Methanogenesis from acetate by cell-free extracts of the thermophilic acetotrophic methanogen Methanothrix thermophila CALS-1

High rates of methanogenesis from acetate and ATP were observed from cell-free extracts of the thermophilic acetotrophic methanogen Methanothrix (Methanosaeta) thermophila strain CALS-1 when cultures were grown in a pH auxostat fed with acetic acid. Specific methanogenic activities ranged from 50–300 nmol min^–1 (mg protein)^–1, which was comparable to those for whole cells. In contrast to results with Methanosarcina spp., the reaction did not require high levels of H₂ in the headspace. CO was inhibitory to methanogenesis from acetate. The inhibition by CO and the lack of effect of H₂ on methanogenesis from acetate resemble previous results with whole cells of CALS-1. Protein concentrations in extracts > 5 mg/ml were required for good activity, and the optimum temperature for the methanogenesis was near 65° C. ATP was required in substrate quantities and was converted mainly to AMP. The maximum CH₄/ATP stoichiometry obtained was near 1.0, consistent with acetate activation using an acetyl-CoA synthetase mechanism that converts ATP to AMP and pyrophosphate. Methanogenesis in extracts was inhibited by bromoethane sulfonate and cyanide, indicating the involvement of methylcoenzyme M methylreductase and a carbon monoxide dehydrogenase complex with methanogenesis from acetate. These results are consistent with acetyl-coenzyme A (CoA) as the form of activated acetate involved in methanogenesis from acetate in strain CALS-1, but no activity could be obtained from extracts using acetyl-CoA as a substrate. Received: 18 March 1996 / Accepted: 14 June 1996     

Carbon Monoxide, Hydrogen, and Formate Metabolism during Methanogenesis from Acetate by Thermophilic Cultures of Methanosarcina and Methanothrix Strains

CO and H₂ have been implicated in methanogenesis from acetate, but it is unclear whether they are directly involved in methanogenesis or electron transfer in acetotrophic methanogens. We compared metabolism of H₂, CO, and formate by cultures of the thermophilic acetotrophic methanogens Methanosarcina thermophila TM-1 and Methanothrix sp. strain CALS-1. M. thermophila accumulated H₂ to partial pressures of 40 to 70 Pa (1 Pa = 0.987 × 10^-5 atm), as has been previously reported for this and other Methanosarcina cultures. In contrast, Methanothrix sp. strain CALS-1 accumulated H₂ to maximum partial pressures near 1 Pa. Growing cultures of Methanothrix sp. strain CALS-1 initially accumulated CO, which reached partial pressures near 0.6 Pa (some CO came from the rubber stopper) during the middle of methanogenesis; this was followed by a decrease in CO partial pressures to less than 0.01 Pa by the end of methanogenesis. Accumulation or consumption of CO by cultures of M. thermophila growing on acetate was not detected. Late-exponential-phase cultures of Methanothrix sp. strain CALS-1, in which the CO partial pressure was decreased by flushing with N₂-CO₂, accumulated CO to 0.16 Pa, whereas cultures to which ca. 0.5 Pa of CO was added consumed CO until it reached this partial pressure. Cyanide (1 mM) blocked CO consumption but not production. High partial pressures of H₂ (40 kPa) inhibited methanogenesis from acetate by M. thermophila but not by Methanothrix sp. strain CALS-1, and 2 kPa of CO was not inhibitory to M. thermophila but was inhibitory to Methanothrix sp. strain CALS-1. Levels of CO dehydrogenase, hydrogenase, and formate dehydrogenase in Methanothrix sp. strain CALS-1 were 9.1, 0.045, and 5.8 μmol of viologen reduced min^-1 mg of protein^-1. These results suggest that CO plays a role in Methanothrix sp. strain CALS-1 similar to that of H₂ in M. thermophila and are consistent with the conclusion that CO is an intermediate in a catabolic or anabolic pathway in Methanothrix sp. strain CALS-1; however, they could also be explained by passive equilibration of CO with a metabolic intermediate.     

