Taxonomic and functional diversity of the microbiome in a jet fuel contaminated site as revealed by combined application of in situ microcosms with metagenomic analysis.

Natural attenuation represents all processes that govern contaminant mass removal, which mainly occurs via microbial degradation in the environment. Although this process is intrinsic its rate and efficiency depend on multiple factors. This study aimed to characterize the microbial taxonomic and functional diversity in different aquifer sediments collected in the saturated zone and in situ microcosms (BACTRAP®s) amended with hydrocarbons (13C-labeled and non-labeled benzene, toluene and naphthalene) using 16S rRNA gene and "shotgun" Illumina high throughput sequencing at a jet-fuel contaminated site. The BACTRAP®s were installed to assess hydrocarbon metabolism by native bacteria. Results indicated that Proteobacteria, Actinobacteria and Firmicutes were the most dominant phyla (~98%) in the aquifer sediment samples. Meanwhile, in the benzene- and toluene-amended BACTRAP®s the phyla Firmicutes and Proteobacteria accounted for about 90% of total community. In the naphthalene-amended BACTRAP®, members of the SR-FBR-L83 family (Order Ignavibacteriales) accounted for almost 80% of bacterial community. Functional annotation of metagenomes showed that only the sediment sample located at the source zone border and with the lowest BTEX concentration, has metabolic potential to degrade hydrocarbons aerobically. On the other hand, in situ BACTRAP®s allowed enrichment of hydrocarbon-degrading bacteria. Metagenomic data suggest that fumarate addition is the main mechanism for hydrocarbon activation of toluene. Also, indications for methylation, hydroxylation and carboxylation as activation mechanisms for benzene anaerobic conversion were found. After 120 days of exposure in the contaminated groundwater, the isotopic analysis of fatty acids extracted from BACTRAP®s demonstrated the assimilation of isotopic labeled compounds in the cells of microbes expressed by strong isotopic enrichment. We propose that the microbiota in this jet-fuel contaminated site has metabolic potential to degrade benzene and toluene by a syntrophic process, between members of the families Geobacteraceae and Peptococcaceae (genus Pelotomaculum), coupled to nitrate, iron and/or sulfate reduction.

Microaerophilic Fe(II)-Oxidizing Zetaproteobacteria Isolated from Low-Fe Marine Coastal Sediments: Physiology and Composition of Their Twisted Stalks.

Microaerophilic Fe(II) oxidizers are commonly found in habitats containing elevated Fe(II) and low O2 concentrations and often produce characteristic Fe mineral structures, so-called twisted stalks or tubular sheaths. Isolates originating from freshwater habitats are all members of the Betaproteobacteria, while isolates from marine habitats belong almost exclusively to the Zetaproteobacteria So far, only a few isolates of marine microaerophilic Fe(II) oxidizers have been described, all of which are obligate microaerophilic Fe(II) oxidizers and have been thought to be restricted to Fe-rich systems. Here, we present two new isolates of marine microaerophilic Fe(II)-oxidizing Zetaproteobacteria that originate from typical coastal marine sediments containing only low Fe concentrations (2 to 11 mg of total Fe/g of sediment [dry weight]; 70 to 100 µM dissolved Fe2+ in the porewater). The two novel Zetaproteobacteria share characteristic physiological properties of the Zetaproteobacteria group, even though they come from low-Fe environments: the isolates are obligate microaerophilic Fe(II) oxidizers and, like most isolated Zetaproteobacteria, they produce twisted stalks. We found a low organic carbon content in the stalks (∼0.3 wt%), with mostly polysaccharides and saturated aliphatic chains (most likely lipids). The Fe minerals in the stalks were identified as lepidocrocite and possibly ferrihydrite. Immobilization experiments with Ni2+ showed that the stalks can function as a sink for trace metals. Our findings show that obligate microaerophilic Fe(II) oxidizers belonging to the Zetaproteobacteria group are not restricted to Fe-rich environments but can also be found in low-Fe marine environments, which increases their overall importance for the global biogeochemical Fe cycle.IMPORTANCE So far, only a few isolates of benthic marine microaerophilic Fe(II) oxidizers belonging to the Zetaproteobacteria exist, and most isolates were obtained from habitats containing elevated Fe concentrations. Consequently, it was thought that these microorganisms are important mainly in habitats with high Fe concentrations. The two novel isolates of Zetaproteobacteria that are presented in the present study were isolated from typical coastal marine sediments that do not contain elevated Fe concentrations. This increases the knowledge about possible habitats in which Zetaproteobacteria can exist. Furthermore, we show that the physiology and the typical organo-mineral structures (twisted stalks) that are produced by the isolates do not notably differ from the physiology and the cell-mineral structures of isolates from environments with high Fe concentrations. We also showed that the organo-mineral structures can function as a sink for trace metals.

