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A lithoautotrophic, Fe(II) oxidizing, nitrate-reducing bacterium, strain 2002 (ATCC BAA-1479; =DSM 18807), was isolated as part of a study on nitrate-dependent Fe(II) oxidation in freshwater lake sediments. Here we provide an in-depth phenotypic and phylogenetic description of the isolate. Strain 2002 is a gram-negative, non-spore forming, motile, rod-shaped bacterium which tested positive for oxidase, catalase, and urease. Analysis of the complete 16S rRNA gene sequence placed strain 2002 in a clade within the family Neisseriaceae in the order Nessieriales of the Betaproteobacteria 99.3% similar to Pseudogulbenkiania subflava. Similar to P. sublfava, predominant whole cell fatty acids were identified as 16:17c, 42.4%, and 16:0, 34.1%. Whole cell difference spectra of the Fe(II) reduced minus nitrate oxidized cyctochrome content revealed a possible role of c-type cytochromes in nitrate-dependent Fe(II) oxidation. Strain 2002 was unable to oxidize aqueous or solid-phase Mn(II) with nitrate as the electron acceptor. In addition to lithotrophic growth with Fe(II), strain 2002 could alternatively grow heterotrophically with long-chain fatty acids, simple organic acids, carbohydrates, yeast extract, or casamino acids. Nitrate, nitrite, nitrous oxide, and oxygen also served as terminal electron acceptors with acetate as the electron donor.

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.

The permanently frozen freshwater Lake Fryxell, located in the Dry Valleys of Antarctica, exhibits an ideal geochemistry for microbial sulfate reduction. To investigate the population of sulfate-reducing bacteria in Lake Fryxell, both 16S rRNA gene and metabolic primer sets targeting the dsrA gene for the dissimilatory sulfite reductase alpha subunit were employed to analyze environmental DNA obtained from the water column and sediments of Lake Fryxell. In addition, enrichment cultures of sulfate-reducing bacteria established at 4°C from Lake Fryxell water were also screened using the dsrA primer set. The sequence information obtained showed that a diverse group of sulfate-reducing prokaryotes of the domain Bacteria inhabit Lake Fryxell. With one exception, the enrichment culture sequences were not represented within the environmental sequences. Sequence data were compared with the geochemical profile of Lake Fryxell to identify possible connections between the diversity of sulfate-reducing bacteria and limnological conditions. Several clone groups were highly localized with respect to lake depth and, therefore, experienced specific physiochemical conditions. However, all sulfate-reducing bacteria inhabiting Lake Fryxell must function under the constantly cold conditions characteristic of this extreme environment.

Although anoxygenic photosynthesis is thought to play an important role in the primary productivity of permanently frozen lakes in the Antarctic dry valleys, the bacterial communities responsible for this metabolism remain uncharacterized. Here we report the composition and activity of phototrophic purple bacteria in Lake Fryxell, Antarctica, as determined by analysis of a photosynthesis-specific gene, pufM . The results revealed an extensive diversity and highly stratified distribution of purple nonsulfur bacteria in Lake Fryxell and showed which phylotypes produced pufM transcripts in situ. Enrichment cultures for purple bacteria yielded two morphotypes, each with a pufM signature identical to signatures detected by environmental screening. The isolates also contained gas vesicles, buoyancy structures previously unknown in purple nonsulfur bacteria, that may be necessary for these organisms to position themselves at specific depths within the nearly freezing water column.

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.

A B S T R A C T 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.

Recent studies on the diversity and ubiquity of Fe(III)-reducing organisms in different environments led to the isolation and identification of four new dissimilatory Fe(III)-reducers (strains H-2T, 172T, TACP-2T and TACP-5). All four isolates are non-motile, Gram-negative, freshwater, mesophilic, strict anaerobes with morphology identical to that of Geobacter metallireducens strain GS-15T. Analysis of the 16S rRNA sequences indicated that the new isolates belong to the genus Geobacter, in the delta-Proteobacteria. Significant differences in phenotypic characteristics, DNA-DNA homology and G+C content indicated that the four isolates represent three new species of the genus. The names Geobacter hydrogenophilus sp. nov. (strain H-2T), Geobacter chapellei sp. nov. (strain 172T) and Geobacter grbiciae sp. nov. (strains TACP-2T and TACP-5) are proposed. Geobacter hydrogenophilus and Geobacter chapellei were isolated from a petroleum-contaminated aquifer and a pristine, deep, subsurface aquifer, respectively. Geobacter grbiciae was isolated from aquatic sediments. All of the isolates can obtain energy for growth by coupling the oxidation of acetate to the reduction of Fe(III). The four isolates also coupled Fe(III) reduction to the oxidation of other simple, volatile fatty acids. In addition, Geobacter hydrogenophilus and Geobacter grbiciae were able to oxidize aromatic compounds such as benzoate, whilst Geobacter grbiciae was also able to use the monoaromatic hydrocarbon toluene.

