Isolation,Characterization and Kinetics of Perchlorate Reducing Magnetospirillum Species

Perchlorate reducing bacteria reduce perchlorate to chlorate (ClO₃?), which, in turn, is reduced to chlorite (ClO₂?) and ultimately to chloride (Cl^?). Magnetospirillum strains are reported to use chlorate/perchlorate as electron acceptors. This study describes the perchlorate reducing property of strain VITRJS5, a Magnetopsirillum isolated from freshwater sediment collected from Chelur freshwater lake, Kerala, India. The strain was microaerophile and was phylogenetically related to a Magnetospirillum sp., a member of the α-subclass of the class Proteobacteria. The placement of the isolate in the genus Magnetospirillum has further confirmed the presence of four key magnetosome membrane genes. PCR amplification and phylogenetic analysis of central metabolic genes such as nifH (nitrogenase) and cbbM (type II RubisCo) displayed the highest similarity (97% and 81%, respectively) with Magnetospirillum sp. BB-1 The growth kinetic parameters of the isolate were studied with acetate as the electron donor in batch experiments. Monod's substrate utilization model has been established with oxygen, nitrate and perchlorate as electron acceptors separately. The maximum specific growth rate (µ_max) and half-saturation constant (k_s^conc) for the bacterium varied while utilizing different electron acceptors. The maximum specific growth rate was 0.226, 0.190 and 0.096 per hour and half-velocity constant K_s was 25.09, 33.36 and 65.37 mg acetate/l for oxygen, nitrate and perchlorate, respectively. The reduction of perchlorate has been analyzed using kinetic studies of the substrate uptake by the bacteria and the half-velocity constant K_s was found to be 52.8 mg/l. The results indicate that the strain VITRJS5 effectively reduces perchlorate by using it as an electron acceptor.     

Limitations to Natural Bioremediation of Perchlorate in a Contaminated Site

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

Degradation of phenol and phenolic compounds by a defined denitrifying bacterial culture

Phenol, a major pollutant in several industrial waste waters is often used as a model compound for studies on biodegradation. This study investigated the anoxic degradation of phenol and other phenolic compounds by a defined mixed culture of Alcaligenes faecalis and Enterobacter species. The culture was capable of degrading high concentrations of phenol (up to 600 mg/l) under anoxic conditions in a simple minimal mineral medium at an initial cell mass of 8 mg/l. However, the lag phase in growth and phenol removal increased with increase in phenol concentration. Dissolved CO₂ was an absolute requirement for phenol degradation. In addition to nitrate, nitrite and oxygen could be used as electron acceptors. The kinetic constants, maximum specific growth rate _max; inhibition constant, K _i and saturation constant, K _s were determined to be 0.206 h^–1, 113 and 15 mg phenol/l respectively. p-Hydroxybenzoic acid was identified as an intermediate during phenol degradation. Apart from phenol, the culture utilized few other monocyclic aromatic compounds as growth substrates. The defined culture has remained stable with consistent phenol-degrading ability for more than 3 years and thus shows promise for its application in anoxic treatment of industrial waste waters containing phenolic compounds.     

Transformation of (per)chlorate into chloride by a newly isolated bacterium: reduction and dismutation

 Bacterial strain GR-1 was isolated from activated sludge for its ability to oxidize acetate with perchlorate as electron acceptor. Sequencing of 16S rDNA revealed the isolate to belong to the β subgroup of Proteobacteria. When strain GR-1 was grown on acetate and perchlorate, the release of chloride was proportional to the disappearance of perchlorate, showing that this compound was completely reduced. In addition to perchlorate, strain GR-1 used chlorate, oxygen, nitrate and Mn(IV) as electron acceptor. The oxidation of acetate is coupled to the reduction of perchlorate and chlorate, whereas chlorite reduction is not affected by the addition of acetate. Strain GR-1 disproportionates chlorite into molecular oxygen and chloride. As a consequence, the strain oxidizes acetate by simultaneously reducing perchlorate to chlorite and molecular oxygen to water. Comparison of growth yields with oxygen, chlorate and perchlorate and calculated ΔG ^0′ values confirms this finding. Received: 26 June 1995/Received revision: 11 October 1995/Accepted: 16 October 1995     

New insights into the energy metabolism and taxonomy of Deferribacteres revealed by the characterization of a new isolate from a hypersaline microbial mat

