Imprints of ecological processes in the taxonomic core community: an analysis of naturally replicated microbial communities enclosed in oil.

It is widely assumed that a taxonomic core community emerges among microbial communities from similar habitats because similar environments select for the same taxa bearing the same traits. Yet, a core community itself is no indicator of selection because it may also arise from dispersal and neutral drift, i.e. by chance. Here, we hypothesize that a core community produced by either selection or chance processes should be distinguishable. While dispersal and drift should produce core communities with similar relative taxon abundances, especially when the proportional core community, i.e. the sum of the relative abundances of the core taxa, is large, selection may produce variable relative abundances. We analyzed the core community of 16S rRNA gene sequences of 193 microbial communities occurring in tiny water droplets enclosed in heavy oil from the Pitch Lake, Trinidad and Tobago. These communities revealed highly variable relative abundances along with a large proportional core community (68.0 ± 19.9%). A dispersal-drift null model predicted a negative relationship of proportional core community and compositional variability along a range of dispersal probabilities and was largely inconsistent with the observed data, suggesting a major role of selection for shaping the water droplet communities in the Pitch Lake.

Insights into the effects of anthropogenic activities on oil reservoir microbiome and metabolic potential.

Microbial communities have long been observed in oil reservoirs, where the subsurface conditions are major drivers shaping their structure and functions. Furthermore, anthropogenic activities such as water flooding during oil production can affect microbial activities and community compositions in oil reservoirs through the injection of recycled produced water, often associated with biocides. However, it is still unclear to what extent the introduced chemicals and microbes influence the metabolic potential of the subsurface microbiome. Here we investigated an onshore oilfield in Germany (Field A) that undergoes secondary oil production along with biocide treatment to prevent souring and microbially induced corrosion (MIC). With the integrated approach of 16 S rRNA gene amplicon and shotgun metagenomic sequencing of water-oil samples from 4 production wells and 1 injection well, we found differences in microbial community structure and metabolic functions. In the injection water samples, amplicon sequence variants (ASVs) belonging to families such as Halanaerobiaceae, Ectothiorhodospiraceae, Hydrogenophilaceae, Halobacteroidaceae, Desulfohalobiaceae, and Methanosarcinaceae were dominant, while in the production water samples, ASVs of families such as Thermotogaceae, Nitrospiraceae, Petrotogaceae, Syntrophaceae, Methanobacteriaceae, and Thermoprotei were also dominant. The metagenomic analysis of the injection water sample revealed the presence of C1-metabolism, namely, genes involved in formaldehyde oxidation. Our analysis revealed that the microbial community structure of the production water samples diverged slightly from that of injection water samples. Additionally, a metabolic potential for oxidizing the applied biocide clearly occurred in the injection water samples indicating an adaptation and buildup of degradation capacity or resistance against the added biocide.

Naphthalene Carboxylation in the Sulfate-Reducing Enrichment Culture N47 Is Proposed to Proceed via 1,3-Dipolar Cycloaddition to the Cofactor Prenylated Flavin Mononucleotide.

