Archaea catalyze iron-dependent anaerobic oxidation of methane

Anaerobic oxidation of methane (AOM) is crucial for controlling the emission of this potent greenhouse gas to the atmosphere. Nitrite-, nitrate-, and sulfate-dependent methane oxidation is well-documented, but AOM coupled to the reduction of oxidized metals has so far been demonstrated only in environmental samples. Here, using a freshwater enrichment culture, we show that archaea of the order Methanosarcinales, related to “Candidatus Methanoperedens nitroreducens,” couple the reduction of environmentally relevant forms of Fe^(3+) and Mn^(4+) to the oxidation of methane. We obtained an enrichment culture of these archaea under anaerobic, nitrate-reducing conditions with a continuous supply of methane. Via batch incubations using [^(13)C]methane, we demonstrated that soluble ferric iron (Fe^(3+), as Fe-citrate) and nanoparticulate forms of Fe^(3+) and Mn^(4+) supported methane-oxidizing activity. CO_2 and ferrous iron (Fe^(2+)) were produced in stoichiometric amounts. Our study connects the previous finding of iron-dependent AOM to microorganisms detected in numerous habitats worldwide. Consequently, it enables a better understanding of the interaction between the biogeochemical cycles of iron and methane

Effects of nitrogen fertilization on diazotrophic activity of microorganisms associated with Sphagnum magellanicum

In pristine ombrotrophic Sphagnum-dominated peatland ecosystems nitrogen (N) is often a limiting nutrient, which is replenished by biological N-2 fixation and atmospheric N deposition. It is, however, unclear which impact long-term N deposition has on microbial N-2 fixing activity and diazotrophic diversity, and whether phosphorus (P) modulates the response. Therefore, we studied the impact of increased N deposition and N depletion on microbial N-2 fixation and diazotrophic diversity associated with the peat moss Sphagnum magellanicum, and their interaction with P availability.Nitrogenase activities of S. magellanicum-associated microorganisms were determined by acetylene reduction assays (ARA) and N-15(2) tracer methods on mosses from two geographically distinct locations with different N deposition histories, high or low N deposition, and in samples depleted in N (grown 3 years in the greenhouse) versus recent field samples. The short-term response to increased N deposition was tested for mosses differing in N and P fertilization histories. In addition, diversity of diazotrophic microorganisms was assessed by nifH gene amplicon sequencing of N-depleted mosses.We showed distinct and persistent differences in diazotrophic communities and their activities associated with S. magellanicum from sites with high versus low N deposition. Initially, diazotrophic activity was six times higher for the low N site. During incubation and repeated ARA, however, this activity strongly decreased, while it remained stable for the high N site. Activity for the high N site could not be increased by long-term experimental N deprivation. Short-term, experimental N application had an inhibitory effect on N-2 fixation for both sites, which was not observed in mosses with high indirect P availability.We conclude that although N deposition negatively affects N-2 fixation as also shown in previous studies, long-term effects of N deprivation on the diazotrophic activity and community are more complex. Furthermore, our results indicated that P availability might be an important factor in modulating the response of Sphagnum-associated diazotrophs to N deposition.</p

Substratabhängige Aktivität und Diversität methanogener Archaeen in Sedimenten zweier Seen - ein jahreszeitlicher Vergleich

