Formation of Aniline as a Transient Metabolite During the Metabolism of Tetryl by a Sulfate-Reducing Bacterial Consortium

A laboratory study was conducted to determine whether tetryl (2,4,6-trinitrophenylmethylnitramine) can be degraded by an anaerobic process. The results indicated that the metabolic conversion of tetryl to aniline is possible by a sulfate-reducing bacterial (SRB) consortium. This SRB consortium metabolized tetryl by co-metabolism with pyruvate as a growth substrate. For every mole of tetryl metabolized, 1 mole of aniline was produced, and the aniline was further metabolized. This metabolic conversion of tetryl is likely to be of value in the anaerobic treatment of tetryl-contaminated soil and ground water, such as found at many military ammunition sites. Received: 18 August 1999 / Accepted: 15 September 1999     

Linked Redox Precipitation of Sulfur and Selenium under Anaerobic Conditions by Sulfate-Reducing Bacterial Biofilms

A biofilm-forming strain of sulfate-reducing bacteria (SRB), isolated from a naturally occurring mixed biofilm and identified by 16S rDNA analysis as a strain of Desulfomicrobium norvegicum, rapidly removed 200 μM selenite from solution during growth on lactate and sulfate. Elemental selenium and elemental sulfur were precipitated outside SRB cells. Precipitation occurred by an abiotic reaction with bacterially generated sulfide. This appears to be a generalized ability among SRB, arising from dissimilatory sulfide biogenesis, and can take place under low redox conditions and in the dark. The reaction represents a new means for the deposition of elemental sulfur by SRB under such conditions. A combination of transmission electron microscopy, environmental scanning electron microscopy, and cryostage field emission scanning electron microscopy were used to reveal the hydrated nature of SRB biofilms and to investigate the location of deposited sulfur-selenium in relation to biofilm elements. When pregrown SRB biofilms were exposed to a selenite-containing medium, nanometer-sized selenium-sulfur granules were precipitated within the biofilm matrix. Selenite was therefore shown to pass through the biofilm matrix before reacting with bacterially generated sulfide. This constitutes an efficient method for the removal of toxic concentrations of selenite from solution. Implications for environmental cycling and the fate of sulfur and selenium are discussed, and a general model for the potential action of SRB in selenium transformations is presented.     

Successional Development of Sulfate-Reducing Bacterial Populations and Their Activities in a Wastewater Biofilm Growing under Microaerophilic Conditions

A combination of fluorescence in situ hybridization, microprofiles, denaturing gradient gel electrophoresis of PCR-amplified 16S ribosomal DNA fragments, and 16S rRNA gene cloning analysis was applied to investigate successional development of sulfate-reducing bacteria (SRB) community structure and in situ sulfide production activity within a biofilm growing under microaerophilic conditions (dissolved oxygen concentration in the bulk liquid was in the range of 0 to 100 μM) and in the presence of nitrate. Microelectrode measurements showed that oxygen penetrated 200 μm from the surface during all stages of biofilm development. The first sulfide production of 0.32 μmol of H₂S m⁻² s⁻¹ was detected below ca. 500 μm in the 3rd week and then gradually increased to 0.70 μmol H₂S m⁻² s⁻¹ in the 8th week. The most active sulfide production zone moved upward to the oxic-anoxic interface and intensified with time. This result coincided with an increase in SRB populations in the surface layer of the biofilm. The numbers of the probe SRB385- and 660-hybridized SRB populations significantly increased to 7.9 × 10⁹ cells cm⁻³ and 3.6 × 10⁹ cells cm⁻³, respectively, in the surface 400 μm during an 8-week cultivation, while those populations were relatively unchanged in the deeper part of the biofilm, probably due to substrate transport limitation. Based on 16S rRNA gene cloning analysis data, clone sequences that related to Desulfomicrobium hypogeium (99% sequence similarity) and Desulfobulbus elongatus (95% sequence similarity) were most frequently found. Different molecular analyses confirmed that Desulfobulbus, Desulfovibrio, and Desulfomicrobium were found to be the numerically important members of SRB in this wastewater biofilm.     

