Publications (63)211.99 Total impact
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Article: Elucidation of in situ polycyclic aromatic hydrocarbon degradation by functional metaproteomics (protein-SIP).
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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.Proteomics 04/2013; · 4.43 Impact Factor -
Article: Influence of mass transfer on stable isotope fractionation.
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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. · 3.42 Impact Factor -
Article: Protein-SIP enables time-resolved analysis of the carbon flux in a sulfate-reducing, benzene-degrading microbial consortium.
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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.The ISME Journal 07/2012; · 7.38 Impact Factor -
Article: Effects of high CO2 concentrations on ecophysiologically different microorganisms.
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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).Environmental pollution (Barking, Essex: 1987) 06/2012; 169:27-34. · 3.43 Impact Factor -
Article: Simulation of a reactive tracer experiment using stochastic hydraulic conductivity fields
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ABSTRACT: Reactive tracer tests are performed to derive flow, transport and in situ biodegradation parameters. This paper describes the 3D simulation of a reactive tracer test using the transition probability geostatistical approach. Fifty different equally probable aquifer realizations were generated based on the geological information of 107 boreholes in an area of 62,500m2. One realization was chosen for the reactive transport simulation based on the results of groundwater flow modeling and on particle tracking calculations for the site. Field velocities at the site vary between 0.4 and 3m/d. The transport of the reactive tracers deuterium ring labeled toluene-d5 and fully deuterated toluene-d8 was simulated and first-order biodegradation rates of 0.017 d−1 for toluene-d5 and 0.012 d−1 for toluene-d8 were determined.Environmental Geology 04/2012; 55(6):1255-1261. · 1.13 Impact Factor -
Article: Experimental investigation of nitrogen and oxygen isotope fractionation in nitrate and nitrite during denitrification
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ABSTRACT: In batch experiments, we studied the isotope fractionation of nitrogen and oxygen during denitrification of two bacterial strains (Azoarcus sp. strain DSM 9056 and Pseudomonas pseudoalcaligenes strain F10). Denitrification experiments were conducted with succinate and toluene as electron donor in three waters with a distinct oxygen isotope composition. Nitrate consumption was observed in all batch experiments. Reaction rates for succinate experiments were more than six times higher than those for toluene experiments. Nitrogen and oxygen isotopes became progressively enriched in the remaining nitrate pool in the course of the experiments; the nitrogen and oxygen isotope fractionation varied between 8.6–16.2 and 4.0–7.3‰, respectively. Within this range, neither electron donors nor the oxygen isotope composition of the medium affected the isotope fractionation process. The experimental results provide evidence that the oxygen isotope fractionation during nitrate reduction is controlled by a kinetic isotope effect which can be quantified using the Rayleigh model. The isotopic examination of nitrite released upon denitrification revealed that nitrogen isotope fractionation largely follows the fractionation of the nitrate pool. However, the oxygen isotope values of nitrite are clearly influenced by a rapid isotope equilibration with the oxygen of the ambient water. Even though this equilibration may in part be due to storage, it shows that under certain natural conditions (re-oxidation of nitrite) the nitrate pool may also be indirectly affected by an isotope equilibration. KeywordsNitrate–Nitrite–Denitrification–Nitrogen isotopes–Oxygen isotopes–Kinetic isotope fractionation–Isotope exchangeBiogeochemistry 04/2012; 103(1):371-384. · 3.07 Impact Factor -
Article: Critical evaluation of the 2D-CSIA scheme for distinguishing fuel oxygenate degradation reaction mechanisms.
