Stable carbon isotope fractionation by acetotrophic sulfur-reducing bacteria

Acetate is the most important intermediate in anaerobic degradation of organic matter. The carbon isotope effects associated with the oxidation of acetate (ɛ_ac) were examined for four acetotrophic sulfur reducers, Desulfuromonas acetoxidans, Desulfuromonas thiophila, Desulfurella acetivorans , and Hippea maritima . During the consumption of acetate and sulfur, acetate was enriched in ¹³C by 11.5 and 11.2‰ in Desulfuromonas acetoxidans and Desulfuromonas thiophila , respectively. By contrast, isotope fractionation in D. acetivorans and H. maritima resulted in isotope enrichment factors of ɛ_ac=−6.3‰ and −8.4‰, respectively. These sulfur-reducing bacteria all metabolize acetate via the tricarboxylic acid cycle, but have different mechanisms for the initial activation of acetate. In Desulfuromonas acetoxidans , acetyl-CoA is formed by succinyl-CoA : acetate-CoA-transferase, and in D. acetivorans by acetate kinase and phosphate acetyltransferase. Hence, values of ɛ_ac seem to be characteristic for the type of activation of acetate to acetyl-CoA in acetotrophic sulfur reducers. Summarizing ɛ_ac-values in anaerobic acetotrophic microorganisms, it appears that isotope fractionation depends on the mechanism of acetate activation to acetyl-CoA, on the key enzyme of the acetate dissimilation pathway, and on the bioavailability of acetate, which all have to be considered when using δ¹³C of acetate in environmental samples for diagnosis of the involved microbial populations.     

Stable carbon isotope ratios of lipid biomarkers of sulfate-reducing bacteria

We examined the potential use of natural-abundance stable carbon isotope ratios of lipids for determining substrate usage by sulfate-reducing bacteria (SRB). Four SRB were grown under autotrophic, mixotrophic, or heterotrophic growth conditions, and the delta13C values of their individual fatty acids (FA) were determined. The FA were usually 13C depleted in relation to biomass, with Deltadelta13C(FA - biomass) of -4 to -17 per thousand; the greatest depletion occurred during heterotrophic growth. The exception was Desulfotomaculum acetoxidans, for which substrate limitation resulted in biomass and FA becoming isotopically heavier than the acetate substrate. The delta13C values of FA in Desulfotomaculum acetoxidans varied with the position of the double bond in the monounsaturated C16 and C18 FA, with FA becoming progressively more 13C depleted as the double bond approached the methyl end. Mixotrophic growth of Desulfovibrio desulfuricans resulted in little depletion of the i17:1 biomarker relative to biomass or acetate, whereas growth with lactate resulted in a higher proportion of i17:1 with a greater depletion in 13C. The relative abundances of 10Me16:0 in Desulfobacter hydrogenophilus and Desulfobacterium autotrophicum were not affected by growth conditions, yet the Deltadelta13C(FA - substrate) values of 10Me16:0 were considerably greater during autotrophic growth. These experiments indicate that FA delta13C values can be useful for interpreting carbon utilization by SRB in natural environments.     

Stable carbon isotope fractionation during degradation of dichloromethane by methylotrophic bacteria

Stable carbon isotope fractionation during dichloromethane (DCM) degradation by methylotrophic bacteria was investigated under aerobic and nitrate-reducing conditions. The strains studied comprise several Hyphomicrobium strains, Methylobacterium, Methylopila, Methylophilus and Methylorhabdus spp. that are considered to degrade DCM by a glutathione (GSH)-dependent dehalogenase enzyme system in the initial step. The stable carbon isotope fractionation factors (alphaC) of the strains varied under aerobic conditions between 1.043 and 1.071 and under nitrate-reducing conditions between 1.048 and 1.065. Comparison of isotope fractionation under aerobic and nitrate-reducing conditions by individual strains revealed only minor to no differences. The variability in isotope fractionation among strains was found to be related to the polymorphism of the functional genes encoding the DCM dehalogenase.     

Stable carbon isotope fractionation by marine phytoplankton during photosynthesis

Abstract. In the marine environment, the range of values of carbon isotope fractionation between particulate tissue of phytoplankton and inorganic carbon can be more than 20‰ (− 35‰ < δ¹³C < − 14‰). This review considers the influence of seawater temperature, lipid content of phytoplanktonic cells, kinetic fractionation, and carbon pathway on δ¹³C values observed at sea.
In order to study the contribution of carboxylases (RUBISCO and the β-carboxylases phosphoenolpyruvate carboxylase, phosphoenoplpyruvate carboxykinase and pyruvate carboxylase) to variations of particulate δ¹³C values at sea, we present results obtained simultenously on carboxylase activities and δ¹³C in various environmental conditions. The lowest δ¹³C values are clearly associated with predominance of ribulose-1.5-bisphosphate carboxylase activity, but it was more difficult to explain the high δ¹³C values. Different hypotheses are discussed.     