Stable Isotopic Studies of n-Alkane Metabolism by a Sulfate-Reducing Bacterial Enrichment Culture

Gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy were used to study the metabolism of deuterated n-alkanes (C₆ to C₁₂) and 1-¹³C-labeled n-hexane by a highly enriched sulfate-reducing bacterial culture. All substrates were activated via fumarate addition to form the corresponding alkylsuccinic acid derivatives as transient metabolites. Formation of d₁₄-hexylsuccinic acid in cell extracts from exogenously added, fully deuterated n-hexane confirmed that this reaction was the initial step in anaerobic alkane metabolism. Analysis of resting cell suspensions amended with 1-¹³C-labeled n-hexane confirmed that addition of the fumarate occurred at the C-2 carbon of the parent substrate. Subsequent metabolism of hexylsuccinic acid resulted in the formation of 4-methyloctanoic acid, and 3-hydroxy-4-methyloctanoic acid was tentatively identified. We also found that ¹³C nuclei from 1-¹³C-labeled n-hexane became incorporated into the succinyl portion of the initial metabolite in a manner that indicated that ¹³C-labeled fumarate was formed and recycled during alkane metabolism. Collectively, the findings obtained with a sulfate-reducing culture using isotopically labeled alkanes augment and support the previously proposed pathway (H. Wilkes, R. Rabus, T. Fischer, A. Armstroff, A. Behrends, and F. Widdel, Arch. Microbiol. 177:235-243, 2002) for metabolism of deuterated n-hexane by a denitrifying bacterium.     

Oxidation of Polycyclic Aromatic Hydrocarbons under Sulfate-Reducing Conditions

[(sup14)C]naphthalene and phenanthrene were oxidized to (sup14)CO(inf2) without a detectable lag under strict anaerobic conditions in sediments from San Diego Bay, San Diego, Calif., that were heavily contaminated with polycyclic aromatic hydrocarbons (PAHs) but not in less contaminated sediments. Sulfate reduction was necessary for PAH oxidation. These results suggest that the self-purification capacity of PAH-contaminated sulfate-reducing environments may be greater than previously recognized.     

Biodegradation of Phenanthrene by a Halophilic Bacterial Consortium Under Aerobic Conditions

A halophilic bacterial consortium that degraded phenanthrene was developed from oil-contaminated saline soil containing 10% salinity. The biodegradation of phenanthrene occurred at 5%, 10%, and 15% salinity, whereas no biodegradation took place at 0.1% and 20% salinity. A 16S rRNA gene analysis showed that all sequences from the denaturing gradient gel electrophoresis profile were similar to those of halophilic bacteria. This is the first report of a halophilic bacterial consortium capable of degrading phenanthrene under hypersaline conditions.     

Transformation of 3- and 4-Picoline under Sulfate-Reducing Conditions

A microbial population which transformed 3- and 4-picoline under sulfate-reducing conditions was isolated from a subsurface soil which had been previously exposed to different N-substituted aromatic compounds for several years. In the presence of sulfate, the microbial culture transformed 3- and 4-picoline (0.4 mM) within 30 days. From the amounts of ammonia released and of sulfide that were determined during the transformation of 3-picoline, it can be concluded that the parent compound was mineralized to carbon dioxide and ammonia. During the transformation of 4-picoline, a UV-absorbing intermediate accumulated in the culture medium. This metabolite was identified as 2-hydroxy-4-picoline by gas chromatography-mass spectrometry and nuclear magnetic resonance analysis, and its further transformation was detected only after an additional month of incubation. The small amount of sulfide produced during the oxidation of 4-picoline and the generation of the hydroxylated metabolite indicated that the initial step in the metabolic pathway of 4-picoline was a monohydroxylation at position 2 of the heterocyclic aromatic ring. The 3- and 4-picoline-degrading cultures could also transform benzoic acid; however, the other methylated pyridine derivatives, 2-picoline, dimethyl-pyridines, and trimethylpyridines, were not degraded.     

