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.     

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.     

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.     

Effect of nitrate, acetate, and hydrogen on native perchlorate-reducing microbial communities and their activity in vadose soil

The effect of nitrate, acetate, and hydrogen on native perchlorate-reducing bacteria (PRB) was examined by conducting microcosm tests using vadose soil collected from a perchlorate-contaminated site. The rate of perchlorate reduction was enhanced by hydrogen amendment and inhibited by acetate amendment, compared with unamendment. Nitrate was reduced before perchlorate in all amendments. In hydrogen-amended and unamended soils, nitrate delayed perchlorate reduction, suggesting that the PRB preferentially use nitrate as an electron acceptor. In contrast, nitrate eliminated the inhibitory effect of acetate amendment on perchlorate reduction and increased the rate and the extent, possibly because the preceding nitrate reduction/denitrification decreased the acetate concentration that was inhibitory to the native PRB. In hydrogen-amended and unamended soils, perchlorate reductase gene (pcrA) copies, representing PRB densities, increased with either perchlorate or nitrate reduction, suggesting that either perchlorate or nitrate stimulates the growth of the PRB. In contrast, in acetate-amended soil pcrA increased only when perchlorate was depleted: a large portion of the PRB may have not utilized nitrate in this amendment. Nitrate addition did not alter the distribution of the dominant pcrA clones in hydrogen-amended soil, likely because of the functional redundancy of PRB as nitrate-reducers/denitrifiers, whereas acetate selected different pcrA clones from those with hydrogen amendment.     

Quantitative Detection of Perchlorate-Reducing Bacteria by Real-Time PCR Targeting the Perchlorate Reductase Gene

A quantitative real-time PCR assay targeting the pcrA gene, encoding the catalytic subunit of perchlorate reductase, detected pcrA genes from perchlorate-reducing bacteria in three different genera and from soil microbial communities. Partial pcrA sequences indicated differences in the composition of perchlorate-reducing bacterial communities following exposure to different electron donors.     

Changes in the structure and function of microbial communities in drinking water treatment bioreactors upon addition of phosphorus

Phosphorus was added as a nutrient to bench-scale and pilot-scale biologically active carbon (BAC) reactors operated for perchlorate and nitrate removal from contaminated groundwater. The two bioreactors responded similarly to phosphorus addition in terms of microbial community function (i.e., reactor performance), while drastically different responses in microbial community structure were detected. Improvement in reactor performance with respect to perchlorate and nitrate removal started within a few days after phosphorus addition for both reactors. Microbial community structures were evaluated using molecular techniques targeting 16S rRNA genes. Clone library results showed that the relative abundance of perchlorate-reducing bacteria (PRB) Dechloromonas and Azospira in the bench-scale reactor increased from 15.2% and 0.6% to 54.2% and 11.7% after phosphorus addition, respectively. Real-time quantitative PCR (qPCR) experiments revealed that these increases started within a few days after phosphorus addition. In contrast, after phosphorus addition, the relative abundance of Dechloromonas in the pilot-scale reactor decreased from 7.1 to 0.6%, while Zoogloea increased from 17.9 to 52.0%. The results of this study demonstrated that similar operating conditions for bench-scale and pilot-scale reactors resulted in similar contaminant removal performances, despite dramatically different responses from microbial communities. These findings suggest that it is important to evaluate the microbial community compositions inside bioreactors used for drinking water treatment, as they determine the microbial composition in the effluent and impact downstream treatment requirements for drinking water production. This information could be particularly relevant to drinking water safety, if pathogens or disinfectant-resistant bacteria are detected in the bioreactors.     

Community structure and function in a H2-based membrane biofilm reactor capable of bioreduction of selenate and chromate

Two different H₂-based, denitrifying membrane-biofilm reactors (MBfRs) initially reduced Se(VI) or Cr(VI) stably to Se⁰ or Cr(III). When the oxidized contaminants in the influent were switched, each new oxidized contaminant was reduced immediately, and its reduction soon was approximately the same or greater than it had been in its original MBfR. The precipitation of reduced selenium and chromium in the biofilm was verified by scanning electron microscopy and energy dispersive X-ray analysis. These results on selenate and chromate reduction are consistent with the interpretation that the H₂-based biofilm community had a high level of functional diversity. The communities’ structures were assessed by cloning analysis. Dechloromonas spp., a known perchlorate-reducing bacteria, dominated the clones from both reactors during selenate and chromate reductions, which suggests that it may have functional diversity capable of reducing selenate and chromate as secondary and dissimilatory acceptors.     