Polycyclic aromatic hydrocarbons are persistent pollutants of anthropogenic or natural origin in the environment and accumulate in anoxic habitats. In this study, we investigated the mechanism of the enzyme naphthalene carboxylase as a model reaction for polycyclic aromatic hydrocarbon activation by carboxylation. An enzyme assay was established with cell extracts of the highly enriched culture N47. In assays without addition of ATP, naphthalene carboxylase catalyzed a stable isotope exchange of the carboxyl group of naphthoate with 13C-labeled bicarbonate buffer, which can only occur via a partial backwards reaction of the naphthalene carboxylase reaction to an intermediate that does not include the carboxyl group. Hence, a new carboxyl group from the labeled bicarbonate is added upon forward reaction to the naphthoate. This indicates that the reaction mechanism consists of two or more steps and that at least the latter steps are reversible and ATP independent. Naphthalene carboxylation assays were carried out in deuterated buffer and revealed the incorporation of 0, 1, 2, or 3 deuterium atoms in the final product naphthoyl-coenzyme A, indicating that the reaction is fully reversible. Putative reaction mechanisms were tested by quantum mechanical calculations. The proposed mechanism of the reaction consists of three steps: the activation of the naphthalene by 1,3-dipolar cycloaddition of the cofactor prFMN to naphthalene, release of a proton and rearomatization producing a stable intermediate, and a carboxylation with a reverse 1,3-dipolar cycloaddition and cleavage of the bond to the cofactor producing 2-naphthoate. IMPORTANCE Pollution with polycyclic aromatic hydrocarbons poses a great hazard to humans and animals, with considerable long-term effects. The anaerobic degradation of polycyclic aromatic hydrocarbons in anoxic zones and anaerobic growth of such organisms is very slow, leading to only poor investigation of the degradation pathways, so far. In this work, we elucidated the mechanism of naphthalene carboxylase, a key enzyme in anaerobic naphthalene degradation. This is the first mechanism proposed for a carboxylase targeting nonsubstituted (polycyclic) aromatic compounds and can serve as a model for the initial activation reaction in the anaerobic degradation of benzene or nonsubstituted polycyclic aromatic hydrocarbons, as well as similar enzymatic reactions from the expanding class of UbiD-like (de)carboxylases.

Remediation of zinc-contaminated groundwater by iron oxide in situ adsorption barriers - From lab to the field.

Heavy metals such as zinc cannot be degraded by microorganisms and form long contaminant plumes in groundwater. Conventional methods for remediating heavy metal-contaminated sites are for example excavation and pump-and-treat, which is expensive and requires very long operation times. This induced interest in new technologies such as in situ adsorption barriers for immobilization of heavy metal contamination. In this study, we present steps and criteria from laboratory tests to field studies, which are necessary for a successful implementation of an in situ adsorption barrier for immobilizing zinc. Groundwater and sediment samples from a contaminated site were brought to the lab, where the adsorption of zinc to Goethite nanoparticles was studied in batch and in flow-through systems mimicking field conditions. The Goethite nanoparticles revealed an in situ adsorption capacity of approximately 23 mg Zn per g Goethite. Transport experiments in sediment columns indicated an expected radius of influence of at least 2.8 m for the injection of Goethite nanoparticles. These findings were validated in a pilot-scale field study, where an in situ adsorption barrier of ca. 11 m × 6 m × 4 m was implemented in a zinc-contaminated aquifer. The injected nanoparticles were irreversibly deposited at the desired location within <24 h, and were not dislocated with the groundwater flow. Despite a constantly increasing inflow of zinc to the barrier and the short contact time between Goethite and zinc in the barrier, the dissolved zinc was effectively immobilized for ca. 90 days. Then, the zinc concentrations increased slowly downstream of the barrier, but the barrier still retained most of the zinc from the inflowing groundwater. The study demonstrated the applicability of Goethite nanoparticles to immobilize heavy metals in situ and highlights the criteria for upscaling laboratory-based determinants to field-scale.

Indigenous microbial communities in heavy oil show a threshold response to salinity.

Microbial degradation influences the quality of oil resources. The environmental factors that shape the composition of oil microbial communities are largely unknown because most samples from oil fields are impacted by anthropogenic oil production, perturbing the native ecosystem with exogenous fluids and microorganisms. We investigated the relationship between formation water geochemistry and microbial community composition in undisturbed oil samples. We isolated 43 microliter-sized water droplets naturally enclosed in the heavy oil of the Pitch Lake, Trinidad and Tobago. The water chemistry and microbial community composition within the same water droplet were determined by ion chromatography and 16S rRNA gene amplicon sequencing, respectively. The results revealed a high variability in ion concentrations and community composition between water droplets. Microbial community composition was mostly affected by the chloride concentration, which ranged from freshwater to brackish-sea water. Remarkably, microbial communities did not respond gradually to increasing chloride concentration but showed a sudden change to less diverse and uneven communities when exceeding a chloride concentration of 57.3 mM. The results reveal a threshold-regulated response of microbial communities to salinity, offering new insights into the microbial ecology of oil reservoirs.

