Microbial treatment of high-strength perchlorate wastewater

To treat wastewater containing high concentrations of perchlorate, a perchlorate reducing-bacterial consortium was obtained by enrichment culture grown on high-strength perchlorate (1200 mg L⁻¹) feed medium, and was characterized in a sequence batch reactor (SBR) over a long-time operation. The consortium removed perchlorate in the SBR with high reduction rates (35-90 mg L⁻¹ h⁻¹) and stable removal efficiency over 200-day operations. The maximum specific perchlorate reduction rate (q_max), half saturation constant (K_s), and optimal pH range were 0.67 mg-perchlorate mg-dry cell weight⁻¹ h⁻¹, 193.8 mg-perchlorate L⁻¹, and pH 7-9, respectively. The perchlorate reduction yield was 0.48 mol-perchlorate mol-acetate⁻¹. A clone library prepared using the amplicons of cld gene encoding chlorate dismutase showed that the dominant (per)chlorate reducing bacteria in the consortium were Dechlorosoma sp. (53%), Ideonella sp. (28%), and Dechloromonas sp. (19%).     

Carbon isotopic fractionation in heterotrophic microbial metabolism

Differences in the natural-abundance carbon stable isotopic compositions between products from aerobic cultures of Escherichia coli K-12 were measured. Respired CO2 was 3.4% depleted in 13C relative to the glucose used as the carbon source, whereas the acetate was 12.3% enriched in 13C. The acetate 13C enrichment was solely in the carboxyl group. Even though the total cellular carbon was only 0.6% depleted in 13C, intracellular components exhibited a significant isotopic heterogeneity. The protein and lipid fractions were -1.1 and -2.7%, respectively. Aspartic and glutamic acids were -1.6 and +2.7%, respectively, yet citrate was isotopically identical to the glucose. Probable sites of carbon isotopic fractionation include the enzyme, phosphotransacetylase, and the Krebs cycle.     

Carbon isotopic fractionation in heterotrophic microbial metabolism.

Differences in the natural-abundance carbon stable isotopic compositions between products from aerobic cultures of Escherichia coli K-12 were measured. Respired CO2 was 3.4% depleted in 13C relative to the glucose used as the carbon source, whereas the acetate was 12.3% enriched in 13C. The acetate 13C enrichment was solely in the carboxyl group. Even though the total cellular carbon was only 0.6% depleted in 13C, intracellular components exhibited a significant isotopic heterogeneity. The protein and lipid fractions were -1.1 and -2.7%, respectively. Aspartic and glutamic acids were -1.6 and +2.7%, respectively, yet citrate was isotopically identical to the glucose. Probable sites of carbon isotopic fractionation include the enzyme, phosphotransacetylase, and the Krebs cycle.     

Comparison of isotopic fractionation in lactic acid and ethanol fermentations

Pure D(-) and L(+) enantiomers of lactic acid were prepared by fermentation reactions with specific bacteria. In addition, naturally deuterated ethanol was prepared and converted into diastereoisomers using mandelic acid. Various sugars and nutrients were fermented into lactic acid in water having different deuterium contents and ethanol samples were obtained from yeast fermentation of sugars from different botanical origins. The methine and methylene groups in lactic acid and ethanol respectively show similar deuterium contents which are related to that found in the fermentation water. However, the methyl groups of both molecules are significantly different whatever the botanical origin of the carbon source in the fermentation medium.     

Carbon and hydrogen isotopic fractionation during anaerobic biodegradation of benzene

Compound-specific isotope analysis has the potential to distinguish physical from biological attenuation processes in the subsurface. In this study, carbon and hydrogen isotopic fractionation effects during biodegradation of benzene under anaerobic conditions with different terminal-electron-accepting processes are reported for the first time. Different enrichment factors (epsilon ) for carbon (range of -1.9 to -3.6 per thousand ) and hydrogen (range of -29 to -79 per thousand ) fractionation were observed during biodegradation of benzene under nitrate-reducing, sulfate-reducing, and methanogenic conditions. These differences are not related to differences in initial biomass or in rates of biodegradation. Carbon isotopic enrichment factors for anaerobic benzene biodegradation in this study are comparable to those previously published for aerobic benzene biodegradation. In contrast, hydrogen enrichment factors determined for anaerobic benzene biodegradation are significantly larger than those previously published for benzene biodegradation under aerobic conditions. A fundamental difference in the previously proposed initial step of aerobic versus proposed anaerobic biodegradation pathways may account for these differences in hydrogen isotopic fractionation. Potentially, C-H bond breakage in the initial step of the anaerobic benzene biodegradation pathway may account for the large fractionation observed compared to that in aerobic benzene biodegradation. Despite some differences in reported enrichment factors between cultures with different terminal-electron-accepting processes, carbon and hydrogen isotope analysis has the potential to provide direct evidence of anaerobic biodegradation of benzene in the field.     

