Dissimilatory Perchlorate-reducing Bacteria Research Articles

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.

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.

Functional Redundancy in Perchlorate and Nitrate Electron Transport Chains and Rewiring Respiratory Pathways to Alter Terminal Electron Acceptor Preference.

Most dissimilatory perchlorate reducing bacteria (DPRB) are also capable of respiratory nitrate reduction, and preferentially utilize nitrate over perchlorate as a terminal electron acceptor. The similar domain architectures and phylogenetic relatedness of the nitrate and perchlorate respiratory complexes suggests a common evolutionary history and a potential for functionally redundant electron carriers. In this study, we identify key genetic redundancies in the electron transfer pathways from the quinone pool(s) to the terminal nitrate and perchlorate reductases in Azospira suillum PS (hereafter referred to as PS). We show that the putative quinol dehydrogenases, (PcrQ and NapC) and the soluble cytochrome electron carriers (PcrO and NapO) are functionally redundant under anaerobic growth conditions. We demonstrate that, when grown diauxically with both nitrate and perchlorate, the endogenous expression of NapC and NapO during the nitrate reduction phase was sufficient to completely erase any growth defect in the perchlorate reduction phase caused by deletion of pcrQ and/or pcrO. We leveraged our understanding of these genetic redundancies to make PS mutants with altered electron acceptor preferences. Deletion of the periplasmic nitrate reductase catalytic subunit, napA, led to preferential utilization of perchlorate even in the presence of equimolar nitrate, and deletion of the electron carrier proteins napQ and napO, resulted in concurrent reduction of nitrate and perchlorate. Our results demonstrate that nitrate and perchlorate respiratory pathways in PS share key functionally redundant electron transfer proteins and that mutagenesis of these proteins can be utilized as a strategy to alter the preferential usage of nitrate over perchlorate.

Biotechnological Applications of Microbial (Per)chlorate Reduction.

While the microbial degradation of a chloroxyanion-based herbicide was first observed nearly ninety years ago, only recently have researchers elucidated the underlying mechanisms of perchlorate and chlorate [collectively, (per)chlorate] respiration. Although the obvious application of these metabolisms lies in the bioremediation and attenuation of (per)chlorate in contaminated environments, a diversity of alternative and innovative biotechnological applications has been proposed based on the unique metabolic abilities of dissimilatory (per)chlorate-reducing bacteria (DPRB). This is fueled in part by the unique ability of these organisms to generate molecular oxygen as a transient intermediate of the central pathway of (per)chlorate respiration. This ability, along with other novel aspects of the metabolism, have resulted in a wide and disparate range of potential biotechnological applications being proposed, including enzymatic perchlorate detection; gas gangrene therapy; enhanced xenobiotic bioremediation; oil reservoir bio-souring control; chemostat hygiene control; aeration enhancement in industrial bioreactors; and, biogenic oxygen production for planetary exploration. While previous reviews focus on the fundamental science of microbial (per)chlorate reduction (for example see Youngblut et al., 2016), here, we provide an overview of the emerging biotechnological applications of (per)chlorate respiration and the underlying organisms and enzymes to environmental and biotechnological industries.

Control of sulfidogenesis through bio-oxidation of H2S coupled to (per)chlorate reduction.

We investigated H2S attenuation by dissimilatory perchlorate-reducing bacteria (DPRB). All DPRB tested oxidized H2S coupled to (per)chlorate reduction without sustaining growth. H2S was preferentially utilized over organic electron donors resulting in an enriched (34S)-elemental sulfur product. Electron microscopy revealed elemental sulfur production in the cytoplasm and on the cell surface of the DPRB Azospira suillum. Based on our results, we propose a novel hybrid enzymatic-abiotic mechanism for H2S oxidation similar to that recently proposed for nitrate-dependent Fe(II) oxidation. The results of this study have implications for the control of biosouring and biocorrosion in a range of industrial environments.

