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PubMed2006

Identification of acetate- or methanol-assimilating bacteria under nitrate-reducing conditions by stable-isotope probing.

Stable-isotope probing (SIP) was used to identify acetate- or methanol-assimilating bacteria under nitrate-reducing conditions in activated sludge. A sludge sample obtained from wastewater treatment systems was incubated in a denitrifying batch reactor fed with synthetic wastewater containing [(13)C]acetate or [(13)C]methanol as the main carbon source and nitrate as the electron acceptor. We analyzed how growth of bacterial populations was stimulated by acetate or methanol as the external carbon source in nitrogen-removal systems. Most of the acetate- or methanol-assimilating bacteria identified by SIP have been known as denitrifiers in wastewater treatment systems. When acetate was used as the carbon source, 16S rRNA gene sequences retrieved from (13)C-labeled DNA were closely related to the 16S rRNA genes of Comamonadaceae (e.g., Comamonas and Acidovorax) and Rhodocyclaceae (e.g., Thauera and Dechloromonas) of the Betaproteobacteria, and Rhodobacteraceae (e.g., Paracoccus and Rhodobacter) of the Alphaproteobacteria. When methanol was used as the carbon source, 16S rRNA gene sequences retrieved from (13)C-DNA were affiliated with Methylophilaceae (e.g., Methylophilus, Methylobacillus, and Aminomonas) and Hyphomicrobiaceae. Rarefaction curves for clones retrieved from (13)C-DNA showed that the diversity levels for methanol-assimilating bacteria were considerably lower than those for acetate-assimilating bacteria. Furthermore, we characterized nitrite reductase genes (nirS and nirK) as functional marker genes for denitrifier communities in acetate- or methanol-assimilating populations and detected the nirS or nirK sequence related to that of some known pure cultures, such as Alcaligenes, Hyphomicrobium, and Thauera. However, most of the nirS or nirK sequences retrieved from (13)C-DNA were clustered in some unidentified groups. On the basis of 16S rRNA gene clone libraries retrieved from (13)C-DNA, these unidentified nir sequences might be identified by examining the nir gene in candidates for true denitrifiers (e.g., the families Comamonadaceae, Hyphomicrobiaceae, Methylophilaceae, and Rhodobacteraceae).

Acetates↗
PubMed2006

Denitratisoma oestradiolicum gen. nov., sp. nov., a 17beta-oestradiol-degrading, denitrifying betaproteobacterium.

A Gram-negative, motile, denitrifying bacterium (strain AcBE2-1(T)) was isolated from activated sludge of a municipal wastewater treatment plant using 17beta-oestradiol (E2) as sole source of carbon and energy. Cells were curved rods, 0.4-0.8 x 0.8-2.0 microm in size, non-fermentative, non-spore-forming, oxidase-positive and catalase-negative. E2 was oxidized completely to carbon dioxide and water by reduction of nitrate to a mixture of dinitrogen monoxide and dinitrogen, with the intermediate accumulation of nitrite. Electron recoveries were between 90 and 100 %, taking assimilated E2 into account. With nitrate as the electron acceptor, the bacterium also grew on fatty acids (C(2) to C(6)), isobutyrate, crotonate, dl-lactate, pyruvate, fumarate and succinate. Phylogenetic analysis of its 16S rRNA gene sequence revealed that strain AcBE2-1(T) represents a separate line of descent within the family Rhodocyclaceae (Betaproteobacteria). The closest relatives are the cholesterol-degrading, denitrifying bacteria Sterolibacterium denitrificans DSM 13999(T) and strain 72Chol (=DSM 12783), with <93.9 % sequence similarity. The G+C content of the DNA was 61.4 mol%. Detection of a quinone system with ubiquinone Q-8 as the predominant compound and a fatty acid profile that included high concentrations of C(16 : 1)omega7c/iso-C(15 : 0) 2-OH and C(16 : 0), in addition to C(18 : 1)omega7c and small amounts of C(8 : 0) 3-OH, supported the results of the phylogenetic analysis. On the basis of 16S rRNA gene sequence data in combination with chemotaxonomic and physiological data, strain AcBE2-1(T) (=DSM 16959(T)=JCM 12830(T)) is placed in a new genus Denitratisoma gen. nov. as the type strain of the type species Denitratisoma oestradiolicum gen. nov., sp. nov.

