Rapid Consumption of Dihydrogen Injected into a Shallow Aquifer by Ecophysiologically Different Microbes.

The envisaged future dihydrogen (H2) economy requires a H2 gas grid as well as large deep underground stores. However, the consequences of an unintended spread of H2 through leaky pipes, wells, or subterranean gas migrations on groundwater resources and their ecosystems are poorly understood. Therefore, we emulated a short-term leakage incident by injecting gaseous H2 into a shallow aquifer at the TestUM test site and monitored the subsequent biogeochemical processes in the groundwater system. At elevated H2 concentrations, an increase in acetate concentrations and a decrease in microbial α-diversity with a concomitant change in microbial ß-diversity were observed. Additionally, microbial H2 oxidation was indicated by temporally higher abundances of taxa known for aerobic or anaerobic H2 oxidation. After H2 concentrations diminished below the detection limit, α- and ß-diversity approached baseline values. In summary, the emulated H2 leakage resulted in a temporally limited change of the groundwater microbiome and associated geochemical conditions due to the intermediate growth of H2 consumers. The results confirm the general assumption that H2, being an excellent energy and electron source for many microorganisms, is quickly microbiologically consumed in the environment after a leakage.

Sulfidic acetate mineralization at 45°C by an aquifer microbial community: key players and effects of heat changes on activity and community structure.

High-Temperature Aquifer Thermal Energy Storage (HT-ATES) is a sustainable approach for integrating thermal energy from various sources into complex energy systems. Temperatures ≥45°C, which are relevant in impact zones of HT-ATES systems, may dramatically influence the structure and activities of indigenous aquifer microbial communities. Here, we characterized an acetate-mineralizing, sulfate-reducing microbial community derived from an aquifer and adapted to 45°C. Acetate mineralization was strongly inhibited at temperatures ≤25°C and 60°C. Prolonged incubation at 12°C and 25°C resulted in acetate mineralization recovery after 40-80 days whereas acetate was not mineralized at 60°C within 100 days. Cultures pre-grown at 45°C and inhibited for 28 days by incubation at 12°C, 25°C, or 60°C recovered quickly after changing the temperature back to 45°C. Phylotypes affiliated to the order Spirochaetales and to endospore-forming sulfate reducers of the order Clostridiales were highly abundant in microcosms being active at 45°C highlighting their key role. In summary, prolonged incubation at 45°C resulted in active microbial communities mainly consisting of organisms adapted to temperatures between the typical temperature range of mesophiles and thermophiles and being resilient to temporary heat changes.

Characterization of Hexachlorocyclohexane Isomer Dehydrochlorination by LinA1 and LinA2 Using Multi-element Compound-Specific Stable Isotope Analysis.

Dehydrochlorination is one of the main (thus far discovered) processes for aerobic microbial transformation of hexachlorocyclohexane (HCH) which is mainly catalyzed by LinA enzymes. In order to gain a better understanding of the reaction mechanisms, multi-element compound-specific stable isotope analysis was applied for evaluating α- and γ-HCH transformations catalyzed by LinA1 and LinA2 enzymes. The isotopic fractionation (εE) values for particular elements of (+)α-HCH (εC = -10.8 ± 1.0‰, εCl = -4.2 ± 0.5‰, εH = -154 ± 16‰) were distinct from the values for (-)α-HCH (εC = -4.1 ± 0.7‰, εCl = -1.6 ± 0.2‰, εH = -68 ± 10‰), whereas the dual-isotope fractionation patterns were almost identical for both enantiomers (ΛC-Cl = 2.4 ± 0.4 and 2.5 ± 0.2, ΛH-C = 12.9 ± 2.4 and 14.9 ± 1.1). The εE of γ-HCH transformation by LinA1 and LinA2 were -7.8 ± 1.0‰ and -7.5 ± 0.8‰ (εC), -2.7 ± 0.3‰ and -2.5 ± 0.4‰ (εCl), -170 ± 25‰ and -150 ± 13‰ (εH), respectively. Similar ΛC-Cl values (2.7 ± 0.2 and 2.9 ± 0.2) were observed as well as similar ΛH-C values (20.1 ± 2.0 and 18.4 ± 1.9), indicating a similar reaction mechanism by both enzymes during γ-HCH transformation. This is the first data set on 3D isotope fractionation of α- and γ-HCH enzymatic dehydrochlorination, which gave a more precise characterization of the bond cleavages, highlighting the potential of multi-element compound-specific stable isotope analysis to characterize different transformation processes (e.g., dehydrochlorination and reductive dehalogenation).