Field-scale demonstration of in situ immobilization of heavy metals by injecting iron oxide nanoparticle adsorption barriers in groundwater.

Remediation of heavy metal-contaminated aquifers is a challenging process because they cannot be degraded by microorganisms. Together with the usually limited effectiveness of technologies applied today for treatment of heavy metal contaminated groundwater, this creates a need for new remediation technologies. We therefore developed a new treatment, in which permeable adsorption barriers are established in situ in aquifers by the injection of colloidal iron oxides. These adsorption barriers aim at the immobilization of heavy metals in aquifers groundwater, which was assessed in a large-scale field study in a brownfield site. Colloidal iron oxide (goethite) nanoparticles were used to install an in situ adsorption barrier in a very heterogeneous, contaminated aquifer of a brownfield in Asturias, Spain. The groundwater contained high concentrations of heavy metals with up to 25 mg/L zinc, 1.3 mg/L lead, 40 mg/L copper, 0.1 mg/L nickel and other minor heavy metal pollutants below 1 mg/L. High amounts of zinc (>900 mg/kg), lead (>2000 mg/kg), nickel (>190 mg/kg) were also present in the sediment. Ca. 1500 kg of goethite nanoparticles of 461 ± 266 nm diameter were injected at low pressure (< 0.6 bar) into the aquifer through nine screened injection wells. For each injection well, a radius of influence of at least 2.5 m was achieved within 8 h, creating an in situ barrier of 22 × 3 × 9 m. Despite the extremely high heavy metal contamination and the strong heterogeneity of the aquifer, successful immobilization of contaminants was observed in the tested area. The contaminant concentrations were strongly reduced immediately after the injection and the abatement of the heavy metals continued for a total post-injection monitoring period of 189 days. The iron oxide particles were found to adsorb heavy metals even at pH-values between 4 and 6, where low adsorption would have been expected. The study demonstrated the applicability of iron oxide nanoparticles for installing adsorption barriers for containment of heavy metals in contaminated groundwater under real conditions.

Oil reservoirs, an exceptional habitat for microorganisms.

Microorganisms are present in oil reservoirs around the world where they degrade oil and lead to changes in oil quality. Unfortunately, our knowledge about processes in deep oil reservoirs is limited due to the lack of undisturbed samples. In this review, we discuss the distribution of microorganisms at the oil-water transition zone as well as in water saturated parts of the oil leg and their possible physiological adaptations to abiotic and biotic ecological factors such as temperature, salinity and viruses. We show the importance of studying the water phase within the oil, because small water inclusions and pockets within the oil leg provide an exceptional habitat for microorganisms within a natural oil reservoir and concurrently enlarge the zone of oil biodegradation. Environmental factors such as temperature and salinity control oil biodegradation. Temperature determines the type of microorganisms which are able to inhabit the reservoir. Proteobacteria and Euryarchaeota, are ubiquitous in oil reservoirs over all temperature ranges, whereas some others are tied to specific temperatures. It is proposed that biofilm formation is the dominant way of life within oil reservoirs, enhancing nutrient uptake, syntrophic interactions and protection against environmental stress. Literature shows that viruses are abundant in oil reservoirs and the possible impact on microbial community composition due to control of microbial activity and function is discussed.

Biological effects of four iron-containing nanoremediation materials on the green alga Chlamydomonas sp.

