Perchlorate-reducing Bacteria Research Articles

Methane-Driven Perchlorate Reduction by a Microbial Consortium.

The phenomenon of methane oxidation linked to perchlorate reduction has been reported in multiple studies; yet, the underlying microbial mechanisms remain unclear. Here, we enriched suspended cultures by performing methane-driven perchlorate reduction under oxygen-limiting conditions in a membrane bioreactor (MBR). Batch test results proved that perchlorate reduction was coupled to methane oxidation, in which acetate was predicted as the potential intermediate and oxygen played an essential role in activating methane. By combining DNA-based stable isotope probing incubation and high-throughput sequencing analyses of 16S rRNA gene and functional genes (pmoA, pcrA, and narG), we found that synergistic interactions between aerobic methanotrophs (Methylococcus and Methylocystis) and perchlorate-reducing bacteria (PRB; Denitratisoma and Dechloromonas) played active roles in mediating methane-driven perchlorate reduction. This partnership was further demonstrated by coculture experiments in which the aerobic methanotroph could produce acetate to support PRB to complete perchlorate reduction. Our findings advance the understanding of the methane-driven perchlorate reduction process and have implications for similar microbial consortia linking methane and chlorine biogeochemical cycles in natural environments.

Bioprospecting of extremophilic perchlorate-reducing bacteria: report of promising Bacillus spp. isolated from sediments of the bay of Cartagena, Colombia

Three extremophile bacterial strains (BBCOL-009, BBCOL-014 and BBCOL-015), capable of degrading high concentrations of perchlorate at a range of pH (6.5 to 10.0), were isolated from Colombian Caribbean Coast sediments. Morphological features included Gram negative strain bacilli with sizes averaged of 1.75 × 0.95, 2.32 × 0.65 and 3.08 × 0.70 μm, respectively. The reported strains tolerate a wide range of pH (6.5 to 10.0); concentrations of NaCl (3.5 to 7.5% w/v) and KClO4− (250 to 10000 mg/L), reduction of KClO4− from 10 to 25%. LB broth with NaCl (3.5–30% w/v) and KClO4ˉ (250-10000 mg/L) were used in independent trials to evaluate susceptibility to salinity and perchlorate, respectively. Isolates increased their biomass at 7.5 % (w/v) NaCl with optimal development at 3.5 % NaCl. Subsequently, ClO4ˉ reduction was assessed using LB medium with 3.5% NaCl and 10000 mg/L ClO4ˉ. BBCOL-009, BBCOL-014 and BBCOL-015 achieved 10%, 17%, and 25% reduction of ClO4ˉ, respectively. The 16 S rRNA gene sequence grouped them as Bacillus flexus T6186-2, Bacillus marisflavi TF-11 (T), and Bacillus vietnamensis 15 − 1 (T) respectively, with < 97.5% homology. In addition, antimicrobial resistance to ertapenem, vancomycine, amoxicillin clavulanate, penicillin, and erythromycin was present in all the isolates, indicating their high adaptability to stressful environments. The isolated strains from marine sediments in Cartagena Bay, Colombia are suitable candidates to reduce perchlorate contamination in different environments. Although the primary focus of the study of perchlorate-reducing and resistant bacteria is in the ecological and agricultural realms, from an astrobiological perspective, perchlorate-resistant bacteria serve as models for astrobiological investigations.

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.

The horizontal gene transfer of perchlorate reduction genomic island in three bacteria from an ecological niche.