Nutritional Requirements of Methanosarcina sp. Strain TM-1

Methanosarcina sp. strain TM-1, an acetotrophic, thermophilic methanogen isolated from an anaerobic sludge digestor, was originally reported to require an anaerobic sludge supernatant for growth. It was found that the sludge supernatant could be replaced with yeast extract (1 g/liter), 6 mM bicarbonate-30% CO₂, and trace metals, with a doubling time on methanol of 14 h. For growth on either methanol or acetate, yeast extract could be replaced with CaCl₂ · 2H₂O (13.6 μM minimum) and the vitamin p-aminobenzoic acid (PABA, ca. 3 nM minimum), with a doubling time on methanol of 8 to 9 h. Filter-sterilized folic acid at 0.3 μM could not replace PABA. The antimetabolite sulfanilamide (20 mM) inhibited growth of and methanogenesis by Methanosarcina sp. strain TM-1, and this inhibition was reversed by the addition of 0.3 μM PABA. When a defined medium buffered with 20 mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid was used, it was shown that Methanosarcina sp. strain TM-1 required 6 mM bicarbonate-30% CO₂ for optimal growth and methanogenesis from methanol. Cells growing on acetate were less dependent on bicarbonate-CO₂. When we used a defined medium in which the only organic compounds present were methanol or acetate, nitrilotriacetic acid (0.2 mM), and PABA, it was possible to limit batch cultures of Methanosarcina sp. strain TM-1 for nitrogen at NH₄⁺ concentrations at or below 2.0 mM, in marked contrast with Methanosarcina barkeri 227, which fixes dinitrogen when grown under NH₄⁺ limitation.     

Isolation and characterization of a thermophilic acetotrophic strain of Methanothrix

An enrichment culture which converted acetate to methane at 60°C was obtained from a thermophilic anaerobic bioreactor. The predominant morphotype in the enrichment was a sheathed gas-vacuolated rod with marked resemblence to the mesophile Methanothrix soehngenii. This organism was isolated using vancomycin treatments and serial dilutions and was named Methanothrix sp. strain CALS-1. Strain CALS-1 grew as filaments typically 2–5 cells long, and cultures showed opalescent turbidity rather than macroscopic clumps. The cells were enclosed in a striated subunit-type sheath and there were distinct cross-walls between the cells, similar to M. soehngenii. The gas vesicles in cells were typically 70 nm in diameter and up to 0.5 m long, and were collapsed by pressures over 3 atm (ca. 300 kPa). Stationary-phase cells tended to have a higher vesicle content than did growing cells, and occasionally bands of cells were seen floating at the top of the liquid in stationary-phase cultures. Acetate was the only substrate of those tested which was used for methanogenesis by strain CALS-1, and acetate was decarboxylated by the aceticlastic reaction. The optimum temperature for growth of strain CALS-1 was near 60°C (doubling time=24–26 h), with no growth occurring at 70°C and 37°C. The optimum pH value for growth was near 6.5 in bicarbonate/CO₂ buffered medium and no growth occurred at pH 5.5 or pH 8.4. No growth was obtained below pH 7 when the medium was buffered with 20 mM phosphate. Strain CALS-1 grew in a chemically defined medium and required biotin. Sulfide concentrations over 1 mM were inhibitory to the culture, and growth was more rapid with 1 mM 2-mercaptoethane sulfonate (coenzyme M) or 1 mM titanium citrate as an accessory reductant than with 1 mM cysteine. It is likely that strain CALS-1 represents a new species in the genus Methanothrix.     

Acetyl-coenzyme A synthetase in the thermophilic, acetate-utilizing methanogen Methanothrix sp. strain CALS-1

Abstract There is considerable evidence that acetyl-CoA synthetase (acetate thiokinase, ACS, EC 6.2.1) is responsible for acetate activation in the mesophilic acetotrophic methanogen Methanothrix soehngenii . If the pyrophosphate produced by ACS is simply cleaved, two high-energy phosphodiester bonds are expended in acetate activation. Hi High ACS activity (2–4 μmol min⁻¹ mg protein⁻¹) was present in cell-free extracts of the thermophile Methanothrix sp. strain CALS-1. The 23-fold purified enzyme had a molecular mass near 165 kDa and a subunit molecular mass near 78 kDa, suggesting that the enzyme is a homodimer. The temperature optimum for ACS was near 70°C and the apparent K _m values were 2–4 mM for acetate and 5.5 mM for MgATP. Coenzyme A at concentrations greater than 0.2 mM inhibited ACS, while acetyl-CoA was not inhibitory. AMP and pyrophosphate inhibited ACS with K _i values of 5 mM and 1.5 mM respectively. Other divalent cations could replace Mg²⁺, with Mn²⁺ showing the highest activity. Activity with ITP was 20% of that with ATP, and other nucleotides tested were considerably less active. Since Methanothrix sp. strain CALS-1 has an active soluble pyrophosphatase, it appears that it uses the same energetically costly method for acetate activation as M. soehngenii .     