液体培养条件下细菌浓度两种测定方法比较

液体培养细菌后,分别测定培养液、培养液沉淀 无菌盐水的吸光度,结果存在明显差异。采用稀释平板法和显微计数法测定了培养液中细菌数量,并推导出嗜水气单胞菌活菌数与吸光度的关系为logI0/I=6.848×10-25·C。     

Anaerobic Degradation of Hexadecan-2-one by a Microbial Enrichment Culture under Sulfate-Reducing Conditions

A microbial enrichment culture from marine sediment was able to grow on hexadecan-2-one as the sole source of carbon and energy under sulfate-reducing conditions. Oxidation of the ketone involved carboxylation reactions and was coupled to sulfide production. This enrichment culture also grew on 6,10,14-trimethylpentadecan-2-one.     

Bacterial Adhesion under Static and Dynamic Conditions

The deposition of various pseudomonads and coryneform bacteria with different hydrophobicities (water contact angles) and negative cell surface charges on negatively charged Teflon and glass surfaces was investigated. The levels of deposition varied between 5.0 × 10⁴ and 1.6 × 10⁷ cells cm^-2 and between 5.0 × 10⁴ and 3.6 × 10⁷ cells cm^-2 for dynamic column and static batch systems, respectively, indicating that there was a wide variation in physicochemical interactions. Batch and column results were compared in order to better distinguish between hydrodynamic and other system-dependent influences and method-independent physicochemical interactions. Despite the shorter suspension-solid contact time in columns (1 h) than in batch systems (4 h), the level of deposition (expressed as the number of cells that adhered) divided by the applied ambient cell concentration was 4.12 ± 1.63 times higher in columns than in batch sytems for 15 of 22 strain-surface combinations studied. This demonstrates that transport of microbial particles from bulk liquid to surfaces is more efficient in dynamic columns (transport dominated by convection and diffusion) than in static batch systems (transport by diffusion only). The relative constancy of this ratio for the 15 combinations shows that physicochemical interactions affect adhesion similarly in the two systems. The deviating deposition behavior of the other seven strain-surface combinations could be attributed to method-dependent effects resulting from specific cell characteristics (e.g., to the presence of capsular polymers, to an ability to aggregate, to large cell sizes, or to a tendency to desorb after passage through an air-liquid interface).     

Carbon Isotope Fractionation during Anaerobic Degradation of Methyl tert-Butyl Ether under Sulfate-Reducing and Methanogenic Conditions

Methyl tert-butyl ether (MTBE), an octane enhancer and a fuel oxygenate in reformulated gasoline, has received increasing public attention after it was detected as a major contaminant of water resources. Although several techniques have been developed to remediate MTBE-contaminated sites, the fate of MTBE is mainly dependent upon natural degradation processes. Compound-specific stable isotope analysis has been proposed as a tool to distinguish the loss of MTBE due to biodegradation from other physical processes. Although MTBE is highly recalcitrant, anaerobic degradation has been demonstrated under different anoxic conditions and may be an important process. To accurately assess in situ MTBE degradation through carbon isotope analysis, carbon isotope fractionation during MTBE degradation by different cultures under different electron-accepting conditions needs to be investigated. In this study, carbon isotope fractionation during MTBE degradation under sulfate-reducing and methanogenic conditions was studied in anaerobic cultures enriched from two different sediments. Significant enrichment of ¹³C in residual MTBE during anaerobic biotransformation was observed under both sulfate-reducing and methanogenic conditions. The isotopic enrichment factors () estimated for each enrichment were almost identical (−13.4 to −14.6; r² = 0.89 to 0.99). A value of −14.4 ± 0.7 was obtained from regression analysis (r² = 0.97, n = 55, 95% confidence interval), when all data from our MTBE-transforming anaerobic cultures were combined. The similar magnitude of carbon isotope fractionation in all enrichments regardless of culture or electron-accepting condition suggests that the terminal electron-accepting process may not significantly affect carbon isotope fractionation during anaerobic MTBE degradation.     