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.

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 × 10 3 to 2.4 × 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 μmol 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.

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, i...

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.

Microorganisms in diverse terrestrial surface and subsurface environments can anaerobically catalyze the oxidative dissolution of uraninite. While a limited quantity (∼5 to 12 μmol liter −1 ) of uranium is oxidatively dissolved in pure culture studies, the metabolism is coupled to electron transport, providing the potential of uraninite to support indigenous microbial populations and to solubilize uranium.

A halotolerant, alkaliphilic dissimilatory Fe(III)-reducing bacterium, strain SFB, was isolated from salt flat sediments collected from Soap Lake, WA. 16S ribosomal ribonucleic acid gene sequence analysis identified strain SFB as a novel Bacillus sp. most similar to Bacillus agaradhaerens (96.7% similarity). Strain SFB, a fermentative, facultative anaerobe, fermented various hexoses including glucose and fructose. The fructose fermentation products were lactate, acetate, and formate. Under fructose-fermenting conditions in a medium amended with Fe(III), Fe(II) accumulated concomitant with a stoichiometric decrease in lactate and an increase in acetate and CO(2). Strain SFB was also capable of respiratory Fe(III) reduction with some unidentified component(s) of Luria broth as an electron donor. In addition to Fe(III), strain SFB could also utilize nitrate, fumarate, or O(2) as alternative electron acceptors. Optimum growth was observed at 30 degrees C and pH 9. Although the optimal salinity for growth was 0%, strain SFB could grow in a medium with up to 15% NaCl by mass. These studies describe a novel alkaliphilic, halotolerant organism capable of dissimilatory Fe(III) reduction under extreme conditions and demonstrate that Bacillus species can contribute to the microbial reduction of Fe(III) in environments at elevated pH and salinity, such as soda lakes.

The identification and analysis of the genes encoding perchlorate reductase and chlorite dismutase has provided not only a building block for pathway understanding, but has also provided a tool for bioremediative and phylogenetic studies. On-going genome sequencing will further facilitate transcriptional profiling under perchlorate-reducing conditions via microarray analyses. This analysis will give a more inclusive look into transcriptional expression patterns associated with the perchlorate metabolism. While further advancements in the genetic analysis of perchlorate-reducing bacteria continue, the recent development of a genetic system in D. aromatica will provide an invaluable tool for corroborating microarray results and solidifying hypotheses regarding microbial perchlorate metabolism.

The field of microbial perchlorate reduction has clearly advanced significantly in a very short period from a poorly understood metabolism to a burgeoning scientific field of discovery. As outlined above, there is now a much greater appreciation of the microbiology involved and the application of the knowledge to the successful treatment of contaminated environments. Overall, the future is promising even though research in this field is still in its infancy. Nothing is known of the evolutionary root of this metabolism. From a biogeochemical perspective, a better understanding of how perchlorate is formed in the natural environment and what geochemical conditions are required for its formation might give some insight into plotting the metabolism against a realistic evolutionary timeline. From a microbial perspective, it will be important to look for this metabolism in more extreme environments such as hypersaline or hyperthermophilic environments to obtain DPRB isolates across a broader phylogeny to establish a broad-base molecular chronometer. With the development of this field comes a better understanding of the ideal electron donors available and the individual factors which truly control the activity of the these organisms in-situ allowing for the design of more effective and robust enhanced in situ bioremediation technologies.

The objective of the studies described in this report were to gain a better understanding of the microbiology involved in microbial perchlorate reduction with an aim of enhancing the application of this novel metabolism to the attenuation of perchlorate contaminated environments.