Isotope fractionation of benzene during partitioning - Revisited.

Isotope fractionation between benzene-D0 and benzene-D6 caused by multi-step partitioning of the benzenes between water and two organic solvents, n-octane and 1-octanol, as well as between water and the gas phase, was measured. The obtained fractionation factors αH = KH/KD are αH = 1.080 ± 0.015 and αH = 1.074 ± 0.015 for extraction into n-octane and 1-octanol, respectively, and αH = 1.049 ± 0.010 for evaporation from aqueous solution. The comparison of solvent- and gas-phase partitioning reveals that about 2/3 of the driving force of fractionation is due to different interactions in the aqueous phase, whereas 1/3 is due to different interactions in the organic phase. The heavy benzene isotopologue behaves more 'hydrophilically' and the light one more 'hydrophobically'. This synergistic alignment gives rise to relatively large fractionation effects in partitioning between water and non-polar organic matter. In contrast to a previous study, there is no indication of strong fractionation by specific interactions between benzene and octanol. Partitioning under non-equilibrium conditions yields smaller apparent fractionation effects due to opposite trends of thermodynamic and kinetic fractionation parameters, i.e. partition and diffusion coefficients of the isotopologues. This may have consequences which should be taken into account when considering isotope fractionation due to sorption in environmental compartments.

The ecological and physiological responses of the microbial community from a semiarid soil to hydrocarbon contamination and its bioremediation using compost amendment.

The linkage between phylogenetic and functional processes may provide profound insights into the effects of hydrocarbon contamination and biodegradation processes in high-diversity environments. Here, the impacts of petroleum contamination and the bioremediation potential of compost amendment, as enhancer of the microbial activity in semiarid soils, were evaluated in a model experiment. The analysis of phospholipid fatty-acids (PLFAs) and metaproteomics allowed the study of biomass, phylogenetic and physiological responses of the microbial community in polluted semiarid soils. Petroleum pollution induced an increase of proteobacterial proteins during the contamination, while the relative abundance of Rhizobiales lowered in comparison to the non-contaminated soil. Despite only 0.55% of the metaproteome of the compost-treated soil was involved in biodegradation processes, the addition of compost promoted the removal of polycyclic aromatic hydrocarbons (PAHs) and alkanes up to 88% after 50 days. However, natural biodegradation of hydrocarbons was not significant in soils without compost. Compost-assisted bioremediation was mainly driven by Sphingomonadales and uncultured bacteria that showed an increased abundance of catabolic enzymes such as catechol 2,3-dioxygenases, cis-dihydrodiol dehydrogenase and 2-hydroxymuconic semialdehyde. For the first time, metaproteomics revealed the functional and phylogenetic relationships of petroleum contamination in soil and the microbial key players involved in the compost-assisted bioremediation.

Carbon and hydrogen isotope fractionation of benzene and toluene during hydrophobic sorption in multistep batch experiments.