Multiphasic Kinetics of Transformation of 1,2,4-Trichlorobenzene at Nano- and Micromolar Concentrations by Burkholderia sp. Strain PS14

The transformation of 1,2,4-trichlorobenzene (1,2,4-TCB) at initial concentrations in nano- and micromolar ranges was studied in batch experiments with Burkholderia sp. strain PS14. 1,2,4-TCB was metabolized from nano- and micromolar concentrations to below its detection limit of 0.5 nM. At low initial 1,2,4-TCB concentrations, a first-order relationship between specific transformation rate and substrate concentration was observed with a specific affinity (a⁰_A) of 0.32 liter · mg (dry weight)⁻¹ · h⁻¹ followed by a second one at higher concentrations with an a^o_A of 0.77 liter · mg (dry weight)⁻¹ · h⁻¹. This transition from the first-order kinetics at low initial 1,2,4-TCB concentrations to the second first-order kinetics at higher 1,2,4-TCB concentrations was shifted towards higher initial 1,2,4-TCB concentrations with increasing cell mass. At high initial concentrations of 1,2,4-TCB, a maximal transformation rate of approximately 37 nmol · min⁻¹ · mg (dry weight)⁻¹ was measured, irrespective of the cell concentration.     

Kinetic studies of acetate fermentation by Methanosarcina sp. MSTA-1

Summary The effect of three parameters (initial acetate concentration, temperature and pH) on the acetoclastic reaction was studied with the thermophilic methanogenic bacterium Methanosarcina sp. MSTA-1. The optimum temperature for growth ranged around 55° C, and optimum pH was 6.5–7.5, giving a minimum generation time of 12.6–13.9 h (µ_max = 0.050–0.055 h^–1) and a maximum value of the specific acetate consumption rate (q _infs ^supps ) of 14–20 mmol/g cells per hour. Contrary to the methane yield, the growth yield was found to be dependent on culture conditions, especially on incubation temperature. Methanosarcina sp. MSTA-1 showed a low affinity for acetate substrate. Growth at 55° C and at constant pH 7 resulted in a K _m value and a threshold acetate concentration of 10.7 mM and 0.7 mM, respectively. Offprint requests to: R. Moletta     

Effects of Temperature on Methanogenesis in a Thermophilic (58°C) Anaerobic Digestor

The short-term effects of temperature on methanogenesis from acetate or CO₂ in a thermophilic (58°C) anaerobic digestor were studied by incubating digestor sludge at different temperatures with ¹⁴C-labeled methane precursors (¹⁴CH₃COO⁻ or ¹⁴CO₂). During a period when Methanosarcina sp. was numerous in the sludge, methanogenesis from acetate was optimal at 55 to 60°C and was completely inhibited at 65°C. A Methanosarcina culture isolated from the digestor grew optimally on acetate at 55 to 58°C and did not grow or produce methane at 65°C. An accidental shift of digestor temperature from 58 to 64°C during this period caused a sharp decrease in gas production and a large increase in acetate concentration within 24 h, indicating that the aceticlastic methanogens in the digestor were the population most susceptible to this temperature increase. During a later period when Methanothrix sp. was numerous in the digestor, methanogenesis from ¹⁴CH₃COO⁻ was optimal at 65°C and completely inhibited at 75°C. A partially purified Methanothrix enrichment culture derived from the digestor had a maximum growth temperature near 70°C. Methanogenesis from ¹⁴CO₂ in the sludge was optimal at 65°C and still proceeded at 75°C. A CO₂-reducing Methanobacterium sp. isolated from the digestor was capable of methanogenesis at 75°C. During the period when Methanothix sp. was apparently dominant, sludge incubated for 24 h at 65°C produced more methane than sludge incubated at 60°C, and no acetate accumulated at 65°C. Methanogenesis was severely inhibited in sludge incubated at 70°C, but since neither acetate nor H₂ accumulated, production of these methanogenic substrates by fermentative bacteria was probably the most temperature-sensitive process. Thus, there was a correlation between digestor performance at different temperatures and responses to temperature by cultures of methanogens believed to play important roles in the digestor.     

