[Show abstract][Hide abstract] ABSTRACT: A universal application of compound-specific isotope analysis of chlorine was thus far limited by the availability of suitable analysis techniques. In this study, gas chromatography in combination with high-temperature conversion interface (GC-HTC) - converting organic chlorine in presence of H2 to gaseous HCl - was coupled to a dual-detection system, combining ion trap mass spectrometer (MS) and isotope-ratio mass spectrometer (IRMS). The combination of the MS/IRMS detection enabled a detailed characterization, optimization and on-line monitoring of the high-temperature conversion process via ion trap MS, as well as a simultaneous chlorine isotope analysis by the IRMS. Using GC-HTC-MS/IRMS, chlorine isotope analysis at optimized conversion conditions resulted in very accurate isotope values (δ37ClSMOC) for measured reference material with known isotope composition, including chlorinated ethylene, chloromethane, hexachlorocyclohexane and trichloroacetic acids methyl ester. Respective detection limits were determined to be < 15 nmol Cl on column with achieved precision of < 0.3‰.
[Show abstract][Hide abstract] ABSTRACT: This study aimed to ascertain the functional and phylogenetic relationships within an m-xylene degrading sulfate-reducing enrichment culture, which had been maintained for several years in the laboratory with m-xylene as the sole source of carbon and energy. Previous studies indicated that a phylotype affiliated to the Desulfobacteraceae was the main m-xylene assimilating organism. In the present study, genes and gene products were identified by a metaproteogenomic approach using LC-MS/MS analysis of the microbial community, and 2426 peptides were identified from 576 proteins. In the metagenome of the community, gene clusters encoding enzymes involved in fumarate addition to a methyl moiety of m-xylene (nms, bss), as well as gene clusters coding for enzymes involved in modified beta-oxidation to (3-methyl)benzoyl-CoA (bns), were identified in two separate contigs. Additionally, gene clusters containing homologues to bam genes encoding benzoyl-CoA reductase (Bcr) class II, catalyzing the dearomatization of (3-methyl)benzoyl-CoA, were identified. Time-resolved protein stable isotope probing (protein-SIP) experiments using (13)C-labeled m-xylene showed that the respective gene products were highly (13)C-labeled. The present data suggested the identification of gene products that were similar to those involved in methylnaphthalene degradation even though the consortium was not capable of growing in the presence of naphthalene, methylnaphthalene or toluene as substrates. Thus, a novel branch of enzymes was found that was probably specific for anaerobic m-xylene degradation.
Systematic and Applied Microbiology 08/2014; · 3.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Anaerobic reductive dechlorination of hexachlorobenzene (HCB) and three isomers of tetrachlorobenzene (TeCB) (1,2,3,4-, 1,2,3,5- and 1,2,4,5-TeCB) was investigated in microcosms containing chloroaromatic contaminated river sediment. All chlorobenzenes were dechlorinated to dichlorobenzene (DCB) or monochlorobenzene. From the sediment, a methanogenic sediment-free culture was obtained which dechlorinated HCB, pentachlorobenzene, three TeCB isomers, three trichlorobenzene (TCB) isomers (1,2,3-, 1,2,4- and 1,3,5-TCB) and 1,2-DCB. Dechlorination involved multiple pathways including the removal of doubly flanked, singly flanked and isolated chlorine substituents. 454-pyrosequencing of partial bacterial 16S rRNA genes amplified from selected chlorobenzene dechlorinating sediment-free enrichment cultures revealed the presence of a variety of bacterial species, including Dehalobacter and Dehalococcoides mccartyi, that were previously documented as organohalide respiring bacteria. A genus with apparent close relationship to Desulfitobacterium that also has been associated with organohalide respiration, composed the major fraction of the operational taxonomic units (OTUs). Another major OTU was linked with Sedimentibacter sp., a genus that was previously identified in strict co-cultures of consortia reductively dehalogenating chlorinated compounds. Our data point towards the existence of multiple interactions within highly chlorinated benzene dechlorinating communities.