As nanoremediation strategies for in-situ groundwater treatment extend beyond nanoiron-based applications to adsorption and oxidation, ecotoxicological evaluations of newly developed materials are required. The biological effects of four new materials with different iron (Fe) speciations ([i] FerMEG12 - pristine flake-like milled Fe(0) nanoparticles (nZVI), [ii] Carbo-Iron® - Fe(0)-nanoclusters containing activated carbon (AC) composite, [iii] Trap-Ox® Fe-BEA35 (Fe-zeolite) - Fe-doped zeolite, and [iv] Nano-Goethite - 'pure' FeOOH) were studied using the unicellular green alga Chlamydomonas sp. as a model test system. Algal growth rate, chlorophyll fluorescence, efficiency of photosystem II, membrane integrity and reactive oxygen species (ROS) generation were assessed following exposure to 10, 50 and 500 mg L-1 of the particles for 2 h and 24 h. The particles had a concentration-, material- and time-dependent effect on Chlamydomonas sp., with increased algal growth rate after 24 h. Conversely, significant intracellular ROS levels were detected after 2 h, with much lower levels after 24 h. All Fe-nanomaterials displayed similar Z-average sizes and zeta-potentials at 2 h and 24 h. Effects on Chlamydomonas sp. decreased in the order FerMEG12 > Carbo-Iron® > Fe-zeolite > Nano-Goethite. Ecotoxicological studies were challenged due to some particle properties, i.e. dark colour, effect of constituents and a tendency to agglomerate, especially at high concentrations. All particles exhibited potential to induce significant toxicity at high concentrations (500 mg L-1), though such concentrations would rapidly decrease to mg or µg L-1 in aquatic environments, levels harmless to Chlamydomonas sp. The presented findings contribute to the practical usage of particle-based nanoremediation in environmental restoration.

Anaerobic degradation of 1-methylnaphthalene by a member of the Thermoanaerobacteraceae contained in an iron-reducing enrichment culture.

An anaerobic culture (1MN) was enriched with 1-methylnaphthalene as sole source of carbon and electrons and Fe(OH)3 as electron acceptor. 1-Naphthoic acid was produced as a metabolite during growth with 1-methylnaphthalene while 2-naphthoic acid was detected with naphthalene and 2-methylnaphthalene. This indicates that the degradation pathway of 1-methylnaphthalene might differ from naphthalene and 2-methylnaphthalene degradation in sulfate reducers. Terminal restriction fragment length polymorphism and pyrosequencing revealed that the culture is mainly composed of two bacteria related to uncultured Gram-positive Thermoanaerobacteraceae and uncultured gram-negative Desulfobulbaceae. Stable isotope probing showed that a 13C-carbon label from 13C10-naphthalene as growth substrate was mostly incorporated by the Thermoanaerobacteraceae. The presence of putative genes involved in naphthalene degradation in the genome of this organism was confirmed via assembly-based metagenomics and supports that it is the naphthalene-degrading bacterium in the culture. Thermoanaerobacteraceae have previously been detected in oil sludge under thermophilic conditions, but have not been shown to degrade hydrocarbons so far. The second member of the community belongs to the Desulfobulbaceae and has high sequence similarity to uncultured bacteria from contaminated sites including recently proposed groundwater cable bacteria. We suggest that the gram-positive Thermoanaerobacteraceae degrade polycyclic aromatic hydrocarbons while the Desulfobacterales are mainly responsible for Fe(III) reduction.

Reconstructing metabolic pathways of a member of the genus Pelotomaculum suggesting its potential to oxidize benzene to carbon dioxide with direct reduction of sulfate.

The enrichment culture BPL is able to degrade benzene with sulfate as electron acceptor and is dominated by an organism of the genus Pelotomaculum. Members of Pelotomaculum are usually known to be fermenters, undergoing syntrophy with anaerobic respiring microorganisms or methanogens. By using a metagenomic approach, we reconstructed a high-quality genome (∼2.97 Mbp, 99% completeness) for Pelotomaculum candidate BPL. The proteogenomic data suggested that (1) anaerobic benzene degradation was activated by a yet unknown mechanism for conversion of benzene to benzoyl-CoA; (2) the central benzoyl-CoA degradation pathway involved reductive dearomatization by a class II benzoyl-CoA reductase followed by hydrolytic ring cleavage and modified ß-oxidation; (3) the oxidative acetyl-CoA pathway was utilized for complete oxidation to CO2. Interestingly, the genome of Pelotomaculum candidate BPL has all the genes for a complete sulfate reduction pathway including a similar electron transfer mechanism for dissimilatory sulfate reduction as in other Gram-positive sulfate-reducing bacteria. The proteome analysis revealed that the essential enzymes for sulfate reduction were all formed during growth with benzene. Thus, our data indicated that, besides its potential to anaerobically degrade benzene, Pelotomaculum candidate BPL is the first member of the genus that can perform sulfate reduction.