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ABSTRACT: Although the uniform initial hydroxylation of methyl tert-butyl ether (MTBE) and other oxygenates during aerobic biodegradation has already been proven by molecular tools, variations in carbon and hydrogen enrichment factors (ε(C) and ε(H)) have still been associated with different reaction mechanisms (McKelvie et al. Environ. Sci. Technol. 2009, 43, 2793-2799). Here, we present new laboratory-derived ε(C) and ε(H) data on the initial degradation mechanisms of MTBE, ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME) by chemical oxidation (permanganate, Fenton reagents), acid hydrolysis, and aerobic bacteria cultures (species of Aquincola, Methylibium, Gordonia, Mycobacterium, Pseudomonas, and Rhodococcus). Plotting of Δδ(2)H/ Δδ(13)C data from chemical oxidation and hydrolysis of ethers resulted in slopes (Λ values) of 22 ± 4 and between 6 and 12, respectively. With A. tertiaricarbonis L108, R. zopfii IFP 2005, and Gordonia sp. IFP 2009, ε(C) was low (<|-1|‰) and ε(H) was insignificant. Fractionation obtained with P. putida GPo1 was similar to acid hydrolysis and M. austroafricanum JOB5 and R. ruber DSM 7511 displayed Λ values previously only ascribed to anaerobic attack. The fractionation patterns rather correlate with the employment of different P450, AlkB, and other monooxygenases, likely catalyzing ether hydroxylation via different transition states. Our data questions the value of 2D-CSIA for a simple distinguishing of oxygenate biotransformation mechanisms, therefore caution and complementary tools are needed for proper interpretation of groundwater plumes at field sites.Environmental Science & Technology 03/2012; 46(9):4757-66. · 4.80 Impact Factor -
Article: Protein-based stable isotope probing (protein-SIP) in functional metaproteomics.
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ABSTRACT: The community phenotype as the sum of molecular functions of organisms living in consortia strongly depends on interactions within these communities. Therefore, the analyses of the most significant molecules in terms of the phenotype, the proteins, have to be performed on samples without disrupting the meta-species environment. Due to the increasing genomic information, proteins provide insights into a potential molecular function and the phylogenetic structure of the community. Unfortunately, the lists of identified proteins are often based first on the technical capacity of the used methods or instruments, and second on the interpretation of them by the assignment of molecular functions to proteins in databases. Especially in non-model organisms the functions of many proteins are often not known and an increasing number of studies indicate a significant amount of uncertainty. To decrease the dependency on assumptions and to enable functional insights by metaproteome approaches, the metabolic labeling from an isotopically labeled substrate can be used. Since the metabolites deriving from the substrate are very rarely species-specific, the incorporation of the stable isotope into proteins can be used as a surrogate marker for metabolic activity. The degree of incorporation can be determined accurately on the peptide level by mass spectrometry; additionally, the peptide sequence provides information on the metabolic active species. Thereby, protein-stable isotope probing (protein-SIP) adds functional information to metaproteome approaches. The classical metaproteome approaches will be reviewed with an emphasis on their attempts towards functional interpretation. The gain from functional insights into metaproteomics by using metabolic labeling of stable isotopes of carbon, nitrogen, and sulfur is reviewed with a focus on the techniques of measurement, calculation of incorporation and data processing. © 2012 Wiley Periodicals, Inc. Mass Spec Rev 31:683-697, 2012.Mass Spectrometry Reviews 03/2012; 31(6):683-97. · 10.46 Impact Factor -
Article: Functional analysis of an anaerobic m-xylene-degrading enrichment culture using protein-based stable isotope probing.
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ABSTRACT: A sulfate-reducing consortium maintained for several years in the laboratory with m-xylene as sole source of carbon and energy was characterized by terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of PCR-amplified 16S rRNA genes and stable isotope probing of proteins (Protein-SIP). During growth upon m-xylene or methyl-labeled m-xylene (1,3-dimethyl-(13)C(2)-benzene), a phylotype affiliated to the family Desulfobacteriaceae became most abundant. A second dominant phylotype was affiliated to the phylum Epsilonproteobacteria. In cultures grown with methyl-labeled m-xylene, 331 proteins were identified by LC-MS/MS analysis. These proteins were either not (13)C-labeled (23%) or showed a (13)C-incorporation of 19-22 atom% (13)C (77%), the latter demonstrating that methyl groups of m-xylene were assimilated. (13)C-labeled proteins were involved in anaerobic m-xylene biodegradation, in sulfate reduction, in the Wood-Ljungdahl-pathway, and in general housekeeping functions. Thirty-eight percent of the labeled proteins were affiliated to Deltaproteobacteria. Probably due to a lack of sequence data from Epsilonproteobacteria, only 14 proteins were assigned to this phylum. Our data suggest that m-xylene is assimilated by the Desulfobacteriaceae phylotype, whereas the role of the Epsilonproteobacterium in the consortium remained unclear.FEMS Microbiology Ecology 02/2012; 81(1):134-44. · 3.41 Impact Factor -
Article: A novel online approach to the determination of isotopic ratios for organically bound chlorine, bromine and sulphur.