Uptake and Metabolization of HCH Isomers in Trees Examined over an Annual Growth Period by Compound-Specific Isotope Analysis and Enantiomer Fractionation.

To understand the role of plants for natural attenuation, a field study was conducted to characterize the fate of HCH in trees over an annual growth period using compound-specific isotope analysis and enantiomer fractionation. Stable and slightly higher δ13C and δ37Cl values of HCH of host soil samples compared to the muck (consisting nearly exclusively of HCH) revealed that masking isotope effects caused by the limited bioavailability may underestimate the real extent of HCH transformation in soil. In contrast, an increase of δ13C and δ37Cl values in trees indicated the transformation of HCH. A large variability of δ13C and δ37Cl values in trees over the growth period was observed, representing different transformation extents among different growth times, which is further supported by the shift of the enantiomer fraction (EF), indicating the preferential transformation of enantiomers also varied over the different growth periods. Based on dual-element isotope analysis, different predominant transformation mechanisms were observed during the growing seasons. Our observation implies that plants are acting as biological pumps driving a cycle of uptake and metabolization of HCH and refeed during littering to soil catalyzing their transformation. The changes of the transformation mechanism in different seasons have implications for phytoscreening and shed new light on phytoremediation of HCH at field sites.

Metabolic history and metabolic fitness as drivers of anabolic heterogeneity in isogenic microbial populations.

Microbial populations often display different degrees of heterogeneity in their substrate assimilation, that is, anabolic heterogeneity. It has been shown that nutrient limitations are a relevant trigger for this behaviour. Here we explore the dynamics of anabolic heterogeneity under nutrient replete conditions. We applied time-resolved stable isotope probing and nanoscale secondary ion mass spectrometry to quantify substrate assimilation by individual cells of Pseudomonas putida, P. stutzeri and Thauera aromatica. Acetate and benzoate at different concentrations were used as substrates. Anabolic heterogeneity was quantified by the cumulative differentiation tendency index. We observed two major, opposing trends of anabolic heterogeneity over time. Most often, microbial populations started as highly heterogeneous, with heterogeneity decreasing by various degrees over time. The second, less frequently observed trend, saw microbial populations starting at low or very low heterogeneity, and remaining largely stable over time. We explain these trends as an interplay of metabolic history (e.g. former growth substrate or other nutrient limitations) and metabolic fitness (i.e. the fine-tuning of metabolic pathways to process a defined growth substrate). Our results offer a new viewpoint on the intra-population functional diversification often encountered in the environment, and suggests that some microbial populations may be intrinsically heterogeneous.

Soil from a Hexachlorocyclohexane Contaminated Field Site Inoculates Wheat in a Pot Experiment to Facilitate the Microbial Transformation of ß-Hexachlorocyclohexane Examined by Compound-Specific Isotope Analysis.

ß-Hexachlorocyclohexane (ß-HCH) is a remnant from former HCH pesticide production. Its removal from the environment gained attention in the last few years since it is the most stable HCH isomer. However, knowledge about the transformation of ß-HCH in soil-plant systems is still limited. Therefore, experiments with a contaminated field soil were conducted to investigate the transformation of ß-HCH in soil-plant systems by compound specific isotope analysis (CSIA). The results showed that the δ13C and δ37Cl values of ß-HCH in the soil of the planted control remained stable, revealing no transformation due to a low bioavailability. Remarkably, an increase of the δ13C and δ37Cl values in soil and plant tissues of the spiked treatments were observed, indicating the transformation of ß-HCH in both the soil and the plant. This was surprising as previously it was shown that wheat is unable to transform ß-HCH when growing in hydroponic culture or garden soil. Thus, results of this work indicate for the first time that a microbial community of the soil inoculated the wheat and then facilitated the transformation of ß-HCH in the wheat, which may have implications for the development of phytoremediation concepts. A high abundance of HCH degraders belonging to Sphingomonas sp., Mycobacterium sp., and others was detected in the ß-HCH-treated bulk and rhizosphere soil, potentially supporting the biotransformation.

