Genes essential for phototrophic growth by a purple alphaproteobacterium

Tn‐seq was used to identify genes essential for phototrophic growth by the purple bacterium Rhodopseudomonas palustris. About 167 genes required for anaerobic growth on acetate in light were identified, 35 of which are annotated as photosynthesis genes. The essentiality of many of these genes by analysing the phenotypes of independently generated mutants that had altered pigmentation was verified. Three genes were identified, two possibly involved in biogenesis of the membrane‐bound photosynthetic apparatus and one for phosphatidylcholine biosynthesis, that were not known to be essential for phototrophic growth. Site‐directed mutagenesis was used to show that the NADH:quinone oxidoreductase complex I_E was essential for phototrophic growth under strictly anaerobic conditions and appeared to play a role in reverse electron transport to generate NADH. A homologous NADH:quinone oxidoreductase complex I_A likely operates in the opposite direction to oxidize NADH. The operation of the two enzymes in opposition would allow R. palustris to maintain redox balance. As a complement to the genetic data, proteomics experiments were carried out in which it was found that 408 proteins were present in significantly higher amounts in cells grown anaerobically in light compared with aerobically. Among these were proteins encoded by subset of the phototrophic growth‐essential genes.     

Influence of electron acceptor,carbon, nitrogen,and phosphorus on polyhydroxyalkanoate (PHA) production by <Emphasis Type="Italic">Brachymonas</Emphasis> sp. P12

Polyhydroxyalkanoates (PHAs), intracellular carbon and energy reserve compounds in many bacteria, have been used extensively in biodegradable plastics. PHA formation is influenced by nutrient limitations and growth conditions. To characterize the PHA accumulation in a new denitrifying phosphorus-removing bacterium Brachymonas sp. P12, batch experiments were conducted in which the electron acceptor (oxygen or nitrate) was varied and different concentrations of carbon (acetate), nitrogen (NH₄Cl), and phosphorus (KH₂PO₄) were used. Polyhydroxybutyrate (PHB) was the dominant product during PHA formation when acetate was the sole carbon source. The PHB content of aerobically growing cells increased from 431 to 636 mg PHB g⁻¹ biomass, but the PHB concentration of an anoxic culture decreased (−218 mg PHB g⁻¹ biomass), when PHB was utilized simultaneously with acetate as an electron donor for anoxic denitrification. The specific PHB production rate of the carbon-limited batch, 158.2 mg PHB g⁻¹ biomass h⁻¹, was much greater than that of batches with normal or excess carbon. The effects of phosphorus and nitrogen concentrations on PHB accumulation were clearly less than the effect of carbon concentration. According to the correlation between the specific PHB production rate and the specific cell growth rate, PHB accumulation by Brachymonas sp. P12 is enhanced by nutrient limitation, is growth-associated, and provides additional energy for the biosynthesis of non-PHB cell constituents to increase the cell growth rate beyond the usual level.     

Metabolism of 2,4,6-trinitrotoluene (TNT) by Desulfovibrio sp. (B strain)

A sulfate-reducing bacterium, Desulfovibrio sp. (B strain) isolated from an anaerobic reactor treating furfural-containing waste-water was studied for its ability to metabolize trinitrotoluene (TNT). The result showed that this isolate could transform 100 ppm TNT within 7 to 10 days of incubation at 37°C, when grown with 30 mm pyruvate as the primary carbon source and 20 mm sulfate as electron acceptor. Under these conditions, the main intermediate produced was 2,4-diamino-6-nitrotoluene. Under culture conditions where TNT served as the sole source of nitrogen for growth with pyruvate as electron donor and sulfate as electron acceptor, TNT was first converted to 2,4-diamino-6-nitrotoluene within 10 days of incubation. This intermediate was further converted to toluene by a reductive deamination process via triaminotoluene. Apart from pyruvate, various other carbon sources such as ethanol, lactate, formate and H₂ + CO₂ were also studied as potential electron donors for TNT metabolism. The rate of TNT biotransformation by Desulfovibrio sp. (B strain) was compared with other sulfate-reducing bacteria and the results were evaluated. This new strain may be useful in decontaminating TNT-contaminated soil and water under anaerobic conditions in conjunction with toluene-degrading denitrifiers (Pseudomonas spp.) or toluene-degrading sulfate reducers in a mixed culture system. Correspondence to: R. Boopathy     