The application of compound-specific stable isotope analysis (CSIA) for evaluating degradation of organic pollutants in the field implies that other processes affecting pollutant concentration are minor with respect to isotope fractionation. Sorption is associated with minor isotope fractionation and pollutants may undergo successive sorption-desorption steps during their migration in aquifers. However, little is known about isotope fractionation of BTEX compounds after consecutive sorption steps. Here, we show that partitioning of benzene and toluene between water and organic sorbents (i.e. 1-octanol, dichloromethane, cyclohexane, hexanoic acid and Amberlite XAD-2) generally exhibits very small carbon and hydrogen isotope effects in multistep batch experiments. However, carbon and hydrogen isotope fractionation was observed for the benzene-octanol pair after several sorption steps (Δδ(13)C=1.6 ± 0.3‰ and Δδ(2)H=88 ± 3‰), yielding isotope fractionation factors of αC=1.0030 ± 0.0005 and αH=1.195 ± 0.026. Our results indicate that the cumulative effect of successive hydrophobic partitioning steps in an aquifer generally results in insignificant isotope fractionation for benzene and toluene. However, significant carbon and hydrogen isotope fractionation cannot be excluded for specific sorbate-sorbent pairs, such as sorbates with π-electrons and sorbents with OH-groups. Consequently, functional groups of sedimentary organic matter (SOM) may specifically interact with BTEX compounds migrating in an aquifer, thereby resulting in potentially relevant isotope fractionation.

Using compound-specific isotope analysis to assess the degradation of chloroacetanilide herbicides in lab-scale wetlands.

Compound-specific isotope analysis (CSIA) is a promising tool to study the environmental fate of a wide range of contaminants including pesticides. In this study, a novel CSIA method was developed to analyse the stable carbon isotope signatures of widely used chloroacetanilide herbicides. The developed method was applied in combination with herbicide concentration and hydrochemical analyses to investigate in situ biodegradation of metolachlor, acetochlor and alachlor during their transport in lab-scale wetlands. Two distinct redox zones were identified in the wetlands. Oxic conditions prevailed close to the inlet of the four wetlands (oxygen concentration of 212±24µM), and anoxic conditions (oxygen concentrations of 28±41µM) prevailed towards the outlet, where dissipation of herbicides mainly occurred. Removal of acetochlor and alachlor from inlet to outlet of wetlands was 56% and 51%, whereas metolachlor was more persistent (23% of load dissipation). CSIA of chloroacetanilides at the inlet and outlet of the wetlands revealed carbon isotope fractionation of alachlor (εbulk=-2.0±0.3‰) and acetochlor (εbulk=-3.4±0.5‰), indicating that biodegradation contributes to the dissipation of both herbicides. This study is a first step towards the application of CSIA to evaluate the transport and degradation of chloroacetanilide herbicides in the environment.

Influences of the substrate feeding regime on methanogenic activity in biogas reactors approached by molecular and stable isotope methods.

In order to better understand the effects of the substrate feeding regime on methanogenesis during anaerobic digestion in biogas reactors, four continuous stirred tank reactors operated under mesophilic conditions were investigated. In addition to standard physicochemical parameters, the stable isotopic signatures of CH4 and CO2 before and after daily feeding were analyzed. The activity of the methanogens was assessed by methyl coenzyme M reductase alpha-subunit (mcrA/mrtA) gene transcript analysis. Two different feeding regimes i.e. single vs. double consecutive feeding of the otherwise same daily maize silage load were investigated. During the first phase, a single feeding of the whole daily dose increased the biogas production within 70-80 min from around 0.5 to 2.0 L/h. This increase was associated with a transient increase of the acetic acid concentration and a corresponding decrease of the pH. Only moderate increase in biogas yield and VFA concentration (mainly acetate) was observed when the daily substrate was apportioned into two feedings. However, the overall daily gas production was similar in both cases. Regardless of the feeding regime, significantly depleted δ(13)CH4 and minor changes in the CO2 content of biogas were observed after feeding, which were followed by enrichment of δ(13)CH4. This period was associated with detectable changes in activity of methanogenic communities monitored by terminal restriction fragment length polymorphism analysis based on the transcripts of mcrA/mrtA genes. Methanoculleus and Methanobacterium spp. were the predominant methanogens in all reactors, while Methanosarcina spp. activity was only significant in two reactors. The activity of Methanoculleus and Methanosarcina spp. increased after the feeding in these reactors, which was followed by a depletion of δ(13)C in the produced gas. In both reactors, the less depleted isotopic values were detected before the second feeding, when Methanobacterium was the most active genus. Variations in reactor performance and methanogenic community characteristics were attributed to inoculum heterogeneity and stochastic factors during the reactor set up.