Ungewöhnlich negative 13C-Signaturen von Chironomidenlarven in Seesedimenten wiesen auf biogenes Methan als bedeutende Kohlenstoffquelle ihrer Nahrung hin. Um die Basis der vermuteten Nahrungskette von Methan produzierenden Archaea über methanoxidierende Bakterien genauer zu untersuchen, wurden zwei morphologisch unterschiedlichen Seen hinsichtlich der Methanproduktion, -verfügbarkeit und -isotopensignatur sowie Diversität der Methanogenen verglichen. Der Große Binnensee ist ein polytropher, ungeschichteter Flachsee mit geringfügig marinem Einfluss und aerober Sedimentoberfläche; der Holzsee ein kleiner, eutropher See mit stabiler Schichtung im Sommer und währenddessen anoxischem Hypolimnion. Ungestörte Sedimentproben wurden im April, Juni und August genommen und in 3 Tiefenstufen aufgeteilt, deren potentielle Methanproduktion und Substratspektrum in Inkubationsexperimenten mit Substrat- und Hemmstoff-Zugaben untersucht wurde. Zusätzlich wurde es hinsichtlich chemischer und physikalischer Parameter charakterisiert. Von in vitro produziertem und in situ aufgefangenem Methan wurde die 13C-Signatur bestimmt. Mittels DNA-Extraktion, PCR-gestützter Amplifikation archaealer 16S-rRNA-Gene sowie des Methanogen-spezifischen MCRα-Gens und anschließender Allelauftrennung mit Denaturierender Gradienten-Gelelektrophorese wurde die Diversität methanogener Archaea im Seen- und Jahreszeiten-Vergleich nachvollzogen. Die Inkubationsexperimente ergaben durchgehend um ein vielfaches höhere Methanproduktionsraten in Holzsee-Sedimenten als in denen des Großen Binnensees. In beiden Seen war die Methanogenese substratlimitiert und konnte durch Zugabe von Acetat und mehr noch durch H2+CO2 stimuliert werden. Die Hemmstoff-Versuche legen für den Holzsee eine dominante Rolle von Acetat nahe, für den Großen Binnensee von H2+CO2. Methanol wurde in beiden Seesedimenten erst nach so langer Gewöhnungszeit zu Methan umgesetzt, dass es im Freiland kein wichtiges methanogenes Substrat zu sein scheint. Jahreszeitliche Schwankungen in beiden Seen waren geringer als Tiefenunterschiede, wobei Produktionsminima in der Regel in der untersten untersuchten Tiefe (12-20 cm) auftraten. Die Hauptunterschiede der beiden Sedimente lagen in deutlich höheren Sulfat- und Makronährstoff-Konzentrationen im Großen Binnensee, jedoch oberflächennah sehr viel geringeren Konzentrationen gelösten Methans. Acetat und andere Fettsäuren waren in beiden Seen nicht nachweisbar. Die Isotopenmessung von in vitro produzierten Methan ergab durchgängig negativere δ13C-Werte für den Holzsee als für den Großen Binnensee, während diese Unterschiede im in situ aufgefangenen Methan geringer ausfielen. Das DGGE-Bandenmuster des 16S-rRNA-Gens der Archaea war undeutlich und zeigte beim Großen Binnensee keine Tiefen- und nur schwache jahreszeitliche, beim Holzsee gar keineUnterschiede zwischen verschiedenen Proben. Die Diversität der MCRα-Allele war sehr gering: Im Großen Binnensee konnten jahreszeitenabhängig zwei verschiedene Banden, im Holzsee lediglich eine gefunden werden. Unusually depleted 13C signatures of Chironomid midge larvae in lake sediments indicated an important contribution of biogenic methane to their diet. To determine the basis of the suggested food chain from methane producing archaea via methane oxidising bacteria two morphologically different lakes were compared concerning their methane production, availability, isotopic signature and diversity of methanogens. Lake Großer Binnensee is a hypereutrophic, non-stratified shallow lake under slight marine influence having an aerobic sediment surface while Lake Holzsee is a small, eutrophic lake with stable stratification during summer and corresponding anoxic hypolimnion. Undisturbed sediment cores were taken in April, June, and August and divided into three depth layers. Through incubation experiments with addition of substrates or inhibitors their potential methane production and substrate spectrum was examined. Additionally, the sediment was characterised with respect to chemical and physical parameters. The 13C isotopic signature of methane trapped in situ and produced in vitro was determined. Diversity of methanogenic archaea in comparison between lakes and seasons was monitored via DNA extraction, PCR