Base Composition↗
PubMed2006

Methyloversatilis universalis gen. nov., sp. nov., a novel taxon within the Betaproteobacteria represented by three methylotrophic isolates.

The taxonomic positions and phylogenetic relationships of two new methylotrophic isolates from Lake Washington (USA) sediment, FAM5T and 500, and the previously described methylotrophic strain EHg5 isolated from contaminated soil in Estarreja (Portugal) were investigated. All three strains were facultative methylotrophs capable of growth on a variety of C1 and multicarbon compounds. Optimal growth occurred at pH 7.5-8 and 30-37 degrees C. The major fatty acids were C16:1omega7c and C16:0. The major quinone was ubiquinone Q8. Neither methanol dehydrogenase nor methanol oxidase activities were detectable in cells grown on methanol, suggesting an alternative, as-yet unknown, mechanism for methanol oxidation. The isolates assimilated C1 units at the level of formaldehyde, via the serine cycle. The DNA G+C content of the strains ranged between 64 and 65 mol%. 16S rRNA gene sequence similarity between the three new isolates was 99.85-100%, but was below 94% with other members of the Betaproteobacteria, indicating that the isolates represent a novel taxon. Based on physiological, phenotypic and genomic characteristics of the three isolates, a new genus, Methyloversatilis gen. nov., is proposed within the family Rhodocyclaceae. The type strain of Methyloversatilis universalis gen. nov., sp. nov. is FAM5T (=CCUG 52030T=JCM 13912T).

Alcohol Oxidoreductases↗
PubMed2005

Investigation of an acetate-fed denitrifying microbial community by stable isotope probing, full-cycle rRNA analysis, and fluorescent in situ hybridization-microautoradiography.

The acetate-utilizing microbial consortium in a full-scale activated sludge process was investigated without prior enrichment using stable isotope probing (SIP). [13C]acetate was used in SIP to label the DNA of the denitrifiers. The [13C]DNA fraction that was extracted was subjected to a full-cycle rRNA analysis. The dominant 16S rRNA gene phylotypes in the 13C library were closely related to the bacterial families Comamonadaceae and Rhodocyclaceae in the class Betaproteobacteria. Seven oligonucleotide probes for use in fluorescent in situ hybridization (FISH) were designed to specifically target these clones. Application of these probes to the sludge of a continuously fed denitrifying sequencing batch reactor (CFDSBR) operated for 16 days revealed that there was a significant positive correlation between the CFDSBR denitrification rate and the relative abundance of all probe-targeted bacteria in the CFDSBR community. FISH-microautoradiography demonstrated that the DEN581 and DEN124 probe-targeted cells that dominated the CFDSBR were capable of taking up [14C]acetate under anoxic conditions. Initially, DEN444 and DEN1454 probe-targeted bacteria also dominated the CFDSBR biomass, but eventually DEN581 and DEN124 probe-targeted bacteria were the dominant bacterial groups. All probe-targeted bacteria assessed in this study were denitrifiers capable of utilizing acetate as a source of carbon. The rapid increase in the number of organisms positively correlated with the immediate increase in denitrification rates observed by plant operators when acetate is used as an external source of carbon to enhance denitrification. We suggest that the impact of bacteria on activated sludge subjected to intermittent acetate supplementation should be assessed prior to the widespread use of acetate in the wastewater industry to enhance denitrification.

Acetates↗
PubMed2006

A nifH-based oligonucleotide microarray for functional diagnostics of nitrogen-fixing microorganisms.