Warming the phycosphere: Differential effect of temperature on the use of diatom-derived carbon by two copiotrophic bacterial taxa.

Heterotrophic bacteria associated with microphytoplankton, particularly those colonizing the phycosphere, are major players in the remineralization of algal-derived carbon. Ocean warming might impact dissolved organic carbon (DOC) uptake by microphytoplankton-associated bacteria with unknown biogeochemical implications. Here, by incubating natural seawater samples at three different temperatures, we analysed the effect of experimental warming on the abundance and C and N uptake activity of Rhodobacteraceae and Flavobacteria, two bacterial groups typically associated with microphytoplankton. Using a nano-scale secondary ion mass spectrometry (nanoSIMS) single-cell analysis, we quantified the temperature sensitivity of these two taxonomic groups to the uptake of algal-derived DOC in the microphytoplankton associated fraction with 13 C-bicarbonate and 15 N-leucine as tracers. We found that cell-specific 13 C uptake was similar for both groups (~0.42 fg C h-1 µm-3 ), but Rhodobacteraceae were more active in 15 N-leucine uptake. Due to the higher abundance of Flavobacteria associated with microphytoplankton, this group incorporated fourfold more carbon than Rhodobacteraceae. Cell-specific 13 C uptake was influenced by temperature, but no significant differences were found for 15 N-leucine uptake. Our results show that the contribution of Flavobacteria and Rhodobacteraceae to C assimilation increased up to sixfold and twofold, respectively, with an increase of 3°C above ambient temperature, suggesting that warming may differently affect the contribution of distinct copiotrophic bacterial taxa to carbon cycling.

Simultaneous Compound-Specific Analysis of δ33S and δ34S in Organic Compounds by GC-MC-ICPMS Using Medium- and Low-Mass-Resolution Modes.

Compound-specific isotope analysis of sulfur (δ34S-CSIA) in organic compounds was established in the last decade employing gas chromatography connected to multiple-collector inductively coupled plasma mass spectrometry (GC-MC-ICPMS). However, δ33S-CSIA has not yet been reported so far. In this study, we present a method for the simultaneous determination of δ33S and δ34S in organic compounds by GC-MC-ICPMS applying medium- and also low-mass-resolution modes. The method was validated using the international isotope reference materials IAEA-S-1, IAEA-S-2, and IAEA-S-3. Overall analytical uncertainty including normalization and reproducibility for δ33S and δ34S was usually better than ±0.2 mUr (σ) for analytes containing at least 100 pmol of S. Further, it is demonstrated that, despite small isobaric interferences, results obtained at low mass resolution are indistinguishable from medium mass resolution offering the benefit of increased sensitivity and versatility of this method. Additionally, the method was applied for the δ33S and δ34S isotope analysis of industrially produced organic compounds to investigate potential mass-independent fractionation (MIF). The relation between δ34S and δ33S in these compounds followed a mass-dependent fractionation trend (MDF; Δ33S ≤ ±0.2 mUr). Degradation of dimethyl disulfide by direct photolysis caused a small but significant MIF (Δ33S = 0.55 ± 0.04 mUr, n = 3), demonstrating sufficient sensitivity of the method for these types of studies.

Compound-Specific Isotope Analysis and Enantiomer Fractionation to Characterize the Transformation of Hexachlorocyclohexane Isomers in a Soil-Wheat Pot System.

The uptake by plants from soil is one of the first steps for hexachlorocyclohexane (HCH) isomers to enter the food web. However, the HCH transformation associated with the uptake process is still not well understood. Therefore, a soil-wheat pot experiment was conducted to characterize the HCH transformation during wheat growth using compound-specific isotope analysis (CSIA) and enantiomer fractionation. The results showed that the δ13C and δ37Cl values of ß-HCH remained stable in soil and wheat, revealing no transformation. In contrast, an increase of δ13C and δ37Cl values of α-HCH indicated its transformation in soil and wheat. A shift of the enantiomer fraction (EF) (-) from 0.50 to 0.35 in soil at the jointing stage and 0.35 to 0.57 at the harvest stage suggested that the preferential transformation of enantiomers varied at different growth stages. Based on the dual element isotope analysis, the transformation mechanism in the soil-wheat system was different from that in wheat in hydroponic systems. The high abundance of HCH degraders, Sphingomonas sp. and Novosphingobium sp., was detected in the α-HCH-treated rhizosphere soil, supporting the potential for biotransformation. The application of CSIA and EF allows characterizing the transformation of organic pollutants such as HCHs in the complex soil-plant systems.