based amplification of archaeal 16S rRNA genes as well as the methanogen-specific MCRα genes followed by denaturing gradient gel electrophoresis (DGGE). Incubation experiments resulted generally in higher rates of methane production in sediments of Holzsee compared to those of Großer Binnensee. Methanogenesis in sediments of both lakes was substrate-limited and could be stimulated by the addition of acetate and to, a greater extent, by hydrogen and carbon dioxide. Inhibitor experiments suggested for Holzsee a dominant role of acetate while for Großer Binnensee of hydrogen and carbon dioxide. Conversion of methanol to methane took such a long time in both lake sediments that it does not seem to be a significant methanogenic substrate in situ. Seasonal variation in both lakes was of less importance than differences between the depth layers, whereas methane production minima occurred usually in the deepest examined layer (12-20 cm). The main differences between the two sediments were a clearly higher sulphate and macronutrient concentration in Großer Binnensee, which, on the other hand, showed much lower methane concentrations near the sediment surface. Isotope measurements of methane produced in vitro resulted in consistently more negative δ13C values in Holzsee than in Großer Binnensee, while these differences were less pronounced in methane trapped in situ. The DGGE banding pattern of the archaeal 16S-rRNA gene was indistinct showing no depth and only weak seasonal differences in Großer Binnensee and no variation among different samples from Holzsee. Diversity of MCRα alleles was minimal: in Großer Binnensee two bands depending from season occurred while in Holzsee only one throughout the year.1 Einleitung 1 1.1 Methan in der Seen-Forschung 2 1.2 Methan als Kohlenstoffquelle in limnischen Nahrungsketten 2 1.3 Zielsetzung der Arbeit 3 2 Untersuchungsgebiet 4 3 Material und Methoden 7 3.1 Probenahmen 7 3.1.1 Entnahme der Sedimentkerne 7 3.1.2 Beprobung der Wassersäule 8 3.1.3 Auffangen von Gasblasen 8 3.2 Weiterverarbeitung der Sedimentproben 9 3.2.1 Aufteilen des Sedimentes in Tiefenstufen 9 3.2.2 Teilproben für Sulfat- und Fettsäurebestimmung 9 3.2.3 Gewinnung von Porenwasser aus Sediment 9 3.3 Bestimmung von potentiellen Methanproduktionsraten 10 3.3.1 Ansetzen des Hemmstoff-Substrat-Versuches 11 3.3.2 Gaschromatographische Messung 12 3.3.3 Berechnung der Methanproduktionsraten 12 3.4 Messung chemisch-physikalischer Parameter 15 3.4.1 Bestimmung von Trockengewicht und Glühverlust 15 3.4.2 Messung von gelöstem Methan 15 3.4.3 Messung der Fettsäuren- und Sulfatkonzentration 16 3.4.4 Messung von Phosphor- und Stickstoffverbindungen sowie gelöstem organischem Kohlenstoff 17 3.4.5 Isotopenmessungen in Gasproben 17 3.5 Molekularbiologische Untersuchungen 18 3.5.1 DNA-Extraktion 18 3.5.2 Agarose-Gelelektrophorese 19 3.5.3 Reinigung der DNA-Extrakte 20 3.5.4 Polymerase-Kettenreaktion (Polymerase chain reaction, PCR) 20 3.5.5 Denaturierende Gradienten-Gelelektrophorese (DGGE) 23 3.6 Statistische Auswertung 25 4 Ergebnisse 26 4.1 Charakterisierung der Seen 26 4.1.1 Sauerstoff- u. Temperaturprofile, pH-Wert 26 4.1.2 Methankonzentration im Sediment 27 4.1.3 Sulfat 27 4.1.4 Phosphor- und Stickstoffverbindungen 30 4.1.5 Organischer Kohlenstoff 32 4.1.6 Fettsäuren 32 4.2 Methanproduktion und Substratpräferenzen 34 4.2.1 Großer Binnensee 34 4.2.2 Holzsee 37 4.3 Isotopensignatur von Methan und Kohlendioxid 41 4.4 Molekularbiologische Untersuchungen 44 4.4.1 Allelauftrennung des 16S-rRNA-Gens der Archaea 44 4.4.2 Allelauftrennung des MCR-Gens der Methanogenen 46 5 Diskussion 49 5.1 Einflussgrößen auf die Methanproduktion 49 5.1.1 Sauerstoffhaushalt der Seen 49 5.1.2 Makronährstoffe 49 5.1.3 Sulfat als alternativer Elektronenakzeptor 51 5.1.4 Organisches Material als Grundlage für methanogene Substrate 52 5.1.5 Acetat und H2+CO2, die wichtigsten Substrate der Methanogenese 53 5.1.6 Methanol 57 5.1.7 Temperatur 58 5.2 Verfügbarkeit von Methan in den Seesedimenten 59 5.3 Diversität 60 5.4 Methodische Anmerkungen und Anregungen 62 5.4.1 Messung physikalisch-chemischer Parameter 62 5.4.2 Molekularbiologisch Analysen 62 5.4.3 Hemmstoff-Substrat-Versuch 63 6 Zusammenfassung 66 7 Summary 68 8 Literaturverzeichnis 6