Strain L21-Ace-BES^T, isolated from a lithifying cyanobacterial mat, could be assigned to a novel species and genus within the class Deferribacteres. It is an important model organism for the study of anaerobic acetate degradation under hypersaline conditions. The metabolism of strain L21-Ace-BES^T was characterized by biochemical studies, comparative genome analyses, and the evaluation of gene expression patterns. The central metabolic pathway is the citric acid cycle, which is mainly controlled by the enzyme succinyl-CoA:acetate-CoA transferase. The potential use of a reversed oxidative citric acid cycle to fix CO₂ has been revealed through genome analysis. However, no autotrophic growth was detected in this strain, whereas sulfide and H₂ can be used mixotrophically. Preferred electron acceptors for the anaerobic oxidation of acetate are nitrate, fumarate and dimethyl sulfoxide, while oxygen can be utilized only under microoxic conditions. Aerotolerant growth by fermentation was observed at higher oxygen concentrations. The redox cycling of sulfur/sulfide enables the generation of reducing power for the assimilation of acetate during growth and could prevent the over-reduction of cells in stationary phase. Extracellular electron transfer appears to be an essential component of the respiratory metabolism in this clade of Deferribacteres and may be involved in the reduction of nitrite to ammonium.     

Behavioral response of dissimilatory perchlorate-reducing bacteria to different electron acceptors

The response behavior of three dissimilatory perchlorate-reducing bacteria to different electron acceptors (nitrate, chlorate, and perchlorate) was investigated with two different assays. The observed response was species-specific, dependent on the prior growth conditions, and was inhibited by oxygen. We observed attraction toward nitrate when Dechloromonas aromatica strain RCB and Azospira suillum strain PS were grown with nitrate. When D. aromatica and Dechloromonas agitata strain CKB were grown with perchlorate, both responded to nitrate, chlorate, and perchlorate. When A. suillum was grown with perchlorate, the organism responded to chlorate and perchlorate but not nitrate. A gene replacement mutant in the perchlorate reductase subunit (pcrA) of D. aromatica resulted in a loss of the attraction response toward perchlorate but had no impact on the nitrate response. Washed-cell suspension studies revealed that the perchlorate grown cells of D. aromatica reduced both perchlorate and nitrate, while A. suillum cells reduced perchlorate only. Based on these observations, energy taxis was proposed as the underlying mechanism for the responses to (per)chlorate by D. aromatica. To the best of our knowledge, this study represents the first investigation of the response behavior of perchlorate-reducing bacteria to environmental stimuli. It clearly demonstrates attraction toward chlorine oxyanions and the unique ability of these organisms to distinguish structurally analogous compounds, nitrate, chlorate, and perchlorate and respond accordingly.     

Kinetics of Perchlorate- and Chlorate-Respiring Bacteria

Ten chlorate-respiring bacteria were isolated from wastewater and a perchlorate-degrading bioreactor. Eight of the isolates were able to degrade perchlorate, and all isolates used oxygen and chlorate as terminal electron acceptors. The growth kinetics of two perchlorate-degrading isolates, designated “Dechlorosoma” sp. strains KJ and PDX, were examined with acetate as the electron donor in batch tests. The maximum observed aerobic growth rates of KJ and PDX (0.27 and 0.28 h⁻¹, respectively) were only slightly higher than the anoxic growth rates obtained by these isolates during growth with chlorate (0.26 and 0.21 h⁻¹, respectively). The maximum observed growth rates of the two non-perchlorate-utilizing isolates (PDA and PDB) were much higher under aerobic conditions (0.64 and 0.41 h⁻¹, respectively) than under anoxic (chlorate-reducing) conditions (0.18 and 0.21 h⁻¹, respectively). The maximum growth rates of PDX on perchlorate and chlorate were identical (0.21 h⁻¹) and exceeded that of strain KJ on perchlorate (0.14 h⁻¹). Growth of one isolate (PDX) was more rapid on acetate than on lactate. There were substantial differences in the half-saturation constants measured for anoxic growth of isolates on acetate with excess perchlorate (470 mg/liter for KJ and 45 mg/liter for PDX). Biomass yields (grams of cells per gram of acetate) for strain KJ were not statistically different in the presence of the electron acceptors oxygen (0.46 ± 0.07 [n = 7]), chlorate (0.44 ± 0.05 [n = 7]), and perchlorate (0.50 ± 0.08 [n = 7]). These studies provide evidence that facultative microorganisms with the capability for perchlorate and chlorate respiration exist, that not all chlorate-respiring microorganisms are capable of anoxic growth on perchlorate, and that isolates have dissimilar growth kinetics using different electron donors and acceptors.     