Stable hydrogen and carbon isotope fractionation during microbial toluene degradation: mechanistic and environmental aspects

Primary features of hydrogen and carbon isotope fractionation during toluene degradation were studied to evaluate if analysis of isotope signatures can be used as a tool to monitor biodegradation in contaminated aquifers. D/H hydrogen isotope fractionation during microbial degradation of toluene was measured by gas chromatography. Per-deuterated toluene-d(8) and nonlabeled toluene were supplied in equal amounts as growth substrates, and kinetic isotope fractionation was calculated from the shift of the molar ratios of toluene-d(8) and nondeuterated toluene. The D/H isotope fractionation varied slightly for sulfate-reducing strain TRM1 (slope of curve [b] = -1.219), Desulfobacterium cetonicum (b = -1.196), Thauera aromatica (b = -0.816), and Geobacter metallireducens (b = -1.004) and was greater for the aerobic bacterium Pseudomonas putida mt-2 (b = -2.667). The D/H isotope fractionation was 3 orders of magnitude greater than the (13)C/(12)C carbon isotope fractionation reported previously. Hydrogen isotope fractionation with nonlabeled toluene was 1.7 and 6 times less than isotope fractionation with per-deuterated toluene-d(8) and nonlabeled toluene for sulfate-reducing strain TRM1 (b = -0.728) and D. cetonicum (b = -0.198), respectively. Carbon and hydrogen isotope fractionation during toluene degradation by D. cetonicum remained constant over a growth temperature range of 15 to 37 degrees C but varied slightly during degradation by P. putida mt-2, which showed maximum hydrogen isotope fractionation at 20 degrees C (b = -4.086) and minimum fractionation at 35 degrees C (b = -2.138). D/H isotope fractionation was observed only if the deuterium label was located at the methyl group of the toluene molecule which is the site of the initial enzymatic attack on the substrate by the bacterial strains investigated in this study. Use of ring-labeled toluene-d(5) in combination with nondeuterated toluene did not lead to significant D/H isotope fractionation. The activity of the first enzyme in the anaerobic toluene degradation pathway, benzylsuccinate synthase, was measured in cell extracts of D. cetonicum with an initial activity of 3.63 mU (mg of protein)(-1). The D/H isotope fractionation (b = -1.580) was 30% greater than that in growth experiments with D. cetonicum. Mass spectroscopic analysis of the product benzylsuccinate showed that H atoms abstracted from the toluene molecules by the enzyme were retained in the same molecules after the product was released. Our findings revealed that the use of deuterium-labeled toluene was appropriate for studying basic features of D/H isotope fractionation. Similar D/H fractionation factors for toluene degradation by anaerobic bacteria, the lack of significant temperature dependence, and the strong fractionation suggest that analysis of D/H fractionation can be used as a sensitive tool to assess degradation activities. Identification of the first enzyme reaction in the pathway as the major fractionating step provides a basis for linking observed isotope fractionation to biochemical reactions.     

Stable isotope fractionation concepts for characterizing biotransformation of organohalides

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Stable sulfur isotope fractionation during the reduction of thiosulfate by Dethiosulfovibrio russensis

Stable sulfur isotope fractionation was investigated during reduction of thiosulfate by growing batch cultures of Dethiosulfovibrio russensis at a cell-specific reduction rate of 2.4 +/- 0.72 fmol cell(-1) d(-1) (28 degrees C). Citrate was used as carbon and energy source. The hydrogen sulfide produced by this sulfur- and thiosulfate-reducing bacterium was depleted in 34S by 11% compared to total thiosulfate sulfur, in agreement with previous results observed for sulfate-reducing bacteria. This indicates the operation of a similar pathway for thiosulfate reduction in these phylogenetically different bacteria.     