Three new strains of dissimilatory perchlorate-reducing bacteria (DPRB), QD19-16, QD1-5, and P3-1, were isolated from an active sludge. Phylogenetic trees based on 16S rRNA genes indicated that QD19-16, QD1-5, and P3-1 belonged to Brucella, Acidovorax, and Citrobacter, respectively, expanding the distribution of DPRB in the Proteobacteria. The three strains were gram-negative and facultative anaerobes with rod-shaped cells without flagella, which were 1.0-1.6μm long and 0.5-0.6μm wide. The three DPRB strains utilized similar broad spectrum of electron donors and acceptors and demonstrated a similar capability to reduce perchlorate within 6days. The enzyme activity of perchlorate reductase in QD19-16 toward chlorate was higher than that toward perchlorate. The high sequence similarity of the perchlorate reductase operon and chlorite dismutase genes in the perchlorate reduction genomic islands (PRI) of the three strains implied that they were monophyletic origin from a common ancestral PRI. Two transposase genes (tnp1 and tnp2) were found in the PRIs of strain QD19-16 and QD1-5, but were absent in the strain P3-1 PRI. The presence of fragments of IR sequences in the P3-1 PRI suggested that P3-1 PRI had previously contained these two tnp genes. Therefore, it is plausible to suggest that a common ancestral PRI transferred across the strains Brucella sp. QD19-16, Acidovorax sp. QD1-5, and Citrobacter sp. P3-1 through horizontal gene transfer, facilitated by transposases. These results provided a direct evidence of horizontal gene transfer of PRI that could jump across phylogenetically unrelated bacteria through transposase. KEY POINTS: • Three new DPRB strains can effectively remove high concentration of perchlorate. • The PRIs of three DPRB strains are acquired from a single ancestral PRI. • PRIs are incorporated into different bacteria genome through HGT by transposase.

A description of the genus Denitromonas nom. rev.: Denitromonas iodatirespirans sp. nov., a novel iodate-reducing bacterium, and two novel perchlorate-reducing bacteria, Denitromonas halophila and Denitromonas ohlonensis, isolated from San Francisco Bay intertidal mudflats.

The genus Denitromonas is currently a non-validated taxon that has been identified in several recent publications as members of microbial communities arising from marine environments. Very little is known about the biology of Denitromonas spp., and no pure cultures are presently found in any culture collections. The current epitaph of Denitromonas was given to the organism under the assumption that all members of this genus are denitrifying bacteria. This study performs phenotypic and genomic analyses on three new Denitromonas spp. isolated from tidal mudflats in the San Francisco Bay. We demonstrate that Denitromonas spp. are indeed all facultative denitrifying bacteria that utilize a variety of carbon sources such as acetate, lactate, and succinate. In addition, individual strains also use the esoteric electron acceptors perchlorate, chlorate, and iodate. Both 16S and Rps/Rpl phylogenetic analyses place Denitromonas spp. as a deep branching clade in the family Zoogloeaceae, separate from either Thauera spp., Azoarcus spp., or Aromatoleum spp. Genome sequencing reveals a G + C content ranging from 63.72% to 66.54%, and genome sizes range between 4.39 and 5.18 Mb. Genes for salt tolerance and denitrification are distinguishing features that separate Denitromonas spp. from the closely related Azoarcus and Aromatoleum genera. IMPORTANCE The genus Denitromonas is currently a non-validated taxon that has been identified in several recent publications as members of microbial communities arising from marine environments. Very little is known about the biology of Denitromonas spp., and no pure cultures are presently found in any culture collections. The current epitaph of Denitromonas was given to the organism under the assumption that all members of this genus are denitrifying bacteria. This study performs phenotypic and genomic analyses on three Denitromonas spp., Denitromonas iodatirespirans sp. nov.-a novel iodate-reducing bacterium-and two novel perchlorate-reducing bacteria, Denitromonas halophila and Denitromonas ohlonensis, isolated from San Francisco Bay intertidal mudflats.

Coremoval of Energetics and Oxyanions via the In Situ Coupling of Catalytic and Enzymatic Destructions: A Solution to Ammunition Wastewater Treatment.

