Chlorate-reducing Bacteria Research Articles

Dechloromonas hortensis sp. nov. and strain ASK-1, two novel (per)chlorate-reducing bacteria, and taxonomic description of strain GR-1

Recent studies on the occurrence of (per)chlorate-reducing bacteria have resulted in the characterization of strains capable of dissimilatory (per)chlorate reduction. Phylogenetic analysis has shown that these bacteria are members of the Proteobacteria. Strains have been isolated from polluted and pristine sites, but only strains from polluted sites have been characterized in detail and deposited in culture collections. Herein we describe the isolation and characterization of perchlorate-reducing bacterium strain MA-1(T) and chlorate-reducing bacterium strain ASK-1, respectively isolated from a pristine and a chlorate-polluted site. Both isolates are members of the Proteobacteria. The 16S rRNA gene sequence similarity of MA-1(T) to Dechloromonas agitata DSM 13637(T) is 97.6%, but the relatedness in DNA-DNA reassociation is only 37%. Therefore, we propose to classify strain MA-1(T) (=DSM 15637(T)=ATCC BAA-776(T)) as the type strain of a novel species, Dechloromonas hortensis sp. nov. Strain ASK-1 and a previously described strain GR-1 show 100 and 99% 16S rRNA gene sequence similarity to Pseudomonas chloritidismutans DSM 13592(T) and Dechlorosoma suillum DSM 13638(T), respectively. DNA-DNA hybridization studies indicated that strains ASK-1 and GR-1 are related at the species level to P. chloritidismutans DSM 13592(T) (79%) and Dechlorosoma suillum DSM 13638(T) (85%), respectively. As suggested previously, Dechlorosoma suillum appears to be a later heterotypic synonym of Azospira oryzae. Although strain ASK-1 is identified as P. chloritidismutans, its morphology and growth requirements are different from those of the type strain.

Molecular analysis of microbial community in a groundwater sample polluted by landfill leachate and seawater

Seashore landfill aquifers are environments of special physicochemical conditions (high organic load and high salinity), and microbes in leachate-polluted aquifers play a significant role for intrinsic bioremediation. In order to characterize microbial diversity and look for clues on the relationship between microbial community structure and hydrochemistry, a culture-independent examination of a typical groundwater sample obtained from a seashore landfill was conducted by sequence analysis of 16S rDNA clone library. Two sets of universal 16S rDNA primers were used to amplify DNA extracted from the groundwater so that problems arising from primer efficiency and specificity could be reduced. Of 74 clones randomly selected from the libraries, 30 contained unique sequences whose analysis showed that the majority of them belonged to bacteria (95.9%), with Proteobacteria (63.5%) being the dominant division. One archaeal sequence and one eukaryotic sequence were found as well. Bacterial sequences belonging to the following phylogenic groups were identified: Bacteroidetes (20.3%), beta, gamma, delta and epsilon-subdivisions of Proteobacteria (47.3%, 9.5%, 5.4% and 1.3%, respectively), Firmicutes (1.4%), Actinobacteria (2.7%), Cyanobacteria (2.7%). The percentages of Proteobacteria and Bacteroides in seawater were greater than those in the groundwater from a non-seashore landfill, indicating a possible influence of seawater. Quite a few sequences had close relatives in marine or hypersaline environments. Many sequences showed affiliations with microbes involved in anaerobic fermentation. The remarkable abundance of sequences related to (per)chlorate-reducing bacteria (ClRB) in the groundwater was significant and worthy of further study.

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.

Universal immunoprobe for (per)chlorate-reducing bacteria.