Investigation of Factors Affecting the Bioregeneration Process for Perchlorate-Laden Gel-Type Anion-Exchange Resin

Ion exchange is the most common process for perchlorate removal from waters. Selective ion-exchange resins are widely used for perchlorate removal from waters, but are incinerated after one-time use, making the ion-exchange process incomplete for perchlorate removal. As perchlorate ions are readily biodegradable, direct contact of spent ion-exchange resins with perchlorate-reducing bacteria for its regeneration has been studied recently. In this research, some factors affecting the bioregeneration of perchlorate-laden gel-type anion-exchange resin were investigated. Bioregeneration is a sustainable process when compared to one-time use of resin and disposal by incineration. Batch bioregeneration experiments were performed to determine (a) the effect of initial perchlorate load in the resin, (b) the effect of microbial concentration, and (c) the effect of nitrate load on the degradation of perchlorate in the resin bead. The results of the bioregeneration tests suggested that the bioregeneration process may be controlled by both kinetics and diffusion. Higher perchlorate load in the resin had a positive effect on perchlorate degradation rates, whereas varying microbial concentration did not have a significant effect on perchlorate degradation in gel-type resin. The presence of nitrate suppressed perchlorate degradation initially, but once all nitrate was utilized, perchlorate degradation took place.     

Direct enrichment of perchlorate-reducing microbial community for efficient electroactive perchlorate reduction in biocathodes

Biological reduction of perchlorate (ClO₄ ^?) has emerged as a promising solution for the removal of perchlorate in contaminated water and soils. In this work, we demonstrate a simple process to enrich perchlorate-reducing microbial communities separately using acetate as electron donor and the municipal aerobic membrane bioreactor sludge as inoculum. Inoculation of cathodes in microbial fuel cells (MFCs) with these enrichments, and further electrochemical enrichment at constant resistance operation of the MFCs, led to perchlorate-reducing biocathodes with peak reduction rates of 0.095 mM/day (2 mg/m²/day). Analysis of the microbial diversity of perchlorate-reducing biocathodes using PCR-DGGE revealed unique community profiles when compared to the denitrifying biocathode communities. More importantly, the total time taken for enrichment of the electroactive communities was reduced from several months reported previously in literature to less than a month in this work.     

Modeling the Biodegradation Kinetics of Perchlorate in the Presence of Oxygen and Nitrate as Competing Electron Acceptors

A mathematical model was developed to describe the biodegradation kinetics of perchlorate in the presence of nitrate and oxygen as competing electron acceptors. The rate of perchlorate degradation is described as a function of the electron donor (acetate) degradation rate, the concentration of the alternate electron acceptors, and rates of biomass growth and decay. The kinetics of biomass growth are described using a modified Monod model, and inhibition factors are incorporated to describe the influence of oxygen and nitrate on perchlorate degradation. In order to develop input parameters for the model, a series of batch biodegradation studies were performed using Azospira suillum JPLRND, a perchlorate-degrading strain isolated from groundwater. This strain is capable of utilizing oxygen, nitrate, or perchlorate as terminal electron acceptors. The maximum specific growth rate (μ_max) and half-saturation constant (K _S ^don) for the bacterium when utilizing either perchlorate or nitrate were similar; 0.16 per h and 158 mg acetate/L, respectively. However, these parameters were different when the strain was growing on oxygen. In this case, μ_max and K _S ^don were 0.22 per h and 119 mg acetate/L, respectively. The batch experiments also revealed that nitrate inhibits perchlorate biodegradation by this strain. This finding was incorporated into the model by applying an inhibition coefficient (K _i ^nit) value of 25 mg nitrate/L. Combined with appropriate groundwater transport models, this model can be used to predict perchlorate biodegradation during in situ remediation efforts.     