Physiological and Genetic Description of Dissimilatory Perchlorate Reduction by the Novel Marine Bacterium Arcobacter sp. Strain CAB

ABSTRACTA novel dissimilatory perchlorate-reducing bacterium (DPRB), Arcobacter sp. strain CAB, was isolated from a marina in Berkeley, CA. Phylogenetically, this halophile was most closely related to Arcobacter defluvii strain SW30-2 and Arcobacter ellisii. With acetate as the electron donor, strain CAB completely reduced perchlorate (ClO4−) or chlorate (ClO3−) [collectively designated (per)chlorate] to innocuous chloride (Cl−), likely using the perchlorate reductase (Pcr) and chlorite dismutase (Cld) enzymes. When grown with perchlorate, optimum growth was observed at 25 to 30°C, pH 7, and 3% NaCl. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) preparations were dominated by free-swimming straight rods with 1 to 2 polar flagella per cell. Strain CAB utilized a variety of organic acids, fructose, and hydrogen as electron donors coupled to (per)chlorate reduction. Further, under anoxic growth conditions strain CAB utilized the biogenic oxygen produced as a result of chlorite dismutation to oxidize catechol via the meta-cleavage pathway of aerobic catechol degradation and the catechol 2,3-dioxygenase enzyme. In addition to (per)chlorate, oxygen and nitrate were alternatively used as electron acceptors. The 3.48-Mb draft genome encoded a distinct perchlorate reduction island (PRI) containing several transposases. The genome lacks the pcrC gene, which was previously thought to be essential for (per)chlorate reduction, and appears to use an unrelated Arcobacter c-type cytochrome to perform the same function.

Magnetospirillum bellicus sp. nov., a Novel Dissimilatory Perchlorate-Reducing Alphaproteobacterium Isolated from a Bioelectrical Reactor

Previously isolated dissimilatory perchlorate-reducing bacteria (DPRB) have been primarily affiliated with the Betaproteobacteria. Enrichments from the cathodic chamber of a bioelectrical reactor (BER) inoculated from creek water in Berkeley, CA, yielded a novel organism most closely related to a previously described strain, WD (99% 16S rRNA gene identity). Strain VDY(T) has 96% 16S rRNA gene identity to both Magnetospirillum gryphiswaldense and Magnetospirillum magnetotacticum, and along with strain WD, distinguishes a clade of perchlorate-reducing Magnetospirillum species in the Alphaproteobacteria. In spite of the phylogenetic location of VDY(T), attempted PCR for the key magnetosome formation genes mamI and mamL was negative. Strain VDY(T) was motile, non-spore forming, and, in addition to perchlorate, could use oxygen, chlorate, nitrate, nitrite, and nitrous oxide as alternative electron acceptors with acetate as the electron donor. Transient chlorate accumulation occurred during respiration of perchlorate. The organism made use of fermentation end products, such as acetate and ethanol, as carbon sources and electron donors for heterotrophic growth, and in addition, strain VDY(T) could grow chemolithotrophically with hydrogen serving as the electron donor. VDY(T) contains a copy of the RuBisCo cbbM gene, which was expressed under autotrophic but not heterotrophic conditions. DNA-DNA hybridization with strain WD confirmed VDY(T) as a separate species (46.2% identity), and the name Magnetospirillum bellicus sp. nov. (DSM 21662, ATCC BAA-1730) is proposed.

Description of the novel perchlorate-reducing bacteria Dechlorobacter hydrogenophilus gen. nov., sp. nov. and Propionivibrio militaris, sp. nov.

Novel dissimilatory perchlorate-reducing bacteria (DPRB) were isolated from enrichments conducted under conditions different from those of all previously described DPRB. Strain LT-1T was enriched using medium buffered at pH 6.6 with 2-(N-morpholino)ethanesulfonic acid (MES) and had only 95% 16S rRNA gene identity with its closest relative, Azonexus caeni. Strain MPT was enriched in the cathodic chamber of a perchlorate-reducing bioelectrical reactor (BER) and together with an additional strain, CR (99% 16S rRNA gene identity), had 97% 16S rRNA gene identity with Propionivibrio limicola. The use of perchlorate and other electron acceptors distinguished strains MPT and CR from P. limicola physiologically. Strain LT-1T had differences in electron donor utilization and optimum growth temperatures from A. caeni. Strains LT-1T and MPT are the first DPRB to be described in the Betaproteobacteria outside of the Dechloromonas and Azospira genera. On the basis of phylogenetic and physiological features, strain LT-1T represents a novel genus in the Rhodocyclaceae; strain MPT represents a novel species within the genus Propionivibrio. The names Dechlorobacter hydrogenophilus gen. nov., sp. nov and Propionivibrio militaris sp. nov. are proposed.