[Show abstract][Hide abstract] ABSTRACT: Natural wetlands are transition zones between anoxic ground and oxic surface water which may enhance the (bio)transformation potential for recalcitrant chloro-organic contaminants due to the unique geochemical conditions and gradients. Monochlorobenzene (MCB) is a frequently detected groundwater contaminant which is toxic and was thought to be persistent under anoxic conditions. Furthermore, to date, no degradation pathways for anoxic MCB removal have been proven in the field. Hence, it is important to investigate MCB biodegradation in the environment, as groundwater is an important drinking water source in many European countries. Therefore, two pilot-scale horizontal subsurface-flow constructed wetlands, planted and unplanted, were used to investigate the processes in situ contributing to the biotransformation of MCB in these gradient systems. The wetlands were fed with anoxic MCB-contaminated groundwater from a nearby aquifer in Bitterfeld, Germany. An overall MCB removal was observed in both wetlands, whereas just 10% of the original MCB inflow concentration was detected in the ponds. In particular in the gravel bed of the planted wetland, MCB removal was highest in summer season with 73 ± 9% compared to the unplanted one with 40 ± 5%. Whereas the MCB concentrations rapidly decreased in the transition zone of unplanted gravel to the pond, a significant MCB removal was already determined in the anoxic gravel bed of the planted system. The investigation of hydro-geochemical parameters revealed that iron and sulphate reduction were relevant redox processes in both wetlands. In parallel, the addition of ferric iron or nitrate stimulated the mineralisation of MCB in laboratory microcosms with anoxic groundwater from the same source, indicating that the potential for anaerobic microbial degradation of MCB is present at the field site.
Science of The Total Environment 02/2014; 472:185–193. · 3.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Northern Baffin Bay between Greenland and Canada is a remote Arctic area restricted in primary production by seasonal ice cover, with presumably low sedimentation rates, carbon content and microbial activities in its sediments. Our aim was to study the so far unknown subseafloor geochemistry and microbial populations driving seafloor ecosystems. Shelf sediments had the highest organic carbon content, numbers of Bacteria and Archaea, and microcosms inoculated from Shelf sediments showed highest sulfate reduction and methane production rates. Sediments in the central deep area and on the southern slope contained less organic carbon and overall lower microbial numbers. Similar 16S rRNA gene copy numbers of Archaea and Bacteria were found for the majority of the sites investigated. Sulfate in pore water correlated with dsrA copy numbers of sulfate-reducing prokaryotes and differed between sites. No methane was found as free gas in the sediments, and mcrA copy numbers of methanogenic Archaea were low. Methanogenic and sulfate-reducing cultures were enriched on a variety of substrates including hydrocarbons. In summary, the Greenlandic shelf sediments contain vital microbial communities adapted to their specific environmental conditions.
[Show abstract][Hide abstract] ABSTRACT: The anaerobic degradation of propane and butane is typically initiated by activation via addition to fumarate. Here we investigated the mechanism of activation under sulfate-reducing conditions by one pure culture (strain BuS5) and three enrichment cultures employing stable isotope analysis. Stable isotope fractionation was compared for cultures incubated with or without substrate diffusion limitation. Bulk enrichment factors were significantly higher in mixed vs. static incubations. Two dimensional factors, given by the correlation of stable isotope fractionation of both carbon and hydrogen at their reactive positions (Lambda reactive position , Λrp ), were compared to analyse the activation mechanisms. A characteristic reactive position isotope fractionation pattern was observed, distinct from aerobic degradation. Λrp values ranged from 10.5 to 11.8 for propane and from 7.8 to 9.4 for butane. Incubations of strain BuS5 with deuterium-labelled n-alkanes indicated that butane was activated solely at the subterminal C atom. In contrast, propane was activated mainly at the subterminal C atom but also significantly at the terminal C atoms. A conservative estimate suggests that about 70% of the propane activation events occurred at the subterminal C atom and about 30% at the terminal C atoms.
[Show abstract][Hide abstract] ABSTRACT: Anaerobic methanotrophic (ANME) mats host methane oxidizing archaea and sulfate reducing prokaryotes. Little is known about the nitrogen cycle in these communities. Here, we link the anaerobic oxidation of methane (AOM) to the nitrogen cycle in microbial mats of the Black Sea by using stable isotope probing (SIP). We used 4 different 15N-labelled sources of nitrogen: dinitrogen, nitrate, nitrite, and ammonium. We assessed the nitrogen incorporation rates into the total biomass and the methyl coenzyme M reductase (MCR). Dinitrogen played an insignificant role as nitrogen source . Assimilatory and dissimilatory nitrate reduction occurred. High rates of nitrate reduction to dinitrogen were stimulated by methane and sulfate, suggesting that oxidation of reduced sulfur compounds like sulfides was necessary for AOM with nitrate as electron acceptor. Nitrate reduction to dinitrogen occurred also in the absence of methane as electron donor but at 6-times slower rates . Dissimilatory nitrate reduction to ammonium (DNRA) was independent from AOM. Ammonium was used for biomass synthesis under all conditions. The pivotal enzyme in AOM coupled to sulfate reduction, MCR, was synthesized from nitrate and ammonium. Results show that AOM coupled to sulfate reduction along with biomass decomposition drive the nitrogen cycle in the ANME mats of the Black Sea and that MCR enzymes are involved in this process. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: The recent development of metaproteomics has enabled the direct identification and quantification of expressed proteins from microbial communities in situ, without the need for microbial enrichment. This became possible by (1) significant increases in quality and quantity of metagenome data and by improvements of (2) accuracy and (3) sensitivity of modern mass spectrometers (MS). The identification of physiologically relevant enzymes can help to understand the role of specific species within a community or an ecological niche. Beside identification, relative and absolute quantitation is also crucial. We will review label-free and label-based methods of quantitation in MS-based proteome analysis and the contribution of quantitative proteome data to microbial ecology. Additionally, approaches of protein-based stable isotope probing (protein-SIP) for deciphering community structures are reviewed. Information on the species-specific metabolic activity can be obtained when substrates or nutrients are labeled with stable isotopes in a protein-SIP approach. The stable isotopes ((13)C, (15)N, (36)S) are incorporated into proteins and the rate of incorporation can be used for assessing the metabolic activity of the corresponding species. We will focus on the relevance of the metabolic and phylogenetic information retrieved with protein-SIP studies and for detecting and quantifying the carbon flux within microbial consortia. Furthermore, the combination of protein-SIP with established tools in microbial ecology such as other stable isotope probing techniques are discussed.The ISME Journal advance online publication, 16 May 2013; doi:10.1038/ismej.2013.78.