Autotrophic, Hydrogen-Oxidizing, Denitrifying Bacteria in Groundwater, Potential Agents for Bioremediation of Nitrate Contamination

Addition of hydrogen or formate significantly enhanced the rate of consumption of nitrate in slurried core samples obtained from an active zone of denitrification in a nitrate-contaminated sand and gravel aquifer (Cape Cod, Mass.). Hydrogen uptake by the core material was immediate and rapid, with an apparent K_m of 0.45 to 0.60 μM and a V_max of 18.7 nmol cm^-3 h^-1 at 30°C. Nine strains of hydrogen-oxidizing denitrifying bacteria were subsequently isolated from the aquifer. Eight of the strains grew autotrophically on hydrogen with either oxygen or nitrate as the electron acceptor. One strain grew mixotrophically. All of the isolates were capable of heterotrophic growth, but none were similar to Paracoccus denitrificans, a well-characterized hydrogen-oxidizing denitrifier. The kinetics for hydrogen uptake during denitrification were determined for each isolate with substrate depletion progress curves; the K_ms ranged from 0.30 to 3.32 μM, with V_maxs of 1.85 to 13.29 fmol cell^-1 h^-1. Because these organisms appear to be common constituents of the in situ population of the aquifer, produce innocuous end products, and could be manipulated to sequentially consume oxygen and then nitrate when both were present, these results suggest that these organisms may have significant potential for in situ bioremediation of nitrate contamination in groundwater.     

The Soil Flagellate Heteromita globosa Accelerates Bacterial Degradation of Alkylbenzenes through Grazing and Acetate Excretion in Batch Culture

The impact of grazing by soil flagellates Heteromita globosa on aerobic biodegradation of benzene by Pseudomonas strain PS+ was examined in batch culture. Growth of H. globosa on these bacteria obeyed Monod kinetics (_max, 0.17 ± 0.03 h^–1; K_s, 1.1 ± 0.2 × 10⁷ bacteria mL^–1) and was optimal at a bacteria/ flagellate ratio of 2000. Carbon mass balance showed that 5.2% of total [ring-U-¹⁴C]benzene fed to bacteria was subsequently incorporated into flagellate biomass. Growth-inhibiting concentrations (IC₅₀) of alkylbenzenes (benzene, toluene, ethylbenzene) were inversely related with their octanol/ water partitioning coefficients, and benzene was least toxic for bacteria and flagellates with IC₅₀ values of 4392 (± 167) M and 2770 (± 653) M, respectively. The first-order rate constant for benzene degradation (k₁, 0.48 ± 0.12 day^–1) was unaffected by the presence or absence of flagellates in cultures. However, the rate of benzene degradation by individual bacteria averaged three times higher in the presence of flagellates (0.73 ± 0.13 fmol cell^–1 h^–1) than in their absence (0.26 ± 0.03 fmol cell^–1 h^–1). Benzene degradation also coincided with higher levels of dissolved oxygen and a higher rate of nitrate reduction in the presence of flagellates (p < 0.02). Grazing by flagellates may have increased the availability of dissolved oxygen to a smaller surviving population of bacteria engaged in the aerobic reactions initiating benzene degradation. In addition, flagellates may also have increased the rate of nitrate reduction through the excretion of acetate as an additional electron donor for these bacteria. Indeed, acetate was shown to progressively accumulate in cultures where flagellates grazed on heat-killed bacteria. This study provided evidence that grazing flagellates stimulate bacterial degradation of alkylbenzenes and provide a link for carbon cycling to consumers at higher trophic levels. This may have important implications for bioremediation processes.     

Model based evaluation of a contaminant plume development under aerobic and anaerobic conditions in 2D bench-scale tank experiments

The influence of transverse mixing on competitive aerobic and anaerobic biodegradation of a hydrocarbon plume was investigated using a two-dimensional, bench-scale flow-through laboratory tank experiment. In the first part of the experiment aerobic degradation of increasing toluene concentrations was carried out by the aerobic strain Pseudomonas putida F1. Successively, ethylbenzene (injected as a mixture of unlabeled and fully deuterium-labeled isotopologues) substituted toluene; nitrate was added as additional electron acceptor and the anaerobic denitrifying strain Aromatoleum aromaticum EbN1 was inoculated to study competitive degradation under aerobic / anaerobic conditions. The spatial distribution of anaerobic degradation was resolved by measurements of compound-specific stable isotope fractionation induced by the anaerobic strain as well as compound concentrations. A fully transient numerical reactive transport model was employed and calibrated using measurements of electron donors, acceptors and isotope fractionation. The aerobic phases of the experiment were successfully reproduced using a double Monod kinetic growth model and assuming an initial homogeneous distribution of P. putida F1. Investigation of the competitive degradation phase shows that the observed isotopic pattern cannot be explained by transverse mixing driven biodegradation only, but also depends on the inoculation process of the anaerobic strain. Transient concentrations of electron acceptors and donors are well reproduced by the model, showing its ability to simulate transient competitive biodegradation.     