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ABSTRACT: A novel approach for the measurement of (37)Cl, (81)Br and (34)S in organic compounds containing chlorine, bromine, and sulphur is presented to overcome some of the major drawbacks of existing methods. Contemporary methods either require reference materials with the exact molecular compositions of the substances to be tested, or necessitate several laborious offline procedures prior to isotope analysis. In our online setup, organic compounds are separated by gas chromatography (GC) coupled to a high-temperature reactor. Using hydrogen as a makeup gas, the reactor achieves quantitative conversion of chlorinated, brominated and sulphurated organic compounds into gaseous hydrogen chloride (HCl), hydrogen bromide (HBr), and hydrogen sulphide (H(2)S), respectively. In this study, the GC interface was coupled to a quadrupole mass spectrometer operated in single-ion mode. The ion traces of either H(35)Cl (m/z 36) and H(37)Cl (m/z 38), H(79)Br (m/z 80) and H(81)Br (m/z 82), or H(2)(32)S (m/z 34) and H(2)(34)S (m/z 36), were recorded to determine the isotopic ratios of chlorine, bromine, and sulphur isotopes. The conversion interface presented here provides a basis for a novel method for compound-specific isotope analysis of halogenated and sulphur-containing compounds. Rapid online measurements of organic chlorine-, bromine- and sulphur-containing mixtures will facilitate the isotopic analysis of compounds containing these elements, and broaden their usage in fields of environmental forensics employing isotopic concepts.Rapid Communications in Mass Spectrometry 10/2011; 25(20):3114-22. · 2.79 Impact Factor -
Article: Development of an enantiomer-specific stable carbon isotope analysis (ESIA) method for assessing the fate of α-hexachlorocyclo-hexane in the environment.
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ABSTRACT: α-Hexachlorocyclohexane (α-HCH) is the only chiral isomer of the eight 1,2,3,4,5,6-HCHs and we have developed an enantiomer-specific stable carbon isotope analysis (ESIA) method for the evaluation of its fate in the environment. The carbon isotope ratios of the α-HCH enantiomers were determined for a commercially available α-HCH sample using a gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) system equipped with a chiral column. The GC-C-IRMS measurements revealed δ-values of -32.5 ± 0.8‰ and -32.3 ± 0.5‰ for (-) α-HCH and (+) α-HCH, respectively. The isotope ratio of bulk α-HCH was estimated to be -32.4 ± 0.6‰ which was in accordance with the δ-values obtained by GC-C-IRMS (-32.7 ± 0.2‰) and elemental analyzer-isotope ratio mass spectrometry (EA-IRMS) of the bulk α-HCH (-32.1 ± 0.1‰). The similarity of the isotope ratio measurements of bulk α-HCH by EA-IRMS and GC-C-IRMS indicates the accuracy of the chiral GC-C-IRMS method. The linearity of the α-HCH ESIA method shows that carbon isotope ratios can be obtained for a signal size above 100 mV. The ESIA measurements exhibited standard deviations (2σ) that were mostly < ± 0.5‰. In order to test the chiral GC-C-IRMS method, the isotope compositions of individual enantiomers in biodegradation experiments of α-HCH with Clostridium pasteurianum and samples from a contaminated field site were determined. The isotopic compositions of the α-HCH enantiomers show a range of enantiomeric and isotope patterns, suggesting that enantiomeric and isotope fractionation can serve as an indicator for biodegradation and source characterization of α-HCH in the environment.Rapid Communications in Mass Spectrometry 05/2011; 25(10):1363-72. · 2.79 Impact Factor -
Article: Combined application of PCR-based functional assays for the detection of aromatic-compound-degrading anaerobes.
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ABSTRACT: To explore the reliability of assays that detect aromatic-compound-degrading anaerobes, a combination of three functional-gene-targeting assays was applied to microcosms from benzene-contaminated aquifers. Results of the assays were consistent and suggest that species related to the genera Azoarcus and Geobacter dominated benzene degradation at the individual sites.Applied and environmental microbiology 05/2011; 77(14):5056-61. · 3.69 Impact Factor -
Article: Stable carbon isotope enrichment factors for cis-1,2-dichloroethene and vinyl chloride reductive dechlorination by Dehalococcoides.