Ammunition wastewater contains toxic nitrated explosives like RDX and oxyanions like nitrate and perchlorate. Its treatment is challenged by low efficiency due to contaminant recalcitrance and high cost due to multiple processes needed for separately removing different contaminant types. This paper reports a H2-based low-energy strategy featuring the treatment of explosives via catalytic denitration followed by microbial mineralization coupled with oxyanion reduction. After a nitrate- and perchlorate-reducing biofilm incapable of RDX biodegradation was coated with palladium nanoparticles (Pd0NPs), RDX was rapidly denitrated with a specific catalytic activity of 8.7 gcat-1 min-1, while biological reductions of nitrate and perchlorate remained efficient. In the subsequent 30-day continuous test, >99% of RDX, nitrate, and perchlorate were coremoved, and their effluent concentrations were below their respective regulation levels. Detected intermediates and shallow metagenome analysis suggest that the intermediates after Pd-catalytic denitration of RDX ultimately were enzymatically utilized by the nitrate- and perchlorate-reducing bacteria as additional electron donor sources.

Perchlorate reduction kinetics and genome-resolved metagenomics identify metabolic interactions in acclimated saline lake perchlorate-reducing consortia

Perchlorate is a widely detected environmental contaminant in surface and underground water, that seriously impacts human health by inhibiting the uptake of thyroidal radioiodine. Perchlorate reduction due to saline lake microorganisms is not as well understood as that in marine environments. In this study, we enriched a perchlorate-reducing microbial consortium collected from saline lake sediments and found that the perchlorate reduction kinetics of the enriched consortium fit the Michaelis–Menten kinetics well, with a maximum specific substrate reduction rate (qmax) of 0.596 ± 0.001 mg ClO4−/mg DW/h and half-saturation constant (Ks) of 16.549 ± 0.488 mg ClO4−/L. Furthermore, we used improved metagenome binning to reconstruct high-quality metagenome-assembled genomes from the metagenomes of the microbial consortia, including the perchlorate-reducing bacteria (PRB) Dechloromonas agitata and Wolinella succinogenes, with the genome of W. succinogenes harboring complete functional genes for perchlorate reduction being the first recovered. Given that the electrons were directly transferred to the electronic carrier cytochrome c-553 from the quinone pool, the electron transfer pathway of W. succinogenes was shorter and more efficient than the canonical pattern. This finding provides a theoretical basis for microbial remediation of sites contaminated by high concentrations of perchlorate. Metagenomic binning and metatranscriptomic analyses revealed the gene transcription variation of perchlorate reductase pcr and chlorite dismutase cld by PRB and the synergistic metabolic mechanism.

Construction of heterotrophic-sulfur autotrophic integrated fluidized bed reactor for simultaneous and efficient removal of compound pollution of perchlorate and nitrate in water

A heterotrophic sulfur autotrophic integrated fluidized bed reactor was established for simultaneous and efficient removal of ClO4− and NO3− from water. The optimum operating conditions forecasted through the response surface method (RSM) were the hydraulic retention time (HRT) of 0.50 h, the influent acetate (CH3COO−) concentration of 55 mg/L and the reflux ratio of 14, contributing to ClO4− and NO3− removal of 98.99% and 99.96%, respectively, without secondary pollution caused by residual carbon (NPOC <3.89 mg/L). Meanwhile, the effluent pH fluctuated in a range of 6.70–8.02 and sulfur-containing by-products (i.e., SO42− and S2−) could be controlled by adjusting operation conditions throughout the experimental stage. The increase of the influent CH3COO− concentration reduced the load borne by autotrophic reduction process and further reduced SO42− production. Shortening HRT, increasing the influent CH3COO− concentration and decreasing the reflux ratio could all reduce alkalinity consumption. Shortening HRT and decreasing the reflux ratio could shorten contact time between sulfur and water and thus inhibit S0 disproportionation. High-throughput sequencing result showed that Proteobacteria and Chlorobi were the dominant bacteria. Sulfurovum, Sulfuricurvum and Ignavibacterium were the major heterotrophic denitrifying bacteria (DB)/perchlorate reducing bacteria (PRB), Ferritrophicum and Geothrix were DB, and Chlorobaculum was S0 disproportionation bacteria.