Characterization of the Reduction of Selenate and Tellurite by Nitrate Reductases

Preliminary studies showed that the periplasmic nitrate reductase (Nap) of Rhodobacter sphaeroides and the membrane-bound nitrate reductases of Escherichia coli are able to reduce selenate and tellurite in vitro with benzyl viologen as an electron donor. In the present study, we found that this is a general feature of denitrifiers. Both the periplasmic and membrane-bound nitrate reductases of Ralstonia eutropha, Paracoccus denitrificans, and Paracoccus pantotrophus can utilize potassium selenate and potassium tellurite as electron acceptors. In order to characterize these reactions, the periplasmic nitrate reductase of R. sphaeroides f. sp. denitrificans IL106 was histidine tagged and purified. The V_max and K_m were determined for nitrate, tellurite, and selenate. For nitrate, values of 39 μmol · min⁻¹ · mg⁻¹ and 0.12 mM were obtained for V_max and K_m, respectively, whereas the V_max values for tellurite and selenate were 40- and 140-fold lower, respectively. These low activities can explain the observation that depletion of the nitrate reductase in R. sphaeroides does not modify the MIC of tellurite for this organism.     

<Emphasis Type="Italic">Desulfurispirillum alkaliphilum</Emphasis> gen. nov. sp. nov., a novel obligately anaerobic sulfur- and dissimilatory nitrate-reducing bacterium from a full-scale sulfide-removing bioreactor

Strain SR 1^T was isolated under anaerobic conditions using elemental sulfur as electron acceptor and acetate as carbon and energy source from the Thiopaq bioreactor in Eerbeek (The Netherlands), which is removing H₂S from biogas by oxidation to elemental sulfur under oxygen-limiting and moderately haloalkaline conditions. The bacterium is obligately anaerobic, using elemental sulfur, nitrate and fumarate as electron acceptors. Elemental sulfur is reduced to sulfide through intermediate polysulfide, while nitrate is dissimilatory reduced to ammonium. Furthermore, in the presence of nitrate, strain SR 1^T was able to oxidize limited amounts of sulfide to elemental sulfur during anaerobic growth with acetate. The new isolate is mesophilic and belongs to moderate haloalkaliphiles, with a pH range for growth (on acetate and nitrate) from 7.5 to 10.25 (optimum 9.0), and a salt range from 0.1 to 2.5 M Na⁺ (optimum 0.4 M). According to phylogenetic analysis, SR 1^T is a member of a deep bacterial lineage, distantly related to Chrysiogenes arsenatis (Macy et al. 1996). On the basis of the phenotypic and genetic data, the novel isolate is placed into a new genus and species, Desulfurispirillum alkaliphilum (type strain SR^T = DSM 18275 = UNIQEM U250). Nucleotide sequence accession number: the GenBank/EMBL accession number of the 16S rRNA gene sequence of strain SR 1^T is DQ666683.     

Biodegradation of aromatic compounds under mixed oxygen/denitrifying conditions: a review

Bioremediation of aromatic hydrocarbons in groundwater and sediments is often limited by dissolved oxygen. Many aromatic hydrocarbons degrade very slowly or not at all under anaerobic conditions. Nitrate is a good alternative electron acceptor to oxygen, and denitrifying bacteria are commonly found in the subsurface and in association with contaminated aquifer materials. Providing both nitrate and microaerophilic levels of oxygen may result in oxidation of the stable benzene rings in aromatic contaminants and allow for the intermediates of this oxidation to degrade via denitrification. The effects of using mixed electron acceptors on biodegradation of subsurface contaminants is unclear. Below some critical oxygen threshold, aerobic biodegradation is inhibited, however high levels of oxygen inhibit denitrification. The mechanisms which regulate electron transfer to oxygen and nitrate are complex. This review: 1) describes the factors which may affect the utilization of oxygen and nitrate as dual electron acceptors during biodegradation; 2) summarizes the incidence of dual use of nitrate and oxygen (aerobic denitrification); and 3) presents evidence of the effectiveness of bioremediation under mixed oxygen/nitrate conditions. Received 08 November 1995/ Accepted in revised form 09 June 1996     