Limitations to Natural Bioremediation of Perchlorate in a Contaminated Site

Perchlorate (ClO₄ ^?) has been detected in many drinking water supplies in the United States, including the Las Vegas Wash and Lake Mead, Nevada. These locations are highly contaminated and contribute perchlorate to Lake Mead and the Colorado River system. Essential elements for perchlorate bioremediation at these locations were examined, including the presence of perchlorate-reducing bacteria (PRB), sufficient electron donors, occurrence of competing electron acceptors, and ability of PRB to utilize a variety of electron donors. Enumeration of PRB was performed anoxically using most probable number (MPN). Values ranged from ≤20 to 230 PRB/100 ml or ≤20 to ≥ 1.6× 10⁵ PRB/g for Lake Mead water samples and Las Vegas Wash sediments, respectively. 16S rRNA sequences revealed that isolates were γ -proteobacteria, Aeromonas, Dechlorosoma, Rahnella and Shewanella. A screening of potential electron donors using BIOLOG^TM demonstrated that all isolates were capable of metabolic versatility. Measurements of total organic carbon (TOC), nitrate and dissolved oxygen (DO) indicated limited presence of electron donor at all sites, whereas the electron acceptors varied throughout the Wash and Lake Mead. The persistence of perchlorate in the sites is attributed to lack of available electron donor and/or the presence of competing electron acceptors. A location has been identified where perchlorate biodegradation could be implemented thereby halting the transport of perchlorate to Lake Mead and the Colorado River.     

Perchlorate reduction by hydrogen autotrophic bacteria and microbial community analysis using high-throughput sequencing

Hydrogen autotrophic reduction of perchlorate have advantages of high removal efficiency and harmless to drinking water. But so far the reported information about the microbial community structure was comparatively limited, changes in the biodiversity and the dominant bacteria during acclimation process required detailed study. In this study, perchlorate-reducing hydrogen autotrophic bacteria were acclimated by hydrogen aeration from activated sludge. For the first time, high-throughput sequencing was applied to analyze changes in biodiversity and the dominant bacteria during acclimation process. The Michaelis–Menten model described the perchlorate reduction kinetics well. Model parameters q _max and K _s were 2.521–3.245 (mg ClO₄ ^?/gVSS h) and 5.44–8.23 (mg/l), respectively. Microbial perchlorate reduction occurred across at pH range 5.0–11.0; removal was highest at pH 9.0. The enriched mixed bacteria could use perchlorate, nitrate and sulfate as electron accepter, and the sequence of preference was: NO₃ ^? > ClO₄ ^? > SO₄ ^2?. Compared to the feed culture, biodiversity decreased greatly during acclimation process, the microbial community structure gradually stabilized after 9 acclimation cycles. The Thauera genus related to Rhodocyclales was the dominated perchlorate reducing bacteria (PRB) in the mixed culture.     

Perchlorate reduction by a novel chemolithoautotrophic,hydrogen-oxidizing bacterium

Water treatment technologies are needed that can remove perchlorate from drinking water without introducing organic chemicals that stimulate bacterial growth in water distribution systems. Hydrogen is an ideal energy source for bacterial degradation of perchlorate as it leaves no organic residue and is sparingly soluble. We describe here the isolation of a perchlorate-respiring, hydrogen-oxidizing bacterium (Dechloromonas sp. strain HZ) that grows with carbon dioxide as sole carbon source. Strain HZ is a Gram-negative, rod-shaped facultative anaerobe that was isolated from a gas-phase anaerobic packed-bed biofilm reactor treating perchlorate-contaminated groundwater. The ability of strain HZ to grow autotrophically with carbon dioxide as the sole carbon source was confirmed by demonstrating that biomass carbon (100.9%) was derived from CO2. Chemolithotrophic growth with hydrogen was coupled with complete reduction of perchlorate (10 mM) to chloride with a maximum doubling time of 8.9 h. Strain HZ also grew using acetate as the electron donor and chlorate, nitrate, or oxygen (but not sulphate) as an electron acceptor. Phylogenetic analysis of the 16S rRNA sequence placed strain HZ in the genus Dechloromonas within the beta subgroup of the Proteobacteria. The study of this and other novel perchlorate-reducing bacteria may lead to new, safe technologies for removing perchlorate and other chemical pollutants from drinking water.     

Simultaneous Bio-reduction of Nitrate,Perchlorate, Selenate,Chromate, Arsenate,and Dibromochloropropane Using a Hydrogen-based Membrane Biofilm Reactor