[Show abstract][Hide abstract] ABSTRACT: Current knowledge of the physiology and phylogeny of polycyclic aromatic hydrocarbon (PAH) degrading bacteria often relies on laboratory enrichments and isolations. In the present study, in situ microcosms consisting of activated carbon pellets (BACTRAP®s) were loaded with either (13) C-naphthalene or (13) C-fluorene and were subsequently exposed in the contaminant source and plume fringe region of an PAH contaminated aquifer. Metaproteomic analysis and protein-SIP revealed Burkholderiales, Actinomycetales and Rhizobiales as the most active microorganisms in the groundwater communities. Proteins identified of the naphthalene degradation pathway showed a relative (13) C isotope abundance of approximately 50 atom% demonstrating that the identified naphthalene degrading bacteria gained at least 80% of their carbon by PAH degradation. Although the microbial community grown on the fluorene-BACTRAPs showed a structure similar to the naphthalene-BACTRAPs, the identification of fluorene degraders and degradation pathways failed in situ. In complementary laboratory microcosms, a clear enrichment in proteins related to Rhodococcus and possible fluorene degradation enzymes was observed. This result demonstrates the impact of laboratory conditions on microbial community structure and activity of certain species and underlines the need on in situ exploration of microbial community functions. In situ microcosms in combination with protein-SIP may be a significant tool for in situ identification of metabolic key players as well as degradation pathways. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Biodegradation of contaminants is a common remediation strategy for subsurface environments. To monitor the success of such remediation means a quantitative assessment of biodegradation at the field scale is required. Nevertheless, the reliable quantification of the in situ biodegradation process it is still a major challenge. Compound-specific stable isotope analysis has become an established method for the qualitative analysis of biodegradation in the field and this method is also proposed for a quantitative analysis. However, to use stable isotope data to obtain quantitative information on in situ biodegradation requires among others knowledge on the influence of mass transfer processes on the observed stable isotope fractionation. This paper reviews recent findings on the influence of mass transfer processes on stable isotope fractionation and on the quantitative interpretation of isotope data. Focus will be given on small-scale mass transfer processes controlling the bioavailability of contaminants. Such bioavailability limitations are known to affect the biodegradation rate and have recently been shown to affect stable isotope fractionation, too. Theoretical as well as experimental studies addressing the link between bioavailability and stable isotope fractionation are reviewed and the implications for assessing biodegradation in the field are discussed.
Applied Microbiology and Biotechnology 01/2013; 97:441-452. · 3.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Artificial carbon dioxide leakage into a shallow aquifer was monitored using stable carbon isotope measurements at a field site near the town of Wittstock, Brandenburg, Germany. Approximately 400 000 L of CO(2) were injected into a shallow aquifer at 18 m depth over 10 days. The (13)C/ (12)C ratios of the CO(2) were measured in both groundwater and soil gas samples to monitor the distribution of the injected CO(2) plume and to evaluate the feasibility and reliability of this approach to detect potential CO(2) leakage, for example from carbon capture and storage (CCS) sites. The isotopic composition of the injected CO(2) (δ(13)C -30.5 ‰) was differentiable from the background CO(2) (δ(13)C -21.9 ‰) and the artificial CO(2) plume was monitored over a period spanning more than 204 days. The results demonstrate that this stable isotope monitoring approach can be used to identify CO(2) sources and detect potential CO(2) migration from CCS sites into overlying shallow aquifers or even into the upper subsurface. A significant difference between the isotope ratios of the natural background and the injected CO(2) is required for this monitoring approach to be effective.