Isolation and preliminary characterization of a respiratory nitrate reductase from hydrocarbon-degrading bacterium <Emphasis Type="Italic">Gordonia alkanivorans</Emphasis> S7

Gordonia alkanivorans S7 is an efficient degrader of fuel oil hydrocarbons that can simultaneously utilize oxygen and nitrate as electron acceptors. The respiratory nitrate reductase (Nar) from this organism has been isolated using ion exchange chromatography and gel filtration, and then preliminarily characterized. PAGE, SDS-PAGE and gel filtration chromatography revealed that Nar consisted of three subunits of 103, 53 and 25 kDa. The enzyme was optimally active at pH 7.9 and 40°C. K _m values for NO₃ ⁻ (110 μM) and for ClO₃ ⁻ (138 μM) were determined for a reduced viologen as an electron donor. The purified Nar did not use NADH as the electron donor to reduce nitrate or chlorate. Azide was a strong inhibitor of its activity. Our results imply that enzyme isolated from G. alkanivorans S7 is a respiratory membrane-bound nitrate reductase. This is the first report of purification of a nitrate reductase from Gordonia species.     

Flexible bacterial strains that oxidize arsenite in anoxic or aerobic conditions and utilize hydrogen or acetate as alternative electron donors

Arsenic is a carcinogenic compound widely distributed in the groundwater around the world. The fate of arsenic in groundwater depends on the activity of microorganisms either by oxidizing arsenite (As^III), or by reducing arsenate (As^V). Because of the higher toxicity and mobility of As^III compared to As^V, microbial-catalyzed oxidation of As^III to As^V can lower the environmental impact of arsenic. Although aerobic As^III-oxidizing bacteria are well known, anoxic oxidation of As^III with nitrate as electron acceptor has also been shown to occur. In this study, three As^III-oxidizing bacterial strains, Azoarcus sp. strain EC1-pb1, Azoarcus sp. strain EC3-pb1 and Diaphorobacter sp. strain MC-pb1, have been characterized. Each strain was tested for its ability to oxidize As^III with four different electron acceptors, nitrate, nitrite, chlorate and oxygen. Complete As^III oxidation was achieved with both nitrate and oxygen, demonstrating the novel ability of these bacterial strains to oxidize As^III in either anoxic or aerobic conditions. Nitrate was only reduced to nitrite. Different electron donors were used to study their suitability in supporting nitrate reduction. Hydrogen and acetate were readily utilized by all the cultures. The flexibility of these As^III-oxidizing bacteria to use oxygen and nitrate to oxidize As^III as well as organic and inorganic substrates as alternative electron donors explains their presence in non-arsenic-contaminated environments. The findings suggest that at least some As^III-oxidizing bacteria are flexible with respect to electron-acceptors and electron-donors and that they are potentially widespread in low arsenic concentration environments.     

Isolation and characterization of Desulfovibrio senezii sp. nov., a halotolerant sulfate reducer from a solar saltern and phylogenetic confirmation of Desulfovibrio fructosovorans as a new species

A new halotolerant Desulfovibrio, strain CVL^T (T = type strain), was isolated from a solar saltern in California. The curved, gram-negative, nonsporeforming cells (0.3 × 1.0–1.3 μm) occurred singly, in pairs, or in chains, were motile by a single polar flagellum and tolerated up to 12.5% NaCl. Strain CVL^T had a generation time of 60 min when grown in lactate-yeast extract medium under optimal conditions (37°C, pH 7.6, 2.5% NaCl). It used lactate, pyruvate, cysteine, or H₂/CO₂ + acetate as electron donors, and sulfate, sulfite, thiosulfate, or fumarate as electron acceptors. Elemental sulfur, nitrate, or oxygen were not used. Sulfite and thiosulfate were disproportionated to sulfate and sulfide. The G+C content of the DNA was 62 mol%. Phylogenetic analysis revealed that Desulfovibrio fructosovorans was the nearest relative. Strain CVL^T is clearly different from other Desulfovibrio species, and is designated Desulfovibrio senezii sp. nov. (DSM 8436). Received: 27 February 1998 / Accepted: 15 June 1998     