Archaea were detected in molecular diversity studies of the permanently frozen Lake Fryxell, Antarctica. Two clusters of methanogens were detected in the sediments, and another cluster of possibly methanotrophic Euryarchaeota was detected in the anoxic water column just above the sediments. One crenarchaeote was detected in water just below the oxycline. The Archaea present in Lake Fryxell are likely involved in the major biogeochemical cycles that occur there.

Microbial nitrate-dependent Fe(II) oxidation is known to contribute to iron biogeochemical cycling; however, the microorganisms responsible are virtually unknown. In an effort to elucidate this microbial metabolic process in the context of an environmental system, a 14-cm sediment core was collected from a freshwater lake and geochemically characterized concurrently with the enumeration of the nitrate-dependent Fe(II)-oxidizing microbial community and subsequent isolation of a nitrate-dependent Fe(II)-oxidizing microorganism. Throughout the sediment core, ambient concentrations of Fe(II) and nitrate were observed to coexist. Concomitant most probable number enumeration revealed the presence of an abundant nitrate-dependent Fe(II)-oxidizing microbial community (2.4 × 10 3 to 1.5 × 10 4 cells g −1 wet sediment) from which a novel anaerobic, lithoautotrophic, Fe(II)-oxidizing bacterium, strain 2002, was isolated. Analysis of the complete 16S rRNA gene sequence revealed that strain 2002 was a member of the beta subclass of the proteobacteria with 94.8% similarity to Chromobacterium violaceum , a bacterium not previously recognized for the ability to oxidize nitrate-dependent Fe(II). Under nongrowth conditions, both strain 2002 and C. violaceum incompletely reduced nitrate to nitrite with Fe(II) as the electron donor, while under growth conditions nitrate was reduced to gaseous end products (N 2 and N 2 O). Lithoautotrophic metabolism under nitrate-dependent Fe(II)-oxidizing conditions was verified by the requirement of CO 2 for growth as well as the assimilation of 14 C-labeled CO 2 into biomass. The isolation of strain 2002 represents the first example of an anaerobic, mesophilic, neutrophilic Fe(II)-oxidizing lithoautotroph isolated from freshwater samples. Our studies further demonstrate the abundance of nitrate-dependent Fe(II) oxidizers in freshwater lake sediments and provide further evidence for the potential of microbially mediated Fe(II) oxidation in anoxic environments.

A morphologically distinct heliobacterium, strain HH, was isolated from Lake El Hamra, a soda lake in the Wadi El Natroun region of northwest Egypt. Strain HH consisted of ring-shaped cells that remained attached after cell division to yield coils of various lengths. Strain HH showed several of the physiological properties of known heliobacteria and grouped in the Heliorestis clade by virtue of its phylogeny and alkaliphily. The closest relative of strain HH was the filamentous alkaliphilic heliobacterium Heliorestis daurensis. However, genomic DNA:DNA hybridization results clearly indicated that strain HH was a distinct species of Heliorestis. Based on its unique phenotypic and genetic properties we describe strain HH here as a new species of the genus Heliorestis, H. convoluta sp. nov.