Desulfotomaculum geothermicum sp. nov., a thermophilic,fatty acid-degrading,sulfate-reducing bacterium isolated with H2 from geothermal ground water

A strictly anaerobic, thermophilic, fatty acids-degrading, sporulating sulfate-reducing bacterium was isolated from geothermal ground water. The organism stained Gram-negative and formed gas vacuoles during sporulation. Lactate, ethanol, fructose and saturated fatty acids up to C₁₈ served as electron donors and carbon sources with sulfate as external electron acceptor. Benzoate was not used. Stoichiometric measurements revealed a complete oxidation of part of butyrate although growth with acetate as only electron donor was not observed. The rest of butyrate was oxidized to acetate. The strain grew chemolithoautotrophically with hydrogen plus sulfate as energy source and carbon dioxide as carbon source without requirement of additional organic carbon like acetate. The strain contained a c-type cytochrome and presumably a sulfite reductase P582. Optimum temperature, pH and NaCl concentration for growth were 54°C, pH 7.3–7.5 and 25 to 35 g NaCl/l. The G+C content of DNA was 50.4 mol %. Strain BSD is proposed as a new species of the spore-forming sulfate-reducing genus Desulfotomaculum, D. geothermicum.     

Effects of electron donor and microbial concentration on the enhanced dechlorination of carbon tetrachloride by anaerobic consortia

 Reductive dechlorination of carbon tetra-chloride (CCl₄) by anaerobic bacterial communities from anaerobic digester sludge with the amendment of low concentrations of electron donors and microorganisms was undertaken to evaluate the influence of electron donors and microbial concentration on the rate of dechlorination of CCl₄. Humic acid, acetate, and glucose were selected to examine the feasibility of the electron donor with respect to the remediation of a contaminated subsurface. The addition of an electron donor and microorganisms significantly enhanced the dechlorination rate of carbon tetrachloride. The addition of an electron donor increased the cell numbers of anaerobic consortia, thereby increasing the rate of dechlorination. Glucose was a better electron donor than acetate and humic acid under reducing environments. The pseudo-first-order degradation rate constants of CCl₄ ranged from 0.0057 day^-1 to 0.135 day^-1, depending on the conditions of the electron donor and biomass supplemented. Furthermore, the addition of the electron donor in the batches amended with 0.56 mg volatile suspended solids (VSS)/l biomass had a higher enhanced efficiency than those with 1.7 mg VSS/l biomass. These results suggest that there is a potential for stimulating the dechlorinating capability of anaerobic consortia to remedy the chlorinated hydrocarbons in the oligotrophic environment if the conditions of the supplementing electron donor are properly selected. Received: 14 August 1995/Received last revision: 15 March 1996/Accepted: 15 April 1996     

Fermentative degradation of dipicolinic acid (pyridine-2,6-dicarboxylic acid) by a defined coculture of strictly anaerobic bacteria

Degradation of dipicolinic acid (pyridine-2,6-dicarboxylic acid) under strictly anaerobic conditions was studied in enrichment cultures from marine and freshwater sediments. In all cases, dipicolinic acid was completely degraded. From an enrichment culture from a marine sediment, a defined coculture of two bacteria was isolated. The dipicolinic acid-fermenting bacterium was a Gram-negative, non-sporeforming strictly anaerobic short rod which utilized dipicolinic acid as sole source of carbon, energy, and nitrogen, and fermented it to acetate, propionate, ammonia, and 2CO₂. No other substrate was fermented. This bacterium could be cultivated only in coculture with another Gram-negative, non-sporeforming rod from the same enrichment culture which oxidized acetate to CO₂ with fumarate, malate, or elemental sulfur as electron acceptor, similar to Desulfuromonas acetoxidans. Since this metabolic activity is not important in substrate degradation by the coculture, the basis of the dependence of the dipicolinic acid-degrading bacterium on the sulfur reducer may be sought in the assimilatory metabolism.     