Perchlorate-reducing bacteria from Antarctic marine sediments.

Perchlorate is a contaminant that can persist in groundwater and soil, and is frequently detected in different ecosystems at concentrations relevant to human health. This study isolated and characterised halotolerant bacteria that can potentially perform perchlorate reduction. Bacterial microorganisms were isolated from marine sediments on Deception, Horseshoe and Half Moon Islands of Antarctica. The results of the 16S ribosomal RNA (rRNA) gene sequence analysis indicated that the isolates were phylogenetically related to Psychrobacter cryohalolentis, Psychrobacter urativorans, Idiomarina loihiensis, Psychrobacter nivimaris, Sporosarcina aquimarina and Pseudomonas lactis. The isolates grew at a sodium chloride concentration of up to 30% and a perchlorate concentration of up to 10,000mg/L, which showed their ability to survive in saline conditions and high perchlorate concentrations. Between 21.6 and 40% of perchlorate was degraded by the isolated bacteria. P. cryohalolentis and P. urativorans degraded 30.3% and 32.6% of perchlorate, respectively. I. loihiensis degraded 40% of perchlorate, and P. nivimaris, S. aquimarina and P. lactis degraded 22%, 21.8% and 21.6% of perchlorate, respectively. I. loihiensis had the highest reduction in perchlorate, whereas P. lactis had the lowest reduction. This study is significant as it is the first finding of P. cryohalolentis and. P. lactis on the Antarctic continent. In conclusion, these bacteria isolated from marine sediments on Antarctica offer promising resources for the bioremediation of perchlorate contamination due to their ability to degrade perchlorate, showing their potential use as a biological system to reduce perchlorate in high-salinity ecosystems.

Chlorate addition enhances perchlorate reduction in denitrifying membrane-biofilm reactors.

Perchlorate is a widespread drinking water contaminant with regulatory standards ranging from 2 to 18μg/L. The hydrogen-based membrane-biofilm reactor (MBfR) can effectively reduce perchlorate, but it is challenging to achieve low-µg/L levels. We explored chlorate addition to increase the abundance of perchlorate-reducing bacteria (PRB) and improve removals. MBfR reactors were operated with and without chlorate addition. Results show that chlorate doubled the abundance of putative PRB (e.g., Rhodocyclales) and improved perchlorate reduction to 23 ± 17µg/L, compared to 53 ± 37µg/L in the control. Sulfate reduction was substantially inhibited during chlorate addition, but quickly recovered once suspended. Our results suggest that chlorate addition can enhance perchlorate reduction by providing a selective pressure for PRB. It also decreases net sulfate reduction. KEY POINTS: • Chlorate increased the abundance of perchlorate-reducing bacteria • Chlorate addition improved perchlorate removal • Chlorate appeared to suppress sulfate reduction.

Efficient nitrate and perchlorate removal from aqueous solution via a novel electro-dialysis ion-exchange membrane bioreactor

The performance of a novel Electro-dialysis ion-exchange membrane bioreactor (EDIMB) for simultaneous removal of perchlorate and nitrate from aqueous solution was studied. EDIMB combines electrodialysis (ED) with bioreduction to remove contaminants while avoiding secondary contamination of organic matter and microorganisms. The removal process of pollutants in water compartment and bio-compartment can be well simulated by the First-order kinetic model and the established Zero-Michaelis-Menten model. Furthermore, compared with ED (VSS = 0.00 g/L), when the microbial concentration was 3.00 g/L, the total current efficiency was increased by 43.5% in first 60 min. The biological reaction takes place in the bio-compartment, which reduces the resistance of pollutant transmembrane migration and promotes ion migration. Microorganisms played a promoting role in the process of pollutant migration. High-throughput sequencing analysis of microorganisms showed that Thauera and Macellibacteroides were the main denitrifying and perchlorate-reducing bacteria.