Can Alkaline Hydrolysis of γ-HCH Serve as a Model Reaction to Study Its Aerobic Enzymatic Dehydrochlorination by LinA?

Hexachlorocyclohexane (HCH) isomers constitute a group of persistent organic pollutants. Their mass production and treatment have led to a global environmental problem that continues to this day. The characterization of modes of degradation of HCH by isotope fractionation is a current challenge. Multi isotope fractionation analysis provides a concept to characterize the nature of enzymatic and chemical transformation reactions. The understanding of the kinetic isotope effects (KIE) on bond cleavage reaction contributes to analyses of the mechanism of chemical and enzymatic reactions. Herein, carbon, chlorine, and hydrogen kinetic isotope effects are measured and predicted for the dehydrochlorination reaction of γ-HCH promoted by the hydroxyl ion in aqueous solution. Quantum mechanical (QM) microsolvation with an implicit solvation model and path integral formalism in combination with free-energy perturbation and umbrella sampling (PI-FEP/UM) and quantum mechanical/molecular mechanical QM/MM potentials for including solvent effects as well as calculating isotope effects are used and analyzed with respect to their performance in reproducing measured values. Reaction characterization is discussed based on the magnitudes of obtained isotope effects. The comparative analysis between the chemical dehydrochlorination of γ-HCH in aqueous media and catalyzed reaction by dehydrochlorinase, LinA is presented and discussed. Based on the values of isotope effects, these two processes seem to occur via the same net mechanism.

Individual stages of bacterial dichloromethane degradation mapped by carbon and chlorine stable isotope analysis.

The fractionation of carbon and chlorine stable isotopes of dichloromethane (CH2Cl2, DCM) upon dechlorination by cells of the aerobic methylotroph Methylobacterium extorquens DM4 and by purified DCM dehalogenases of the glutathione S-transferase family was analyzed. Isotope effects for individual steps of the multi-stage DCM degradation process, including transfer across the cell wall from the aqueous medium to the cell cytoplasm, dehalogenase binding, and catalytic reaction, were considered. The observed carbon and chlorine isotope fractionation accompanying DCM consumption by cell supensions and enzymes was mainly determined by the breaking of CCl bonds, and not by inflow of DCM into cells. Chlorine isotope effects of DCM dehalogenation were initially masked in high density cultures, presumably due to inverse isotope effects of non-specific DCM oxidation under conditions of oxygen excess. Glutathione cofactor supply remarkably affected the correlation of variations of DCM carbon and chlorine stable isotopes (Δδ13C/Δδ37Cl), increasing corresponding ratio from 7.2-8.6 to 9.6-10.5 under conditions of glutathione deficiency. This suggests that enzymatic reaction of DCM with glutathione thiolate may involve stepwise breaking and making of bonds with the carbon atom of DCM, unlike the uncatalyzed reaction, which is a one-stage process, as shown by quantum-chemical modeling.

Distinct Carbon Isotope Fractionation Signatures during Biotic and Abiotic Reductive Transformation of Chlordecone.

Chlordecone is a synthetic organochlorine pesticide, extensively used in banana plantations of the French West Indies from 1972 to 1993. Due to its environmental persistence and bioaccumulation, it has dramatic public health and socio-economic impact. Here we describe a method for carbon-directed compound specific isotope analysis (CSIA) for chlordecone and apply it to monitor biotic and abiotic reductive transformation reactions, selected on the basis of their distinct product profiles (polychloroindenes versus lower chlorinated hydrochlordecones). Significant carbon isotopic enrichments were observed for all microbially mediated transformations (εbulk = -6.8‰ with a Citrobacter strain and εbulk = -4.6‰ with a bacterial consortium) and for two abiotic transformations (εbulk = -4.1‰ with zerovalent iron and εbulk = -2.6‰ with sodium sulfide and vitamin B12). The reaction with titanium(III) citrate and vitamin B12, which shows the product profile most similar to that observed in biotic transformation, led to low carbon isotope enrichment (εbulk =-0.8‰). The CSIA protocol was also applied on representative chlordecone formulations previously used in the French West Indies, giving similar chlordecone δ13C values from -31.1 ± 0.2‰ to -34.2 ± 0.2‰ for all studied samples. This allows the in situ application of CSIA for the assessment of chlordecone persistence.