Nitrogen fixation is an important process in biogeochemical cycles exclusively carried out by prokaryotes, mostly by an evolutionarily conserved nitrogenase protein complex, of which one of the structural genes (nifH) is highly valuable for phylogenetic and diversity analyses. We developed a nifH-based short oligonucleotide microarray (nifH diagnostic microarray) as a rapid tool to effectively monitor nitrogen-fixing diazotrophic populations in a wide range of environments. Taking account of the overwhelming predominance of environmental nifH fragments from uncultivated microorganisms in public databases, our nifH microarray is mainly based on nifH sequences from as yet unidentified prokaryotes. Standard conditions for microarray performance were determined, and criteria for the design of specific oligonucleotides were defined. A primary set of 56 oligonucleotides was validated with fluorescence-labeled single-stranded nifH targets from five reference strains, 26 environmental clones, and artificial mixtures of reference strains. The nifH microarray was applied to analyze the diversity (based on DNA) and activity (based on mRNA) of diazotrophs in roots of wild rice samples from Namibia. Results demonstrated that only a small subset of diazotrophs being present in the sample were actually fixing nitrogen actively. Our data suggest that the developed nifH microarray is a highly reproducible and semiquantitative method for mapping the variability of diazotrophic diversity, allowing rapid comparisons of the relative abundance and activity of diazotrophic prokaryotes in the environment. A further refined nifH microarray comprising of 194 oligonucleotide probes now covers more than 90% of sequences in our nifH database.

Azoarcus↗
PubMed2005

Enzymes responsible for chlorate reduction by Pseudomonas sp. are different from those used for perchlorate reduction by Azospira sp.

Pseudomonas sp. PDA is an unusual bacterium due to its ability to respire using chlorate under aerobic conditions. The chlorate reductase produced by PDA was shown to be intrinsically different from the enzyme responsible for chlorate and perchlorate [(per)chlorate] reduction produced by Azospira sp. KJ based on subunit composition and other enzyme properties. The perchlorate reductase from strain KJ appeared to have two subunits (100 and 40 kDa) while the chlorate reductase from PDA had three subunits (60, 48, and 27 kDa). N-terminal amino acid sequencing of the 100 kDa protein from strain KJ showed a 77% similarity with the perchlorate reductase alpha subunit from another perchlorate-respiring bacterium, Dechloromonas agitata, while the N-terminus amino acid sequence of the 60 kDa protein from strain PDA did not show a similarity to previously isolated chlorate or perchlorate reductases.

Amino Acid Sequence↗
PubMed2006

Supplementing Bacillus sp. RS1 with Dechloromonas sp. HZ for enhancing selenate reduction in agricultural drainage water.

Cost and efficiency are two important factors considered in the remediation of Se-contaminated agricultural drainage water through bacterial reduction of soluble Se(VI) to insoluble Se(0). Bacillus sp. RS1 isolated from rice straw was assessed for its ability to use inexpensive molasses to reduce Se(VI) in agricultural drainage water containing NO3- levels of 0, 50, 100, 250, and 500 mg/L. The results showed that Se(VI) (1000 microg/L) was almost entirely reduced to Se(IV) (62.7%) and Se(0) (36.4%) by Bacillus sp. RS1 in synthetic agricultural drainage (SAD) water without the presence of NO3-. The reduction Se(VI) to Se(0) was limited in the SAD water with NO3- levels of 100, 250, and 500 mg/L. The addition of Dechloromonas sp., a NO3- reducer, to the SAD water not only increased NO3- removal, but also enhanced Se(VI) reduction by Bacillus sp. RS1. During an 8-day experiment, 98-99% of the added Se(VI) was reduced to Se(0) with small amounts of Se(IV) and Se(-II) in the SAD water containing 100 and 250 mg/L NO3-. The addition of Dechloromonas sp. HZ to the natural agricultural drainage water also significantly increased the reduction of Se(VI) (748 microg/L) by Bacillus sp. RS1, with a production of Se(0) (65%) and Se(-II) (32%). These results suggest that a combination of Bacillus sp. RS1 with Dechloromonas sp. HZ has great potential with the use of inexpensive molasses to remediate Se-contaminated agricultural drainage water containing relatively high NO3- levels.