Effects of substrate limitation on product distribution and H2/CO2 ratio inKlebsiella pneumoniae during anaerobic fermentation of glycerol

Product formation during anaerobic degradation of glycerol byKlebsiella pneumoniae DSM 2026, under glycerol limitation and glycerol excess in continugius cultures, has been investigated. Major and minor products and by-products as well as gaseous products were measured. The results indicated a positive correlation between specific glycerol uptake and most product formation rates under glycerol limitation. The production of 1,3-propanediol, lactate, formate, acetate, succinate and the by-products of anaerobic glycerol degradation byK. pneumoniae, acetoin and 2,3-butanediol, was favoured by glycerol excess, while hydrogen generation and ethanol formation were best under glycerol limitation. It was also found that under glycerol limitation the rate of hydrogen evolution was generally higher than the CO₂ production rate while under excess glycerol the reverse was true. Hence, on the basis of the ratio of the specific rates of evolution of H₂ and CO₂ (q _{H 2}/q _{CO 2}), it is possible to infer the existence of glycerol limitation. On the basis of the carbon and available electron balances, which are independent of metabolic pathways, the data are consistent. The NADH₂ balance, which took into consideration the pathways of product formation, was also tested to check the validity of the assumed pathways and to check critically the consistency of the data. Good balances were also obtained.[     

Growth of Rhodopseudomonas palustris under phototrophic and non-phototrophic conditions and its CO-dependent H2 production

A new hydrogen producing bacterium, Rhodopseudomonas palustris P4, originally isolated under an anaerobic/phototrophic condition, grew well under aerobic/chemoheterotrophic or anaerobic/chemoheterotrophic conditions and showed CO-dependent, H₂ production activity when transferred to anaerobic conditions. Cell growth was best under an aerobic/chemoheterotrophic condition as the doubling time of 1 h, while the H₂ production activity was highest in the cells grown under an aerobic/chemoheterotrophic condition at 20 mmol g^–1 cell^–1 h^–1.     

The path of electron transfer to nitrogenase in a phototrophic alpha‐proteobacterium

The phototrophic alpha‐proteobacterium, Rhodopseudomonas palustris, is a model for studies of regulatory and physiological parameters that control the activity of nitrogenase. This enzyme produces the energy‐rich compound H₂, in addition to converting N₂ gas to NH₃. Nitrogenase is an ATP‐requiring enzyme that uses large amounts of reducing power, but the electron transfer pathway to nitrogenase in R. palustris was incompletely known. Here, we show that the ferredoxin, Fer1, is the primary but not sole electron carrier protein encoded by R. palustris that serves as an electron donor to nitrogenase. A flavodoxin, FldA, is also an important electron donor, especially under iron limitation. We present a model where the electron bifurcating complex, FixABCX, can reduce both ferredoxin and flavodoxin to transfer electrons to nitrogenase, and we present bioinformatic evidence that FixABCX and Fer1 form a conserved electron transfer pathway to nitrogenase in nitrogen‐fixing proteobacteria. These results may be useful in the design of strategies to reroute electrons generated during metabolism of organic compounds to nitrogenase to achieve maximal activity.     

Bioconversion of synthesis gas to hydrogen using a light-dependent photosynthetic bacterium,Rhodospirillum rubrum

Biological hydrogen production from synthesis gas was carried out in batch culture. The phototrophic anaerobic bacterium, Rhodospirillum rubrum was used to oxidize CO and water to CO₂ and hydrogen. The bacteria were grown under anaerobic conditions in liquid medium; also acetate was used as carbon source in presence of synthesis gas. Biological hydrogen production was catalysed by R. rubrum via the water–gas shift reaction. A light-dependent cell growth modelled with a desired rate of hydrogen production and CO uptake was determined. The effect of light intensity on microbial cell growth was also studied at 500, 1,000 and 1,500 m.cd. A complete conversion of CO to hydrogen and maximum light efficiency were obtained with an acetate concentration of 1 g/l and light intensity of 500 m.cd. Utilization of the carbon monoxide from the gas phase was often considered as a mass transfer limited process, which needed to diffuse through the gas–liquid interface and then further diffuse into liquid medium prior to reaction. The results from this study showed that maximum cell propagation and hydrogen production were achieved with a limited light intensity of 1,000 m.cd. It was also found that high-light intensity may interfere with cell metabolism. In low-light intensity and substrate concentration, no inhibition was observed, however at extreme conditions, non-competitive inhibition was identified. The adverse effect of high-light intensity was shown at 5,000 m.cd, where the CO conversion drastically dropped to as low as 21%. Maximum CO conversion of 98% and maximum yield of 86% with an acetate concentration of 1.5 g/l and a light intensity of 1,000 m.cd were achieved.     