The influence of oxygen and methane on nitrogen fixation in subarctic Sphagnum mosses

Contains fulltext : 191474.pdf (publisher's version ) (Open Access)9 p

Stratification of Diversity and Activity of Methanogenic and Methanotrophic Microorganisms in a Nitrogen-Fertilized Italian Paddy Soil

Contains fulltext : 179231.pdf (publisher's version ) (Open Access)Paddy fields are important ecosystems, as rice is the primary food source for about half of the world’s population. Paddy fields are impacted by nitrogen fertilization and are a major anthropogenic source of methane. Microbial diversity and methane metabolism were investigated in the upper 60cm of a paddy soil by qPCR, 16S rRNA gene amplicon sequencing and anoxic 13C-CH4 turnover with a suite of electron acceptors. The bacterial community consisted mainly of Acidobacteria, Chloroflexi, Proteobacteria, Planctomycetes and Actinobacteria. Among archaea, Euryarchaeota and Bathyarchaeota dominated over Thaumarchaeota in the upper 30cm of the soil. Bathyarchaeota constituted up to 45% of the total archaeal reads in the top 5cm. In the methanogenic community, Methanosaeta were generally more abundant than the versatile Methanosarcina. The measured maximum methane production rate was 444 nmol gdwh-1, and the maximum rates of nitrate-, nitrite- and iron-dependent anaerobic oxidation of methane (AOM) were 57 nmol, 55 nmol and 56 nmol gdwh-1, respectively, at different depths. qPCR revealed a higher abundance of ‘Candidatus Methanoperedens nitroreducens’ than methanotrophic NC10 phylum bacteria at all depths, except at 60cm. These results demonstrate that there is substantial potential for anaerobic oxidation of methane in fertilized paddy fields, with ‘Candidatus Methanoperedens nitroreducens’ archaea as a potential important contributor.15 p

McrA primers for the detection and quantification of the anaerobic archaeal methanotroph ‘Candidatus Methanoperedens nitroreducens’