Evaluation of stable isotope fingerprinting techniques for the assessment of the predominant methanogenic pathways in anaerobic digesters.

Laboratory biogas reactors were operated under various conditions using maize silage, chicken manure, or distillers grains as substrate. In addition to the standard process parameters, the hydrogen and carbon stable isotopic composition of biogas was analyzed to estimate the predominant methanogenic pathways as a potential process control tool. The isotopic fingerprinting technique was evaluated by parallel analysis of mcrA genes and their transcripts to study the diversity and activity of methanogens. The dominant hydrogenotrophs were Methanomicrobiales, while aceticlastic methanogens were represented by Methanosaeta and Methanosarcina at low and high organic loading rates, respectively. Major changes in the relative abundance of mcrA transcripts were observed compared to the results obtained from DNA level. In agreement with the molecular results, the isotope data suggested the predominance of the hydrogenotrophic pathway in one reactor fed with chicken manure, while both pathways were important in the other reactors. Short-term changes in the isotopic composition were followed, and a significant change in isotope values was observed after feeding a reactor digesting maize silage. This ability of stable isotope fingerprinting to follow short-term activity changes shows potential for indicating process failures and makes it a promising technology for process control.

Biodegradation of chlorobenzene in a constructed wetland treating contaminated groundwater.

Monochlorobenzene (MCB) is an important groundwater contaminant world-wide. In this study, a horizontal subsurface flow constructed wetland with an integrated water compartment was fed with MCB contaminated groundwater originating from the local aquifer. Analysis of spatial concentration dynamics of MCB and oxygen was combined with isotope composition analysis of MCB for assessing in situ biodegradation. Removal of MCB was most effective in the upper layer of the soil filter, reaching up to 77.1%. Trace oxygen concentrations below 0.16 mg L(-1) were observed throughout the wetland transect, suggesting a considerable limitation of aerobic microbial MCB degradation. Enrichment of 13C in the residual MCB fraction at increasing distance from the inflow point indicated microbial MCB degradation in the wetland. The observed isotope shift was higher than expected for aerobic MCB degradation and thus pointed out a significant contribution of an anaerobic degradation pathway to the overall biodegradation.

In situ assessment of biodegradation potential using biotraps amended with 13C-labeled benzene or toluene.

Stable isotope fractionation analysis of an aquifer heavily contaminated with benzene (up to 850 mg L(-1)) and toluene (up to 50 mg L(-1)) at a former hydrogenation plant in Zeitz (Saxonia, Germany) has suggested that significant biodegradation of toluene was occurring. However, clear evidence of benzene biodegradation has been lacking at this site. Determining the fate of benzene is often a determining factor in regulatory approval of a risk-based management strategy. The objective of the work described here was the demonstration of a new tool that can be used to provide proof of biodegradation of benzene or other organics by indigenous microorganisms under actual aquifer conditions. Unique in situ biotraps containing Bio-Sep beads, amended with 13C-labeled or 12C nonlabeled benzene and toluene, were deployed at the Zeitz site for 32 days in an existing groundwater monitoring well and used to collect and enrich microbial biofilms. Lipid biomarkers or remaining substrate was extracted from the beads and analyzed by mass spectrometry and molecular methods. Isotopic analysis of the remaining amounts of 13C-labeled contaminants (about 15-18% of the initial loading) showed no alteration of the 12C/13C ratio during incubation. Therefore, no measurable exchange of labeled compounds in the beads by the nonlabeled compounds in the aquifer materials occurred. Isotopic ratio analysis of microbial lipid fatty acids (as methyl ester derivatives) from labeled benzene- and toluene-amended biotraps showed 13C enrichment in several fatty acids of up to delta (13C) 13400%o, clearly verifying benzene and toluene biodegradation and the transformation of the labeled carbon into biomass by indigenous organisms under aquifer conditions. Fatty acid profiles of total lipid fatty acids and the phospholipid fatty acid fraction and their isotopic composition showed significant differences between benzene- and toluene-amended biotraps, suggesting that different microbial communities were involved in the biodegradation of the two compounds.