Swine production is an important industry in the United States, with an annual value of more than 12 billion dollars. In 1997, the annual nationwide production was more than 100 million pigs, with a resultant production of 8.5 × 106 tons (dry weight) of manure per year requiring treatment and disposal (21, 28a). The low profit margins associated with swine production and the difficulties associated with poor public acceptance of hog facilities in many regions have resulted in a significant increase in the number of high-density-confinement rearing facilities in an attempt to improve efficiency based on economies of scale. Although hog wastes can be used as a good nutritional source of nitrogen and phosphorous for agronomic crops, the practice of land spreading has come under intensive scrutiny in the last decade because of runoff contamination of water resources and the odor of the untreated slurry. In addition, swine manures contain high concentrations of metals such as iron, copper, zinc, cobalt, and cadmium that are present as both soluble organic complexes and insoluble oxides and can accumulate in a soil environment as a result of land spreading (34). Thus, major air, soil, and water pollution problems are associated with storage and disposal of swine waste. Odor management has become a crucial issue for the swine industry, and the sustainability, productivity, and profitability of a producer depend on the extent to which odor emissions can be controlled (62). Malodorous components of swine waste can be divided into four classes: volatile fatty acids (VFAs), indoles and phenols, ammonia and volatile amines, and volatile sulfur compounds (63). Each of these components is primarily microbially formed through the activity of fermentative bacteria that degrade the complex organics present in the waste (56, 62). These compounds accumulate in storage facilities, where the mixtures of feces and urine collected from under-floor collection pits decompose under the prevailing anaerobic conditions. Although these pits are left exposed to the atmosphere, the high organic content of swine waste ensures that oxygen diffusing from the atmosphere is rapidly removed biologically (10). As such, although the malodorous components are readily biodegraded by many respiratory microbial species, the activity of these organisms is inhibited by the limiting availability of suitable electron acceptors or by the organisms' low rate of metabolism under methanogenic conditions (29, 48). Swine waste can be treated microbially in aerobic activated sludge systems; however, these systems are energy intensive, and there is a large production of microbial biomass (1.0 to 1.5 mol mol−1 waste treated) (33) that also requires treatment and disposal. Alternatively, anaerobic treatment processes, such as the use of methanogenic bioreactors, can be applied for the removal of odor and the generation of combustible biogases. This type of treatment has the advantages over aerobic systems of lower energy input and of much lower yield of biomass per liter of treated waste (0.032 mol mol−1 waste treated) (25). However, traditional methanogenic systems are slow due to the long doubling times of the fatty acid-degrading, syntrophic bacteria whose activity is central to the process (46). Alternative treatment systems based on sulfate- or nitrate-reducing bacteria could potentially be faster, due to the favorable thermodynamics of these metabolisms (46, 58). However, both of these systems can produce noxious and toxic products (e.g., sulfide, nitrite, and nitrogen oxides). Here, we describe an alternative approach for treatment of the malodorous compounds associated with hog waste by stimulating their removal through Fe(III) supplementation and bioaugmentation with a novel dissimilatory Fe(III)-reducing organism. This process can be used to treat hog waste directly in primary and secondary lagoons without the need to construct dedicated bioreactors. Microbial Fe(III) reduction is an energetically favorable process, and in the natural environment, Fe(III)-reducing bacteria (FeRB) can outcompete and inhibit both sulfate-reducing and methanogenic bacteria (10, 38, 44, 60). FeRB also have diverse metabolisms, and many pure-culture examples exist that can completely oxidize straight- and branched-chain fatty acids and aromatic organics without the need for the activity of the rate-limiting syntrophic bacteria (14, 37). The respiratory end product of microbial Fe(III) reduction, Fe(II), is nontoxic and can be recycled after abiotic reoxidation through its reaction with O2. In addition, added iron will abiotically react with malodorous HS− ions, forming non-odor-causing metal sulfide precipitates. This approach has the potential for the long-term and sustainable removal of the major odor-causing components of hog manure.

The reduction of perchlorate to chlorite, the first enzymatic step in the bacterial reduction of perchlorate, is catalyzed by perchlorate reductase. The genes encoding perchlorate reductase ( pcrABCD ) in two Dechloromonas species were characterized. Sequence analysis of the pcrAB gene products revealed similarity to α- and β-subunits of microbial nitrate reductase, selenate reductase, dimethyl sulfide dehydrogenase, ethylbenzene dehydrogenase, and chlorate reductase, all of which are type II members of the microbial dimethyl sulfoxide (DMSO) reductase family. The pcrC gene product was similar to a c -type cytochrome, while the pcrD gene product exhibited similarity to molybdenum chaperone proteins of the DMSO reductase family members mentioned above. Expression analysis of the pcrA gene from Dechloromonas agitata indicated that transcription occurred only under anaerobic (per)chlorate-reducing conditions. The presence of oxygen completely inhibited pcrA expression regardless of the presence of perchlorate, chlorate, or nitrate. Deletion of the pcrA gene in Dechloromonas aromatica abolished growth in both perchlorate and chlorate but not growth in nitrate, indicating that the pcrABCD genes play a functional role in perchlorate reduction separate from nitrate reduction. Phylogenetic analysis of PcrA and other α-subunits of the DMSO reductase family indicated that perchlorate reductase forms a monophyletic group separate from chlorate reductase of Ideonella dechloratans . The separation of perchlorate reductase as an activity distinct from chlorate reductase was further supported by DNA hybridization analysis of (per)chlorate- and chlorate-reducing strains using the pcrA gene as a probe.

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.