Methylamine as a nitrogen source for microorganisms from a coastal marine environment.

Nitrogen is a key limiting resource for biomass production in the marine environment. Methylated amines, released from the degradation of osmolytes, could provide a nitrogen source for marine microbes. Thus far, studies in aquatic habitats on the utilization of methylamine, the simplest methylated amine, have mainly focussed on the fate of the carbon from this compound. Various groups of methylotrophs, microorganisms that can grow on one-carbon compounds, use methylamine as a carbon source. Non-methylotrophic microorganisms may also utilize methylamine as a nitrogen source, but little is known about their diversity, especially in the marine environment. In this proof-of-concept study, stable isotope probing (SIP) was used to identify microorganisms from a coastal environment that assimilate nitrogen from methylamine. SIP experiments using 15 N methylamine combined with metagenomics and metaproteomics facilitated identification of active methylamine-utilizing Alpha- and Gammaproteobacteria. The draft genomes of two methylamine utilizers were obtained and their metabolism with respect to methylamine was examined. Both bacteria identified in these SIP experiments used the γ-glutamyl-methylamide pathway, found in both methylotrophs and non-methylotrophs, to metabolize methylamine. The utilization of 15 N methylamine also led to the release of 15 N ammonium that was used as nitrogen source by other microorganisms not directly using methylamine.

Differential sensitivity of total and active soil microbial communities to drought and forest management.

Climate change will affect semiarid ecosystems through severe droughts that increase the competition for resources in plant and microbial communities. In these habitats, adaptations to climate change may consist of thinning-that reduces competition for resources through a decrease in tree density and the promotion of plant survival. We deciphered the functional and phylogenetic responses of the microbial community to 6 years of drought induced by rainfall exclusion and how forest management affects its resistance to drought, in a semiarid forest ecosystem dominated by Pinus halepensis Mill. A multiOMIC approach was applied to reveal novel, community-based strategies in the face of climate change. The diversity and the composition of the total and active soil microbiome were evaluated by 16S rRNA gene (bacteria) and ITS (fungal) sequencing, and by metaproteomics. The microbial biomass was analyzed by phospholipid fatty acids (PLFAs), and the microbially mediated ecosystem multifunctionality was studied by the integration of soil enzyme activities related to the cycles of C, N, and P. The microbial biomass and ecosystem multifunctionality decreased in drought-plots, as a consequence of the lower soil moisture and poorer plant development, but this decrease was more notable in unthinned plots. The structure and diversity of the total bacterial community was unaffected by drought at phylum and order level, but did so at genus level, and was influenced by seasonality. However, the total fungal community and the active microbial community were more sensitive to drought and were related to ecosystem multifunctionality. Thinning in plots without drought increased the active diversity while the total diversity was not affected. Thinning promoted the resistance of ecosystem multifunctionality to drought through changes in the active microbial community. The integration of total and active microbiome analyses avoids misinterpretations of the links between the soil microbial community and climate change.

Validation of GC-IRMS techniques for δ13C and δ2H CSIA of organophosphorus compounds and their potential for studying the mode of hydrolysis in the environment.

Compound-specific stable isotope analysis (CSIA) is among the most promising tools for studying the fate of organic pollutants in the environment. However, the feasibility of multidimensional CSIA was limited by the availability of a robust method for precise isotope analysis of heteroatom-bearing organic compounds. We developed a method for δ 13C and δ 2H analysis of eight organophosphorus compounds (OPs) with different chemical properties. In particular, we aimed to compare high-temperature conversion (HTC) and chromium-based HTC (Cr/HTC) units to explore the limitations of hydrogen isotope analysis of heteroatom-bearing compounds. Analysis of the amount dependency of the isotope values (linearity analysis) of OPs indicated that the formation of HCl was a significant isotope fractionation process leading to inaccurate δ 2H analysis in HTC. In the case of nonchlorinated OPs, by-product formation of HCN, H2S, or PH3 in HTC was observed but did not affect the dynamic range of reproducible isotope values above the limit of detection. No hydrogen-containing by-products were found in the Cr/HTC process by use of ion trap mass spectrometry analysis. The accuracy of gas chromatography - isotope ratio mass spectrometry was validated in comparison with elemental analyzer - isotope ratio mass spectrometry. Dual-isotope fractionation yielded Λ values of 0 ± 0 at pH 7, 7 ± 1 at pH 9, and 30 ± 6 at pH 12, indicating the potential of 2D CSIA to characterize the hydrolysis mechanisms of OPs. This is the first report on the combination of δ 2H and δ 13C isotope analysis of OPs, and this is the first study providing a systematic evaluation of HTC and Cr/HTC for hydrogen isotope analysis using OPs as target compounds. Graphical Abstract Comparison of δ2H measurement of non-chlorinated and chlorinated OPs via GC-Cr/HTC-IRMS and GC-HTC-IRMS system.