Anaerobic Degradation of Benzene and Polycyclic Aromatic Hydrocarbons.

Aromatic hydrocarbons such as benzene and polycyclic aromatic hydrocarbons (PAHs) are very slowly degraded without molecular oxygen. Here, we review the recent advances in the elucidation of the first known degradation pathways of these environmental hazards. Anaerobic degradation of benzene and PAHs has been successfully documented in the environment by metabolite analysis, compound-specific isotope analysis and microcosm studies. Subsequently, also enrichments and pure cultures were obtained that anaerobically degrade benzene, naphthalene or methylnaphthalene, and even phenanthrene, the largest PAH currently known to be degradable under anoxic conditions. Although such cultures grow very slowly, with doubling times of around 2 weeks, and produce only very little biomass in batch cultures, successful proteogenomic, transcriptomic and biochemical studies revealed novel degradation pathways with exciting biochemical reactions such as for example the carboxylation of naphthalene or the ATP-independent reduction of naphthoyl-coenzyme A. The elucidation of the first anaerobic degradation pathways of naphthalene and methylnaphthalene at the genetic and biochemical level now opens the door to studying the anaerobic metabolism and ecology of anaerobic PAH degraders. This will contribute to assessing the fate of one of the most important contaminant classes in anoxic sediments and aquifers.

Selective elimination of bacterial faecal indicators in the Schmutzdecke of slow sand filtration columns.

Slow sand filtration (SSF) is an effective low-tech water treatment method for pathogen and particle removal. Yet despite its application for centuries, it has been uncertain to which extent pathogenic microbes are removed by mechanical filtration or due to ecological interactions such as grazing and competition for nutrients. In this study, we quantified the removal of bacterial faecal indicators, Escherichia coli and Enterococcus faecalis, from secondary effluent of a wastewater treatment plant and analysed the microbial community composition in compartments of laboratory model SSF columns. The columns were packed with different sand grain sizes and eliminated 1.6-2.3 log units of faecal indicators, which translated into effluents of bathing water quality according to the EU directive (<500 colony forming units of E. coli per 100 ml) for columns with small grain size. Most of that removal occurred in the upper filter area, the Schmutzdecke. Within that same zone, total bacterial numbers increased however, thus suggesting a specific elimination of the faecal indicators. The analysis of the microbial communities also revealed that some taxa were removed more from the wastewater than others. These results accentuate the contribution of biological mechanisms to water purification in SSF.

Oil biodegradation. Water droplets in oil are microhabitats for microbial life.

Anaerobic microbial degradation of hydrocarbons, typically occurring at the oil-water transition zone, influences the quality of oil reservoirs. In Pitch Lake, Trinidad and Tobago--the world's largest asphalt lake--we found that microorganisms are metabolically active in minuscule water droplets (1 to 3 microliters) entrapped in oil. Pyrotag sequencing of individual droplet microbiomes revealed complex methanogenic microbial communities actively degrading the oil into a diverse range of metabolites, as shown by nuclear magnetic resonance and Fourier transform ion cyclotron resonance mass spectrometry. High salinity and water-stable isotopes of the droplets indicate a deep subsurface origin. The 13.5% water content and the large surface area of the droplets represent an underestimated potential for biodegradation of oil away from the oil-water transition zone.

Iron oxide nanoparticles in geomicrobiology: from biogeochemistry to bioremediation.

Iron oxides are important constituents of soils and sediments and microbial iron reduction is considered to be a significant anaerobic respiration process in the subsurface, however low microbial reduction rates of macroparticulate Fe oxides in laboratory studies led to an underestimation of the role of Fe oxides in the global Fe redox cycle. Recent studies show the high potential of nano-sized Fe oxides in the environment as, for example, electron acceptor for microbial respiration, electron shuttle between different microorganisms, and scavenger for heavy metals. Biotic and abiotic reactivity of iron macroparticles differ significantly from nano-sized Fe oxides, which are usually much more reactive. Factors such as particle size, solubility, ferrous iron, crystal structure, and organic molecules were identified to influence the reactivity. This review discusses factors influencing the microbial reactivity of Fe oxides. It highlights the differences between natural and synthetic Fe oxides especially regarding the presence of organic molecules such as humic acids and natural organic matter. Attention is given to the transport behavior of Fe oxides in laboratory systems and in the environment, because of the high affinity of different contaminants to Fe oxide surfaces and associated co-transport of pollutants. The high reactivity of Fe oxides and their potential as adsorbents for different pollutants are discussed with respect to application and development of remediation technologies.