A moderately psychrophilic purple non-sulfur bacterium, Rhodoferax antarcticus strain Fryx1, is described. Strain Fryx1 was isolated from the water column under the ice of the permanently frozen Lake Fryxell, Antarctica. Cells of Fryx1 are long thin rods and contain gas vesicles, the first report of such structures in purple non-sulfur bacteria. Gas vesicles are clustered at 2-4 sites per cell. Surprisingly, the 16S rRNA gene sequence of strain Fryx1 is nearly identical to that of Rfx. antarcticus strain AB, a short, vibrio-shaped phototroph isolated from an Antarctic microbial mat. Although showing physiological parallels, strains AB and Fryx1 differ distinctly in their morphology and absorption spectra. DNA-DNA hybridization shows that the genomes of strains AB and Fryx1 are highly related, yet distinct. We conclude that although strains AB and Fryx1 may indeed be the same species, their ecologies are quite different. Unlike strain AB, strain Fryx1 has adapted to a planktonic existence in the nearly freezing water column of Lake Fryxell.

It is less than 7 years since perchlorate, a predominantly man-made toxic anion, was first identified as a significant water contaminant throughout the United States. Owing to its solubility and non-reactivity, bioremediation was targeted as the most promising solution for the problem of perchlorate contamination. Since 1996, concerted efforts have resulted in significant advances in our understanding of the microbiology, biochemistry and genetics of the microorganisms that are capable of reductively transforming perchlorate into innocuous chloride. The recent completion of the whole-genome sequence of the perchlorate-reducing microorganism Dechloromonas aromatica offers further insight into the evolution and regulation of this unique metabolic pathway. Several in situ and ex situ bioremediative processes have been engineered, and many monitoring tools that are based on immunology, molecular biology and stable isotope content are now available. As such, the rapid scientific response to this emerging contaminant offers great hope for its successful elimination from contaminated environments in the future.

Bioremediation of perchlorate-contaminated groundwater can occur via bacterial reduction of perchlorate to chloride. Although perchlorate reduction has been demonstrated in bacterial pure cultures, little is known about the efficacy of using perchlorate-reducing bacteria as inoculants for bioremediation in the field. A pilot-scale, fixed-bed bioreactor containing plastic support medium was used to treat perchlorate-contaminated groundwater at a site in Southern California. The bioreactor was inoculated with a field-grown suspension of the perchlorate-respiring bacterium Dechlorosoma sp. strain KJ and fed groundwater containing indigenous bacteria and a carbon source amendment. Because the reactor was flushed weekly to remove accumulated biomass, only bacteria capable of growing in biofilms in the reactor were expected to survive. After 26 days of operation, perchlorate was not detected in bioreactor effluent. Perchlorate remained undetected by ion chromatography (detection limit 4 mug L(-1)) during 6 months of operation, after which the reactor was drained. Plastic medium was subsampled from top, middle, and bottom locations of the reactor for shipment on blue ice and storage at -80 degrees C prior to analysis. Microbial community DNA was extracted from successive washes of thawed biofilm material for PCR-based community profiling by 16S-23S ribosomal intergenic spacer analysis (RISA). No DNA sequences characteristic of strain KJ were recovered from any RISA bands. The most intense bands yielded DNA sequences with high similarities to Dechloromonas spp., a closely related but different genus of perchlorate-respiring bacteria. Additional sequences from RISA profiles indicated presence of representatives of the low G+C gram-positive bacteria and the Cytophaga-Flavobacterium-Bacteroides group. Confocal scanning laser microscopy and fluorescence in situ hybridization (FISH) were also used to examine biofilms using genus-specific 16S ribosomal RNA probes. FISH was more sensitive than RISA profiling in detecting possible survivors from the initial inoculum. FISH revealed that bacteria hybridizing to Dechlorosoma probes constituted1% of all cells in the biofilms examined, except in the deepest portions where they represented 3-5%. Numbers of bacteria hybridizing to Dechloromonas probes decreased as biofilm depth increased, and they were most abundant at the biofilm surface (23% of all cells). These spatial distribution differences suggested persistence of low numbers of the inoculated strain Dechlorosoma sp. KJ in parts of the biofilm nearest to the plastic medium, concomitant with active colonization or growth by indigenous Dechloromonas spp. in the biofilm exterior. This study demonstrated the feasibility of post hoc analysis of frozen biofilms following completion of field remediation studies.

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.