Stable carbon isotope fractionation during aerobic and anaerobic transformation of trichlorobenzene.

Fractionation of stable carbon isotopes upon degradation of trichlorobenzenes was studied under aerobic and anaerobic conditions. Mineralization of 1,2,4-trichlorobenzene by the aerobic strain Pseudomonas sp. P51 which uses a dioxygenase for the initial enzymatic reaction was not accompanied by a significant isotope fractionation. In contrast, reductive dehalogenation by the anaerobic strain Dehalococcoides sp. strain CBDB1 revealed average isotope enrichment factors (eta) between -3.1 and -3.7 for 1,2,3- and 1,2,4-trichlorobenzene, respectively. The significant isotope fractionation during reductive dehalogenation would allow tracing the in situ biodegradation of halogenated benzenes in contaminated anoxic aquifers, whereas the lack of isotope fractionation during aerobic transformation limits the use of this approach in oxic environments.

In-situ biodegradation of tetrachloroethene and trichloroethene in contaminated aquifers monitored by stable isotope fractionation.

Stable carbon isotope analysis of tetrachloroethene (PCE) and trichloroethene (TCE) was applied to evaluatenatural attenuation processes in the upper Quaternary and lower Tertiary aquifer in the area of a former dry-cleaning plant located in Leipzig, Germany. Groundwater samples were taken during one monitoring campaign in 2001. The 13C enrichment in contaminants along the water flow path suggested that both, PCE and TCE were degraded in the Quaternary aquifer. The enrichment of 13C in the residual PCE fraction and an isotope fractionation factor from laboratory experiments were used to calculate the extent of biodegradation in the Quatemary aquifer. These calculations indicated that a major portion of PCE was biodegraded in the course of the plume. In the Tertiary aquifer the carbon isotope ratios of PCE and TCE indicated that the decreasing concentrations of these contaminants were probably not caused by microbial processes.

Isotope fractionation of toluene: a perspective to characterise microbial in situ degradation.

A concept to assess in situ biodegradation of organic contaminants in aquifers is presented. The alteration of the carbon isotope composition of contaminants along the groundwater flow path indicates microbial degradation processes and can be used as an indicator for in situ biodegradation. The Rayleigh equation was applied to calculate the percentage of the in situ biodegradation (B[%]) using the change in the isotopic composition of contaminants (Rt/R0) along the ground water flow path and a kinetic carbon isotope fractionation factor (alphaC) derived from defined biodegradation experiments in the laboratory. When the groundwater hydrology is known and a representative source concentration (C0) for a groundwater flow path can be determined, the extent of in situ biodegradation can be quantified.

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

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

Anaerobic degradation of 2-methylnaphthalene by a sulfate-reducing enrichment culture.