The nitrogen and methane cycles are important biogeochemical processes. Recently, ‘Candidatus Methanoperedens nitroreducens,’ archaea that catalyze nitrate-dependent anaerobic oxidation of methane (AOM), were enriched, and their genomes were analyzed. Diagnostic molecular tools for the sensitive detection of ‘Candidatus M. nitroreducens’ are not yet available. Here, we report the design of two novel mcrA primer combinations that specifically target the alpha sub-unit of the methyl-coenzyme M reductase (mcrA) gene of ‘Candidatus M. nitroreducens’. The first primer pair produces a fragment of 186-bp that can be used to quantify ‘Candidatus M. nitroreducens’ cells, whereas the second primer pair yields an 1191-bp amplicon that is with sufficient length and well suited for more detailed phylogenetic analyses. Six different environmental samples were evaluated with the new qPCR primer pair, and the abundances were compared with those determined using primers for the 16S rRNA gene. The qPCR results indicated that the number of copies of the ‘Candidatus M. nitroreducens’ mcrA gene was highest in rice field soil, with 5.6 ± 0.8 × 106 copies g−1 wet weight, whereas Indonesian river sediment had only 4.6 ± 2.7 × 102 copies g−1 wet weight. In addition to freshwater environments, sequences were also detected in marine sediment of the North Sea, which contained approximately 2.5 ± 0.7 × 104 copies g−1 wet weight. Phylogenetic analysis revealed that the amplified 1191-bp mcrA gene sequences from the different environments all clustered together with available genome sequences of mcrA from known ‘Candidatus M. nitroreducens’ archaea. Taken together, these results demonstrate the validity and utility of the new primers for the quantitative and sensitive detection of the mcrA gene sequences of these important nitrate-dependent AOM archaea. Furthermore, the newly obtained mcrA sequences will contribute to greater phylogenetic resolution of ‘Candidatus M. nitroreducens’ sequences, which have been only poorly captured by general methanogenic mcrA primers.BT/Environmental Biotechnolog

Enrichment and Molecular Detection of Denitrifying Methanotrophic Bacteria of the NC10 Phylum▿

Anaerobic methane oxidation coupled to denitrification was recently assigned to bacteria belonging to the uncultured phylum NC10. In this study, we incubated sediment from a eutrophic ditch harboring a diverse community of NC10 bacteria in a bioreactor with a constant supply of methane and nitrite. After 6 months, fluorescence in situ hybridization showed that NC10 bacteria dominated the resulting population. The enrichment culture oxidized methane and reduced nitrite to dinitrogen gas. We assessed NC10 phylum diversity in the inoculum and the enrichment culture, compiled the sequences currently available for this bacterial phylum, and showed that of the initial diversity, only members of one subgroup had been enriched. The growth of this subgroup was monitored by quantitative PCR and correlated to nitrite-reducing activity and the total biomass of the culture. Together, the results indicate that the enriched subgroup of NC10 bacteria is responsible for anaerobic methane oxidation coupled to nitrite reduction. Due to methodological limitations (a strong bias against NC10 bacteria in 16S rRNA gene clone libraries and inhibition by commonly used stopper material) the environmental distribution and importance of these bacteria could be largely underestimated at present

A new intra-aerobic metabolism in the nitrite-dependent anaerobic methane-oxidizing bacterium Candidatus 'Methylomirabilis oxyfera'

Wu ML, Ettwig KF, Jetten MSM, Strous M, Keltjens JT, van Niftrik L. A new intra-aerobic metabolism in the nitrite-dependent anaerobic methane-oxidizing bacterium Candidatus 'Methylomirabilis oxyfera'. Biochemical Society Transactions. 2011;39(1):243-248.Biological methane oxidation proceeds either through aerobic or anaerobic pathways. The newly discovered bacterium Candidatus 'Methylomirabilis oxyfera' challenges this dichotomy. This bacterium performs anaerobic methane oxidation coupled to denitrification, but does so in a peculiar way. Instead of scavenging oxygen from the environment, like the aerobic methanotrophs, or driving methane oxidation by reverse methanogenesis, like the methanogenic archaea in sulfate-reducing systems, it produces its own supply of oxygen by metabolizing nitrite via nitric oxide into oxygen and dinitrogen gas. The intracellularly produced oxygen is then used for the oxidation of methane by the classical aerobic methane oxidation pathway involving methane mono-oxygenase. The present mini-review summarizes the current knowledge about this process and the micro-organism responsible for it