Dissimilatory Iodate Reduction by Marine Pseudomonas sp. Strain SCT

Bacterial iodate (IO₃⁻) reduction is poorly understood largely due to the limited number of available isolates as well as the paucity of information about key enzymes involved in the reaction. In this study, an iodate-reducing bacterium, designated strain SCT, was newly isolated from marine sediment slurry. SCT is phylogenetically closely related to the denitrifying bacterium Pseudomonas stutzeri and reduced 200 μM iodate to iodide (I⁻) within 12 h in an anaerobic culture containing 10 mM nitrate. The strain did not reduce iodate under the aerobic conditions. An anaerobic washed cell suspension of SCT reduced iodate when the cells were pregrown anaerobically with 10 mM nitrate and 200 μM iodate. However, cells pregrown without iodate did not reduce it. The cells in the former category showed methyl viologen-dependent iodate reductase activity (0.31 U mg⁻¹), which was located predominantly in the periplasmic space. Furthermore, SCT was capable of anaerobic growth with 3 mM iodate as the sole electron acceptor, and the cells showed enhanced activity with respect to iodate reductase (2.46 U mg⁻¹). These results suggest that SCT is a dissimilatory iodate-reducing bacterium and that its iodate reductase is induced by iodate under anaerobic growth conditions.     

Formation of Hydride-Meisenheimer Complexes of Picric Acid (2,4,6-Trinitrophenol) and 2,4-Dinitrophenol during Mineralization of Picric Acid by Nocardioides sp. Strain CB 22-2

There are only a few examples of microbial conversion of picric acid (2,4,6-trinitrophenol). None of the organisms that have been described previously is able to use this compound as a sole source of carbon, nitrogen, and energy at high rates. In this study we isolated and characterized a strain, strain CB 22-2, that was able to use picric acid as a sole source of carbon and energy at concentrations up to 40 mM and at rates of 1.6 mmol · h⁻¹ · g (dry weight) of cells⁻¹ in continuous cultures and 920 μmol · h⁻¹ · g (dry weight) of cells⁻¹ in flasks. In addition, this strain was able to use picric acid as a sole source of nitrogen at comparable rates in a nitrogen-free medium. Biochemical characterization and 16S ribosomal DNA analysis revealed that strain CB 22-2 is a Nocardioides sp. strain. High-pressure liquid chromatography and UV-visible light data, the low residual chemical oxygen demand, and the stoichiometric release of 2.9 ± 0.1 mol of nitrite per mol of picric acid provided strong evidence that complete mineralization of picric acid occurred. During transformation, the metabolites detected in the culture supernatant were the [H⁻]-Meisenheimer complexes of picric acid and 2,4-dinitrophenol (H⁻-DNP), as well as 2,4-dinitrophenol. Experiments performed with crude extracts revealed that H⁻-DNP formation indeed is a physiologically relevant step in picric acid metabolism.     

Methanogenesis in thermophilic biogas reactors

Methanogenesis in thermophilic biogas reactors fed with different wastes is examined. The specific methanogenic activity with acetate or hydrogen as substrate reflected the organic loading of the specific reactor examined. Increasing the loading of thermophilic reactors stabilized the process as indicated by a lower concentration of volatile fatty acids in the effluent from the reactors. The specific methanogenic activity in a thermophilic pilot-plant biogas reactor fed with a mixture of cow and pig manure reflected the stability of the reactor. The numbers of methanogens counted by the most probable number (MPN) technique with acetate or hydrogen as substrate were further found to vary depending on the loading rate and the stability of the reactor. The numbers of methanogens counted with antibody probes in one of the reactor samples was 10 times lower for the hydrogen-utilizing methanogens compared to the counts using the MPN technique, indicating that other non-reacting methanogens were present. Methanogens that reacted with the probe againstMethanobacterium thermoautotrophicum were the most numerous in this reactor. For the acetate-utilizing methanogens, the numbers counted with the antibody probes were more than a factor of 10 higher than the numbers found by MPN. The majority of acetate utilizing methanogens in the reactor wereMethanosarcina spp. single cells, which is a difficult form of the organism to cultivatein vitro. No reactions were observed with antibody probes raised againstMethanothrix soehngenii orMethanothrix CALS-1 in any of the thermophilic biogas reactors examined. Studies using 2-¹⁴C-labeled acetate showed that at high concentrations (more than approx. 1 mM) acetate was metabolized via the aceticlastic pathway, transforming the methyl-group of acetate into methane. When the concentration of acetate was less than approx. 1 mM, most of the acetate was oxidized via a two-step mechanism (syntrophic acetate oxidation) involving one organism oxidizing acetate into hydrogen and carbon dioxide and a hydrogen-utilizing methanogen forming the products of the first microorganism into methane. In thermophilic biogas reactors, acetate oxidizing cultures occupied the niche ofMethanothrix species, aceticlastic methanogens which dominate at low acetate concentrations in mesophilic systems. Normally, thermophilic biogas reactors are operated at temperatures from 52 to 56° C. Experiments using biogas reactors fed with cow manure showed that the same biogas yield found at 55° C could be obtained at 61° C after a long adaptation period. However, propionate degradation was inhibited by increasing the temperature.     