A Multiplex Immunosensor for Detecting Perchlorate-Reducing Bacteria for Environmental Monitoring and Planetary Exploration.

Perchlorate anions are produced by chemical industries and are important contaminants in certain natural ecosystems. Perchlorate also occurs in some natural and uncontaminated environments such as the Atacama Desert, the high Arctic or the Antarctic Dry Valleys, and is especially abundant on the surface of Mars. As some bacterial strains are capable of using perchlorate as an electron acceptor under anaerobic conditions, their detection is relevant for environmental monitoring on Earth as well as for the search for life on Mars. We have developed an antibody microarray with 20 polyclonal antibodies to detect perchlorate-reducing bacteria (PRB) strains and two crucial and highly conserved enzymes involved in perchlorate respiration: perchlorate reductase and chlorite dismutase. We determined the cross-reactivity, the working concentration, and the limit of detection of each antibody individually and in a multiplex format by Fluorescent Sandwich Microarray Immunoassay. Although most of them exhibited relatively high sensitivity and specificity, we applied a deconvolution method based on graph theory to discriminate between specific signals and cross-reactions from related microorganisms. We validated the system by analyzing multiple bacterial isolates, crude extracts from contaminated reactors and salt-rich natural samples from the high Arctic. The PRB detecting chip (PRBCHIP) allowed us to detect and classify environmental isolates as well as to detect similar strains by using crude extracts obtained from 0.5 g even from soils with low organic-matter levels (<103 cells/g of soil). Our results demonstrated that PRBCHIP is a valuable tool for sensitive and reliable detection of perchlorate-reducing bacteria for research purposes, environmental monitoring and planetary exploration.

Carbon Source Competition in Biological Selenate Reduction under Other Oxyanions Contamination

Selenate removal in drinking water is being vigorously debated due to the various health issues concerned. As a viable treatment option, this study investigated a fixed-bed biofilm reactor (FBBR) with internal recycling. The experimental design tested how hydraulic loading rate and electron donor affect selenate reduction together with other oxyanions. The tested accompanying oxyanions were nitrate and perchlorate and experiments were designed to test how an FBBR responded to the limited electron donor condition. The results showed that the reactor achieved almost complete selenate reduction with the initial hydraulic loading rate of 12 m3/m2/day (influent concentration of 1416 µg SeO42−/L). Increasing the hydraulic loading rates to 16.24 and 48 m3/m2/day led to a gradual decline in selenate removal efficiency. A sufficient external carbon source (C:N of 3.3:1) achieved an almost complete reduction of nitrate as well as selenate. The FBBR acclimated to selenate instantaneously and reduced nitrate via synergistic denitrification. An experiment with another oxyanion addition, perchlorate (459 µg ClO4−/L), revealed that perchlorate-reducing bacteria were more strongly associated with carbon limitation than selenate-reducing bacteria, which can help us to understand parallel reactions in FBBRs. This research provides a framework to further study the use of electron donor-controlled FBBRs for simultaneous reduction of selenate and other oxyanions threatening the drinking water-related environment and public health.

Electro-assisted autohydrogenotrophic reduction of perchlorate and microbial community in a dual-chamber biofilm-electrode reactor