We tested the hypothesis that the H₂-based membrane biofilm reactor (MBfR) is capable of reducing multiple oxidized contaminants, a common situation for groundwater contamination. We conducted bench-scale experiments with three groundwater samples collected from California’s San Joaquin Valley and on two synthetic groundwaters containing selenate and chromate. The actual groundwater sources had nitrate levels exceeding 10 mg-N l⁻¹ and different combinations of anthropogenic perchlorate + chlorate, arsenate, and dibromochloropropane (DBCP). For all actual groundwaters, the MBfR reduced nitrate to less than 0.01 mg-N l⁻¹. Present in two groundwaters, perchlorate + chlorate was reduced to below the California Notification Level, 6 μg-ClO₄ l⁻¹. As(V) was substantially reduced to As(III) for two groundwaters samples, which had influent As(V) concentrations from 3 to 8.8 μg-As l⁻¹. DBCP, present in one groundwater at 1.4 μg l⁻¹, was reduced to below its detection limit of 0.01 μg l⁻¹, which is well below California’s 0.2 μg l⁻¹ MCL for DBCP. For the synthetic groundwaters, two MBfRs initially reduced Se(VI) or Cr(VI) stably to Se° or Cr(III). When we switched the influent oxidized contaminants, the new oxidized contaminant was reduced immediately, and its reduction soon was approximately the same or greater than it had been reduced in its original MBfR. These results support that the H₂-based MBfR can reduce multiple oxidized contaminants simultaneously.     

Effective biofilm control in a membrane biofilm reactor using a quenching bacterium (Rhodococcus sp. BH4)

The biofilm thickness in membrane biofilm reactors (MBfRs) is an important factor affecting system performance because excessive biofilm formation on the membrane surface inhibits gas diffusion to the interior of the biofilm, resulting in a significant reduction in the performance of contaminant removal. This study provides innovative insights into the control of biofilm thickness in O₂-based MBfRs by using the quorum quenching (QQ) method. The study was carried out in MBfRs operated at different gas pressures and hydraulic retention times (HRTs) using QQ beads containing Rhodococcus sp. BH4 at different amounts. The highest performance was observed in reactors operated with 0.21 ml QQ bead/cm² membrane surface area, 12 HRTs and 1.40 atm. Over this period, the performance increase in chemical oxygen demand (COD) removal was 25%, while the biofilm thickness on the membrane surface was determined to be 250 μm. Moreover, acetate and equivalent oxygen flux results reached 6080 and 10 640 mg·m⁻²·d⁻¹ maximum values, respectively. The extracellular polymeric substances of the biofilm decreased significantly with the increase of gas pressure and QQ beads amount. Polymerase chain reaction denaturing gradient gel electrophoresis results showed that the microbial community in the MBfR system changed depending on operating conditions and bead amount. The results showed that the QQ method was an effective method to control the biofilm thickness in MBfR and provide insights for future research.     

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-1^T 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 MP^T 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 MP^T and CR from P. limicola physiologically. Strain LT-1^T had differences in electron donor utilization and optimum growth temperatures from A. caeni. Strains LT-1^T and MP^T 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-1^T represents a novel genus in the Rhodocyclaceae; strain MP^T represents a novel species within the genus Propionivibrio. The names Dechlorobacter hydrogenophilus gen. nov., sp. nov and Propionivibrio militaris sp. nov. are proposed.     

Persistence of Perchlorate and the Relative Numbers of Perchlorate- and Chlorate-Respiring Microorganisms in Natural Waters, Soils, and Wastewater

Cell numbers of perchlorate (PRM)- and chlorate (CRM)-reducing microorganisms and the persistence of perchlorate were determined in samples of soils, natural waters, and wastewater incubated under laboratory conditions. Complete perchlorate reduction in raw wastewater and creek water was achieved in 4 to 7 days and 8 to 29 days, respectively, depending on the individual growth substrate (acetate, lactate, citric acid, or molasses) employed. Perchlorate persisted in most mixed cultures developed with 2 g of “pristine” soil, but declined in mixed cultures developed with 100 g of soil. Less than seven days were required to completely reduce perchlorate in cultures started with 10 g of a perchlorate-contaminated soil obtained from a site in Texas. The concentration of PRM was estimated using a 5-tube most probable number (MPN) procedure. To account for discrepancies due to differences in the total number of bacteria (per mass of sample) in the samples, difficulty in removing bacteria from soil samples, and the lack of an unequivocal method to measure total viable cells in these different systems, we normalized our MPN results on the basis of 10⁶ or 10⁹ total bacteria counted using acridine orange direct counts (AODC). There were more PRM in wastewater samples on a per-cell basis (15 to 350 PRM/10⁶-AODC) than in water samples (0.02 to 0.4 PRM/10⁶-AODC). There were also more PRM in soils from sites exhibiting direct evidence of perchlorate contamination (100 to 200 PRM/10⁹-AODC) than from other sites (nondetectable to 0.77 PRM/10⁹-AODC). These results demonstrate that perchlorate-reducing bacteria are present at perchlorate-contaminated sites, and that perchlorate can be degraded by these microorganisms through the addition of different electron donors, such as acetate and lactate.