Environmental Factors That Control Microbial Perchlorate Reduction

As part of a study to elucidate the environmental parameters that control microbial perchlorate respiration, we investigated the reduction of perchlorate by the dissimilatory perchlorate reducer Dechlorosoma suillum under a diverse set of environmental conditions. Our results demonstrated that perchlorate reduction by D. suillum only occurred under anaerobic conditions in the presence of perchlorate and was dependent on the presence of molybdenum. Perchlorate reduction was dependent on the presence of the enzyme chlorite dismutase, which was induced during metabolism of perchlorate. Anaerobic conditions alone were not enough to induce expression of this enzyme. Dissolved oxygen concentrations less than 2 mg liter⁻¹ were enough to inhibit perchlorate reduction by D. suillum. Similarly to oxygen, nitrate also regulated chlorite dismutase expression and repressed perchlorate reduction by D. suillum. Perchlorate-grown cultures of D. suillum preferentially reduced nitrate in media with equimolar amounts of perchlorate and nitrate. In contrast, an extended (40 h) lag phase was observed if a similar nitrate-perchlorate medium was inoculated with a nitrate-grown culture. Perchlorate reduction commenced only when nitrate was completely removed in either of these experiments. In contrast to D. suillum, nitrate had no inhibitory effects on perchlorate reduction by the perchlorate reducer Dechloromonas agitata strain CKB. Nitrate was reduced to nitrite concomitant with perchlorate reduction to chloride. These studies demonstrate that microbial respiration of perchlorate is significantly affected by environmental conditions and perchlorate reduction is directly dependent on bioavailable molybdenum and the presence or absence of competing electron acceptors. A microbial treatment strategy can achieve and maintain perchlorate concentrations below the recommended regulatory level, but only in environments in which the variables described above can be controlled.     

Peptide biomarkers as evidence of perchlorate biodegradation

Perchlorate is a known health hazard for humans, fish, and other species. Therefore, it is important to assess the response of an ecosystem exposed to perchlorate contamination. The data reported here show that a liquid chromatography-mass spectrometry-based proteomics approach for the detection of perchlorate-reducing enzymes can be used to measure the ability of microorganisms to degrade perchlorate, including determining the current perchlorate degradation status. Signature peptides derived from chlorite dismutase (CD) and perchlorate reductase can be used as biomarkers of perchlorate presence and biodegradation. Four peptides each derived from CD and perchlorate reductase subunit A (PcrA) and seven peptides derived from perchlorate reductase subunit B (PcrB) were identified as signature biomarkers for perchlorate degradation, as these sequences are conserved in the majority of the pure and mixed perchlorate-degrading microbial cultures examined. However, chlorite dismutase signature biomarker peptides from Dechloromonas agitata CKB were found to be different from those in other cultures used and should also be included with selected CD biomarkers. The combination of these peptides derived from the two enzymes represents a promising perchlorate presence/biodegradation biomarker system. The biomarker peptides were detected at perchlorate concentrations as low as 0.1 mM and at different time points both in pure cultures and within perchlorate-reducing environmental enrichment consortia. The peptide biomarkers were also detected in the simultaneous presence of perchlorate and an alternate electron acceptor, nitrate. We believe that this technique can be useful for monitoring bioremediation processes for other anthropogenic environmental contaminants with known metabolic pathways.     

Inhibition of aerobic respiration and dissimilatory perchlorate reduction using cyanide

The effect of low concentrations of cyanide on dissimilatory perchlorate and chlorate reduction and aerobic respiration was examined using pure cultures of Azospira sp. KJ. Cyanide at a concentration of 38 microM inhibited cell growth on perchlorate, chlorate and molecular oxygen, but it did not inhibit the activity of chlorite dismutase. When oxygen accumulation was prevented by adding an oxygen scavenger (Oxyrase or L-cysteine), however, cells completely reduced perchlorate in the presence of cyanide. It was concluded that the inhibition of dissimilative perchlorate reduction by cyanide at this concentration was a consequence of oxygen accumulation, not inhibition of the enzymes used for perchlorate reduction. This finding on the effect of cyanide on respiratory enzymes provides a new method to control and study respiratory enzymes used for 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.     