Threshold Acetate Concentrations for Acetate Catabolism by Aceticlastic Methanogenic Bacteria

Marked differences were found for minimum threshold concentrations of acetate catabolism by Methanosarcina barkeri 227 (1.180 mM), Methanosarcina mazei S-6 (0.396 mM), and a Methanothrix sp. (0.069 mM). This indicates that the aceticlastic methanogens responsible for the conversion of acetate to methane in various ecosystems might be different, depending on the prevailing in situ acetate concentrations.     

Anilofos Tolerance and Its Mineralization by the Cyanobacterium Synechocystis sp. Strain PUPCCC 64

This study deals with anilofos tolerance and its mineralization by the common rice field cyanobacterium Synechocystis sp. strain PUPCCC 64. The organism tolerated anilofos up to 25 mg L⁻¹. The herbicide caused inhibitory effects on photosynthetic pigments of the test organism in a dose-dependent manner. The organism exhibited 60, 89, 96, 85 and 79% decrease in chlorophyll a, carotenoids, phycocyanin, allophycocyanin and phycoerythrin, respectively, in 20 mg L⁻¹ anilofos on day six. Activities of superoxide dismutase, catalase and peroxidase increased by 1.04 to 1.80 times over control cultures in presence of 20 mg L⁻¹ anilofos. Glutathione content decreased by 26% while proline content was unaffected by 20 mg L⁻¹ anilofos. The test organism showed intracellular uptake and metabolized the herbicide. Uptake of herbicide by test organism was fast during initial six hours followed by slow uptake until 120 hours. The organism exhibited maximum anilofos removal at 100 mg protein L⁻¹, pH 8.0 and 30°C. Its growth in phosphate deficient basal medium in the presence of anilofos (2.5 mg L⁻¹) indicated that herbicide was used by the strain PUPCCC 64 as a source of phosphate.     

Saturable, Energy-Dependent Uptake of Phenanthrene in Aqueous Phase by Mycobacterium sp. Strain RJGII-135

The mechanism of uptake of phenanthrene by Mycobacterium sp. strain RJGII-135, a polycyclic hydrocarbon-degrading bacterium, was examined with cultures grown on phenanthrene (induced for phenanthrene metabolism) and acetate (uninduced). Washed cells were suspended in aqueous solutions of [9-¹⁴C]phenanthrene, and then the cells were collected by filtration. Low-level steady-state ¹⁴C concentrations in uninduced cells were achieved within the first 15 s of incubation. This immediate uptake did not show saturation kinetics and was not susceptible to inhibitors of active transport, cyanide and carbonyl cyanide m-chlorophenylhydrazone. These results indicated that phenanthrene enters rapidly into the cells by passive diffusion. However, induced cells showed cumulative uptake over several minutes. The initial uptake rates followed saturation kinetics, with an apparent affinity constant (K_t) of 26 ± 3 nM (mean ± standard deviation). Uptake of phenanthrene by induced cells was strongly inhibited by the inhibitors. Analysis of cell-associated ¹⁴C-labeled compounds revealed that the concurrent metabolism during uptake was rapid and was not saturated at the substrate concentrations tested, suggesting that the saturable uptake observed reflects membrane transport rather than intracellular metabolism. These results were consistent with the presence of a saturable, energy-dependent mechanism for transport of phenanthrene in induced cells. Moreover, the kinetic data for the cumulative uptake suggested that phenanthrene is specifically bound by induced cells, based on its saturation with an apparent dissociation constant (K_d) of 41 ± 21 nM (mean ± standard deviation). Given the low values of K_t and K_d, Mycobacterium sp. strain RJGII-135 may use a high-affinity transport system(s) to take up phenanthrene from the aqueous phase.     