Previous studies have demonstrated that reduced humic substances (HS) can be reoxidized by anaerobic bacteria such as Geobacter , Geothrix , and Wolinella species with a suitable electron acceptor; however, little is known of the importance of this metabolism in the environment. Recently we investigated this metabolism in a diversity of environments including marine and aquatic sediments, forest soils, and drainage ditch soils. Most-probable-number enumeration studies were performed using 2,6-anthrahydroquinone disulfonate (AHDS), an analog for reduced HS, as the electron donor with nitrate as the electron acceptor. Anaerobic organisms capable of utilizing reduced HS as an electron donor were found in all environments tested and ranged from a low of 2.31 × 10 1 in aquifer sediments to a high of 9.33 × 10 6 in lake sediments. As part of this study we isolated six novel organisms capable of anaerobic AHDS oxidation. All of the isolates coupled the oxidation of AHDS to the reduction of nitrate with acetate (0.1 mM) as the carbon source. In the absence of cells, no AHDS oxidation was apparent, and in the absence of AHDS, no cell density increase was observed. Generally, nitrate was reduced to N 2 . Analysis of the AHDS and its oxidized form, 2,6-anthraquinone disulfonate (AQDS), in the medium during growth revealed that the anthraquinone was not being biodegraded as a carbon source and was simply being oxidized as an energy source. Determination of the AHDS oxidized and nitrate reduced accounted for 109% of the theoretical electron transfer. In addition to AHDS, all of these isolates could also couple the oxidation of reduced humic substances to the reduction of nitrate. No HS oxidation occurred in the absence of cells and in the absence of a suitable electron acceptor, demonstrating that these organisms were capable of utilizing natural HS as an energy source and that AHDS serves as a suitable analog for studying this metabolism. Alternative electron donors included simple volatile fatty acids such as propionate, butyrate, and valerate as well as simple organic acids such as lactate and pyruvate. Analysis of the complete sequences of the 16S rRNA genes revealed that the isolates were not closely related to each other and were phylogenetically diverse, with members in the alpha, beta, gamma, and delta subdivisions of the Proteobacteria . Most of the isolates were closely related to known genera not previously recognized for their ability to couple growth to HS oxidation, while one of the isolates represented a new genus in the delta subclass of the Proteobacteria . The results presented here demonstrate that microbial oxidation of HS is a ubiquitous metabolism in the environment. This study represents the first description of HS-oxidizing isolates and demonstrates that microorganisms capable of HS oxidation are phylogenetically diverse.

Benzene contamination is a significant problem. It is used in a wide range of manufacturing processes and is a primary component of petroleum-based fuels. Benzene is a hydrocarbon that is soluble, mobile, toxic and stable, especially in ground and surface waters. It is poorly biodegraded in the absence of oxygen. However, anaerobic benzene biodegradation has been documented under various conditions. Although benzene biomineralization has been demonstrated with nitrate1, Fe(III)2,3,4,5, sulphate6,7 or CO28,9 as alternative electron acceptors, these studies were based on sediments or microbial enrichments. Until now there were no organisms in pure culture that degraded benzene anaerobically. Here we report two Dechloromonas strains, RCB and JJ, that can completely mineralize various mono-aromatic compounds including benzene to CO2 in the absence of O2 with nitrate as the electron acceptor. This is the first example, to our knowledge, of an organism of any type that can oxidize benzene anaerobically, and we demonstrate the potential applicability of these organisms to the treatment of contaminated environments.

Primer sets were designed to target specific 16S ribosomal DNA (rDNA) sequences of photosynthetic bacteria, including the green sulfur bacteria, the green nonsulfur bacteria, and the members of the Heliobacteriaceae (a gram-positive phylum). Due to the phylogenetic diversity of purple sulfur and purple nonsulfur phototrophs, the 16S rDNA gene was not an appropriate target for phylogenetic rDNA primers. Thus, a primer set was designed that targets the pufM gene, encoding the M subunit of the photosynthetic reaction center, which is universally distributed among purple phototrophic bacteria. The pufM primer set amplified DNAs not only from purple sulfur and purple nonsulfur phototrophs but also from Chloroflexus species, which also produce a reaction center like that of the purple bacteria. Although the purple bacterial reaction center structurally resembles green plant photosystem II, the pufM primers did not amplify cyanobacterial DNA, further indicating their specificity for purple anoxyphototrophs. This combination of phylogenetic- and photosynthesis-specific primers covers all groups of known anoxygenic phototrophs and as such shows promise as a molecular tool for the rapid assessment of natural samples in ecological studies of these organisms.

