Hans H. Richnow

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.

Monitoring and assessing processes of organic chemicals removal in constructed wetlands

Physical, chemical and biological processes interact and work in concert during attenuation of organic chemicals in wetland systems. This review summarizes the recent progress made towards understanding how the various mechanisms attributed to organic chemicals removal interact to form a functioning wetland. We also discuss the main degradation pathways for different groups of contaminants and examine some of the key characteristics of constructed wetlands that control the removal of organic chemicals. Furthermore, we address possible comprehensive approaches and recent techniques to follow up in situ processes within the system, especially those involved in the biodegradation processes.

Molecular signals for anaerobic methane oxidation in Black Sea seep carbonates and a microbial mat

Linked to gas seeps on the Ukrainian shelf (northwestern Black Sea), massive authigenic carbonates form as a result of anaerobic methane oxidation. Lipid distributions in these ‘cold seep’ carbonates and an associated microbial mat were investigated for process markers reflecting the presence and metabolic activity of distinctive methane-related biota. The samples contain free, irregular isoprenoid hydrocarbons, namely the tail-to-tail linked acyclic C20-isoprenoid 2,6,11,15-tetramethylhexadecane (crocetane), its C25-homologue 2,6,10,15,19-pentamethylicosane (PMI), and several unsaturated derivatives thereof. Furthermore, specific acyclic and cyclic C40-isoprenoids were released upon ether cleavage of the polar fraction from the carbonate. The abundance of these compounds indicates a pronounced role of particular Archaea in the biogeochemical cycling of carbon at methane seeps. Stable carbon isotopic analyses of these lipids reveal extraordinary depletions in 13C corresponding to δ-values in the range of −100±30‰ PDB, whereas other compounds show isotopic compositions normally observed for marine lipids (around −30‰ PDB). The isotope data imply that the biosynthesis of the archaeal isoprenoids occurred in situ and involved the utilization of isotopically depleted, i.e. methane-derived, carbon. Apart from archaeal markers, the carbonate and the mat contain authigenic, framboidal pyrite and isotopically depleted fatty acids, namely iso-, and anteiso-branched compounds most likely derived from sulphate-reducing bacteria (SRB). The indications for a tight association of these normally competitive organisms support a model invoking a syntrophic relationship of SRB with Archaea responsible for the anaerobic oxidation of methane. The biomarker patterns obtained from the Black Sea samples were further compared to those from a Oligocene seep carbonate (Lincoln Creek Formation, WA, USA) in order to evaluate their biomarker potential for ancient settings. The prominent occurrence of isotopically light crocetane (−112‰) and PMI (−120‰) meets the findings for the contemporary materials. Thus, isotopically depleted isoprenoids provide diagenetically stable fingerprints for the reconstruction of carbon cycling in both, modern and ancient methane seep systems.

Anaerobic Naphthalene Degradation by a Sulfate-Reducing Enrichment Culture

ABSTRACT 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 [1-13C]naphthalene or deuterated D8-naphthalene, all metabolites mentioned derived from the introduced labeled naphthalene. When a [13C]bicarbonate-buffered growth medium was used in conjunction with unlabeled naphthalene, 13C 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

ABSTRACT The thermophilic aerobic bacterium Bacillus thermoleovorans Hamburg 2 grows at 60°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 CO2, was determined by the application of [1-13C]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 thermophilicB. thermoleovorans differs from the known pathways found for mesophilic bacteria.

Stable Hydrogen and Carbon Isotope Fractionation during Microbial Toluene Degradation: Mechanistic and Environmental Aspects

ABSTRACT 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-d8 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-d8 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 putidamt-2 (b = −2.667). The D/H isotope fractionation was 3 orders of magnitude greater than the13C/12C 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-d8 and nonlabeled toluene for sulfate-reducing strain TRM1 (b = −0.728) andD. 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°C but varied slightly during degradation by P. putida mt-2, which showed maximum hydrogen isotope fractionation at 20°C (b = −4.086) and minimum fractionation at 35°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-d5 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

ABSTRACT 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 μM 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.

Carbon and Hydrogen Stable Isotope Fractionation during Aerobic Bacterial Degradation of Aromatic Hydrocarbons

ABSTRACT 13C/12C and D/H stable isotope fractionation during aerobic degradation was determined for Pseudomonas putida strain mt-2, Pseudomonas putida strain F1, Ralstonia pickettii strain PKO1, and Pseudomonas putida strain NCIB 9816 grown with toluene, xylenes, and naphthalene. Different types of initial reactions used by the respective bacterial strains could be linked with certain extents of stable isotope fractionation during substrate degradation.

Current approaches for the assessment of in situ biodegradation

Considering the high costs and technical difficulties associated with conventional remediation strategies, in situ biodegradation has become a promising approach for cleaning up contaminated aquifers. To verify if in situ biodegradation of organic contaminants is taking place at a contaminated site and to determine if these processes are efficient enough to replace conventional cleanup technologies, a comprehensive characterization of site-specific biodegradation processes is essential. In recent years, several strategies including geochemical analyses, microbial and molecular methods, tracer tests, metabolite analysis, compound-specific isotope analysis, and in situ microcosms have been developed to investigate the relevance of biodegradation processes for cleaning up contaminated aquifers. In this review, we outline current approaches for the assessment of in situ biodegradation and discuss their potential and limitations. We also discuss the benefits of research strategies combining complementary methods to gain a more comprehensive understanding of the complex hydrogeological and microbial interactions governing contaminant biodegradation in the field.

Molecular characterization of bacterial communities mineralizing benzene under sulfate‐reducing conditions

The microbial communities of in situ reactor columns degrading benzene with sulfate as an electron acceptor were analyzed based on clone libraries and terminal restriction fragment length polymorphism fingerprinting of PCR-amplified 16S rRNA genes. The columns were filled with either lava granules or sand particles and percolated with groundwater from a benzene-contaminated aquifer. The predominant organisms colonizing the lava granules were related to Magnetobacterium sp., followed by a phylotype affiliated to the genera Cryptanaerobacter/Pelotomaculum and several Deltaproteobacteria. From the sand-filled columns, a stable benzene-degrading consortium was established in sand-filled laboratory microcosms under sulfate-reducing conditions. It was composed of Delta- and Epsilonproteobacteria, Clostridia, Chloroflexi, Actinobacteria and Bacteroidetes. The most prominent phylotype of the consortium was related to the genus Sulfurovum, followed by Desulfovibrio sp. and the Cryptanaerobacter/Pelotomaculum phylotype. The proportion of the latter was similar in both communities and significantly increased after repeated benzene-spiking. During cultivation on aromatic substrates other than benzene, the Cryptanaerobacter/Pelotomaculum phylotype was outcompeted by other community members. Hence, this organism appears to be specific for benzene as a growth substrate and might play a key role in benzene degradation in both communities. Based on the possible functions of the community members and thermodynamic calculations, a functional model for syntrophic benzene degradation under sulfate-reducing conditions is proposed.