Comparison of relative rates of BTEX biodegradation using respirometry

Biodegradation of BTEX by a microbial consortium isolated from a closed municipal landfill was studied using respirometric techniques. The kinetics of biodegradation were estimated from experimental oxygen uptake data using a nonlinear parameter estimation technique. All of the six compounds were rapidly degraded by the microbial culture and no substrate inhibition was observed at the concentration levels examined (200 mg L⁻¹ as COD). Microbial growth and contaminant degradation were adequately described by the Monod equation. Considerable differences were observed in the rates of BTEX biodegradation as seen from the estimates of the kinetic parameters. A three-fold variation was seen in the values of the maximum specific growth rate, μ_max. The highest value of μ_max was 0.389 h⁻¹ for p-xylene while o-xylene was characterized by a μ_max value of 0.14 h⁻¹, the lowest observed in this study. The half saturation coefficient, K _s, and the yield coefficient, Y, varied between 1.288–4.681 mg L⁻¹ and 0.272–0.645 mg mg⁻¹, respectively. Benzene and o-xylene exhibited higher resistance to biodegradation while toluene and p-xylene were rapidly degraded. Ethylbenzene and m-xylene were degraded at intermediate rates. In biodegradation experiments with a multiple substrate matrix, substrate depletion was slower than in single substrate experiments, suggesting an inhibitory nature of substrate interaction. Received 15 February 1998/ Accepted in revised form 5 July 1998     

Environmental Factors That Control Microbial Perchlorate Reduction

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.     

Growth yields and energy generation by Campylobacter sputorum subspecies bubulus during growth in continuous culture with different hydrogen acceptors

Campylobacter sputorum subspecies bubulus was grown in continuous culture with excess of l-lactate or formate, and growth-limiting amounts of oxygen, fumarate, nitrate or nitrite. l-Lactate was oxidized to acetate, fumarate was reduced to succinate, and nitrate and nitrite were reduced to ammonia. The Y _lactate values (g dry weight bacteria/g mol lactate) for the respective hydrogen acceptors were much higher than the Y _formate values. Steady state cultures on formate and nitrite could only be obtained at a low dilution rate and low nitrite concentrations in the growth medium. In H⁺/2e measurements with lactate-grown cells proton ejections were observed with lactate or pyruvate as a hydrogen donor, and oxygen or hydrogen peroxide as a hydrogen acceptor. Proton ejection was also observed with pyruvate and nitrate. Proton ejection did not occur with lactate and nitrate, neither with lactate or pyruvate and fumarate or nitrite. With formate as a hydrogen donor acidification occurred with all hydrogen acceptors mentioned. It has been concluded that during growth on lactate and fumarate or nitrite substrate level phosphorylation at acetate formation is the sole ATP-generating system. Growth on formate and fumarate or nitrite is explained by a proton gradient generated as a result of oxidation of formate at the periplasmic side of the cytoplasmic membrane. With oxygen and nitrate additional ATP is formed by electron transport-linked phosphorylation. The low molar growth yields with formate are explained by the observation that formate-grown cells had a great permeability to protons.Abbreviations H⁺/2e value number of protons ejected per electron pair transported in the respiratory system - P/2e value mol of ATP formed per electron pair transported in the respiratory system - CCCP carbonyl cyanide m-chlorophenyl-hydrazone     

Aerobic denitrification: a controversy revived

During studies on the denitrifying mixotroph, Thiosphaera pantotropha, it has been found that this organism is capable of simultaneously utilizing nitrate and oxygen as terminal electron acceptors in respiration. This phenomenon, termed aerobic denitrification, has been found in cultures maintained at dissolved oxygen concentrations up to 90% of air saturation.The evidence for aerobic denitrification was obtained from a number of independant experiments. Denitrifying enzymes were present even in organisms growing aerobically without nitrate. Aerobic yields on acetate were higher (8.1 g protein/mol) without than with (6.0 g protein/mol) nitrate, while the anaerobic yield with nitrate was even lower (4 g protein/mol). The maximum specific growth rate of Tsa. pantotropha was higher (0.34 h^-1) in the presence of both oxygen (>80% air saturation) and nitrate than in similar cultures not supplied with nitrate (0.27 h^-1), indicating that the rate of electron transport to oxygen was limiting. This was confirmed by oxygen uptake experiments which showed that although the rate of respiration on acetate was not affected by nitrate, the total oxygen uptake was reduced in its presence. The original oxygen uptake could be restored by the addition of denitrification inhibitors.Dedicated to Professor Dr. H.-G. Schlegel on the occasion of his 60th birthday