From enrichment cultures established for purple nonsulfur bacteria using water and sediment samples from Lake Bogoria and Crater Lake, two soda lakes in the African Rift Valley, three strains of purple nonsulfur bacteria were isolated; strain LBB1 was studied in detail. Cells of strain LBB1 were motile and spherical to rod-shaped, suggesting a relationship to Rhodobacter or Rhodovulum species, and the organism was capable of both phototrophic and chemotrophic growth on a wide variety of organic compounds. Phototrophically grown cultures were yellow to yellow-brown in color and grew optimally at pH 9 (pH range 7.5–10) and 1% NaCl (range 0–10%). In physiological studies of strain LBB1, neither photoautotrophy (H2- or sulfide-dependent) nor nitrogen fixation was observed. Absorption spectra revealed that all three strains contained bacteriochlorophyll a and carotenoids of the spheroidene pathway and synthesized only a light-harvesting (LH) I-type photosynthetic antenna complex. Electron microscopy of cells of strain LBB1 revealed a vesicular intracytoplasmic membrane system, although only a few vesicles were observed per cell. The G+C content of strain LBB1 DNA was 59 mol%, significantly lower than that of known Rhodobacter and Rhodovulum species, and its phylogeny as determined by ribosomal RNA gene sequencing placed it within the Rhodobacter/Rhodovulum clade yet distinct from all described species of either of these genera. The unique assemblage of properties observed in strain LBB1 warrants its inclusion in a new genus of purple nonsulfur bacteria and the name Rhodobaca bogoriensis is proposed herein, the genus name reflecting morphological characteristics and the species epithet referring to the habitat.

A new species of purple nonsulfur bacteria isolated from an Antarctic microbial mat is described. The organism, designated strain ANT.BR, was mildly psychrophilic, growing optimally at 15-18 degrees C with a growth temperature range of 0-25 degrees C. Cells of strain ANT.BR were highly motile curved rods and spirals, contained bacteriochlorophyll a, and showed a multicomponent in vivo absorption spectrum. A specific phylogenetic relationship was observed between strain ANT.BR and the purple bacterium Rhodoferax fermentans FR2T, and the two organisms shared several physiological and other phenotypic properties, with the notable exception of growth temperature optimum. Tests of genomic DNA hybridization, however, showed Rfx. fermentans FR2T and strain ANT.BR to be genetically distinct bacteria. Because of its unique set of properties, especially its requirement for low growth temperatures, we propose to recognize strain ANT.BR as a new species of the genus Rhodoferax, Rhodoferax antarcticus, named for its known habitat, the Antarctic.

In 1997, following the development of a highly sensitive analytical method for perchlorate, monitoring studies identified perchlorate as a contaminant of major drinking water resources in the southwestern U.S.1,2,3 The only known natural source of perchlorate is associated with mineral deposits found in Chile where the perchlorate may represent as much as 6–7% of the total mass.4 Although these Chilean deposits have been extensively mined as a mineral and nitrate source for fertilizer manufacture, this is not thought to represent a significant source of perchlorate in the environment. The primary industrial use of perchlorate is as an oxidant in munitions manufacture and the unregulated release of perchlorate-containing wastestreams from munition manufacturing and handling facilities has been identified as the predominant source of this contaminant in the U.S.1,3

Microbial chlorate (ClO3 −) and perchlorate (ClO4 −) reduction has recently been recognized as an important form of microbial metabolism for the removal of chlorine oxyanion contamination in the environment.1 Chlorine oxyanions have many industrial applications including use as bleaching agents by the paper and pulp industry,2,3 as disinfectants and defoliants by the agricultural industry,4 and as components of explosives and rocket propellants by the aerospace and defense industries.5 Chlorates can also be formed as a result of ozonation of drinking waters which have been treated with chlorine6 or photodecomposition of chlorite or chlorine dioxide7 which are used in addition to chlorine for water disinfection.