ATP-dependent/-independent enzymatic ring reductions involved in the anaerobic catabolism of naphthalene.

Polycyclic aromatic hydrocarbons are among the most hazardous environmental pollutants. However, in contrast to aerobic degradation, the respective degradation pathways in anaerobes are greatly unknown which has so far prohibited many environmental investigations. In this work, we studied the enzymatic dearomatization reactions involved in the degradation of the PAH model compounds naphthalene and 2-methylnaphthalene in the sulfate-reducing enrichment culture N47. Cell extracts of N47 grown on naphthalene catalysed the sodium dithionite-dependent four-electron reduction of the key intermediate 2-naphthoyl-coenzyme A (NCoA) to 5,6,7,8-tetrahydro-2-naphthoyl-CoA (THNCoA). The NCoA reductase activity was independent of ATP and was, surprisingly, not sensitive to oxygen. In cell extracts in the presence of various electron donors the product THNCoA was further reduced by a two-electron reaction to most likely a conjugated hexahydro-2-naphthoyl-CoA isomer (HHNCoA). The reaction assigned to THNCoA reductase strictly depended on ATP and was oxygen-sensitive with a half-life time between 30 s and 1 min when exposed to air. The rate was highest with NADH as electron donor. The results indicate that two novel and completely different dearomatizing ring reductases are involved in anaerobic naphthalene degradation. While the THNCoA reducing activity shows some properties of ATP-dependent class I benzoyl-CoA reductases, NCoA reduction appears to be catalysed by a previously unknown class of dearomatizing aryl-carboxyl-CoA reductases.

Reevaluation of colorimetric iron determination methods commonly used in geomicrobiology.

The ferrozine and phenanthroline colorimetric assays are commonly applied for the determination of ferrous and total iron concentrations in geomicrobiological studies. However, accuracy of both methods depends on slight changes in their protocols, on the investigated iron species, and on geochemical variations in sample conditions. Therefore, we tested the performance of both methods using Fe(II)((aq)), Fe(III)((aq)), mixed valence solutions, synthetic goethite, ferrihydrite, and pyrite, as well as microbially-formed magnetite and a mixture of goethite and magnetite. The results were compared to concentrations determined with aqua regia dissolution and inductively coupled plasma atomic emission spectroscopy (ICP-AES). Iron dissolution prior to the photometric assays included dissolution in 1M or 6M HCl, at 21 or 60°C, and oxic or anoxic conditions. Results indicated a good reproducibility of quantitative total iron determinations by the ferrozine and phenanthroline assays for easily soluble iron forms such as Fe(II)((aq)), Fe(III)((aq)), mixed valence solutions, and ferrihydrite. The ferrozine test underestimated total iron contents of some of these samples after dissolution in 1M HCl by 10 to 13%, whereas phenanthroline matched the results determined by ICP-AES with a deviation of 5%. Total iron concentrations after dissolution in 1M HCl of highly crystalline oxides such as magnetite, a mixture of goethite and magnetite, and goethite were underestimated by up to 95% with both methods. When dissolving these minerals in 6M HCl at 60°C, the ferrozine method was more reliable for total iron content with an accuracy of ±5%, related to values determined with ICP-AES. Phenanthroline was more reliable for the determination of total pyritic iron as well as ferrous iron after incubation in 1M HCl at 21°C in the Fe(II)((aq)) sample with a recovery of 98%. Low ferrous iron concentrations of less than 0.5mM were overestimated in a Fe(III) background by up to 150% by both methods. Heating of mineral samples in 6M HCl increased their solubility and susceptibility for both photometric assays which is a need for total iron determination of highly crystalline minerals. However, heating also rendered a subsequent reliable determination of ferrous iron impossible due to fast abiotic oxidation. Due to the low solubility of highly crystalline samples, the determination of total iron is solely possible after dissolution in 6M HCl at 60°C which on the other hand makes determination of ferrous iron impossible. The recommended procedure for ferrous iron determination is therefore incubation at 21°C in 6M HCl, centrifugation, and subsequent measurement of ferrous iron in the supernatant. The different procedures were tested during growth of G. sulfurreducens on synthetic ferrihydrite. Here, the phenanthroline test was more accurate compared to the ferrozine test. However, the latter provided easy handling and seemed preferable for larger amounts of samples.