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ABSTRACT: Compound-specific stable isotope analysis (CSIA) is a promising tool for monitoring in situ microbial activity, and enrichment factors (ε values) determined using CSIA can be employed to estimate compound transformation. Although ε values for some dechlorination reactions catalyzed by Dehalococcoides (Dhc) have been reported, reproducibility between independent experiments, variability between different Dhc strains, and congruency between pure and mixed cultures are unknown. In experiments conducted with pure cultures of Dhc sp. strain BAV1, ε values for 1,1-DCE, cis-DCE, trans-DCE, and VC were -5.1, -14.9, -20.8, and -23.2‰, respectively. The ε value for 1,1-DCE dechlorination was 48.9% higher than the value reported in a previous study, but ε values for other chlorinated ethenes were equal between independent experiments. For the dechlorination of cis-DCE and VC by Dhc strains BAV1, FL2, GT, and VS, average ε values were -18.4 and -23.2‰, respectively. cis-DCE and VC ε values determined in pure Dhc cultures with different reductive dehalogenase genes (e.g., vcrA vs bvcA) varied by less than 36.8 and 8.3%, respectively. In the BDI consortium, ε values for cis-DCE and VC dechlorination were -25.3‰ and -19.9‰, 31.6% higher and 15.3% lower, respectively, compared to the average ε value for Dhc pure cultures. As cis-DCE and VC ε values are all within the same order-of-magnitude and fractionation is always measured during Dhc dechlorination, CSIA may be a valuable approach for monitoring in situ cis-DCE and VC reductive dechlorination.Environmental Science & Technology 03/2011; 45(7):2951-7. · 4.80 Impact Factor -
Article: Anaerobic benzene degradation by bacteria.
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ABSTRACT: Benzene is a widespread and toxic contaminant. The fate of benzene in contaminated aquifers seems to be primarily controlled by the abundance of oxygen: benzene is aerobically degraded at high rates by ubiquitous microorganisms, and the oxygen-dependent pathways for its breakdown were elucidated more than 50 years ago. In contrast, benzene was thought to be persistent under anoxic conditions until 25 years ago. Nevertheless, within the last 15 years, several benzene-degrading cultures have been enriched under varying electron acceptor conditions in laboratories around the world, and organisms involved in anaerobic benzene degradation have been identified, indicating that anaerobic benzene degradation is a relevant environmental process. However, only a few benzene degraders have been isolated in pure culture so far, and they all use nitrate as an electron acceptor. In some highly enriched strictly anaerobic cultures, benzene has been described to be mineralized cooperatively by two or more different organisms. Despite great efforts, the biochemical mechanism by which the aromatic ring of benzene is activated in the absence of oxygen is still not fully elucidated; methylation, hydroxylation and carboxylation are discussed as likely reactions. This review summarizes the current knowledge about the 'key players' of anaerobic benzene degradation under different electron acceptor conditions and the possible pathway(s) of anaerobic benzene degradation.Microbial Biotechnology 03/2011; 4(6):710-24. · 2.53 Impact Factor -
Article: Detection of monochlorobenzene metabolizing bacteria under anoxic conditions by DNA-stable isotope probing.
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ABSTRACT: Cultivation-independent analyses were applied to study the structural diversity of the bacterial community which developed in groundwater inoculated microcosms actively metabolizing monochlorobenzene (MCB) under anaerobic conditions. Addition of (13)C-labelled MCB demonstrated that the community produced (13)CO(2) as a metabolite at slightly increasing rates over a period of 1,051 days while no (13)C-methane evolved. Genetic profiles of partial 16S rRNA genes generated with the single-strand conformation polymorphism (SSCP) technique by PCR from directly extracted total DNA revealed that, despite the long incubation period, six replicate microcosms were characterized by almost the same microbial members. Nine distinguishable contributors to the SSCP-profiles were characterized by DNA sequencing, revealing the presence of different members from the phyla Proteobacteria, Fibrobacteres and from the candidate division OD1. DNA-stable isotope probing (SIP) was applied to distinguish the actual MCB metabolizing bacteria from the other community members. This study reveals for the first time the structural diversity of an anaerobic MCB metabolizing bacterial community. However, it also demonstrates the limitations of SIP to detect bacteria slowly metabolizing carbon sources under anaerobic conditions.Biodegradation 02/2011; 22(5):973-82. · 2.02 Impact Factor -
Article: Accelerated methanogenesis from aliphatic and aromatic hydrocarbons under iron- and sulfate-reducing conditions.