Compound-Specific Stable Isotope Analysis: Implications in Hexachlorocyclohexane in-vitro and Field Assessment.

Assessment of biotic and abiotic degradation reactions by studying the variation in stable isotopic compositions of organic contaminants in contaminated soil and aquifers is being increasingly considered during the last two decades with development of Compound specific stable isotope analysis (CSIA) technique. CSIA has been recognized as a potential tool for evaluating both qualitative and quantitative degradation with measurement of shifts in isotope ratios of contaminants and their degradation products as its basis. Amongst a wide variety of environmental pollutants including monoaromatics, chlorinated ethenes and benzenes etc., it is only recently that its efficacy is being tested for assessing biodegradation of a noxious pollutant namely hexachlorocyclohexane (HCH), by pure microbial cultures as well as directly at the field site. Anticipating the increase in demand of this technique for monitoring the microbial degradation along with natural attenuation, this review highlights the basic problems associated with HCH contamination emphasizing the applicability of emerging CSIA technique to absolve the major bottlenecks in assessment of HCH. To this end, the review also provides a brief overview of this technique with summarizing the recent revelations put forward by both in vitro and in situ studies by CSIA in monitoring HCH biodegradation.

The active microbial diversity drives ecosystem multifunctionality and is physiologically related to carbon availability in Mediterranean semi-arid soils.

Biogeochemical processes and ecosystemic functions are mostly driven by soil microbial communities. However, most methods focus on evaluating the total microbial community and fail to discriminate its active fraction which is linked to soil functionality. Precisely, the activity of the microbial community is strongly limited by the availability of organic carbon (C) in soils under arid and semi-arid climate. Here, we provide a complementary genomic and metaproteomic approach to investigate the relationships between the diversity of the total community, the active diversity and ecosystem functionality across a dissolved organic carbon (DOC) gradient in southeast Spain. DOC correlated with the ecosystem multifunctionality index composed by soil respiration, enzyme activities (urease, alkaline phosphatase and ß-glucosidase) and microbial biomass (phospholipid fatty acids, PLFA). This study highlights that the active diversity (determined by metaprotoemics) but not the diversity of the whole microbial community (evaluated by amplicon gene sequencing) is related to the availability of organic C and it is also connected to the ecosystem multifunctionality index. We reveal that DOC shapes the activities of bacterial and fungal populations in Mediterranean semi-arid soils and determines the compartmentalization of functional niches. For instance, Rhizobales thrived at high-DOC sites probably fuelled by metabolism of one-C compounds. Moreover, the analysis of proteins involved in the transport and metabolism of carbohydrates revealed that Ascomycota and Basidiomycota occupied different nutritional niches. The functional mechanisms for niche specialization were not constant across the DOC gradient.

Pulsed (13)C2-Acetate Protein-SIP Unveils Epsilonproteobacteria as Dominant Acetate Utilizers in a Sulfate-Reducing Microbial Community Mineralizing Benzene.