A novel alkaliphilic heliobacterium was isolated from microbial mats of a low-salt alkaline Siberian soda lake. Cells of the new organism were tightly coiled when grown in coculture with a rod-shaped bacterium, but grew as short filaments when finally obtained in pure culture. The new phototroph, designated strain BT-H1, produced bacteriochlorophyll g and a neurosporene-like pigment, and lacked internal photosynthetic membranes. Similar to other heliobacteria, strain BT-H1 grew photoheterotrophically on a limited range of organic compounds including acetate and pyruvate. Sulfide was oxidized to elemental sulfur and polysulfides under photoheterotrophic conditions; however, photoautotrophic growth was not observed. Cultures of strain BT-H1 were alkaliphilic, growing optimally at pH 9, and unlike other heliobacteria, they grew optimally at a temperature of 25 degrees C rather than at 40 degrees C or above. Analysis of the 16S rRNA gene sequence of the new organism showed that it groups within the heliobacterial clade. However, its branching order was phylogenetically basal to all previously investigated species of heliobacteria. The G+C content of the DNA of strain BT-H1 (44.9 mol%) was also quite distinct from that of other heliobacteria. This unique assemblage of properties implicates strain BT-H1 as a new genus and species of the heliobacteria, Heliorestis daurensis, named for its unusual morphology ("restis" is Latin for "rope") and for the Daur Steppe in Russia in which these soda lakes are located.

A second flavodoxin gene, distinct from the nifF gene encoding nitrogenase flavodoxin, has been isolated from Klebsiella pneumoniae. This flavodoxin gene is a homologue of the E. coli fldA gene and is located 286 bp upstream of the K. pneumoniae fur gene. Primer extension analysis revealed an unusual promoter region upstream of the K. pneumoniae fldA gene that does not match the −35/−10 consensus sequence. Transcriptional analyses using a Fur titration assay and fldA gene fusions demonstrated that, unlike E. coli fldA, the K. pneumoniae fldA gene is not constitutively expressed. Rather, the fldA gene of K. pneumoniae is repressed in high iron conditions by the ferric uptake regulator (Fur) protein. Expression of K. pneumoniae fldA is also induced by heat shock but not by salt stress.

The Fur (ferric uptake regulator) protein controls the expression of a number of bacterial virulence determinants including those involved in iron uptake. The fur gene was cloned and characterized from Klebsiella pneumoniae. The gene is preceded by a single autoregulated promoter whose -10 region overlaps the putative Fur binding site. The autoregulated nature of the K. pneumoniae fur gene and functionality of the encoded Fur repressor were tested in Fur titration and complementation assays. A partial open reading frame upstream from the fur gene was identified as a flavodoxin (fldA) gene. An open reading frame located 50 bases downstream from the fur stop codon appears to be a truncated citA gene that, if functional, would encode only the carboxy terminus of a citrate utilization protein. The fldA-fur arrangement is also present in Escherichia coli. However, the fur-citA arrangement found in K. pneumoniae is novel. It appears that the chromosomal region downstream from the fur gene is unstable and, thus, variable even in closely related bacterial lineages. To assess of the ability of the Fur protein sequence to reflect organismal phylogeny, the Fur protein tree was compared to the tree of 16S rRNA (ribosomal RNA). The Fur dataset comprises almost an order of magnitude fewer characters than the 16S rRNA but is nonetheless able to track the phylogenetic signal reasonably well, suggesting that the fur gene, like the 16S rDNA, may not be subject to horizontal gene transfer in these bacteria.

A new species of purple nonsulfur bacteria isolated from an Antarctic microbial mat is described. The organism, designated strain ANT.BR, was mildly psychrophilic, growing optimally at 15-18 degrees C with a growth temperature range of 0-25 degrees C. Cells of strain ANT.BR were highly motile curved rods and spirals, contained bacteriochlorophyll a, and showed a multicomponent in vivo absorption spectrum. A specific phylogenetic relationship was observed between strain ANT.BR and the purple bacterium Rhodoferax fermentans FR2T, and the two organisms shared several physiological and other phenotypic properties, with the notable exception of growth temperature optimum. Tests of genomic DNA hybridization, however, showed Rfx. fermentans FR2T and strain ANT.BR to be genetically distinct bacteria. Because of its unique set of properties, especially its requirement for low growth temperatures, we propose to recognize strain ANT.BR as a new species of the genus Rhodoferax, Rhodoferax antarcticus, named for its known habitat, the Antarctic.