[Show abstract][Hide abstract] ABSTRACT: A small scale and temporally limited CO2 injection test was performed in a shallow aquifer to investigate the geochemical impact of CO2 upon such aquifers and to apply and verify different monitoring methods. Detailed site investigation coupled with multiphase simulations were necessary to design the injection experiment and to set up the monitoring network, before CO2 was injected over a ten-day period at three injection wells, at a depth of 18 m below surface level into a quaternary sand aquifer located close to the town of Wittstock in Northeast Germany. Monitoring methods comprised groundwater sampling and standard analyses, as well as trace element analyses and isotope analyses; geoelectrical borehole monitoring; passive samplers to analyse temporally integrated for cations and multi-parameter probes that can measure continuously for dissolved CO2, pH and electrical conductivity. Due to CO2 injection, total inorganic carbon concentrations increased and pH decreased down to a level of 5.1. Associated reactions comprised the release of major cations and trace elements. Geoelectrical monitoring, as well as isotope analyses and multi-parameter probes proved to be suitable methods for monitoring injected CO2 and/or the alteration of groundwater.
[Show abstract][Hide abstract] ABSTRACT: Benzene is a major contaminant in various environments, but the mechanisms behind its biodegradation under strictly anoxic conditions are not yet entirely clear. Here we analyzed a benzene-degrading, sulfate-reducing enrichment culture originating from a benzene-contaminated aquifer by a metagenome-based functional metaproteomic approach, using protein-based stable isotope probing (protein-SIP). The time-resolved, quantitative analysis of carbon fluxes within the community supplied with either (13)C-labeled benzene or (13)C-labeled carbonate yielded different functional groups of organisms, with their peptides showing specific time dependencies of (13)C relative isotope abundance indicating different carbon utilization. Through a detailed analysis of the mass spectrometric (MS) data, it was possible to quantify the utilization of the initial carbon source and the metabolic intermediates. The functional groups were affiliated to Clostridiales, Deltaproteobacteria and Bacteroidetes/Chlorobi. The Clostridiales-related organisms were involved in benzene degradation, putatively by fermentation, and additionally used significant amounts of carbonate as a carbon source. The other groups of organisms were found to perform diverse functions, with Deltaproteobacteria degrading fermentation products and Bacteroidetes/Chlorobi being putative scavengers feeding on dead cells. A functional classification of identified proteins supported this allocation and gave further insights into the metabolic pathways and the interactions between the community members. This example shows how protein-SIP can be applied to obtain temporal and phylogenetic information about functional interdependencies within microbial communities.
[Show abstract][Hide abstract] ABSTRACT: We investigated the effect of increasing CO(2) concentrations on the growth and viability of ecophysiologically different microorganisms to obtain information for a leakage scenario of CO(2) into shallow aquifers related to the capture and storage of CO(2) in deep geological sections. CO(2) concentrations in the gas phase varied between atmospheric conditions and 80% CO(2) for the aerobic strains Pseudomonas putida F1 and Bacillus subtilis 168 and up to 100% CO(2) for the anaerobic strains Thauera aromatica K172 and Desulfovibrio vulgaris Hildenborough. Increased CO(2) concentrations caused prolonged lag-phases, and reduced growth rates and cell yields; the extent of this effect was proportional to the CO(2) concentration. Additional experiments with increasing CO(2) concentrations and increasing pressure (1-5000 kPa) simulated situations occurring in deep CO(2) storage sites. Living cell numbers decreased significantly within 24 h at pressures ≥1000 kPa, demonstrating a severe lethal effect for the combination of high pressure and CO(2).
[Show abstract][Hide abstract] ABSTRACT: Carbon flow in a model methanogenic consortium capable of hydrocarbon degradation was investigated using a combination of stable isotope fractionation, protein-based stable isotope probing, and metaproteomics. Overall δ(13) C enrichment for methane and CO2 in the presence and absence of oil suggests that complex microbial interactions occur during methanogenic hydrocarbon mineralization. Specifically, the Δδ(13) C of CO2 was statistically identical in all incubations irrespective of oil presence, but the Δδ(13) C for methane was greater in the presence of oil compared with fatty acids alone. In addition, carbon from uniformly ((13) C) labelled n-fatty acids was distributed evenly among consortium members in the presence of oil, but used by relatively few community members when provided alone. In all incubations, aceticlastic and hydrogenotrophic methanogens were labelled to an equal extent, suggesting that no pathway is overwhelmingly dominant during methane production by the model consortium. Protein-based stable isotope probing identified key enzymes responsible for methanogenesis from CO2 and acetate labelled with 78.0 ± 4.4% and 73.3 ± 1.0% (13) C respectively. Results suggest that acetate was used directly by methanogens in the presence of n-fatty acids alone, and that methanogenesis from CO2 was a secondary process. Proteins capable of catalysing hydrocarbon activation by addition to fumarate were not found. Collectively, this study demonstrates that significant microbial cooperation is required to recover hydrocarbons as methane.