Reduction of Perchlorate and Nitrate by Microbial Communities in Vadose Soil

Perchlorate contamination is a concern because of the increasing frequency of its detection in soils and groundwater and its presumed inhibitory effect on human thyroid hormone production. Although significant perchlorate contamination occurs in the vadose (unsaturated) zone, little is known about perchlorate biodegradation potential by indigenous microorganisms in these soils. We measured the effects of electron donor (acetate and hydrogen) and nitrate addition on perchlorate reduction rates and microbial community composition in microcosm incubations of vadose soil. Acetate and hydrogen addition enhanced perchlorate reduction, and a longer lag period was observed for hydrogen (41 days) than for acetate (14 days). Initially, nitrate suppressed perchlorate reduction, but once perchlorate started to be degraded, the process was stimulated by nitrate. Changes in the bacterial community composition were observed in microcosms enriched with perchlorate and either acetate or hydrogen. Denaturing gradient gel electrophoresis analysis and partial sequencing of 16S rRNA genes recovered from these microcosms indicated that formerly reported perchlorate-reducing bacteria were present in the soil and that microbial community compositions were different between acetate- and hydrogen-amended microcosms. These results indicate that there is potential for perchlorate bioremediation by native microbial communities in vadose soil.     

Reduction of perchlorate and nitrate by microbial communities in vadose soil

Perchlorate contamination is a concern because of the increasing frequency of its detection in soils and groundwater and its presumed inhibitory effect on human thyroid hormone production. Although significant perchlorate contamination occurs in the vadose (unsaturated) zone, little is known about perchlorate biodegradation potential by indigenous microorganisms in these soils. We measured the effects of electron donor (acetate and hydrogen) and nitrate addition on perchlorate reduction rates and microbial community composition in microcosm incubations of vadose soil. Acetate and hydrogen addition enhanced perchlorate reduction, and a longer lag period was observed for hydrogen (41 days) than for acetate (14 days). Initially, nitrate suppressed perchlorate reduction, but once perchlorate started to be degraded, the process was stimulated by nitrate. Changes in the bacterial community composition were observed in microcosms enriched with perchlorate and either acetate or hydrogen. Denaturing gradient gel electrophoresis analysis and partial sequencing of 16S rRNA genes recovered from these microcosms indicated that formerly reported perchlorate-reducing bacteria were present in the soil and that microbial community compositions were different between acetate- and hydrogen-amended microcosms. These results indicate that there is potential for perchlorate bioremediation by native microbial communities in vadose soil.     

Reduction of perchlorate by an anaerobic enrichment culture

Summary A mixed bacterial culture capable of reducing perchlorate stoichiometrically to chloride under naerobic conditions was enriched from municipal digester sludge. The reduction of 10 mM perchlorate resulted in oxidation of the medium and cessation of perchlorate reduction. The activity was recovered on addition of a reducing agent. Addition of air to the culture during perchlorate reduction immediately terminated the process and aeration for 12 h permanently destroyed the ability of the culture to reduce perchlorate. The culture also reduced nitrite, nitrate, chlorite, chlorate and sulfate. The presence of 10 mM nitrite or chlorite completely inhibited perchlorate reduction, whereas the same concentration of chlorate decreased the reduction rate. Nitrate or sulfate did not affect perchlorate reduction. Chlorate and chlorite, suspected intermediates in the reduction of perchlorate to chloride, were not detected in any cultures during reduction of perchlorate.     

Purification and Characterization of (Per)Chlorate Reductase from the Chlorate-Respiring Strain GR-1

Strain GR-1 is one of several recently isolated bacterial species that are able to respire by using chlorate or perchlorate as the terminal electron acceptor. The organism performs a complete reduction of chlorate or perchlorate to chloride and oxygen, with the intermediate formation of chlorite. This study describes the purification and characterization of the key enzyme of the reductive pathway, the chlorate and perchlorate reductase. A single enzyme was found to catalyze both the chlorate- and perchlorate-reducing activity. The oxygen-sensitive enzyme was located in the periplasm and had an apparent molecular mass of 420 kDa, with subunits of 95 and 40 kDa in an alpha(3)beta(3) composition. Metal analysis showed the presence of 11 mol of iron, 1 mol of molybdenum, and 1 mol of selenium per mol of heterodimer. In accordance, quantitative electron paramagnetic resonance spectroscopy showed the presence of one [3Fe-4S] cluster and two [4Fe-4S] clusters. Furthermore, two different signals were ascribed to Mo(V). The K(m) values for perchlorate and chlorate were 27 and <5 microM, respectively. Besides perchlorate and chlorate, nitrate, iodate, and bromate were also reduced at considerable rates. The resemblance of the enzyme to nitrate reductases, formate dehydrogenases, and selenate reductase is discussed.     

Microbial isotopic fractionation of perchlorate chlorine

Perchlorate contamination can be microbially respired to innocuous chloride and thus can be treated effectively. However, monitoring a bioremediative strategy is often difficult due to the complexities of environmental samples. Here we demonstrate that microbial respiration of perchlorate results in a significant fractionation ( approximately -15 per thousand ) of the chlorine stable isotope composition of perchlorate. This can be used to quantify the extent of biotic degradation and to separate biotic from abiotic attenuation of this contaminant.     

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.     