Growth‐independent cross‐feeding modifies boundaries for coexistence in a bacterial mutualism

Nutrient cross‐feeding can stabilize microbial mutualisms, including those important for carbon cycling in nutrient‐limited anaerobic environments. It remains poorly understood how nutrient limitation within natural environments impacts mutualist growth, cross‐feeding levels and ultimately mutualism dynamics. We examined the effects of nutrient limitation within a mutualism using theoretical and experimental approaches with a synthetic anaerobic coculture pairing fermentative Escherichia coli and phototrophic Rhodopseudomonas palustris. In this coculture, E. coli and R. palustris resemble an anaerobic food web by cross‐feeding essential carbon (organic acids) and nitrogen (ammonium) respectively. Organic acid cross‐feeding stemming from E. coli fermentation can continue in a growth‐independent manner during nitrogen limitation, while ammonium cross‐feeding by R. palustris is growth‐dependent. When ammonium cross‐feeding was limited, coculture trends changed yet coexistence persisted under both homogenous and heterogenous conditions. Theoretical modelling indicated that growth‐independent fermentation was crucial to sustain cooperative growth under conditions of low nutrient exchange. In contrast to stabilization at most cell densities, growth‐independent fermentation inhibited mutualistic growth when the E. coli cell density was adequately high relative to that of R. palustris. Thus, growth‐independent fermentation can conditionally stabilize or destabilize a mutualism, indicating the potential importance of growth‐independent metabolism for nutrient‐limited mutualistic communities.     

Isotopic and chemical investigations of Fe (III) reduction by Shewanella putrefaciens strain 200R

Experiments were conducted using the Fe⁺³‐reducing bacterium Shewanella putrefaciens strain 200R to determine the stable carbon isotope fractionation during dissimilatory Fe (III) reduction and associated lactate oxidation at circum‐neutral pH. Previous studies used equilibrium fractionation factors (~14.3‰) between bacterial biomass and synthesized fatty acids to identify the predominant carbon fixation pathways for some of the most frequently isolated microbes including Shewanella under anaerobic conditions. We investigated the carbon isotope disproportionation among organic carbon substrate (lactate), biomass and respired carbon dioxide at the lag to stationary phase of the growth curve. Ferric citrate and sodium lactate were used as electron acceptor and donor, respectively. Sodium bicarbonate or potassium phosphate was used as buffering agent. Iron (II), iron (III), dissolved inorganic carbon (DIC) and carbon isotope ratios were measured for both bicarbonate‐ and phosphate‐buffered systems. Carbon isotope ratio measurements were made on the respired CO₂ (as DIC) and microbial biomass for both buffering conditions. The fraction of lactate consumed was estimated using DIC as a proxy and was verified by direct measurement using HPLC. Our result showed that bicarbonate‐buffered system has an enhancing effect in the reduction process compared to the phosphate system. Both systems resulted in carbon isotope fractionations between the lactate substrate and DIC that could be modelled as a Rayleigh process. The biomass produced under both buffer conditions was depleted on average by ~2‰ relative to the substrate and enriched by ~5‰ relative to the DIC. This translates to an overall isotopic fractionation of 10–12‰ between the biomass and respired CO₂ in both buffering systems.     

Microautoradiographic studies on host-parasite interactions II. The exchange of 3H-lysine between Uromyces phaseoli and Phaseolus vulgaris

A new species of anaerobic bacterium that degrades the even-numbered carbon fatty acids, butyrate, caproate and caprylate, to acetate and H₂ and the odd-numbered carbon fatty acids, valerate and heptanoate, to acetate, propionate and H₂ was obtained in coculture with either an H₂-utilizing methanogen or H₂-utilizing desulfovibrio. The organism could be grown only in syntrophic association with the H₂-utilizer and no other energy sources or combination of electron donor and acceptors were utilized. It was a Gram-negative helical rod with 2 to 8 flagella, about 20 nm in diameter, inserted in a linear fashion about 130 nm or more apart along the concave side of the cell. It grew with a generation time of 84 h in co-culture with Methanospirillum hungatii and was present in numbers of at least 4.5×10^-6 per g of anaerobic digestor sludge.     