Agriculture↗
PubMed2005

Small-scale, hydrogen-oxidizing-denitrifying bioreactor for treatment of nitrate-contaminated drinking water.

Nitrate removal by hydrogen-coupled denitrification was examined using flow-through, packed-bed bioreactors to develop a small-scale, cost effective system for treating nitrate-contaminated drinking-water supplies. Nitrate removal was accomplished using a Rhodocyclus sp., strain HOD 5, isolated from a sole-source drinking-water aquifer. The autotrophic capacity of the purple non-sulfur photosynthetic bacterium made it particularly adept for this purpose. Initial tests used a commercial bioreactor filled with glass beads and countercurrent, non-sterile flow of an autotrophic, air-saturated, growth medium and hydrogen gas. Complete removal of 2 mM nitrate was achieved for more than 300 days of operation at a 2-h retention time. A low-cost hydrogen generator/bioreactor system was then constructed from readily available materials as a water treatment approach using the Rhodocyclus strain. After initial tests with the growth medium, the constructed system was tested using nitrate-amended drinking water obtained from fractured granite and sandstone aquifers, with moderate and low TDS loads, respectively. Incomplete nitrate removal was evident in both water types, with high-nitrite concentrations in the bioreactor output, due to a pH increase, which inhibited nitrite reduction. This was rectified by including carbon dioxide in the hydrogen stream. Additionally, complete nitrate removal was accomplished with wastewater-impacted surface water, with a concurrent decrease in dissolved organic carbon. The results of this study using three chemically distinct water supplies demonstrate that hydrogen-coupled denitrification can serve as the basis for small-scale remediation and that pilot-scale testing might be the next logical step.

Bioreactors↗
PubMed2003

Chlorine isotope fractionation during microbial reduction of perchlorate.

Perchlorate contamination of surface water and groundwater is an emerging public health problem that has adversely affected the drinking water supplies of millions of people in the western United States. Microbial reduction has shown promise as a cost-effective means for in situ bioremediation of perchlorate-contaminated water. Measurements of stable isotope ratios of light elements (H, C, N, O, S, Cl) can often be used to distinguish biodegradation of organic and inorganic molecules from abiotic loss mechanisms such as adsorption, dispersion, or volatilization because of the relatively large kinetic isotope effects accompanying biodegradation. We quantified chlorine isotope fractionation during perchlorate biodegradation by a common perchlorate-reducing bacterium, Dechlorosoma suillum, initially isolated from a perchlorate-contaminated groundwater source in southern California. The values of the chlorine isotopic fractionation factor alpha derived from two microcosm experiments were alpha = 0.9834 +/- 0.0001 (R2 = 0.9999) and alpha = 0.9871 +/- 0.0008 (R2 = 0.9832). These alpha values indicate that the rate of the 35ClO4 reduction is approximately 1.3-1.7% faster than that of the 37ClO4 reduction. This relatively large kinetic isotope effect indicates that chlorine isotope analysis provides a sensitive technique by which to document in situ bioremediation of perchlorate in groundwater.

Chlorine↗
PubMed2006

Perchlorate reduction by autotrophic bacteria in the presence of zero-valent iron.