Variations in microbial isotopic fractionation during soil organic matter decomposition

The soil microbial biomass (SMB) is known to participate in key soil processes such as the decomposition of soil organic matter (SOM). However, its contribution to the isotopic composition of the SOM is not clear yet. Shifts in the ¹³C and ¹⁵N natural abundances of the SMB and SOM fractions (mineralised, water soluble and non-extractable) were investigated by incubating an unamended arable soil for 6 months. Microbial communities were also studied using Fatty Acid Methyl Ester specific isotope analysis. The SMB was significantly ¹³C and ¹⁵N-enriched relative to other fractions throughout the incubation. However, significant isotopic variations with time were also observed due to the rapid consumption of relatively ¹³C-enriched water soluble compounds. The increase in the difference in SMB and water soluble ¹⁵N compositions as the water soluble C/N ratio decreased, indicated a shift from N assimilation to N dissimilation during the incubation. These changes also induced modifications of the microbial community structure. Once the system reached a steady-state (after 1 month), the isotopic trends appeared to corroborate those obtained in long term experiments in the field in that there was a constant microbial isotopic fractionation leading to a ¹³C and ¹⁵N enrichment of the SOM over the long-term. This work also suggests that caution must be exercised when interpreting short term incubation studies since perturbations associated with experimental set-up can have an important effect on C and N dynamics, microbial fractionation of ¹³C and ¹⁵N and microbial community structure.     

Effect of rhizobial strain and host plant on nitrogen isotopic fractionation in legumes

Lotus pedunculatus L., Medicago sativa L., Macroptilium atropurpureum, Glycine max, and Trifolium repens L. were grown in a N-free medium and inoculated with one of ten Rhizobium strains. Dry matter, N content, and δ¹⁵N values were determined for various plant parts.

Nodules, with the exception of those from lucerne, were enriched in ¹⁵N relative to atmospheric N. Considerable variation was found in δ¹⁵N values of plant herbage (−4.5 to +0.8). The extent of isotopic discrimination was dependent on both the host plant and the infecting rhizobial strain. This further complicates, but does not invalidate, the use of small variations in the natural abundance of ¹⁵N to estimate the contribution of symbiotically fixed N₂ to the N in legume herbage. Some other implications of the observed differences are also discussed.

     

Zinc isotopic fractionation in Phragmites australis in response to toxic levels of zinc

Stable isotope signatures of Zn have shown great promise in elucidating changes in uptake and translocation mechanisms of this metal in plants during environmental changes. Here this potential was tested by investigating the effect of high Zn concentrations on the isotopic fractionation patterns of Phragmites australis (Cav.) Trin. ex Steud. Plants were grown for 40?d in a nutritive solution containing 3.2?μM (sufficient) or 2?mM (toxic) Zn. The Zn isotopic composition of roots, rhizomes, shoots, and leaves was analysed. Stems and leaves were sampled at different heights to evaluate the effect of long-distance transport on Zn fractionation. During Zn sufficiency, roots, rhizomes, and shoots were isotopically heavy (δ(66)Zn(JMC Lyon)=0.2‰) while the youngest leaves were isotopically light (-0.5‰). During Zn excess, roots were still isotopically heavier (δ(66)Zn=0.5‰) and the rest of the plant was isotopically light (up to -0.5‰). The enrichment of heavy isotopes at the roots was attributed to Zn uptake mediated by transporter proteins under Zn-sufficient conditions and to chelation and compartmentation in Zn excess. The isotopically lighter Zn in shoots and leaves is consistent with long-distance root to shoot transport. The tolerance response of P. australis increased the range of Zn fractionation within the plant and with respect to the environment.     