Isolation and Characterization of Novosphingobium sp. Strain MT1, a Dominant Polychlorophenol-Degrading Strain in a Groundwater Bioremediation System

A high-rate fluidized-bed bioreactor has been treating polychlorophenol-contaminated groundwater in southern Finland at 5 to 8°C for over 6 years. We examined the microbial diversity of the bioreactor using three 16S ribosomal DNA (rDNA)-based methods: denaturing gradient gel electrophoresis, length heterogeneity-PCR analysis, and restriction fragment length polymorphism analysis. The molecular study revealed that the process was dependent on a stable bacterial community with low species diversity. The dominant organism, Novosphingobium sp. strain MT1, was isolated and characterized. Novosphingobium sp. strain MT1 degraded the main contaminants of the groundwater, 2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol, and pentachlorophenol, at 8°C. The strain carried a homolog of the pcpB gene, coding for the pentachlorophenol-4-monooxygenase in Sphingobium chlorophenolicum. Spontaneous deletion of the pcpB gene homolog resulted in the loss of degradation ability. Phenotypic dimorphism (planktonic and sessile phenotypes), low growth rate (0.14 to 0.15 h⁻¹), and low-copy-number 16S rDNA genes (single copy) were characteristic of strain MT1 and other MT1-like organisms isolated from the bioreactor.     

Natural Electrotransformation of Lightning-Competent Pseudomonas sp. Strain N3 in Artificial Soil Microcosms

The lightning-competent Pseudomonas sp. strain N3, recently isolated from soil, has been used to study the extent of natural electrotransformation (NET) or lightning transformation as a horizontal gene transfer mechanism in soil. The variation of electrical fields applied to the soil with a laboratory-scale lightning system provides an estimate of the volume of soil affected by NET. Based on the range of the electric field that induces NET of Pseudomonas strain N3, the volume of soil, where NET could occur, ranges from 2 to 950 m³ per lightning strike. The influence of DNA parameters (amount, size, and purity) and DNA soil residence time were also investigated. NET frequencies (electrotransformants/recipient cells) ranged from 10⁻⁸ for cell lysate after 1 day of residence in soil to 4 × 10⁻⁷ with a purified plasmid added immediately before the lightning. The electrical field gradient (in kilovolts per cm) also played a role as NET frequencies ranging from 1 × 10⁻⁵ at 2.3 kV/cm to 1.7 × 10⁻⁴ at 6.5 kV/cm.     

Enrichment of acetate-, propionate-, and butyrate-degrading co-cultures from the biofilm of an anaerobic fluidized bed reactor

Summary Scanning electron microphotographs from the biofilm of a pilot scale anaerobic fluid-ized-bed reactor fed with acetate, propionate, and butyrate as carbon sources showed a predominance of filamentous organisms resembling Methanothrix sp. which could be isolated as an al-most pure culture as well as a Methanosarcina strain. Three syntrophic cultures, enriched in the medium of Boone and Xun, contained four or five microscopically distinguishable microorganisms, among them Methanospirillum sp., Methanothrix sp., Methanosarcina sp., and rods of acetogenic bacteria degrading propionate or butyrate effectively.     

Initial Reaction(s) in Biotransformation of CL-20 Is Catalyzed by Salicylate 1-Monooxygenase from Pseudomonas sp. Strain ATCC 29352

CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane) (C₆H₆N₁₂O₁₂), a future-generation high-energy explosive, is biodegradable by Pseudomonas sp. strain FA1 and Agrobacterium sp. strain JS71; however, the nature of the enzyme(s) involved in the process was not understood. In the present study, salicylate 1-monooxygenase, a flavin adenine dinucleotide (FAD)-containing purified enzyme from Pseudomonas sp. strain ATCC 29352, biotransformed CL-20 at rates of 0.256 ± 0.011 and 0.043 ± 0.003 nmol min⁻¹ mg of protein⁻¹ under anaerobic and aerobic conditions, respectively. The disappearance of CL-20 was accompanied by the release of nitrite ions. Using liquid chromatography/mass spectrometry in the negative electrospray ionization mode, we detected a metabolite with a deprotonated mass ion [M − H]⁻ at 345 Da, corresponding to an empirical formula of C₆H₆N₁₀O₈, produced as a result of two sequential N denitration steps on the CL- 20 molecule. We also detected two isomeric metabolites with [M − H]⁻ at 381 Da corresponding to an empirical formula of C₆H₁₀N₁₀O₁₀. The latter was a hydrated product of the metabolite C₆H₆N₁₀O₈ with addition of two H₂O molecules, as confirmed by tests using ¹⁸O-labeled water. The product stoichiometry showed that each reacted CL-20 molecule produced about 1.7 nitrite ions, 3.2 molecules of nitrous oxide, 1.5 molecules of formic acid, and 0.6 ammonium ion. Diphenyliodonium-mediated inhibition of salicylate 1-monooxygenase and a comparative study between native, deflavo, and reconstituted enzyme(s) showed that FAD site of the enzyme was involved in the biotransformation of CL-20 catalyzed by salicylate 1-monooxygenase. The data suggested that salicylate 1-monooxygenase catalyzed two oxygen-sensitive single-electron transfer steps necessary to release two nitrite ions from CL-20 and that this was followed by the secondary decomposition of this energetic chemical.     