In a benzene-degrading and sulfate-reducing syntrophic consortium, a clostridium affiliated to the genus Pelotomaculum was previously described to ferment benzene while various sulfate-reducing Deltaproteobacteria and a member of the Epsilonproteobacteria were supposed to utilize acetate and hydrogen as key metabolites derived from benzene fermentation. However, the acetate utilization network within this community was not yet unveiled. In this study, we performed a pulsed (13)C2-acetate protein stable isotope probing (protein-SIP) approach continuously spiking low amounts of acetate (10 µM per day) in addition to the ongoing mineralization of unlabeled benzene. Metaproteomics revealed high abundances of Clostridiales followed by Syntrophobacterales, Desulfobacterales, Desulfuromonadales, Desulfovibrionales, Archaeoglobales, and Campylobacterales. Pulsed acetate protein-SIP results indicated that members of the Campylobacterales, the Syntrophobacterales, the Archaeoglobales, the Clostridiales, and the Desulfobacterales were linked to acetate utilization in descending abundance. The Campylobacterales revealed the fastest and highest (13)C incorporation. Previous experiments suggested that the activity of the Campylobacterales was not essential for anaerobic benzene degradation in the investigated community. However, these organisms were consistently detected in various hydrocarbon-degrading and sulfate-reducing consortia enriched from the same aquifer. Here, we demonstrate that this member of the Campylobacterales is the dominant acetate utilizer in the benzene-degrading microbial consortium.

Dual Carbon-Bromine Stable Isotope Analysis Allows Distinguishing Transformation Pathways of Ethylene Dibromide.

The present study investigated dual carbon-bromine isotope fractionation of the common groundwater contaminant ethylene dibromide (EDB) during chemical and biological transformations, including aerobic and anaerobic biodegradation, alkaline hydrolysis, Fenton-like degradation, debromination by Zn(0) and reduced corrinoids. Significantly different correlation of carbon and bromine isotope fractionation (ΛC/Br) was observed not only for the processes following different transformation pathways, but also for abiotic and biotic processes with, the presumed, same formal chemical degradation mechanism. The studied processes resulted in a wide range of ΛC/Br values: ΛC/Br = 30.1 was observed for hydrolysis of EDB in alkaline solution; ΛC/Br between 4.2 and 5.3 were determined for dibromoelimination pathway with reduced corrinoids and Zn(0) particles; EDB biodegradation by Ancylobacter aquaticus and Sulfurospirillum multivorans resulted in ΛC/Br = 10.7 and 2.4, respectively; Fenton-like degradation resulted in carbon isotope fractionation only, leading to ΛC/Br ∞. Calculated carbon apparent kinetic isotope effects ((13)C-AKIE) fell with 1.005 to 1.035 within expected ranges according to the theoretical KIE, however, biotic transformations resulted in weaker carbon isotope effects than respective abiotic transformations. Relatively large bromine isotope effects with (81)Br-AKIE of 1.0012-1.002 and 1.0021-1.004 were observed for nucleophilic substitution and dibromoelimination, respectively, and reveal so far underestimated strong bromine isotope effects.

Hydrogen Isotope Fractionation As a Tool to Identify Aerobic and Anaerobic PAH Biodegradation.

Aerobic and anaerobic polycyclic aromatic hydrocarbon (PAH) biodegradation was characterized by compound specific stable isotope analysis (CSIA) of the carbon and hydrogen isotope effects of the enzymatic reactions initiating specific degradation pathways, using naphthalene and 2-methylnaphtalene as model compounds. Aerobic activation of naphthalene and 2-methylnaphthalene by Pseudomonas putida NCIB 9816 and Pseudomonas fluorescens ATCC 17483 containing naphthalene dioxygenases was associated with moderate carbon isotope fractionation (εC = -0.8 ± 0.1‰ to -1.6 ± 0.2‰). In contrast, anaerobic activation of naphthalene by a carboxylation-like mechanism by strain NaphS6 was linked to negligible carbon isotope fractionation (εC = -0.2 ± 0.2‰ to -0.4 ± 0.3‰). Notably, anaerobic activation of naphthalene by strain NaphS6 exhibited a normal hydrogen isotope fractionation (εH = -11 ± 2‰ to -47 ± 4‰), whereas an inverse hydrogen isotope fractionation was observed for the aerobic strains (εH = +15 ± 2‰ to +71 ± 6‰). Additionally, isotope fractionation of NaphS6 was determined in an overlaying hydrophobic carrier phase, resulting in more reliable enrichment factors compared to immobilizing the PAHs on the bottle walls without carrier phase. The observed differences especially in hydrogen fractionation might be used to differentiate between aerobic and anaerobic naphthalene and 2-methylnaphthalene biodegradation pathways at PAH-contaminated field sites.