Carbon isotope fractionation by anoxygenic phototrophic bacteria in euxinic Lake Cadagno

Anoxygenic phototrophic bacteria utilize ancient metabolic pathways to link sulfur and iron metabolism to the reduction of CO₂. In meromictic Lake Cadagno, Switzerland, both purple sulfur (PSB) and green sulfur anoxygenic phototrophic bacteria (GSB) dominate the chemocline community and drive the sulfur cycle. PSB and GSB fix carbon utilizing different enzymatic pathways and these fractionate C‐isotopes to different extents. Here, these differences in C‐isotope fractionation are used to constrain the relative input of various anoxygenic phototrophs to the bulk community C‐isotope signal in the chemocline. We sought to determine whether a distinct isotopic signature of GSB and PSB in the chemocline persists in the settling fraction and in the sediment. To answer these questions, we also sought investigated C‐isotope fractionation in the water column, settling material, and sediment of Lake Cadagno, compared these values to C‐isotope fractionation of isolated anoxygenic phototroph cultures, and took a mass balance approach to investigate relative contributions to the bulk fractionation signature. We found a large C‐isotope fractionation between dissolved inorganic carbon (DIC) and particulate organic carbon (POC) in the Lake Cadagno chemocline. This large fractionation between the DIC and POC was also found in culture experiments carried out with anoxygenic phototrophic bacteria isolated from the lake. In the Lake Cadagno chemocline, anoxygenic phototrophic bacteria controlled the bulk C‐isotope fractionation, but the influence of GSB and PSB differed with season. Furthermore, the contribution of PSB and GSB to bulk C‐isotope fractionation in the chemocline could be traced in the settling fraction and in the sediment. Taken together with other studies, such as lipid biomarker analyzes and investigations of other stratified lakes, these results offer a firmer understanding of diagenetic influences on bacterial biomass.     

Stable carbon isotope analysis of nucleic acids to trace sources of dissolved substrates used by estuarine bacteria

The natural abundance of stable carbon isotopes measured in bacterial nucleic acids extracted from estuarine bacterial concentrates was used to trace sources of organic matter for bacteria in aquatic environments. The stable carbon isotope ratios of Pseudomonas aeruginosa and nucleic acids extracted from cultures resembled those of the carbon source on which bacteria were grown. The carbon isotope discrimination between the substrate and total cell carbon from bacterial cultures averaged 2.3% +/- 0.6% (n = 13). Furthermore, the isotope discrimination between the substrate and nucleic acids extracted from bacterial cultures was 2.4% +/- 0.4% (n = 10), not significantly different from the discrimination between bacteria and the substrate. Estuarine water samples were prefiltered through 1-micron-pore-size cartridge filters. Bacterium-sized particles in the filtrates were concentrated with tangential-flow filtration and centrifugation, and nucleic acids were then extracted from these concentrates. Hybridization with 16S rRNA probes showed that approximately 90% of the nucleic acids extracted on two sample dates were of eubacterial origin. Bacteria and nucleic acids from incubation experiments using estuarine water samples enriched with dissolved organic matter from Spartina alterniflora and Cyclotella caspia had stable carbon isotope values similar to those of the substrate sources. In a survey that compared diverse estuarine environments, stable carbon isotopes of bacteria grown in incubation experiments ranged from -31.9 to -20.5%. The range in isotope values of nucleic acids extracted from indigenous bacteria from the same waters was similar, -27.9 to -20.2%. Generally, the lack of isotope discrimination between bacteria and nucleic acids that was noted in the laboratory was observed in the field.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inorganic carbon fixation by sulfate-reducing bacteria in the Black Sea water column

The Black Sea is the largest anoxic water basin on Earth and its stratified water column comprises an upper oxic, middle suboxic and a lower permanently anoxic, sulfidic zone. The abundance of sulfate-reducing bacteria (SRB) in water samples was determined by quantifying the copy number of the dsrA gene coding for the alpha subunit of the dissimilatory (bi)sulfite reductase using real-time polymerase chain reaction. The dsrA gene was detected throughout the whole suboxic and anoxic zones. The maximum dsrA copy numbers were 5 x 10(2) and 6.3 x 10(2) copies ml(-1) at 95 m in the suboxic and at 150 m in the upper anoxic zone, respectively. The proportion of SRB to total Bacteria was 0.1% in the oxic, 0.8-1.9% in the suboxic and 1.2-4.7% in the anoxic zone. A phylogenetic analysis of 16S rDNA clones showed that most clones from the anoxic zone formed a coherent cluster within the Desulfonema-Desulfosarcina group. A similar depth profile as for dsrA copy numbers was obtained for the concentration of non-isoprenoidal dialkyl glycerol diethers (DGDs), which are most likely SRB-specific lipid biomarkers. Three different DGDs were found to be major components of the total lipid fractions from the anoxic zone. The DGDs were depleted in (13)C relative to the delta(13)C values of dissolved CO(2) (delta(13)C(CO2)) by 14-19 per thousand. Their delta(13)C values [delta(13)C(DGD(II-III))] co-varied with depth showing the least (13)C-depleted values in the top of the sulfidic, anoxic zone and the most (13)C-depleted values in the deep anoxic waters at 1500 m. This co-variation provides evidence for CO(2) incorporation by the DGD(II-III)-producing SRB, while the 1:2 relationship between delta(13)C(CO2) and delta(13)C(DGD(II-III)) indicates the use of an additional organic carbon source.     