Acetate Production from Oil under Sulfate-Reducing Conditions in Bioreactors Injected with Sulfate and Nitrate

Oil production by water injection can cause souring in which sulfate in the injection water is reduced to sulfide by resident sulfate-reducing bacteria (SRB). Sulfate (2 mM) in medium injected at a rate of 1 pore volume per day into upflow bioreactors containing residual heavy oil from the Medicine Hat Glauconitic C field was nearly completely reduced to sulfide, and this was associated with the generation of 3 to 4 mM acetate. Inclusion of 4 mM nitrate inhibited souring for 60 days, after which complete sulfate reduction and associated acetate production were once again observed. Sulfate reduction was permanently inhibited when 100 mM nitrate was injected by the nitrite formed under these conditions. Pulsed injection of 4 or 100 mM nitrate inhibited sulfate reduction temporarily. Sulfate reduction resumed once nitrate injection was stopped and was associated with the production of acetate in all cases. The stoichiometry of acetate formation (3 to 4 mM formed per 2 mM sulfate reduced) is consistent with a mechanism in which oil alkanes and water are metabolized to acetate and hydrogen by fermentative and syntrophic bacteria (K. Zengler et al., Nature 401:266–269, 1999), with the hydrogen being used by SRB to reduce sulfate to sulfide. In support of this model, microbial community analyses by pyrosequencing indicated SRB of the genus Desulfovibrio, which use hydrogen but not acetate as an electron donor for sulfate reduction, to be a major community component. The model explains the high concentrations of acetate that are sometimes found in waters produced from water-injected oil fields.     

Response of the Sulfate-Reducing Community to the Re-establishment of Estuarine Conditions in Two Contrasting Soils: a Mesocosm Approach

We studied the response of the sulfate-reducing prokaryote (SRP) communities to the experimental variation of salinity and tide in an outdoor mesocosm setup. Intact soil monoliths were collected at two areas of the Haringvliet lagoon (The Netherlands): one sampling location consisted of agricultural grassland, drained and fertilized for at least the last century; the other of a freshwater marshland with more recent sea influence. Two factors, i.e., “salinity” (freshwater/oligohaline) and “tide” (nontidal/tidal), were tested in a full-factorial design. Soil samples were collected after 5 months (June–October). Dissimilatory (bi)sulfite reductase β subunit-based denaturing gradient gel electrophoresis (dsrB-DGGE) analysis revealed that the SRP community composition in the agricultural grassland and in the freshwater marshland was represented mainly by microorganisms related to the Desulfobulbaceae and the Desulfobacteraceae, respectively. Desulfovibrio-related dsrB were detected only in the tidal treatments; Desulfomonile-related dsrB occurrence was related to the presence of oligohaline conditions. Treatments did have an effect on the overall SRP community composition of both soils, but not on the sulfate depletion rates in sulfate-amended anoxic slurry incubations. However, initiation of sulfate reduction upon sulfate addition was clearly different between the two soils.     

Structural and Functional Dynamics of Sulfate-Reducing Populations in Bacterial Biofilms

We describe the combined application of microsensors and molecular techniques to investigate the development of sulfate reduction and of sulfate-reducing bacterial populations in an aerobic bacterial biofilm. Microsensor measurements for oxygen showed that anaerobic zones developed in the biofilm within 1 week and that oxygen was depleted in the top 200 to 400 μm during all stages of biofilm development. Sulfate reduction was first detected after 6 weeks of growth, although favorable conditions for growth of sulfate-reducing bacteria (SRB) were present from the first week. In situ hybridization with a 16S rRNA probe for SRB revealed that sulfate reducers were present in high numbers (approximately 10⁸ SRB/ml) in all stages of development, both in the oxic and anoxic zones of the biofilm. Denaturing gradient gel electrophoresis (DGGE) showed that the genetic diversity of the microbial community increased during the development of the biofilm. Hybridization analysis of the DGGE profiles with taxon-specific oligonucleotide probes showed that Desulfobulbus and Desulfovibrio were the main sulfate-reducing bacteria in all biofilm samples as well as in the bulk activated sludge. However, different Desulfobulbus and Desulfovibrio species were found in the 6th and 8th weeks of incubation, respectively, coinciding with the development of sulfate reduction. Our data indicate that not all SRB detected by molecular analysis were sulfidogenically active in the biofilm.     