The electro-assisted autohydrogenotrophic reduction of perchlorate (ClO4−) was investigated in a dual-chamber biofilm-electrode reactor (BER), in which the microbial community was inoculated from natural sediments. To avoid the effect of extreme pH and direct electron transfer on perchlorate reduction, a novel cathode configuration was designed. The pH of the cathode compartment was successfully controlled in the range of 7.2–8.4 during whole experiment. The effective biological autohydrogenotrophic reduction of perchlorate was achieved using hydrogen generated in-situ on the electrode surface, and the removal rate of 10 mg L−1 perchlorate reached 98.16% at HRT of 48 h. The highest perchlorate removal flux reached to 1498.420 mg m−2·d−1 with a 0.410 kW·h g-perchlorate−1 energy consumption. The microbial community evolution in the BER was determined by high-throughput sequencing and the results indicated that the Firmicutes and Bacteroidetes were dominant at phylum level when perchlorate concentration was 10 mg L−1 or lower. And the Proteobacteria became ascendant at the perchlorate concentration of 20 mg L−1. The functional populations for perchlorate reduction were successfully enriched including Nitrosomonas (30%), Thermomonas (9%), Comamonas (8%) and Hydrogenophaga (3%). Meanwhile, the proportion of functional population in biofilm linked to perchlorate concentration. With the increase of influent perchlorate concentration, the perchlorate-reducing bacteria (PRB) were enriched successfully and became ascendant.

Effect of Nitrate and Perchlorate on Selenate Reduction in a Sequencing Batch Reactor

Selenate removal from a water body is being vigorously debated owing to severe health impact, but inhibitions of coexisting anions have been reported. To suggest a viable treatment option, this study investigates the effect of nitrate and perchlorate on selenate reduction in a laboratory-scale sequencing batch reactor. The experimental design tests how competing electron acceptors (NO3− and ClO4−) and electron donor (acetate) limitations affect selenate reduction in the reactor. Results show that the reactor achieves almost complete selenate reduction within the initial concentration ranges of 0.1–1 mM by enriching selenate-reducing bacteria with appropriate temperature (30 °C) and acclimation period (50 days). We monitored simultaneous selenate and nitrate reduction in the reactor without specific inhibition due to a difference in microbial growth strategy related to electron donor status. Lack of perchlorate-reducing bacteria makes perchlorate addition (0.2 mM) not to be closely associated with dissimilative perchlorate reduction. These results provide information that can help us to understand the effect of competing electron acceptors on selenate reduction and the kinetics of potential parallel reactions in the reactor.

Identification of a parasitic symbiosis between respiratory metabolisms in the biogeochemical chlorine cycle.

A key step in the chlorine cycle is the reduction of perchlorate (ClO4-) and chlorate (ClO3-) to chloride by microbial respiratory pathways. Perchlorate-reducing bacteria and chlorate-reducing bacteria differ in that the latter cannot use perchlorate, the most oxidized chlorine compound. However, a recent study identified a bacterium with the chlorate reduction pathway dominating a community provided only perchlorate. Here we confirm a metabolic interaction between perchlorate- and chlorate-reducing bacteria and define its mechanism. Perchlorate-reducing bacteria supported the growth of chlorate-reducing bacteria to up to 90% of total cells in communities and co-cultures. Chlorate-reducing bacteria required the gene for chlorate reductase to grow in co-culture with perchlorate-reducing bacteria, demonstrating that chlorate is responsible for the interaction, not the subsequent intermediates chlorite and oxygen. Modeling of the interaction suggested that cells specialized for chlorate reduction have a competitive advantage for consuming chlorate produced from perchlorate, especially at high concentrations of perchlorate, because perchlorate and chlorate compete for a single enzyme in perchlorate-reducing cells. We conclude that perchlorate-reducing bacteria inadvertently support large populations of chlorate-reducing bacteria in a parasitic relationship through the release of the intermediate chlorate. An implication of these findings is that undetected chlorate-reducing bacteria have likely negatively impacted efforts to bioremediate perchlorate pollution for decades.

Biological perchlorate reduction: which electron donor we can choose?

Biological reduction is an effective method for removal of perchlorate (ClO4-), where perchlorate is transformed into chloride by perchlorate-reducing bacteria (PRB). An external electron donor is required for autotrophic and heterotrophic reduction of perchlorate. Therefore, plenty of suitable electron donors including organic (e.g., acetate, ethanol, carbohydrate, glycerol, methane) and inorganic (e.g., hydrogen, zero-valent iron, element sulfur, anthrahydroquinone) as well as the cathode have been used in biological reduction of perchlorate. This paper reviews the application of various electron donors in biological perchlorate reduction and their influences on treatment efficiency of perchlorate and biological activity of PRB. We discussed the criteria for selection of appropriate electron donor to provide a flexible strategy of electron donor choice for the bioremediation of perchlorate-contaminated water.

