for PRB to reduce ClO4-. Because methanotrophs were present, we cannot rule out that they also were involved in AnMO-PR if they received O2 generated by disproportionation of ClO2- from the PRB.

Bromate and Nitrate Bioreduction Coupled with Poly-ß-hydroxybutyrate Production in a Methane-Based Membrane Biofilm Reactor.

This work demonstrates bromate (BrO3-) reduction in a methane (CH4)-based membrane biofilm reactor (MBfR), and it documents contrasting impacts of nitrate (NO3-) on BrO3- reduction, as well as formation of poly-ß-hydroxybutyrate (PHB), an internal C- and electron-storage material. When the electron donor, CH4, was in ample supply, NO3- enhanced BrO3- reduction by stimulating the growth of denitrifying bacteria ( Meiothermus, Comamonadaceae, and Anaerolineaceae) able to reduce BrO3- and NO3- simultaneously. This was supported by increases in denitrifying enzymes (e.g., nitrate reductase, nitrite reductase, nitrous-oxide reductase, and nitric-oxide reductase) through quantitative polymerase chain reaction (qPCR) analysis and metagenomic prediction of these functional genes. When the electron donor was in limited supply, NO3- was the preferred electron acceptor over BrO3- due to competition for the common electron donor; this was supported by the significant oxidation of stored PHB when NO3- was high enough to cause electron-donor limitation. Methanotrophs (e.g., Methylocystis, Methylomonas, and genera within Comamonadaceae) were implicated as the main PHB producers in the biofilms, and their ability to oxidize PHB mitigated the impacts of competition for CH4.

The effect of electron competition on chromate reduction using methane as electron donor.

We studied the effect of electron competition on chromate (Cr(VI)) reduction in a methane (CH4)-based membrane biofilm reactor (MBfR), since the reduction rate was usually limited by electron supply. A low surface loading of SO42- promoted Cr(VI) reduction. The Cr(VI) removal percentage increased from 60 to 70% when the SO42- loading increased from 0 to 4.7 mg SO42-/m2-d. After the SO42- loading decreased back to zero, the Cr(VI) removal further increased to 90%, suggesting that some sulfate-reducing bacteria (SRB) stayed in the reactor to reduce Cr(VI). However, a high surface loading of SO42- (26.6 mg SO42-/m2-d) significantly slowed down the Cr(VI) reduction to 40% removal, which was probably due to competition between Cr(VI) and SO42- reduction. Similarly, when 0.5 mg/L of Se(VI) was introduced into the MBfR, Cr(VI) removal percentage slightly decreased to 60% and then increased to 80% when input Se(VI) was removed again. The microbial community strongly depended on the loadings of Cr(VI) and SO42-. In the sulfate effect experiment, three genera were dominant. Based on the correlation between the abundances of the three genera and the loadings of Cr(VI) and SO42-, we conclude that Methylocystis, a type II methanotroph, reduced both Cr(VI) and sulfate, Meiothermus only reduced Cr(VI), and Ferruginibacter only reduced SO42-.