Short-term measurement of carbon isotope fractionation in plants

Combustion-based studies of the carbon-13 content of plants give only an integrated, long-term value for the isotope fractionation associated with photosynthesis. A method is described here which permits determination of this isotope fractionation in 2 to 3 hours. To accomplish this, the plant is enclosed in a glass chamber, and the quantity and isotopic content of the CO₂ remaining in the atmosphere are monitored during photosynthesis. Isotope fractionation studies by this method give results consistent with what is expected from combustion studies of C₃, C₄, and Crassulacean acid metabolism plants. This method will make possible a variety of new studies of environmental and species effects in carbon isotope fractionation.     

Stable carbon isotope analysis of nucleic acids to trace sources of dissolved substrates used by estuarine bacteria.

The natural abundance of stable carbon isotopes measured in bacterial nucleic acids extracted from estuarine bacterial concentrates was used to trace sources of organic matter for bacteria in aquatic environments. The stable carbon isotope ratios of Pseudomonas aeruginosa and nucleic acids extracted from cultures resembled those of the carbon source on which bacteria were grown. The carbon isotope discrimination between the substrate and total cell carbon from bacterial cultures averaged 2.3% +/- 0.6% (n = 13). Furthermore, the isotope discrimination between the substrate and nucleic acids extracted from bacterial cultures was 2.4% +/- 0.4% (n = 10), not significantly different from the discrimination between bacteria and the substrate. Estuarine water samples were prefiltered through 1-micron-pore-size cartridge filters. Bacterium-sized particles in the filtrates were concentrated with tangential-flow filtration and centrifugation, and nucleic acids were then extracted from these concentrates. Hybridization with 16S rRNA probes showed that approximately 90% of the nucleic acids extracted on two sample dates were of eubacterial origin. Bacteria and nucleic acids from incubation experiments using estuarine water samples enriched with dissolved organic matter from Spartina alterniflora and Cyclotella caspia had stable carbon isotope values similar to those of the substrate sources. In a survey that compared diverse estuarine environments, stable carbon isotopes of bacteria grown in incubation experiments ranged from -31.9 to -20.5%. The range in isotope values of nucleic acids extracted from indigenous bacteria from the same waters was similar, -27.9 to -20.2%. Generally, the lack of isotope discrimination between bacteria and nucleic acids that was noted in the laboratory was observed in the field.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction of molybdate by sulfate-reducing bacteria

Molybdate is an essential trace element required by biological systems including the anaerobic sulfate-reducing bacteria (SRB); however, detrimental consequences may occur if molybdate is present in high concentrations in the environment. While molybdate is a structural analog of sulfate and inhibits sulfate respiration of SRB, little information is available concerning the effect of molybdate on pure cultures. We followed the growth of Desulfovibrio gigas ATCC 19364, Desulfovibrio vulgaris Hildenborough, Desulfovibrio desulfuricans DSM 642, and D. desulfuricans DSM 27774 in media containing sub-lethal levels of molybdate and observed a red-brown color in the culture fluid. Spectral analysis of the culture fluid revealed absorption peaks at 467, 395 and 314 nm and this color is proposed to be a molybdate–sulfide complex. Reduction of molybdate with the formation of molybdate disulfide occurs in the periplasm D. gigas and D. desulfuricans DSM 642. From these results we suggest that the occurrence of poorly crystalline Mo-sulfides in black shale may be a result from SRB reduction and selective enrichment of Mo in paleo-seawater.     