Behavioral response of dissimilatory perchlorate-reducing bacteria to different electron acceptors

The response behavior of three dissimilatory perchlorate-reducing bacteria to different electron acceptors (nitrate, chlorate, and perchlorate) was investigated with two different assays. The observed response was species-specific, dependent on the prior growth conditions, and was inhibited by oxygen. We observed attraction toward nitrate when Dechloromonas aromatica strain RCB and Azospira suillum strain PS were grown with nitrate. When D. aromatica and Dechloromonas agitata strain CKB were grown with perchlorate, both responded to nitrate, chlorate, and perchlorate. When A. suillum was grown with perchlorate, the organism responded to chlorate and perchlorate but not nitrate. A gene replacement mutant in the perchlorate reductase subunit (pcrA) of D. aromatica resulted in a loss of the attraction response toward perchlorate but had no impact on the nitrate response. Washed-cell suspension studies revealed that the perchlorate grown cells of D. aromatica reduced both perchlorate and nitrate, while A. suillum cells reduced perchlorate only. Based on these observations, energy taxis was proposed as the underlying mechanism for the responses to (per)chlorate by D. aromatica. To the best of our knowledge, this study represents the first investigation of the response behavior of perchlorate-reducing bacteria to environmental stimuli. It clearly demonstrates attraction toward chlorine oxyanions and the unique ability of these organisms to distinguish structurally analogous compounds, nitrate, chlorate, and perchlorate and respond accordingly.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-009-2051-3) contains supplementary material, which is available to authorized users.

Adapting a denitrifying biocathode for perchlorate reduction

Perchlorate is widely used as a propellant in the aerospace and defense industries, and is of environmental concern due to its high mobility and inhibiting effect on thyroid function. An ideal treatment approach is bioreduction to chloride via dissimilatory perchlorate-reducing bacteria (PCRB). PCRB are ubiquitous in the environment, and are mainly facultative anaerobes and denitrifiers. Previous research suggests that PCRB may grow using a cathode as an electron donor, although this research was performed in a half cell with exogenous electron shuttles. We investigated a functioning MFC with a denitrifying biocathode for perchlorate reduction, as a means to confirm the existence of biocathode-utilizing PCRB and the possibility of perchlorate remediation without added shuttles. The biocathode was initially run with 20 mgN/L nitrate. The perchlorate concentration was increased stepwise from 0.1 mg/L to 20 mg/L, while the nitrate concentration was decreased from 20 mgN/L to 5 mgN/L. The maximum perchlorate removal was 12 mg/L-d, contributing 64% to the 0.28 mA produced by the cell. Given the lack of soluble electron donor in the medium, the extent of perchlorate reduction, and the improvement of perchlorate reduction over time, these tests strongly suggest PCRB are utilizing the cathode as an electron donor without exogenous electron shuttles.

Electrochemical Stimulation of Microbial Perchlorate Reduction

As part of our studies into the diversity of dissimilatory perchlorate reducing bacteria (DPRB) we investigated the reduction of perchlorate in the cathodic chamber of a bioelectrical reactor (BER). Our results demonstrated that washed cells of Dechloromonas and Azospira species readily reduced 90 mg L(-1) perchlorate in the BER with 2,6-anthraquinone disulfonate (AQDS) as a mediator. No perchlorate was reduced in the absence of cells or AQDS, or in an open-circuit control. Similar results were observed when a natural microbial community was inoculated into a fed-batch BER. After 70 days of operation, a novel DPRB, strain VDY, was isolated which readily reduced perchlorate in a mediatorless BER. Continuous up-flow BERs (UFBERs) were seeded with active cultures of strain VDY, and perchlorate at a volumetric loading of 60 mg L(-1) day(-1) was successfully removed. Gas phase analysis indicated that low levels of H2 produced at the cathode surface through electrolysis may mediate this metabolism. The results of these studies demonstrate that biological perchlorate remediation can be facilitated through the use of a cathode as the primary electron donor, and that continuous treatment in such a system approaches current industry standards. This has important implications for the continuous treatment of this critical contaminant in industrial waste streams and drinking water.

Metabolic primers for detection of (Per)chlorate-reducing bacteria in the environment and phylogenetic analysis of cld gene sequences.