Anaerobic degradation of 2-methylnaphthalene was investigated with a sulfate-reducing enrichment culture. Metabolite analyses revealed two groups of degradation products. The first group comprised two succinic acid adducts which were identified as naphthyl-2-methyl-succinic acid and naphthyl-2-methylene-succinic acid by comparison with chemically synthesized reference compounds. Naphthyl-2-methyl-succinic acid accumulated to 0.5 microM in culture supernatants. Production of naphthyl-2-methyl-succinic acid was analyzed in enzyme assays with dense cell suspensions. The conversion of 2-methylnaphthalene to naphthyl-2-methyl-succinic acid was detected at a specific activity of 0.020 +/- 0.003 nmol min(-1) mg of protein(-1) only in the presence of cells and fumarate. We conclude that under anaerobic conditions 2-methylnaphthalene is activated by fumarate addition to the methyl group, as is the case in anaerobic toluene degradation. The second group of metabolites comprised 2-naphthoic acid and reduced 2-naphthoic acid derivatives, including 5,6,7,8-tetrahydro-2-naphthoic acid, octahydro-2-naphthoic acid, and decahydro-2-naphthoic acid. These compounds were also identified in an earlier study as products of anaerobic naphthalene degradation with the same enrichment culture. A pathway for anaerobic degradation of 2-methylnaphthalene analogous to that for anaerobic toluene degradation is proposed.

Anaerobic naphthalene degradation by a sulfate-reducing enrichment culture.

Anaerobic naphthalene degradation by a sulfate-reducing enrichment culture was studied by substrate utilization tests and identification of metabolites by gas chromatography-mass spectrometry. In substrate utilization tests, the culture was able to oxidize naphthalene, 2-methylnaphthalene, 1- and 2-naphthoic acids, phenylacetic acid, benzoic acid, cyclohexanecarboxylic acid, and cyclohex-1-ene-carboxylic acid with sulfate as the electron acceptor. Neither hydroxylated 1- or 2-naphthoic acid derivatives and 1- or 2-naphthol nor the monoaromatic compounds ortho-phthalic acid, 2-carboxy-1-phenylacetic acid, and salicylic acid were utilized by the culture within 100 days. 2-Naphthoic acid accumulated in all naphthalene-grown cultures. Reduced 2-naphthoic acid derivatives could be identified by comparison of mass spectra and coelution with commercial reference compounds such as 1,2,3, 4-tetrahydro-2-naphthoic acid and chemically synthesized decahydro-2-naphthoic acid. 5,6,7,8-Tetrahydro-2-naphthoic acid and octahydro-2-naphthoic acid were tentatively identified by their mass spectra. The metabolites identified suggest a stepwise reduction of the aromatic ring system before ring cleavage. In degradation experiments with [1-(13)C]naphthalene or deuterated D(8)-naphthalene, all metabolites mentioned derived from the introduced labeled naphthalene. When a [(13)C]bicarbonate-buffered growth medium was used in conjunction with unlabeled naphthalene, (13)C incorporation into the carboxylic group of 2-naphthoic acid was shown, indicating that activation of naphthalene by carboxylation was the initial degradation step. No ring fission products were identified.

Naphthalene degradation and incorporation of naphthalene-derived carbon into biomass by the thermophile Bacillus thermoleovorans.

The thermophilic aerobic bacterium Bacillus thermoleovorans Hamburg 2 grows at 60 degrees C on naphthalene as the sole source of carbon and energy. In batch cultures, an effective substrate degradation was observed. The carbon balance, including naphthalene, metabolites, biomass, and CO(2), was determined by the application of [1-(13)C]naphthalene. The incorporation of naphthalene-derived carbon into the bulk biomass as well as into specified biomass fractions such as fatty acids and amino acids was confirmed by coupled gas chromatography-mass spectrometry (GC-MS) and isotope analyses. Metabolites were characterized by GC-MS; the established structures allow tracing the degradation pathway under thermophilic conditions. Apart from typical metabolites of naphthalene degradation known from mesophiles, intermediates such as 2, 3-dihydroxynaphthalene, 2-carboxycinnamic acid, and phthalic and benzoic acid were identified for the pathway of this bacterium. These compounds indicate that naphthalene degradation by the thermophilic B. thermoleovorans differs from the known pathways found for mesophilic bacteria.

Tracing the transformation of labelled [1-13C]phenanthrene in a soil bioreactor.