Stable isotope fractionation caused by glycyl radical enzymes during bacterial degradation of aromatic compounds

Stable isotope fractionation was studied during the degradation of m-xylene, o-xylene, m-cresol, and p-cresol with two pure cultures of sulfate-reducing bacteria. Degradation of all four compounds is initiated by a fumarate addition reaction by a glycyl radical enzyme, analogous to the well-studied benzylsuccinate synthase reaction in toluene degradation. The extent of stable carbon isotope fractionation caused by these radical-type reactions was between enrichment factors (epsilon) of -1.5 and -3.9, which is in the same order of magnitude as data provided before for anaerobic toluene degradation. Based on our results, an analysis of isotope fractionation should be applicable for the evaluation of in situ bioremediation of all contaminants degraded by glycyl radical enzyme mechanisms that are smaller than 14 carbon atoms. In order to compare carbon isotope fractionations upon the degradation of various substrates whose numbers of carbon atoms differ, intrinsic epsilon (epsilon(intrinsic)) were calculated. A comparison of epsilon(intrinsic) at the single carbon atoms of the molecule where the benzylsuccinate synthase reaction took place with compound-specific epsilon elucidated that both varied on average to the same extent. Despite variations during the degradation of different substrates, the range of epsilon found for glycyl radical reactions was reasonably narrow to propose that rough estimates of biodegradation in situ might be given by using an average epsilon if no fractionation factor is available for single compounds.     

Dimethylsulfoxide reduction by marine sulfate-reducing bacteria

Abstract Dimethylsulfoxide (DMSO) reduction occurred in five out of nine strains of sulfate-reducing bacteria from marine or saline environments, but not in three freshwater isolates. DMSO reduction supported growth in all positive strains. In Desulfovibrio desulfuricans strain PA2805, DMSO reduction occurred simultaneously with sulfate reduction and was not effectively inhibited by molybdate, a specific inhibitor of sulfate reduction. The growth yield per mol lactate was 26% higher with DMSO than with sulfate as electron acceptor. In extracts of cells of strain PA2805 grown on sulfate, a low level of DMSO-reducing activity was present (0.013 μmol (mg protein)⁻ min⁻); higher levels were found in cells grown on DMSO (0.56 μmol (mg protein)⁻ min⁻). In anoxic marine environments DMSO reduction by sulfate-reducing bacteria may lead to enhanced dimethylsulfide emission rates.     

Diversity of sulfur isotope fractionations by sulfate-reducing prokaryotes

Batch culture experiments were performed with 32 different sulfate-reducing prokaryotes to explore the diversity in sulfur isotope fractionation during dissimilatory sulfate reduction by pure cultures. The selected strains reflect the phylogenetic and physiologic diversity of presently known sulfate reducers and cover a broad range of natural marine and freshwater habitats. Experimental conditions were designed to achieve optimum growth conditions with respect to electron donors, salinity, temperature, and pH. Under these optimized conditions, experimental fractionation factors ranged from 2.0 to 42.0 per thousand. Salinity, incubation temperature, pH, and phylogeny had no systematic effect on the sulfur isotope fractionation. There was no correlation between isotope fractionation and sulfate reduction rate. The type of dissimilatory bisulfite reductase also had no effect on fractionation. Sulfate reducers that oxidized the carbon source completely to CO2 showed greater fractionations than sulfate reducers that released acetate as the final product of carbon oxidation. Different metabolic pathways and variable regulation of sulfate transport across the cell membrane all potentially affect isotope fractionation. Previous models that explained fractionation only in terms of sulfate reduction rates appear to be oversimplified. The species-specific physiology of each sulfate reducer thus needs to be taken into account to understand the regulation of sulfur isotope fractionation during dissimilatory sulfate reduction.     

Anaerobic benzene degradation by Gram-positive sulfate-reducing bacteria

Despite its high chemical stability, benzene is known to be biodegradable with various electron acceptors under anaerobic conditions. However, our understanding of the initial activation reaction and the responsible prokaryotes is limited. In the present study, we enriched a bacterial culture that oxidizes benzene to carbon dioxide under sulfate-reducing conditions. Community analysis using terminal restriction fragment length polymorphism, 16S rRNA gene sequencing and FISH revealed 95% dominance of one phylotype that is affiliated to the Gram-positive bacterial genus Pelotomaculum showing that sulfate-reducing Gram-positive bacteria are involved in anaerobic benzene degradation. In order to get indications of the initial activation mechanism, we tested the substrate utilization, performed cometabolism tests and screened for putative metabolites. Phenol, toluene, and benzoate could not be utilized as alternative carbon sources by the benzene-degrading culture. Cometabolic degradation experiments resulted in retarded rates of benzene degradation in the presence of phenol whereas toluene had no effect on benzene metabolism. Phenol, 2-hydroxybenzoate, 4-hydroxybenzoate, and benzoate were identified as putative metabolites in the enrichment culture. However, hydroxylated aromatics were shown to be formed abiotically. Thus, the finding of benzoate as an intermediate compound supports a direct carboxylation of benzene as the initial activation mechanism but additional reactions leading to its formation cannot be excluded definitely.