Perchlorate bio-reduction in a methane-based membrane biofilm reactor in the presence and absence of oxygen

Perchlorate has been widely detected in various water environments and could cause serious health problems. Methane has been proposed as a promising electron donor to remove perchlorate from contaminated water, yet it is unclear whether and how microbial methane oxidation couples with perchlorate reduction, in particular under anoxic conditions. Here, the feasibility and performance of perchlorate reduction driven by methane in the presence and absence of oxygen were investigated and compared in a lab-scale methane-based membrane biofilm reactor. Long-term operational performance suggested that perchlorate was reduced to chloride, with 4 mg Cl/L/d of perchlorate removal rate under anoxic conditions. Differently, perchlorate removal rate increased to 16 mg Cl/L/d, and volatile fatty acids (VFAs) were produced from methane partial oxidation when a limited oxygen (10 mg/L/d) was externally supplied. Regardless of oxygen conditions, microbial perchlorate reduction driven by methane might be mediated through synergistic interactions by a microbial consortium, but with different key microbial members under both oxygen regimes. Under anoxic conditions, aerobic methanotrophs (likely Methylocystaceae and Methylococcaceae) might micro-aerobically oxidize methane by utilizing internal oxygen from microbial perchlorate reduction, which might be mediated by Rhodocyclaceae. In contrast, under oxygen-limiting conditions, methanogens (e.g., Methanosarcina) and fermenters (e.g., Veillonellaceae) likely jointly converted methane into VFAs, then dissimilatory perchlorate-reducing bacteria (e.g., Rhodocyclaceae) utilized the produced VFAs to reduce perchlorate to chloride. Our findings provide evidence to link methane oxidation with perchlorate reduction under both oxygen regimes, which could be facilitated to design a process to remove perchlorate from groundwater.

Resistance and Resilience of Sulfidogenic Communities in the Face of the Specific Inhibitor Perchlorate.

Hydrogen sulfide is a toxic and corrosive gas, produced by the activity of sulfate-reducing microorganisms (SRM). Owing to the environmental, economic and human-health consequences of sulfide, there is interest in developing specific inhibitors of SRM. Recent studies have identified perchlorate as a promising emerging inhibitor. The aim of this work is to quantitatively dissect the inhibitory dynamics of perchlorate. Sulfidogenic mixed continuous-flow systems were treated with perchlorate. SRM number, sulfide production and community structure were monitored pre-, during and post-treatment. The data generated was compared to a simple mathematical model, where SRM growth slows as a result of inhibition. The experimental data supports the interpretation that perchlorate largely acts to suppress SRM growth rates, rendering planktonic SRM increasingly susceptible to wash-out. Surface-attachment was identified as an important parameter preventing SRM wash-out and thus governing inhibitory dynamics. Our study confirmed the lesser depletion of surface-attached SRM as compared to planktonic SRM during perchlorate treatment. Indirect effects of perchlorate (bio-competitive exclusion of SRM by dissimilatory perchlorate-reducing bacteria, DPRB) were also assayed by amending reactors with DPRB. Indeed, low concentrations of perchlorate coupled with DRPB amendment can drive sulfide concentrations to zero. Further, inhibition in a complex community was compared to that in a pure culture, highlighting similarities and differences between the two scenarios. Finally, we quantified susceptibility to perchlorate across SRM in various culture conditions, showing that prediction of complex behavior in continuous systems from batch results is possible. This study thus provides an overview of the sensitivity of sulfidogenic communities to perchlorate, as well as mechanisms underlying these patterns.

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.