[1-(13)C]-labelled phenanthrene was incubated in a closed bioreactor to study the flux and biotransformation of polycyclic aromatic hydrocarbon (PAH) in contaminated soils on a bulk and molecular level. The degradation of extractable phenanthrene was observed by GC-MS measurements and the mineralisation was monitored by (13)CO(2) production. The transformation of the (13)C-label into non-extractable soil-bound residues was determined by carbon isotopic measurements. With these data we were able to calculate a carbon budget of the (13)C-label. Moreover, the chemical structure of non-extractable bound residues was characterised by applying selective chemical degradation reactions to cleave xenobiotic subunits from the macromolecular organic soil matrix. The obtained low molecular weight products yielded (13)C-labelled compounds which were identified using IRM (isotope ratio monitoring)-GC-MS and structurally characterised with GC-MS. Most of the (13)C-labelled products obtained by chemical degradation of non-extractable bound residues are well-known metabolites of phenanthrene. Thus, metabolites of [1-(13)C]phenanthrene formed during biodegradation appear to be reactive components which are subsequently involved in the bound residue formation. Hydrolysable amino acids of the soil residues were significantly labelled with (13)C as confirmed by IRM-GC-MS measurements. Therefore, phenanthrene-derived carbon was transformed by anabolic microbial processes into typical biologically derived compounds. These substances are likely to be incorporated into humic-like material after cell death.

Methane formation from long-chain alkanes by anaerobic microorganisms.

Biological formation of methane is the terminal process of biomass degradation in aquatic habitats where oxygen, nitrate, ferric iron and sulphate have been depleted as electron acceptors. The pathway leading from dead biomass to methane through the metabolism of anaerobic bacteria and archaea is well understood for easily degradable biomolecules such as carbohydrates, proteins and lipids. However, little is known about the organic compounds that lead to methane in old anoxic sediments where easily degradable biomolecules are no longer available. One class of naturally formed long-lived compounds in such sediments is the saturated hydrocarbons (alkanes). Alkanes are usually considered to be inert in the absence of oxygen, nitrate or sulphate, and the analysis of alkane patterns is often used for biogeochemical characterization of sediments. However, alkanes might be consumed in anoxic sediments below the zone of sulphate reduction, but the underlying process has not been elucidated. Here we used enrichment cultures to show that the biological conversion of long-chain alkanes to the simplest hydrocarbon, methane, is possible under strictly anoxic conditions.

Formation of bound residues during microbial degradation of [14C]anthracene in soil.

Carbon partitioning and residue formation during microbial degradation of polycyclic aromatic hydrocarbons (PAH) in soil and soil-compost mixtures were examined by using [14C]anthracenes labeled at different positions. In native soil 43.8% of [9-14C]anthracene was mineralized by the autochthonous microflora and 45.4% was transformed into bound residues within 176 days. Addition of compost increased the metabolism (67.2% of the anthracene was mineralized) and decreased the residue formation (20. 7% of the anthracene was transformed). Thus, the higher organic carbon content after compost was added did not increase the level of residue formation. [14C]anthracene labeled at position 1,2,3,4,4a,5a was metabolized more rapidly and resulted in formation of higher levels of residues (28.5%) by the soil-compost mixture than [14C]anthracene radiolabeled at position C-9 (20.7%). Two phases of residue formation were observed in the experiments. In the first phase the original compound was sequestered in the soil, as indicated by its limited extractability. In the second phase metabolites were incorporated into humic substances after microbial degradation of the PAH (biogenic residue formation). PAH metabolites undergo oxidative coupling to phenolic compounds to form nonhydrolyzable humic substance-like macromolecules. We found indications that monomeric educts are coupled by C-C- or either bonds. Hydrolyzable ester bonds or sorption of the parent compounds plays a minor role in residue formation. Moreover, experiments performed with 14CO2 revealed that residues may arise from CO2 in the soil in amounts typical for anthracene biodegradation. The extent of residue formation depends on the metabolic capacity of the soil microflora and the characteristics of the soil. The position of the 14C label is another important factor which controls mineralization and residue formation from metabolized compounds.