Growth of Rhodopseudomonas capsulata under anaerobic dark conditions with dimethyl sulfoxide

The photosynthetic bacterium, Rhodopseudomonas capsulata, could be cultured anaerobically in the absence of light on a synthetic medium with glucose as the carbon source only when dimethyl sulfoxide (DMSO) was added. The extent of growth was proportional to both DMSO and glucose concentrations. Optimal growth was achieved with 20 mm DMSO and 0.25% glucose. Under the best conditions, cells divided with a doubling time of 12 h. Pyruvate also supported the anaerobic dark growth of R. capsulata when DMSO was present. R. capsulata, R. sphaeroides, and R. palustris strains were all able to grow under anaerobic dark conditions with DMSO. Experiments using [¹⁴C]DMSO showed that more than 95% of the ¹⁴C was converted by cultures of R. capsulata to a volatile compound, identified as dimethyl sulfide (DMS) by gas chromatography, thus demonstrating that DMSO was being reduced to DMS during growth. These results indicate that R. capsulata requires a terminal electron acceptor for anaerobic dark growth and that DMSO can serve that function.     

Bacterial dissimilatory MnO2 reduction at extremely haloalkaline conditions

A possibility of dissimilatory MnO₂ reduction at extremely high salt and pH was studied in sediments from hypersaline alkaline lakes in Kulunda Steppe (Altai, Russia). Experiments with anaerobic sediment slurries demonstrated a relatively rapid reduction of colloidal MnO₂ in the presence of acetate and formate as electron donor at in situ conditions (i.e., pH 10 and a salt content from 0.6 to 4 M total Na⁺). All reduced Mn at these conditions remained in the solid phase. A single, stable enrichment culture was obtained from the slurries consistently reducing MnO₂ at pH 10 and 0.6 M total Na⁺ with formate. A pure culture of a haloalkaliphilic Mn-reducing bacterium obtained from the positive enrichment was phylogenetically closely related to the anaerobic haloalkaliphilic Bacillus arseniciselenatis isolated from Mono Lake (CA, USA). Bacillus sp. strain AMnr1 was obligately anaerobic, able to grow either by glucose fermentation, or respiring few nonfermentable substrates by using MnO₂ as the electron acceptor. Optimal growth by dissimilatory MnO₂ reduction was achieved with glycerol as electron donor at pH 9.5–10 and salt content between 0.4 and 0.8 M total Na⁺.     

Anaerobic dechlorination of carbon tetrachloride by free-living and attached bacteria under various electron-donor conditions

The dechlorination of carbon tetrachloride (CCl₄) by free-living and attached bacteria under anaerobic conditions was studied to examine the relationship between porous media and electron donor. Two batch-type experiments, the free-living and attached bacterial systems, were conducted with and without addition of 0.5-mm glass beads. Glucose and acetate were selected as the primary electron donors because they are easily biodegradable. Direct epifluorescence technology, the DAPI (4′ 6-diamidino-2-phenylindole) method, was used for counting the microbial activities. Adding glass beads could accelerate the dechlorination rate of CCl₄. Removals of 44 %–57 % were observed in free-living bacterial system. Whereas a two- to fivefold increase in the CCl₄ dechlorination rate was observed in the attached system. Experimental results and thermodynamic calculations indicated that glucose is a better supplementary substrate than acetate for stimulating the dechlorinating capability of microorganisms because of its relatively high available free energy. A higher concentration of substrate provided more reducing power for attached bacteria to initiate the dechlorination reaction. The pseudo-first-order rate constants of CCl₄ dechlorination ranged from 0.007 day⁻¹ to 0.017 day⁻¹ and from 0.011 day⁻¹ to 0.0625 day⁻¹ for free-living and attached bacterial systems respectively. Microscopic observation revealed a three- to eightfold difference of microbial number between the free-living and attached bacterial systems. On the basis of the results in this study, we can conclude that the presence of porous media and an electron donor can change the dechlorination capabilities of the microorganisms. This work will be valuable in the design of in situ bioremediation as it discusses the specific area of the medium and supplementation with an electron donor to stimulate the indigenous microflora. Received: 21 June 1996 / Received revision: 2 September 1996 / Accepted: 29 September 1996     

Ferribacterium limneticum, gen. nov., sp. nov., an Fe(III)-reducing microorganism isolated from mining-impacted freshwater lake sediments