Microbial Isotopic Fractionation of Perchlorate Chlorine

Perchlorate contamination can be microbially respired to innocuous chloride and thus can be treated effectively. However, monitoring a bioremediative strategy is often difficult due to the complexities of environmental samples. Here we demonstrate that microbial respiration of perchlorate results in a significant fractionation (~−15‰) of the chlorine stable isotope composition of perchlorate. This can be used to quantify the extent of biotic degradation and to separate biotic from abiotic attenuation of this contaminant.     

Chemolithotrophic perchlorate reduction linked to the oxidation of elemental sulfur

Perchlorate (ClO(4)(-)) contamination of ground and surface water has been recently recognized as a widespread environmental problem. Biological methods offer promising perspectives of perchlorate remediation. Facultative anaerobic bacteria couple the oxidation of organic and inorganic electron-donating substrates to the reduction of perchlorate as a terminal electron acceptor, converting it completely to the benign end-product, chloride. Insoluble inorganic substrates are of interest for low maintenance bioreactor or permeable reactive barrier systems because they can provide a long-term supply of electron donor without generating organic residuals. The main objective of this research was to investigate the feasibility of utilizing elemental sulfur (S(0)) as an insoluble electron donor for the biological reduction of perchlorate. A chemolithotrophic enrichment culture derived from aerobic activated sludge was obtained which effectively coupled the oxidation of elemental sulfur to sulfate with the reduction of perchlorate to chloride and gained energy from the process for cell growth. The enrichment culture grew at a rate of 0.41 or 0.81 1/d in the absence and presence of added organic carbon for cell growth, respectively. The enrichment culture was also shown to carry out sulfur disproportionation to a limited extent as evidenced by the formation of sulfide and sulfate in the absence of added electron acceptor. When nitrate and perchlorate were added together, the two electron acceptors were removed simultaneously after an initial partial decrease in the nitrate concentration.     

Nitrate and (per)chlorate reduction pathways in (per)chlorate-reducing bacteria

The reduction of (per)chlorate and nitrate in (per)chlorate-reducing bacteria shows similarities and differences. (Per)chlorate reductase and nitrate reductase both belong to the type?II DMSO family of enzymes and have a common bis(molybdopterin guanine dinucleotide)molybdenum cofactor. There are two types of dissimilatory nitrate reductases. With respect to their localization, (per)chlorate reductase is more similar to the dissimilatory periplasmic nitrate reductase. However, the periplasmic, unlike the membrane-bound, respiratory nitrate reductase, is not able to use chlorate. Structurally, (per)chlorate reductase is more similar to respiratory nitrate reductase, since these reductases have analogous subunits encoded by analogous genes. Both periplasmic (per)chlorate reductase and membrane-bound nitrate reductase activities are induced under anoxic conditions in the presence of (per)chlorate and nitrate respectively. During microbial (per)chlorate reduction, molecular oxygen is generated. This is not the case for nitrate reduction, although an atypical reaction in nitrite reduction linked to oxygen formation has been described recently. Microbial oxygen production during reduction of oxyanions may enhance biodegradation of pollutants under anoxic conditions.     

Isolation and characterization of Dechlorospirillum anomalous strain JB116 from a sewage treatment plant

The dissimilatory perchlorate reducers mainly belong to two monophyletic groups, viz. Dechloromonas and Azospira in the beta subclass of Proteobacteria. The present study describes isolation and genetic characterization of Dechlorospirillum anomalous strain JB116 that belongs to alpha subclass of Proteobacteria. The strain JB116 was isolated under facultative anaerobic conditions on a growth medium containing sodium perchlorate and sodium acetate as electron (e(-)) acceptor and e(-) donor, respectively. The strain is a spirillum shaped, dissimilatory perchlorate and nitrate reducer that prefers nitrate to perchlorate. It grows heterotrophically with acetate at temperatures between 25-35 degrees C, NaCl concentrations between 0-0.5% and pH of 7-7.8. The strain JB116 is the second only representative strain within D. anomalous that shares 99% 16S rDNA sequence similarity with the type strain D. anomalous strain WD.     

Complete genome sequence of the anaerobic perchlorate-reducing bacterium Azospira suillum strain PS

Azospira suillum strain PS (formally Dechlorosoma suillum strain PS) is a metabolically versatile betaproteobacterium first identified for its ability to grow by dissimilatory reduction of perchlorate and chlorate [denoted (per)chlorate]. Together with Dechloromonas species, these two genera represent the dominant (per)chlorate-reducing bacteria in mesophilic freshwater environments. In addition to (per)chlorate reduction, A. suillum is capable of the anaerobic oxidation of humic substances and is the first anaerobic nitrate-dependent Fe(II) oxidizer outside the Diaphorobacter and Acidovorax genera for which there is a completed genome sequence.     

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.     

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.