A series of batch experiments were performed to study the combination of zero-valent iron (ZVI) with perchlorate-reducing microorganisms (PRMs) to remove perchlorate from groundwater. In this method, H2 produced during the process of iron corrosion by water is used by PRMs as an electron donor to reduce perchlorate to chloride. Perchlorate degradation rates followed Monod kinetics, with a normalized maximum utilization rate (rmax) of 9200 microg g(-1) (dry wt) h(-1) and a half-velocity constant (Ks) of 8900 microg L(-1). The overall rate of perchlorate reduction was affected by the biomass density within the system. An increase in the OD600 from 0.025 to 0.08 led to a corresponding 4-fold increase of perchlorate reduction rate. PRM adaptation to the local environment and initiation of perchlorate reduction was rapid under neutral pH conditions. At the initial OD600 of 0.015, perchlorate reduction followed pseudo-first-order reaction rates with constants of 0.059 and 0.033 h(-1) at initial pH 7 and 8, respectively. Once perchlorate reduction was established, the bioreductive process was insensitive to the increases of pH from near neutral to 9.0. In the presence of nitrate, perchlorate reduction rate was reduced, but not inhibited completely.

Hydrogen↗
PubMed2004

A third lineage with two-piece tmRNA.

tmRNA combines tRNA and mRNA properties and helps bacteria to cope with stalled ribosomes. Its termini normally pair in the tRNA domain, closing the mRNA portion into a looping domain. A striking variation is a two-piece form that effectively breaks open the mRNA domain loop, resulting from independent gene permutation events in alphaproteobacteria and cyanobacteria. Convergent evolution to a similar form in separate bacterial lineages suggests that loop-opening benefits tmRNA function. This argument is strengthened by the discovery of a third bacterial lineage with a loop-opened two-piece tmRNA. Whereas most betaproteobacteria have one-piece tmRNA, a permuted tmRNA gene was found for Dechloromonas aromatica and close relatives. Correspondingly, two tmRNA pieces were identified, at approximately equal abundance and at a level one-fifteenth that of ribosomes, a 189 nt mRNA piece and a 65 nt aminoacylatable piece. Together these pieces were active with purified Escherichia coli translational components, but not alone. The proposed secondary structure combines common tmRNA features differently from the structures of other two-piece forms. The origin of the gene is unclear; horizontal transfer may be indicated by the similarity of the tRNA domain to that from a cyanobacterial two-piece tmRNA, but such transfer would not appear simple since the mRNA domain is most similar to that of other betaproteobacteria.

Base Sequence↗
PubMed2004

Petrobacter succinatimandens gen. nov., sp. nov., a moderately thermophilic, nitrate-reducing bacterium isolated from an Australian oil well.

A novel Gram-negative, aerobic and moderately thermophilic bacterium, strain 4BON(T), was isolated from a non-water-flooded Australian terrestrial oil reservoir. Cells were non-spore-forming straight rods, which were motile by means of a polar flagellum. The optimum growth conditions were 55 degrees C, pH 6.9 and 0.5 % NaCl. Strain 4BON(T) was oxidase- and catalase-positive; it grew on fumarate, pyruvate, succinate, formate, ethanol and yeast extract in the presence of oxygen or nitrate as terminal electron acceptor. Nitrate was reduced to nitrous oxide. The DNA G+C content of the strain was 58.6 mol%. The closest phylogenetic relative of strain 4BON(T) was Hydrogenophilus thermoluteolus (similarity of 91.8 %), of the beta-Proteobacteria. As strain 4BON(T) is physiologically and phylogenetically different from H. thermoluteolus, it is proposed that it be assigned to a novel species of a novel genus, Petrobacter succinatimandens gen. nov., sp. nov. The type strain is 4BON(T) (=DSM 15512(T)=CIP 107790(T)).

Australia↗
PubMed2005

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.

Chlorates↗
PubMed2006

Azonexus caeni sp. nov., a denitrifying bacterium isolated from sludge of a wastewater treatment plant.