A dissimilatory Fe(III)-reducing bacterium was isolated from mining-impacted lake sediments and designated strain CdA-1. The strain was isolated from a 4-month enrichment culture with acetate and Fe(III)-oxyhydroxide. Strain CdA-1 is a motile, obligately anaerobic rod, capable of coupling the oxidation of acetate and other organic acids to the reduction of ferric iron. Fe(III) reduction was not observed using methanol, ethanol, isopropanol, propionate, succinate, fumarate, H₂, citrate, glucose, or phenol as potential electron donors. With acetate as an electron donor, strain CdA-1 also grew by reducing nitrate or fumarate. Growth was not observed with acetate as electron donor and O₂, sulfoxyanions, nitrite, trimethylamine N-oxide, Mn(IV), As(V), or Se(VI) as potential terminal electron acceptors. Comparative 16 S rRNA gene sequence analyses show strain CdA-1 to be most closely related (93.6% sequence similarity) to Rhodocyclus tenuis. However, R. tenuis did not grow heterotrophically by Fe(III) reduction, nor did strain CdA-1 grow photrophically. We propose that strain CdA-1 represents a new genus and species, Ferribacterium limneticum. Strain CdA-1 represents the first dissimilatory Fe(III) reducer in the β subclass of Proteobacteria, as well as the first Fe(III) reducer isolated from mine wastes. Received: 14 July 1998 / Accepted: 14 December 1998     

Anaerobic Growth of Microorganisms with Chlorate as an Electron Acceptor

The ability of microorganisms to use chlorate (ClO₃^-) as an electron acceptor for respiration under anaerobic conditions was studied in batch and continuous tests. Complex microbial communities were cultivated anaerobically in defined media containing chlorate, all essential minerals, and acetate as the sole energy and carbon source. It was shown that chlorate was reduced to chloride, while acetate was oxidized to carbon dioxide and water and used as the carbon source for synthesis of new biomass. A biomass yield of 1.9 to 3.8 g of volatile suspended solids per equivalent of available electrons was obtained, showing that anaerobic growth with chlorate as an electron acceptor gives a high energy yield. This indicates that microbial reduction of chlorate to chloride in anaerobic systems is coupled with electron transport phosphorylation.     

Effect of different carbon sources on the enhanced biological phosphorus removal in a sequencing batch reactor

The effect of the different carbon sources acetate, acetate/glucose or glucose on the enhanced biological phosphorus removal (EBPR) process was studied by experiments under alternating anaerobic–aerobic conditions in one sequencing batch reactor for each carbon source. The glucose was consumed completely within the first 30 min of the anaerobic phase whereas acetate degradation was slow and incomplete. Phosphate was released independently of the carbon source during the whole anaerobic phase. The highest phosphate release (27 mg P l⁻¹) and polyhydroxyalkanoate (PHA) storage (20 mg C g⁻¹ dry matter (DM)) during the anaerobic phase as well as the highest polyphosphate (poly-P) (8 mg P g⁻¹ DM) and glycogen storage (17 mg C g⁻¹ DM) during the aerobic phase were observed with acetate. In contrast to other investigations, glycogen storage did not increase with glucose as substrate but was significantly smaller than with acetate. The PHA composition was also influenced strongly by the carbon source. The polyhydroxyvalerate (PHV) portion of the PHA was maximal 17% for acetate and 82% for glucose. Due to the strong influence of the carbon source on the PHA concentration and composition, PHA storage seems to regulate mainly the phosphate release and uptake. This revised version was published online in July 2006 with corrections to the Cover Date.     

Anaerobic mineralization of cholesterol by a novel type of denitrifying bacterium

A novel denitrifying bacterium, strain 72Chol, was enriched and isolated under strictly anoxic conditions on cholesterol as sole electron donor and carbon source. Strain 72Chol grew on cholesterol with oxygen or nitrate as electron acceptor. Strictly anaerobic growth in the absence of oxygen was demonstrated using chemically reduced culture media. During anaerobic growth, nitrate was initially reduced to nitrite. At low nitrate concentrations, nitrite was further reduced to nitrogen gas. Ammonia was assimilated. The degradation balance measured in cholesterol-limited cultures and the amounts of carbon dioxide, nitrite, and nitrogen gas formed during the microbial process indicated a complete oxidation of cholesterol to carbon dioxide. A phylogenetic comparison based on total 16S rDNA sequence analysis indicated that the isolated micro-organism, strain 72Chol, belongs to the β2-subgroup in the Proteobacteria and is related to Rhodocyclus, Thauera, and Azoarcus species. Received: 16 July 1996 / Accepted: 5 December 1996