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ABSTRACT: The impact of four electron acceptors on hydrocarbon-induced methanogenesis was studied. Methanogenesis from residual hydrocarbons may enhance the exploitation of oil reservoirs and may improve bioremediation. The conditions to drive the rate-limiting first hydrocarbon-oxidizing steps for the conversion of hydrocarbons into methanogenic substrates are crucial. Thus, the electron acceptors ferrihydrite, manganese dioxide, nitrate or sulfate were added to sediment microcosms acquired from two brackish water locations. Hexadecane, ethylbenzene or 1-(13)C-naphthalene were used as model hydrocarbons. Methane was released most rapidly from incubations amended with ferrihydrite and hexadecane. Ferrihydrite enhanced only hexadecane-dependent methanogenesis. The rates of methanogenesis were negatively affected by sulfate and nitrate at concentrations of more than 5 and 1 mM, respectively. Metal-reducing Geobacteraceae and potential sulfate reducers as well as Methanosarcina were present in situ and in vitro. Ferrihydrite addition triggered the growth of Methanosarcina-related methanogens. Additionally, methane was removed concomitantly by anaerobic methanotrophy. ANME-1 and -2 methyl coenzyme M reductase genes were detected, indicating anaerobic methanotrophy as an accompanying process [Correction added 16 December after online publication: 'methyl coenzyme A' changed to 'methyl coenzyme M' in this sentence]. The experiments presented here demonstrate the feasibility of enhancing methanogenic alkane degradation by ferrihydrite or sulfate addition in different geological settings.FEMS Microbiology Letters 02/2011; 315(1):6-16. · 2.04 Impact Factor -
Article: Protein-based stable isotope probing.
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ABSTRACT: We describe a stable isotope probing (SIP) technique that was developed to link microbe-specific metabolic function to phylogenetic information. Carbon ((13)C)- or nitrogen ((15)N)-labeled substrates (typically with >98% heavy label) were used in cultivation experiments and the heavy isotope incorporation into proteins (protein-SIP) on growth was determined. The amount of incorporation provides a measure for assimilation of a substrate, and the sequence information from peptide analysis obtained by mass spectrometry delivers phylogenetic information about the microorganisms responsible for the metabolism of the particular substrate. In this article, we provide guidelines for incubating microbial cultures with labeled substrates and a protocol for protein-SIP. The protocol guides readers through the proteomics pipeline, including protein extraction, gel-free and gel-based protein separation, the subsequent mass spectrometric analysis of peptides and the calculation of the incorporation of stable isotopes into peptides. Extraction of proteins and the mass fingerprint measurements of unlabeled and labeled fractions can be performed in 2-3 d.Nature Protocol 12/2010; 5(12):1957-66. · 8.36 Impact Factor -
Article: Carbon isotope fractionation during dechlorination of 1,2,3,4-tetrachlorodibenzo-p-dioxin by a Dehalococcoides-containing culture.
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ABSTRACT: Carbon isotope fractionation was observed during dechlorination of 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TeCDD) by a mixed culture containing Dehalococcoides ethenogenes strain 195. Fractionation was examined when 1,2,3,4-TeCDD was added as the only chlorinated compound and when 1,2,3,4-TeCDD was added with a known growth substrate, tetrachloroethene (PCE). The 1,2,3,4-TeCDD was dechlorinated to 1,2,4-trichlorodibenzo-p-dioxin (1,2,4-TrCDD) which was enriched in (13)C relative to 1,2,3,4-TeCDD with isotope separation factors, epsilon(C), of 1.3+/-0.2 per thousand and 1.7+/-0.4 per thousand (average+/-95% confidence interval (CI)) in cultures with and without PCE, respectively. The 1,2,4-TrCDD was further dechlorinated to 1,3-dichlorodibenzo-p-dioxin (1,3-DCDD) which was depleted in (13)C relative to 1,2,4-TrCDD with epsilon(C) of -2.4+/-0.4 per thousand and -2.9+/-0.8 per thousand (average+/-95% CI) in cultures with and without PCE, respectively. This demonstrates carbon isotope fractionation during sequential reductive dechlorination of PCDDs, where isotope fractionation during dechlorination of the intermediate was substantial and a (13)C depleted lightly chlorinated PCDD congener was ultimately formed during dechlorination of more highly chlorinated PCDD congeners. Despite reproducible, statistically significant differences between isotope compositions of the parent, 1,2,3,4-TeCDD and daughter, 1,2,4-TrCDD and 1,3-DCDD congeners in triplicate bottles of both treatments, fractionation factors for 1,2,3,4-TeCDD could not be determined for all replicates by regression analysis of the plot of the Rayleigh equation. It is possible that dissolution of 1,2,3,4-TeCDD imposed a kinetic limitation on dechlorination, thus masking isotope fractionation during its dechlorination.Chemosphere 08/2010; 80(10):1113-9. · 3.21 Impact Factor -
Article: Decimal place slope, a fast and precise method for quantifying 13C incorporation levels for detecting the metabolic activity of microbial species.