Transformation of Low Concentrations of 3-Chlorobenzoate by Pseudomonas sp. Strain B13: Kinetics and Residual Concentrations

The transformation of 3-chlorobenzoate (3CB) and acetate at initial concentrations in the wide range of 10 nM to 16 mM was studied in batch experiments with Pseudomonas sp. strain B13. Transformation rates of 3CB at millimolar concentrations could be described by Michaelis-Menten kinetics (K(infm), 0.13 mM; V(infmax), 24 nmol (middot) mg of protein(sup-1) (middot) min(sup-1)). Experiments with nanomolar and low micromolar concentrations of 3CB indicated the possible existence of two different transformation systems for 3CB. The first transformation system operated above 1 (mu)M 3CB, with an apparent threshold concentration of 0.50 (plusmn) 0.11 (mu)M. A second transformation system operated below 1 (mu)M 3CB and showed first-order kinetics (rate constant, 0.076 liter (middot) g of protein(sup-1) (middot) min(sup-1)), with no threshold concentration in the nanomolar range. A residual substrate concentration, as has been reported for some other Pseudomonas strains, could not be detected for 3CB (detection limit, 1.0 nM) in batch incubations with Pseudomonas sp. strain B13. The addition of various concentrations of acetate as a second, easily degradable substrate neither affected the transformation kinetics of 3CB nor induced a detectable residual substrate concentration. Acetate alone also showed no residual concentration (detection limit, 0.5 nM). The results presented indicate that the concentration limits for substrate conversion obtained by extrapolation from kinetic data at higher substrate concentrations may underestimate the true conversion capacity of a microbial culture.     

Oxygen-Dependent Growth of the Sulfate-Reducing Bacterium Desulfovibrio oxyclinae in Coculture with Marinobacter sp. Strain MB in an Aerated Sulfate-Depleted Chemostat

A chemostat coculture of the sulfate-reducing bacterium Desulfovibrio oxyclinae and the facultatively aerobic heterotroph Marinobacter sp. strain MB was grown for 1 week under anaerobic conditions at a dilution rate of 0.05 h⁻¹. It was then exposed to an oxygen flux of 223 μmol min⁻¹ by gassing the growth vessel with 5% O₂. Sulfate reduction persisted under these conditions, though the amount of sulfate reduced decreased by 45% compared to the amount reduced during the initial anaerobic mode. After 1 week of growth under these conditions, sulfate was excluded from the incoming medium. The sulfate concentration in the growth vessel decreased exponentially from 4.1 mM to 2.5 μM. The coculture consumed oxygen effectively, and no residual oxygen was detected during either growth mode in which oxygen was supplied. The proportion of D. oxyclinae cells in the coculture as determined by in situ hybridization decreased from 86% under anaerobic conditions to 70% in the microaerobic sulfate-reducing mode and 34% in the microaerobic sulfate-depleted mode. As determined by the most-probable-number (MPN) method, the numbers of viable D. oxyclinae cells during the two microaerobic growth modes decreased compared to the numbers during the anaerobic growth mode. However, there was no significant difference between the MPN values for the two modes when oxygen was supplied. The patterns of consumption of electron donors and acceptors suggested that when oxygen was supplied in the absence of sulfate and thiosulfate, D. oxyclinae performed incomplete aerobic oxidation of lactate to acetate. This is the first observation of oxygen-dependent growth of a sulfate-reducing bacterium in the absence of either sulfate or thiosulfate. Cells harvested during the microaerobic sulfate-depleted stage and exposed to sulfate and thiosulfate in a respiration chamber were capable of anaerobic sulfate and thiosulfate reduction.