The fractionation of chlorine isotopes by the aerobic methylotrophic bacterium Methylobacterium dichloromethanicum grown on dichloromethane

Methylobacterium dichloromethanicum was found to be able to utilize dichloromethane (DCM) as the source of carbon and energy with the production of biomass, CO2, and HCl. A comparative analysis of abundances of the major DCM isotopomers 35Cl(2)12C1H2, 35Cl37Cl12C1H2 and 37Cl(2)12CH2 made it possible to estimate the fractionation of chlorine isotopes during the bacterial metabolism of DCM. The kinetic chlorine isotope effects for 35Cl37Cl12C1H2 (m/z 86) and 37Cl(2)12C1H2 (m/z 88) relative to 35Cl(2)12C1H2 (m/z 84) turned out to be alpha 86/84 = 1.006 +/- 0.002 and alpha 88/84 = 1.023 +/- 0.003, respectively. The inference is made that the growth of M. dichloromethanicum on DCM is accompanied by the mass-independent fractionation of the DCM isotopomers.     

Theoretical evaluation of isotopic fractionation factors in oxidation reactions of benzene,phenol and chlorophenols

We have studied theoretically the rate determining steps of reactions of benzene with permanganate, perchlorate, ozone and dioxygen in the gas phase and aqueous solution as well as phenol and dichlorophenol in protonated and unprotonated forms in aqueous solution. Kinetic isotope effects were then calculated for all carbon atoms and based on their values isotopic fractionation factors corresponding to compound specific isotopic analysis have been evaluated. The influence of the oxidant, substituents, environment and protonation on the isotopic fractionation factors has been analyzed.     

Carbon isotopic fractionation in subtropical brazilian grassland soils. Comparison with tropical forest soils

The natural relationship¹³C/¹²C determined in three soil profiles under grass vegetation indicated a depletion in organic¹³C at depth: theδ ¹³C was between −18‰ and −15‰ in the A horizons and ranged from −18 to −22‰ at depth. Previous work showed that in forest soils, whereδ ¹³C was near −28‰ in the upper horizon, there was, on the contrary, a relative enrichment of the lower strata. This meant thatδ ¹³C, initially different in the various topsoils, became more equal at depth. Comparison between dark, deep horizons (sombric horizons), which are certainly of illuvial origine, would confirm this:δ ¹³C of grassland and a forest sombric horizon were almost equal at around −22‰. These results might mean that, in natural ecosystems, the isotopic carbon composition of the soil underlying humus would be independent of the vegetation type. This would have practical implications for the use of¹³C as a tracer for soil organic matter studies.     

Compound-specific isotopic fractionation patterns suggest different carbon metabolisms among Chloroflexus-like bacteria in hot-spring microbial mats

Stable carbon isotope fractionations between dissolved inorganic carbon and lipid biomarkers suggest photoautotrophy by Chloroflexus-like organisms in sulfidic and nonsulfidic Yellowstone hot springs. Where co-occurring, cyanobacteria appear to cross-feed Chloroflexus-like organisms supporting photoheterotrophy as well, although the relatively small 13C fractionation associated with cyanobacterial sugar biosynthesis may sometimes obscure this process.     

Carbon and hydrogen isotopic fractionation during lipid biosynthesis in a higher plant (Cryptomeria japonica)

Compound-specific carbon and hydrogen isotopic compositions of lipid biomolecules (n-alkanes, n-alkanoic acids, n-alkanols, sesquiterpenes, diterpenes, phytol, diterpenols and β-sitosterol), extracted from Cryptomeria japonica leaves, were determined in order to understand isotopic fractionations occurring during lipid biosynthesis in this species. All lipid biomolecules were depleted in both ¹³C and D relative to bulk tissue and ambient water, respectively. n-Alkyl lipids associated with the acetogenic pathway were depleted in ¹³C relative to bulk tissue by 2.4-9.9‰ and depleted in D relative to ambient water by 91-152‰. C₁₅- and C₃₀-isoprenoid lipids (sesquiterpenes, squalene and β-sitosterol) associated with the mevalonic-acid pathway are depleted in ¹³C relative to bulk tissue by 1.7-3.1‰ and depleted in D relative to ambient water by 212-238‰. C₂₀-isoprenoid lipids (phytol and diterpenoids) associated with the non-mevalonic-acid pathway were depleted in ¹³C relative to bulk tissue by 4.6-5.9‰ and depleted in D relative to ambient water by 238-303‰. Phytol was significantly depleted in D by amounts up to 65‰ relative to other C₂₀ isoprenoid lipids. The acetogenic, mevalonic-acid and non-mevalonic-acid pathways were clearly discriminated using a cross-plot between the carbon and hydrogen isotopic fractionations.     