Recent studies in our lab have demonstrated the ubiquity and diversity of microorganisms which couple growth to the reduction of chlorate or perchlorate [(per)chlorate] under anaerobic conditions. We identified two taxonomic groups, the Dechloromonas and the Dechlorosoma groups, which represent the dominant (per)chlorate-reducing bacteria (ClRB) in the environment. As part of these studies we demonstrated that chlorite dismutation is a central step in the reductive pathway of (per)chlorate that is common to all ClRB and which is mediated by the enzyme chlorite dismutase (CD). Initial studies on CD suggested that this enzyme is highly conserved among the ClRB, regardless of their phylogenetic affiliation. As such, this enzyme makes an ideal target for a probe specific for these organisms. Polyclonal antibodies were commercially raised against the purified CD from the ClRB Dechloromonas agitata strain CKB. The obtained antiserum was deproteinated by ammonium sulfate precipitation, and the antigen binding activity was assessed using dot blot analysis of a serial dilution of the antiserum. The titers obtained with purified CD indicated that the antiserum had a high affinity for the CD enzyme, and activity was observed in dilutions as low as 10(-6) of the original antiserum. The antiserum was active against both cell lysates and whole cells of D. agitata, but only if the cells were grown anaerobically with (per)chlorate. No response was obtained with aerobically grown cultures. In addition to D. agitata, dot blot analysis employed with both whole-cell suspensions and cell lysates of several diverse ClRB representing the alpha, beta, and gamma subclasses of Proteobacteria tested positive regardless of phylogenetic affiliation. Interestingly, the dot blot response obtained for each of the ClRB cell lysates was different, suggesting that there may be some differences in the antigenic sites of the CD protein produced in these organisms. In general, no reactions were observed with cells or cell lysates of the organisms closely related to the ClRB which could not grow by (per)chlorate reduction. These studies have resulted in the development of a highly specific and sensitive immunoprobe based on the commonality of the CD enzyme in ClRB which can be used to assess dissimilatory (per)chlorate-reducing populations in environmental samples regardless of their phylogenetic affiliations.

Dechloromonas agitata gen. nov., sp. nov. and Dechlorosoma suillum gen. nov., sp. nov., two novel environmentally dominant (per)chlorate-reducing bacteria and their phylogenetic position.

Previous studies on the ubiquity and diversity of microbial (per)chlorate reduction resulted in the isolation of 20 new strains of dissimilatory (per)chlorate-reducing bacteria. Phylogenetic analysis revealed that all of the isolates were members of the Proteobacteria with representatives in the alpha-, beta- and gamma-subclasses. The majority of the new isolates were located in the beta-subclass and were closely related to each other and to the phototrophic Rhodocyclus species. Here an in-depth analysis of these organisms which form two distinct monophyletic groups within the Rhodocyclus assemblage is presented. Two new genera, Dechloromonas and Dechlorosoma, are proposed for these beta-subclass lineages which represent the predominant (per)chlorate-reducing bacteria in the environment. The type species and strains for these new genera are Dechloromonas agitata strain CKBT and Dechlorosoma suillum strain PST, respectively.

Ubiquity and diversity of dissimilatory (per)chlorate-reducing bacteria.

Environmental contamination with compounds containing oxyanions of chlorine, such as perchlorate or chlorate [(per)chlorate] or chlorine dioxide, has been a constantly growing problem over the last 100 years. Although the fact that microbes reduce these compounds has been recognized for more than 50 years, only six organisms which can obtain energy for growth by this metabolic process have been described. As part of a study to investigate the diversity and ubiquity of microorganisms involved in the microbial reduction of (per)chlorate, we enumerated the (per)chlorate-reducing bacteria (ClRB) in very diverse environments, including pristine and hydrocarbon-contaminated soils, aquatic sediments, paper mill waste sludges, and farm animal waste lagoons. In all of the environments tested, the acetate-oxidizing ClRB represented a significant population, whose size ranged from 2.31 x 10(3) to 2.4 x 10(6) cells per g of sample. In addition, we isolated 13 ClRB from these environments. All of these organisms could grow anaerobically by coupling complete oxidation of acetate to reduction of (per)chlorate. Chloride was the sole end product of this reductive metabolism. All of the isolates could also use oxygen as a sole electron acceptor, and most, but not all, could use nitrate. The alternative electron donors included simple volatile fatty acids, such as propionate, butyrate, or valerate, as well as simple organic acids, such as lactate or pyruvate. Oxidized-minus-reduced difference spectra of washed whole-cell suspensions of the isolates had absorbance maxima close to 425, 525, and 550 nm, which are characteristic of type c cytochromes. In addition, washed cell suspensions of all of the ClRB isolates could dismutate chlorite, an intermediate in the reductive metabolism of (per)chlorate, into chloride and molecular oxygen. Chlorite dismutation was a result of the activity of a single enzyme which in pure form had a specific activity of approximately 1,928 micromol of chlorite per mg of protein per min. Analyses of the 16S ribosomal DNA sequences of the organisms indicated that they all belonged to the alpha, beta, or gamma subclass of the Proteobacteria. Several were closely related to members of previously described genera that are not recognized for the ability to reduce (per)chlorate, such as the genera Pseudomonas and Azospirllum. However, many were not closely related to any previously described organism and represented new genera within the Proteobacteria. The results of this study significantly increase the limited number of microbial isolates that are known to be capable of dissimilatory (per)chlorate reduction and demonstrate the hitherto unrecognized phylogenetic diversity and ubiquity of the microorganisms that exhibit this type of metabolism.