Natural attenuation of the environmental contaminant perchlorate is a cost-effective alternative to current removal methods. The success of natural perchlorate remediation is dependent on the presence and activity of dissimilatory (per)chlorate-reducing bacteria (DPRB) within a target site. To detect DPRB in the environment, two degenerate primer sets targeting the chlorite dismutase (cld) gene were developed and optimized. A nested PCR approach was used in conjunction with these primer sets to increase the sensitivity of the molecular detection method. Screening of environmental samples indicated that all products amplified by this method were cld gene sequences. These sequences were obtained from pristine sites as well as contaminated sites from which DPRB were isolated. More than one cld phylotype was also identified from some samples, indicating the presence of more than one DPRB strain at those sites. The use of these primer sets represents a direct and sensitive molecular method for the qualitative detection of (per)chlorate-reducing bacteria in the environment, thus offering another tool for monitoring natural attenuation. Sequences of cld genes isolated in the course of this project were also generated from various DPRB and provided the first opportunity for a phylogenetic treatment of this metabolic gene. Comparisons of the cld and 16S ribosomal DNA (rDNA) gene trees indicated that the cld gene does not track 16S rDNA phylogeny, further implicating the possible role of horizontal transfer in the evolution of (per)chlorate respiration.

Sequencing and transcriptional analysis of the chlorite dismutase gene of Dechloromonas agitata and its use as a metabolic probe.

The dismutation of chlorite into chloride and O(2) represents a central step in the reductive pathway of perchlorate that is common to all dissimilatory perchlorate-reducing bacteria and is mediated by a single enzyme, chlorite dismutase. The chlorite dismutase gene cld was isolated and sequenced from the perchlorate-reducing bacterium Dechloromonas agitata strain CKB. Sequence analysis identified an open reading frame of 834 bp that would encode a mature protein with an N-terminal sequence identical to that of the previously purified D. agitata chlorite dismutase enzyme. The predicted translation product of the D. agitata cld gene is a protein of 277 amino acids (aa), including a leader peptide of 26 aa. Primer extension analysis identified a single transcription start site directly downstream of an AT-rich region that could represent the -10 promoter region of the D. agitata cld gene. Northern blot analysis indicated that the cld gene was transcriptionally up-regulated when D. agitata cells were grown in perchlorate-reducing versus aerobic conditions. Slot blot hybridizations with a D. agitata cld probe demonstrated the conservation of the cld gene among perchlorate-reducing bacteria. This study represents the first description of a functional gene associated with microbial perchlorate reduction.

Anaerobic biooxidation of Fe(II) by Dechlorosoma suillum.

Anaerobic microbial oxidation of Fe(II) was only recently discovered and very little is known about this metabolism. We recently demonstrated that several dissimilatory perchlorate-reducing bacteria could utilize Fe(II) as an electron donor under anaerobic conditions. Here we report on a more in-depth analysis of Fe(II) oxidation by one of these organisms, Dechlorosoma suillum. Similarly to most known nitrate-dependent Fe(II) oxidizers, D. suillum did not grow heterotrophically or lithoautotrophically by anaerobic Fe(II) oxidation. In the absence of a suitable organic carbon source, cells rapidly lysed even though nitrate-dependent Fe(II) oxidation was still occurring. The coupling of Fe(II) oxidation to a particular electron acceptor was dependent on the growth conditions of cells of D. suillum. As such, anaerobically grown cultures of D. suillum did not mediate Fe(II) oxidation with oxygen as the electron acceptor, while conversely, aerobically grown cultures did not mediate Fe(II) oxidation with nitrate as the electron acceptor. Anaerobic washed cell suspensions of D. suillum rapidly produced an orange/brown precipitate which X-ray diffraction analysis identified as amorphous ferric oxyhydroxide or ferrihydrite. This is similar to all other identified nitrate-dependent Fe(II) oxidizers but is in contrast to what is observed for growth cultures of D. suillum, which produced a mixed-valence Fe(II)-Fe(III) precipitate known as green rust. D. suillum rapidly oxidized the Fe(II) content of natural sediments. Although the form of ferrous iron in these sediments is unknown, it is probably a component of an insoluble mineral, as previous studies indicated that soluble Fe(II) is a relatively minor form of the total Fe(II) content of anoxic environments. The results of this study further enhance our knowledge of a poorly understood form of microbial metabolism and indicate that anaerobic Fe(II) oxidation by D. suillum is significantly different from previously described forms of nitrate-dependent microbial Fe(II) oxidation.