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ABSTRACT: The metabolic incorporation of stable isotopes such as (13)C or (15)N into proteins has become a powerful tool for qualitative and quantitative proteome studies. We recently introduced a method that monitors heavy isotope incorporation into proteins and presented data revealing the metabolic activity of various species in a microbial consortium using this technique. To further develop our method using an liquid chromatography (LC)-mass spectrometry (MS)-based approach, we present here a novel approach for calculating the incorporation level of (13)C into peptides by using the information given in the decimal places of peptide masses obtained by modern high-resolution MS. In the present study, the applicability of this approach is demonstrated using Pseudomonas putida ML2 proteins uniformly labeled via the consumption of [(13)C(6)]benzene present in the medium at concentrations of 0, 10, 25, 50, and 100 atom %. The incorporation of (13)C was calculated on the basis of several labeled peptides derived from one band on an SDS-PAGE gel. The accuracy of the calculated incorporation level depended upon the number of peptide masses included in the analysis, and it was observed that at least 100 peptide masses were required to reduce the deviation below 4 atom %. This accuracy was comparable with calculations of incorporation based on the isotope envelope. Furthermore, this method can be extended to the calculation of the labeling efficiency for a wide range of biomolecules, including RNA and DNA. The technique will therefore allow a highly accurate determination of the carbon flux in microbial consortia with a direct approach based solely on LC-MS.Molecular & Cellular Proteomics 06/2010; 9(6):1221-7. · 7.40 Impact Factor -
Article: Calculation of partial isotope incorporation into peptides measured by mass spectrometry.
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ABSTRACT: Stable isotope probing (SIP) technique was developed to link function, structure and activity of microbial cultures metabolizing carbon and nitrogen containing substrates to synthesize their biomass. Currently, available methods are restricted solely to the estimation of fully saturated heavy stable isotope incorporation and convenient methods with sufficient accuracy are still missing. However in order to track carbon fluxes in microbial communities new methods are required that allow the calculation of partial incorporation into biomolecules. In this study, we use the characteristics of the so-called 'half decimal place rule' (HDPR) in order to accurately calculate the partial13C incorporation in peptides from enzymatic digested proteins. Due to the clade-crossing universality of proteins within bacteria, any available high-resolution mass spectrometry generated dataset consisting of tryptically-digested peptides can be used as reference.We used a freely available peptide mass dataset from Mycobacterium tuberculosis consisting of 315,579 entries. From this the error of estimated versus known heavy stable isotope incorporation from an increasing number of randomly drawn peptide sub-samples (100 times each; no repetition) was calculated. To acquire an estimated incorporation error of less than 5 atom %, about 100 peptide masses were needed. Finally, for testing the general applicability of our method, peptide masses of tryptically digested proteins from Pseudomonas putida ML2 grown on labeled substrate of various known concentrations were used and13C isotopic incorporation was successfully predicted. An easy-to-use script 1 was further developed to guide users through the calculation procedure for their own data series. Our method is valuable for estimating13C incorporation into peptides/proteins accurately and with high sensitivity. Generally, our method holds promise for wider applications in qualitative and especially quantitative proteomics.BMC Research Notes 01/2010; 3:178.
Top co-authors
Top Journals
Institutions
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2005–2012
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Helmholtz-Zentrum für Umweltforschung
- • Department Isotopenbiogeochemie
- • Department Umweltbiotechnologie
- • Department Umweltmikrobiologie
- • Department Proteomik
Leipzig, Saxony, Germany -
Max-Planck-Gesellschaft
München, Bavaria, Germany
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2008–2011
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Bundesanstalt für Geowissenschaften und Rohstoffe
Hannover, Lower Saxony, Germany
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2009
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Georgia Institute of Technology
- School of Civil & Environmental Engineering
Atlanta, GA, USA
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2002
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Eberhard-Karls-Universität Tübingen
- Group of Applied Geosciences
Tübingen, Baden-Wuerttemberg, Germany
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