Hydrocarbon Bioremediative Potential of (Per)Chlorate-Reducing Bacteria

Petroleum contamination of soils and sediments is a national concern due to the toxicity and recalcitrance of the aromatic components in the absence of oxygen. Oxygen can be introduced into the anaerobic zone of a contaminated environment by injection of gaseous O2 to stimulate biodegradation, but this process is costly and inefficient. Other more soluble electron acceptors, such as nitrate or sulfate, may alternatively be used, but the rates of oxidation are slow and not all hydrocarbons are degraded. Here we report that chlorite dismutation by (per)chlorate-reducing bacteria may offer an alternative source of oxygen for contaminant degradation. The dismutation of chlorite is an intermediate step in the microbial reduction of chlorate. Chlorite dismutation can stimulate the rapid oxidation of aromatic hydrocarbons such as benzene or naphthalene in anoxic environments by supplying oxygen to the aerobic hydrocarbon-oxidizing population. Benzene, which is extremely recalcitrant under anaerobic conditions, is rapidly degraded to CO2 even in pristine soils with no prior exposure to hydrocarbons. The (per)chlorate-reducing bacteria grew rapidly in a broad diversity of environmental conditions and survived in significant numbers over the long term in inoculated sediments. In addition, the (per)chlorate-reducer Dechlorimonas agitatus strain CKB could survive starvation, forming a stable ultramicrobacterium with a cell size less than one-tenth that of the vegetative cells. Such ultramicrobacterial cells can readily pass through small pore sizes of subsurface environments preventing near-well plugging in bioaugmentation strategies. The ultramicrobacterial cells formed could readily be recovered as vegetative cells and could be used to stimulate hydrocarbon oxidation after only 68 hours recovery. Our results suggest that chlorite in the presence of (per)chlorate-reducing bacteria may be used as an effective in situ remediation strategy for petroleum contamination.

Reduction of (per)chlorate by a novel organism isolated from paper mill waste.

As part of a study on the microbiology of chlorate reduction, several new dissimilatory chlorate-reducing bacteria were isolated from a broad diversity of environments. One of these, strain CKB, was selected for a more complete characterization. Strain CKB was enriched and isolated from paper mill waste with acetate as the sole electron donor and chlorate as the sole electron acceptor. Strain CKB is a completely oxidizing, non-fermentative, Gram-negative, facultative anaerobe. Cells of strain CKB are 0.5 x 2 microm and are highly motile, with a single polar flagellum. In addition to acetate, strain CKB can use propionate, butyrate, lactate, succinate, fumarate, malate or yeast extract as electron donors, with chlorate as the sole electron acceptor. Strain CKB can also couple chlorate reduction to the oxidation of ferrous iron, sulphide, or the reduced form of the humic substances analogue 2,6-anthrahydroquinone disulphonate. Fe(II) is oxidized to insoluble amorphous Fe(II) oxide, whereas sulphide is oxidized to elemental sulphur. Growth is not associated with this metabolism, even when small quantities of acetate are added as a potential carbon source. In addition to chlorate, strain CKB can also couple acetate oxidation to the reduction of oxygen or perchlorate. Chlorate is completely reduced to chloride. Strain CKB has an optimum temperature of 35 degrees C, a pH optimum of 7.5 and a salinity optimum of 1% NaCl. Strain CKB can grow in chlorate and perchlorate concentrations of 80 or 20 mM respectively. Under anaerobic conditions, strain CKB can dismutate chlorite into chloride and O2, and is only the second organism shown to be capable of this metabolism. Oxidized minus reduced spectra of whole-cell suspensions of strain CKB showed absorbance maxima at 423, 523 and 552nm, which are indicative of the presence of c-type cytochrome(s). Analysis of the complete sequence of the 16S rDNA indicates that strain CKB is a member of the beta subclass of the Proteobacteria. The phototroph Rhodocyclus tenuis is the closest known relative. When tested, strain CKB could not grow by phototrophy and did not contain bacteriochlorophyll. Phenotypically and phylogenetically, strain CKB differs from all other described bacteria and represents the type strain of a new genus and species.