A polyphasic taxonomic study was carried out to determine the taxonomic position of a newly isolated denitrifying bacterium, designated Slu-05T, which had been isolated from sludge from the main aerobic treatment tanks of a municipal sewage treatment plant. Phylogenetic analysis based on comparative 16S rRNA gene sequencing indicated that strain Slu-05T was closely related to Azonexus fungiphilus LMG 19178T (96.4 % sequence similarity), the sole species in the genus Azonexus. Strain Slu-05T comprised Gram-negative, motile, non-spore-forming and slightly curved rods. The predominant respiratory lipoquinone was Q-8. The major fatty acids were C16:1omega7c, C16:0, C18:1 isomers and C10:0 3-OH. The G+C content of the genomic DNA was 65.6 mol%. The results of DNA-DNA hybridization (15.6 %) together with phenotypic determination showed that strain Slu-5T could be distinguished from A. fungiphilus. Moreover, some phenotypic properties concerning enzyme activity, the substrates utilized as carbon sources and growth conditions distinguish strain Slu-5T from A. fungiphilus. On the basis of the results obtained in this study, Slu-05T (=DSM 17719T=KCTC 12530T=CCBAU 10199T) is the type strain of a novel species of Azonexus, for which the name Azonexus caeni sp. nov. is proposed.

Bacterial Typing Techniques↗
PubMed2006

Which are the polyphosphate accumulating organisms in full-scale activated sludge enhanced biological phosphate removal systems in Australia?

AIMS: To see if the compositions of the microbial communities in full scale enhanced biological phosphorus removal activated sludge systems were the same as those from laboratory scale sequencing batch reactors fed a synthetic sewage. METHODS: Biomass samples taken from nine full scale enhanced biological phosphate removal (EBPR) activated sludge plants in the eastern states of Australia were analysed for their populations of polyphosphate (polyP)-accumulating organisms (PAO) using semi-quantitative fluorescence in situ hybridization (FISH) in combination with DAPI (4'-6-diamidino-2-phenylindole) staining for polyP. RESULTS: Very few betaproteobacterial Rhodocyclus related organisms could be detected by FISH in most of the plants examined, and even where present, not all these cells even within a single cluster, stained positively for polyP with DAPI. In some plants in samples from aerobic reactors the Actinobacteria dominated populations containing polyP. CONCLUSIONS: The PAO populations in full-scale EBPR systems often differ to those seen in laboratory scale reactors fed artificial sewage, and Rhodocyclus related organisms, dominating these latter communities may not be as important in full-scale systems. Instead Actinobacteria may be the major PAO. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings illustrate how little is still known about the microbial ecology of EBPR processes and that more emphasis should now be placed on analysis of full-scale plants if microbiological methods are to be applied to monitoring their performances.

Actinobacteria↗
PubMed2006

Ecophysiology of a group of uncultured Gammaproteobacterial glycogen-accumulating organisms in full-scale enhanced biological phosphorus removal wastewater treatment plants.

The presence of glycogen-accumulating organisms (GAOs) in enhanced biological phosphorus removal (EBPR) plants can seriously deteriorate the biological P-removal by out-competing the polyphosphate-accumulating organisms (PAOs). In this study, uncultured putative GAOs (the GB group, belonging to the Gammaproteobacteria) were investigated in detail in 12 full-scale EBPR plants. Fluorescence in situ hybridization (FISH) revealed that the biovolume of the GB bacteria constituted 2-6% of total bacterial biovolume. At least six different subgroups of the GB bacteria were found, and the number of dominant subgroups present in each plant varied between one and five. Ecophysiological investigations using microautoradiography in combination with FISH showed that, under aerobic or anaerobic conditions, all subgroups of the GB bacteria could take up acetate, pyruvate, propionate and some amino acids, while some subgroups in addition could take up formate and thymidine. Glucose, ethanol, butyrate and several other organic substrates were not taken up. Glycolysis was essential for the anaerobic uptake of organic substrates. Polyhydroxyalkanoates (PHA) but not polyphosphate (polyP) granules were detected in all GB bacterial cells. Polyhydroxyalkanoate formation after anaerobic uptake of acetate was confirmed by measuring the increase in fluorescence intensity of PHA granules inside GB bacterial cells after Nile blue staining. One GB subgroup was possibly able to denitrify, and several others were able to reduce nitrate to nitrite. PAOs were also enumerated by FISH in the same treatment plants. Rhodocyclus-related PAOs and Actinobacteria-related PAOs constituted up to 7% and 29% of total bacterial biovolume respectively. Rhodocyclus-related PAOs always coexisted with the GB bacteria and showed many physiological similarities. Factors of importance for the competition between the three groups of important bacteria in EBPR plants are discussed.