Anaerobic naphthalene degradation by Gram-positive, iron-reducing bacteria.

An anaerobic naphthalene-degrading culture (N49) was enriched with ferric iron as electron acceptor. A closed electron balance indicated the total oxidation of naphthalene to CO(2). In all growing cultures, the concentration of the presumed central metabolite of naphthalene degradation, 2-naphthoic acid, increased concomitantly with growth. The first metabolite of anaerobic methylnaphthalene degradation, naphthyl-2-methyl-succinic acid, was not identified in culture supernatants, which does not support a methylation to methylnaphthalene as the initial activation reaction of naphthalene, but rather a carboxylation, as proposed for other naphthalene-degrading cultures. Substrate utilization tests revealed that the culture was able to grow on 1-methyl-naphthalene, 2-methyl-naphthalene, 1-naphthoic acid or 2-naphthoic acid, whereas it did not grow on 1-naphthol, 2-naphthol, anthracene, phenanthrene, indane and indene. Terminal restriction fragment length polymorphism and 16S rRNA gene sequence analyses revealed that the microbial community of the culture was dominated by one bacterial microorganism, which was closely related (99% 16S sequence similarity) to the major organism in the iron-reducing, benzene-degrading enrichment culture BF [ISME J (2007) 1: 643; Int J Syst Evol Microbiol (2010) 60: 686]. The phylogenetic classification supports a new candidate species and genus of Gram-positive spore-forming iron-reducers that can degrade non-substituted aromatic hydrocarbons. It furthermore indicates that Gram-positive microorganisms might also play an important role in anaerobic polycyclic aromatic hydrocarbon-degradation.

Identification of enzymes involved in anaerobic benzene degradation by a strictly anaerobic iron-reducing enrichment culture.

Anaerobic benzene degradation was studied with a highly enriched iron-reducing culture (BF) composed of mainly Peptococcaceae-related Gram-positive microorganisms. The proteomes of benzene-, phenol- and benzoate-grown cells of culture BF were compared by SDS-PAGE. A specific benzene-expressed protein band of 60 kDa, which could not be observed during growth on phenol or benzoate, was subjected to N-terminal sequence analysis. The first 31 amino acids revealed that the protein was encoded by ORF 138 in the shotgun sequenced metagenome of culture BF. ORF 138 showed 43% sequence identity to phenylphosphate carboxylase subunit PpcA of Aromatoleum aromaticum strain EbN1. A LC/ESI-MS/MS-based shotgun proteomic analysis revealed other specifically benzene-expressed proteins with encoding genes located adjacent to ORF 138 on the metagenome. The protein products of ORF 137, ORF 139 and ORF 140 showed sequence identities of 37% to phenylphosphate carboxylase PpcD of A. aromaticum strain EbN1, 56% to benzoate-CoA ligase (BamY) of Geobacter metallireducens and 67% to 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (UbiD/UbiX) of A. aromaticum strain EbN1 respectively. These genes are proposed as constituents of a putative benzene degradation gene cluster (∼ 17 kb) composed of carboxylase-related genes. The identified gene sequences suggest that the initial activation reaction in anaerobic benzene degradation is probably a direct carboxylation of benzene to benzoate catalysed by putative anaerobic benzene carboxylase (Abc). The putative Abc probably consists of several subunits, two of which are encoded by ORFs 137 and 138, and belongs to a family of carboxylases including phenylphosphate carboxylase (Ppc) and 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (UbiD/UbiX).