Phenol Biodegradation and Metal Removal by a Mixed Bacterial Consortium

This paper reports the tolerance and biodegradation of phenol by a heavy metal–adapted environmental bacterial consortium, known as consortium culture (CC). At the highest tolerable phenol concentration of 1200 mg/L, CC displayed specific growth rate of 0.04 h^?1, phenol degradation rate of 6.11 mg L^?1 h^?1 and biomass of 8.45 ± 0.35 (log₁₀ colony-forming units [CFU]/ml) at the end of incubation. Phenol was degraded via the ortho-cleavage pathway catalyzed by cathechol-1,2-dioxygenase with specific activity of 0.083 (µmol min^?1 mg^?1 protein). The different constituent bacterial isolates of CC preferentially grow on benzene, toluene, xylene, ethylbenzene, cresol, and catechol, suggesting a synergistic mechanism involved in the degradation process. Microtox assay showed that phenol degradation was achieved without producing toxic dead-end metabolites. Moreover, lead (Pb) and cadmium (Cd) at the highest tested concentration of 1.0 and 0.1 mg/L, respectively, did not inhibit phenol degradation by CC. Simultaneous metal removal during phenol degradation was achieved using CC. These findings confirmed the dual function of CC to degrade phenol and to remove heavy metals from a mixed-pollutant medium.     

Distribution of the ermG Gene among Bacterial Isolates from Porcine Intestinal Contents

The ermG gene was first found in the soil bacterium Bacillus sphaericus. More recently, it was found in several human intestinal Bacteroides species. We report here the first finding of ermG genes in gram-positive bacteria isolated from porcine feces and from under-barn manure pits used to store porcine wastes. The porcine ermG sequences were identical to the sequence of the B. sphaericus ermG gene except that six of the seven ermG-containing strains contained an insertion sequence element insertion in the C-terminal end of the gene. The porcine ermG genes were found in three different gram-positive genera, an indication that it is possible that the gene is being spread by horizontal gene transfer. A segment of a Bacteroides conjugative transposon that carries an ermG gene cross-hybridized with DNA from six of the seven porcine isolates, but the restriction patterns in the porcine strains were different from that of the Bacteroides conjugative transposon.     

Reduction of Cr(VI) by a Consortium of Sulfate-Reducing Bacteria (SRB III)

A consortium of bacteria with tolerance to high concentrations of Cr(VI) (up to 2,500 ppm) and other toxic heavy metals has been obtained from metal-refinishing wastewaters in Chengdu, People's Republic of China. This consortium consists of a range of gram-positive and gram-negative rods and has the capacity to reduce Cr(VI) to Cr(III) as amorphous precipitates which are associated with the bacterial surfaces. An endospore-producing, gram-positive rod and a gram-negative rod accumulate the most metallic precipitates, and, over time, 80 to 95% of Cr can be removed from concentrations ranging from 50 to 2,000 ppm (0.96 to 38.45 mM). Kinetic studies revealed a first-order constant for Cr removal of 0.1518 h^-1 for an initial concentration of 1,000 ppm (19.3 mM), and the sorption isothermal data could be interpreted by the Freundlich relationship. The sorption was not entirely due to a passive interaction with reactive sites on the bacterial surfaces since gamma-irradiated, killed cells could not immobilize as much metal. When U or Zn was added with the Cr, it was also removed and could even increase the total amount of Cr immobilized. The consortium was tolerant to small amounts of oxygen in the headspace of tubes, but active growth of the bacteria was a requirement for Cr immobilization through Cr(VI) reduction, resulting in the lowering of E_h. Our data suggest that the reduction was via H₂S. This consortium has been named SRB III, and it may be useful for the bioremediation of fluid metal-refining wastes.     

Anaerobic Biotransformation of High Concentrations of Chloroform by an Enrichment Culture and Two Bacterial Isolates