Ecophysiology of 13C and 15N isotopic fractionation in forest fungi and the roots of the saprotrophic-mycorrhizal divide

To quantify and characterize N and C isotopic fractionation effects due to fungal transformation of organic substrates in forest ecosystems, we performed a field study in California and a meta-analysis of three additional studies conducted by others across the Northern Hemisphere. Basidiomycete fungal biomass was consistently enriched for the heavier isotope for C relative to substrate and either enriched or depleted for N relative to atmospheric N. Extent and pattern of fractionation was very variable, but the distinction between ectomycorrhizal and saprotrophic basidiomycetes was strongly supported, particularly when dual isotope analyses were performed. This differentiation, which we call the "EM-SAP Divide" holds for studies within a restricted ecosystem, but becomes less distinct over larger geographical regions, removing the rationale for using direct isotopic values from single specimens as diagnostic of ecophysiological role. For C, the EM-SAP Divide seems to reflect substrate effects, potentially due to differential access to recently synthesized versus recycled organic compounds, rather than distinct physiological pathways. Once substrate and ecophysiological role effects are removed, our meta-analysis suggests the existence of more than one mechanism causing C fractionations in fungi which is found equally in ectomycorrhizal and saprotrophic fungi. Similarly, a multimodal distribution of '¹⁵N values suggests that physiological effects may play a much stronger influence on N natural isotopic distributions in fungi. Our meta-analysis provides a firm statistical base to evaluate fungal ecological statements based on natural isotopic distributions of C and N. We call into question the current practice of using direct isotopic measurements to make statements about trophic relationships of fungi in the absence of other supporting evidence.     

Nitrogen isotopic fractionation and 18O exchange in relation to the mechanism of denitrification of nitrite by Pseudomonas stutzeri

Two types of mechanisms for the enzymatic reduction of NO2- to N2O have been proposed. In one, two NO2- ions are reduced in parallel, with the nitrogen-nitrogen bond formed from reduced intermediates. In the second, the two NO2- ions enter the reaction sequentially, with the nitrogen of at least one of the two having a valence of 3+ when the nitrogen-nitrogen bond is formed. Our objective was to distinguish between these two types of mechanism. Toward that end, the exchange of 18O from H2O to NO2- and the overall nitrogen isotopic fractionation factor (beta obs) were measured. The rate of exchange of oxygen from H2O to NO2-, resulting from a protonation-dehydration step preceding reductive events in both mechanisms, was less than 10% of the rate of denitrification at both low and high [NO2-]. The value of beta obs was 1.010 +/- 0.001 and 1.020 +/- 0.001 at low and high [NO2-], respectively. Expressions for beta obs, as a function of the measured rate of entry of oxygen from H2O into NO2-, were derived for both types of mechanism. The measured dependence of beta obs on substrate concentration, as constrained by the 18O exchange data, is inconsistent with the first type of mechanism, but consistent with the second type. Thus, by combining nitrogen isotopic fractionation and 18O exchange data, we rule out any mechanism in Pseudomonas stutzeri in which NO2- ions are reduced in parallel, with the nitrogen-nitrogen bond being formed from reduced intermediates.     

Differential isotopic fractionation during Cr(VI) reduction by an aquifer-derived bacterium under aerobic versus denitrifying conditions

We studied Cr isotopic fractionation during Cr(VI) reduction by Pseudomonas stutzeri strain RCH2. Despite the fact that strain RCH2 reduces Cr(VI) cometabolically under both aerobic and denitrifying conditions and at similar specific rates, fractionation was markedly different under these two conditions (ε was ～2‰ aerobically and ～0.4‰ under denitrifying conditions).