DNA-SIP identifies sulfate-reducing Clostridia as important toluene degraders in tar-oil-contaminated aquifer sediment.

Global groundwater resources are constantly challenged by a multitude of contaminants such as aromatic hydrocarbons. Especially in anaerobic habitats, a large diversity of unrecognized microbial populations may be responsible for their degradation. Still, our present understanding of the respective microbiota and their ecophysiology is almost exclusively based on a small number of cultured organisms, mostly within the Proteobacteria. Here, by DNA-based stable isotope probing (SIP), we directly identified the most active sulfate-reducing toluene degraders in a diverse sedimentary microbial community originating from a tar-oil-contaminated aquifer at a former coal gasification plant. On incubation of fresh sediments with (13)C(7)-toluene, the production of both sulfide and (13)CO(2) was clearly coupled to the (13)C-labeling of DNA of microbes related to Desulfosporosinus spp. within the Peptococcaceae (Clostridia). The screening of labeled DNA fractions also suggested a novel benzylsuccinate synthase alpha-subunit (bssA) sequence type previously only detected in the environment to be tentatively affiliated with these degraders. However, carbon flow from the contaminant into degrader DNA was only ∼50%, pointing toward high ratios of heterotrophic CO(2)-fixation during assimilation of acetyl-CoA originating from the contaminant by these degraders. These findings demonstrate that the importance of non-proteobacterial populations in anaerobic aromatics degradation, as well as their specific ecophysiology in the subsurface may still be largely ungrasped.

Combined genomic and proteomic approaches identify gene clusters involved in anaerobic 2-methylnaphthalene degradation in the sulfate-reducing enrichment culture N47.

The highly enriched deltaproteobacterial culture N47 anaerobically oxidizes the polycyclic aromatic hydrocarbons naphthalene and 2-methylnaphthalene, with sulfate as the electron acceptor. Combined genome sequencing and liquid chromatography-tandem mass spectrometry-based shotgun proteome analyses were performed to identify genes and proteins involved in anaerobic aromatic catabolism. Proteome analysis of 2-methylnaphthalene-grown N47 cells resulted in the identification of putative enzymes catalyzing the anaerobic conversion of 2-methylnaphthalene to 2-naphthoyl coenzyme A (2-naphthoyl-CoA), as well as the reductive ring cleavage of 2-naphthoyl-CoA, leading to the formation of acetyl-CoA and CO(2). The glycyl radical-catalyzed fumarate addition to the methyl group of 2-methylnaphthalene is catalyzed by naphthyl-2-methyl-succinate synthase (Nms), composed of alpha-, beta-, and gamma-subunits that are encoded by the genes nmsABC. Located upstream of nmsABC is nmsD, encoding the Nms-activating enzyme, which harbors the characteristic [Fe(4)S(4)] cluster sequence motifs of S-adenosylmethionine radical enzymes. The bns gene cluster, coding for enzymes involved in beta-oxidation reactions converting naphthyl-2-methyl-succinate to 2-naphthoyl-CoA, was found four intervening open reading frames further downstream. This cluster consists of eight genes (bnsABCDEFGH) corresponding to 8.1 kb, which are closely related to genes for enzymes involved in anaerobic toluene degradation within the denitrifiers "Aromatoleum aromaticum" EbN1, Azoarcus sp. strain T, and Thauera aromatica. Another contiguous DNA sequence harbors the gene for 2-naphthoyl-CoA reductase (ncr) and 16 additional genes that were found to be expressed in 2-methylnaphthalene-grown cells. These genes code for enzymes that were supposed to catalyze the dearomatization and ring cleavage reactions converting 2-naphthoyl-CoA to acetyl-CoA and CO(2). Comparative sequence analysis of the four encoding subunits (ncrABCD) showed the gene product to have the closest similarity to the Azoarcus type of benzoyl-CoA reductase. The present work provides the first insight into the genetic basis of anaerobic 2-methylnaphthalene metabolism and delivers implications for understanding contaminant degradation.