Acetates↗
PubMed2004

Aerobic and anaerobic toluene degradation by a newly isolated denitrifying bacterium, Thauera sp. strain DNT-1.

A newly isolated denitrifying bacterium, Thauera sp. strain DNT-1, grew on toluene as the sole carbon and energy source under both aerobic and anaerobic conditions. When this strain was cultivated under oxygen-limiting conditions with nitrate, first toluene was degraded as oxygen was consumed, while later toluene was degraded as nitrate was reduced. Biochemical observations indicated that initial degradation of toluene occurred through a dioxygenase-mediated pathway and the benzylsuccinate pathway under aerobic and denitrifying conditions, respectively. Homologous genes for toluene dioxygenase (tod) and benzylsuccinate synthase (bss), which are the key enzymes in aerobic and anaerobic toluene degradation, respectively, were cloned from genomic DNA of strain DNT-1. The results of Northern blot analyses and real-time quantitative reverse transcriptase PCR suggested that transcription of both sets of genes was induced by toluene. In addition, the tod genes were induced under aerobic conditions, whereas the bss genes were induced under both aerobic and anaerobic conditions. On the basis of these results, it is concluded that strain DNT-1 modulates the expression of two different initial pathways of toluene degradation according to the availability of oxygen in the environment.

Aerobiosis↗
PubMed2004

Microautoradiographic study of Rhodocyclus-related polyphosphate-accumulating bacteria in full-scale enhanced biological phosphorus removal plants.

The ecophysiology of uncultured Rhodocyclus-related polyphosphate-accumulating organisms (PAO) present in three full-scale enhanced biological phosphorus removal (EBPR) activated sludge plants was studied by using microautoradiography combined with fluorescence in situ hybridization. The investigations showed that these organisms were present in all plants examined and constituted 5 to 10, 10 to 15, and 17 to 22% of the community biomass. The behavior of these bacteria generally was consistent with the biochemical models proposed for PAO, based on studies of lab-scale investigations of enriched and often unknown PAO cultures. Rhodocyclus-related PAO were able to accumulate short-chain substrates, including acetate, propionate, and pyruvate, under anaerobic conditions, but they could not assimilate many other low-molecular-weight compounds, such as ethanol and butyrate. They were able to assimilate two substrates (e.g., acetate and propionate) simultaneously. Leucine and thymidine could not be assimilated as sole substrates and could only be assimilated as cosubstrates with acetate, perhaps serving as N sources. Glucose could not be assimilated by the Rhodocyclus-related PAO, but it was easily fermented in the sludge to products that were subsequently consumed. Glycolysis, and not the tricarboxylic acid cycle, was the source that provided the reducing power needed by the Rhodocyclus-related PAO to form the intracellular polyhydroxyalkanoate storage compounds during anaerobic substrate assimilation. The Rhodocyclus-related PAO were able to take up orthophosphate and accumulate polyphosphate when oxygen, nitrate, or nitrite was present as an electron acceptor. Furthermore, in the presence of acetate growth was sustained by using oxygen, as well as nitrate or nitrite, as an electron acceptor. This strongly indicates that Rhodocyclus-related PAO were able to denitrify and thus played a role in the denitrification occurring in full-scale EBPR plants.

In Situ Hybridization, Fluorescence↗