A fermentative enrichment culture (designated DHM-1) was developed that is capable of cometabolically biotransforming high concentrations of chloroform (CF) to nontoxic end products. Two Pantoea spp. were isolated from DHM-1 that also possess this dechlorination capability. Following acclimation to increasing levels of CF, corn syrup-grown DHM-1 was able to transform over 500 mg/liter CF in the presence of vitamin B₁₂ (approximately 3% of CF on a molar basis) at a rate as high as 22 mg/liter/day in a mineral salts medium. CO, CO₂, and organic acids were the predominant biodegradation products, suggesting that hydrolytic reactions predominate during CF transformation. DHM-1 was capable of growing on corn syrup in the presence of high concentrations of CF (as may be present near contaminant source zones in groundwater), which makes it a promising culture for bioaugmentation. Strains DHM-1B and DHM-1T transform CF at rates similar to that of the DHM-1 enrichment culture. The ability of these strains to grow in the presence of high concentrations of CF appears to be related to alteration of membrane fluidity or homeoviscous and homeophasic adaptation.Chloroform (CF) is a toxic organic compound that is frequently detected in groundwater. In the 2007 “CERCLA Priority List of Hazardous Substances,” CF ranks eleventh overall and is the third highest among chlorinated organics after vinyl chloride and polychlorinated biphenyls (4). When present at hazardous waste sites, CF is often a focal point for evaluating the feasibility of bioremediation, since it is toxic to many obligate anaerobic prokaryotes (44). For example, 1 mg/liter of CF completely inhibited dechlorination of tetrachloroethene (PCE) by a chlororespiring anaerobic isolate (32). Inhibition of reductive dechlorination of chloroethenes by CF is a general problem for sites cocontaminated with CF, which can only be overcome by first removing the CF (6).In spite of major recent advances in bioremediation of chlorinated organic compounds, treatment of CF, especially at high concentrations (e.g., >100 mg/liter), remains challenging. Although aerobic biotransformation of CF is possible (e.g., cometabolism by a butane-grown strain) (14), CF is more difficult to cometabolize than trichloroethene (42). Biotransformation of CF by mixed or pure cultures under methanogenic (5, 21) and sulfate-reducing (20) conditions has been reported, however, only at low-mg/liter CF concentrations.Corrinoids such as vitamin B₁₂ (i.e., cyanocobalamin) are effective catalysts for increasing the rate of halomethane biotransformation under anaerobic conditions. Addition of vitamin B₁₂ also shifts the pathway away from reductive dechlorination and toward hydrolytic and substitutive reactions, forming CO, CO₂, and organic acids as the major products (8, 23, 24). With low levels of B₁₂ added (3 to 5% molar ratios of CF), an enrichment culture grown on dichloromethane (DCM) as the sole substrate (8) and a lactate-grown sulfate-reducing enrichment culture (18) were able to biotransform up to 260 mg/liter and 270 mg/liter CF, respectively. However, use of a DCM-grown culture is not feasible for in situ bioremediation since DCM is also a regulated contaminant and the culture grows well only at 35°C. A major drawback with using a lactate-grown culture is the accumulation of DCM during CF transformation. Organohalide respiration of CF by a Dehalobacter population was recently reported (19), although DCM is the end product. Further treatment of the DCM would be necessary for bioremediation to be successful, although bioaugmentation cultures for this purpose are not yet available. It is apparent that a strategy for bioremediation of high concentrations of chloroform is still lacking.In a recent microcosm study using soil and groundwater from an industrial site, we demonstrated that bioaugmentation is a technically feasible remediation strategy for high concentrations of chloroform as well as carbon tetrachloride (CT) and trichlorofluoromethane (35). High rates of transformation were achieved with a fermentative enrichment culture that grows on corn syrup, supplemented with B₁₂ (1.3 to 1.4 mol%). The objectives of this study were to characterize the fermentative culture in terms of its maximum rate of CF transformation, its dependence on B₁₂ for transformation of CF, and its ability to grow on corn syrup in the presence of CF; to identify the transformation products from CF; to isolate and identify the microbes in the enrichment culture that are responsible for CF biotransformation; and to investigate the adaptation mechanisms used by the isolates to tolerate the toxicity of high concentrations of CF.     

Metabolism under Microaerobic Conditions of Mitochondria from Cowpea Nodules

A method is described for isolating mitochondria from nodules of cowpea (Vigna unguiculata [L.] Walp.) under completely anaerobic conditions. The mitochondria were immediately active when incubated aerobically with substrates, and their respiration rates were higher than mitochondria prepared in air. The mitochondria lacked fumarate reductase and were not inhibited by 5% CO₂. When incubated under microaerobic conditions, their respiration could be measured by leghemoglobin spectroscopy. Microaerobic respiration was inhibited approximately 50% by 1 millimolar malonate, and was completely inhibited by cyanide. O₂ uptake and the ATP/O ratio declined under microaerobic conditions, and therefore ATP production may be low in the environment of infected nodule cells.