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Methane is a major contributor to the greenhouse effect, its atmospheric concentration being more than doubled since the XIX century. Every year 22 Tg of methane are released to the atmosphere from several natural and anthropogenic sources. Natural sources include geothermal/volcanic areas but the estimation of the total methane emission from these areas is currently not well defined since the balance between emission through degassing and microbial oxidation within the soils is not well known. Microbial oxidation in soils contributes globally for about 3-9% to the removal of methane from the atmosphere and recent studies evidenced methanotrophic activity also in soils of volcanic/geothermal areas despite their harsh environmental conditions (high temperatures, low pH and high concentrations of H2S and NH3). Methanotrophs are a diverse group of bacteria that are able to metabolize methane as their only source of carbon and energy and are found within the Alpha and Gamma classes of Proteobacteria and within the phylum Verrucomicrobia. Our purpose was to study the interaction between methanotrophic communities and the methane emitted from the geothermally most active site of Pantelleria island (Italy), Favara Grande, whose total methane emission has been previously estimated in about 2.5 t/a. Laboratory incubation experiments with soil samples from Favara Grande showed methane consumption values of up to 9500 ng g1 dry soil per hour while soils collected outside the geothermal area consume less than 6 ng g1 h1. The maximum consumption was measured in the shallowest part of the soil profile (1-3 cm) and high values (>100 ng g1 h1) were maintained up to a depht of 15 cm. Furthermore, the highest consumption was measured at 37 C, and a still recognizable consumption (>20 ng g1 h1) at 80 C, with positive correlation with the methane concentration in the incubation atmosphere. These results can be considered a clear evidence of the presence of methanotrophs that were investigated by culturing and culture-independent techniques. The diversity of proteobacterial methanotrophs was investigated by creating a clone library of the amplified methane mono-oxygenase encoding gene, pmoA. Clone sequencing indicates the presence of Gammaproteobacteria in the soils of Favara Grande. Enrichment cultures, on a mineral medium in a CH4-enriched atmosphere, led to the isolation of different strains that were identified as Methylocistis spp., which belong to the Alphaproteobacteria. The presence of Verrucomicrobia was detected by amplification of pmoA gene using newly designed primers. Soils from Favara Grande show therefore the largest spectrum of methanotrophic microorganisms until now detected in a geothermal environment. While the presence of Verrucomicrobia in geothermal soils was predictable due to their thermophilic and acidophilic character, the presence of both Alpha and Gamma proteobacteria was unexpected. Their presence is limited to the shallowest part of the soil were temperatures are lower and is probably favored by a soil pH that is not too low (pH 5) and their contribution to biological methane oxidation at Pantelleria is significant. Understanding the ecology of methanotrophy in geothermal sites will increase our knowledge of the role of soils in methane emissions in such environments.

Methane is released to the atmosphere by a wide number of natural (geological and biological) and anthropogenic sources, and is the second most important greenhouse gas after CO2. Microbial oxidation in soils by methanotrophic bacteria contributes to the removal of CH4 from the atmosphere and methanotrophic activity was also detected in volcanic/geothermal areas where degassing of endogenous gases occurs. Our aim is to describe the methanotrophs at the main exhalative area of Le Favare site at Pantelleria Island, where high CH4 consumption (up to 950 ng/g/ per h) was measured. Total soil bacterial diversity was analysed by TTGE of amplified 16S rRNA genes and the diversity of proteobacterial methanotrophs was investigated by creating a clone library of the amplified methane mono-oxygenase encoding genes, pmmoA. Enrichment cultures on a mineral medium in a CH4-enriched atmosphere led to the isolation of different strains that were identified as Methylocistis spp. Understanding the ecology of methanotrophy in geothermal sites will increase our knowledge of the role of such soils in methane emissions.

Methane plays an important role in the Earth’s atmospheric chemistry and radiative balance being the second most important greenhouse gas after carbon dioxide. Methane is released to the atmosphere by a wide number of sources, both natural and anthropogenic, with the latter being twice as large as the former. It has recently been established that significant amounts of geological methane, produced within the Earth’s crust, are currently released naturally into the atmosphere. Active or recent volcanic/geothermal areas represent one of these sources of geological methane. Microbial oxidation in soils contributes for about 3-9% to the total removal of CH4 from the atmosphere. Recent studies evidenced methanotrophic activity also in soils of volcanic/geothermal areas notwithstanding their harsh environmental conditions (high temperatures, low pH and high concentrations of H2S and NH3). The purpose of our study was to verify the methanotrophic potential and the bacterial diversity of the soils of the main geothermal area of Pantelleria island (Italy). Laboratory incubation experiments with soil samples collected at the main exhalative area showed methane consumption values of up to 9500 ng per g of dry soil per hour while soils collected outside the geothermal area less than 6 ng/g/h. Geothermal soils showed their maximum methane consumption in the shallowest part of the soil profile (0-3 cm) mantaining high values (>100 ng/g/h) at least up to dephts of 15 cm. Furthermore they showed the maximum consumption at about 37°C, showing a still recognizable consumption (>20 ng/g/h) at 80°C, and a positive correlation with the methane concentration in the incubation atmosphere. These results can be considered a clear evidence of the presence of methanotrophs. In order to evaluate the bacterial diversity, soil metagenomic DNA was extracted from Le Favare and analysed using a Temporal Temperature Gradient Electrophoresis (TTGE) analysis of the amplified Bacterial 16S rRNA gene. The amplification of metagenomic DNA with primers targeting Proteobacterial and Verrucomicrobial MMO (methane monooxygenase) genes is in progress. Enrichment cultures on a mineral medium in a CH4-enriched (25%) atmosphere allowed to isolate different strains that are under characterization.

Methane is the most abundant hydrocarbon in the atmosphere and a significant contributor to the radiative forcing with a global warming potential about 21 times that of CO2. Methane is released to the atmosphere by a wide number of sources, both natural and anthropogenic, with the latter being twice as large as the former. Significant amounts of geological methane, produced within the Earth’s crust (e.g. volcanic/geothermal areas), are currently released into the atmosphere (48 Tg CH4/y). Microbial oxidation in soils by methanotrophic bacteria contributes to the removal of CH4 from the atmosphere for about 3-9%. Methanotrophs belong to the Gamma- and Alpha-proteobacteria and to the recently discovered acidophilic Verrucomicrobia. Evidence of methanotrophic activity also in soils of volcanic/geothermal areas has been recently revealed, notwithstanding their harsh environmental conditions (high temperatures, low pH, high concentrations of H2S and NH3). The purpose of our study was to verify the methanotrophic potential and the bacterial diversity of the soils of the main geothermal area of Pantelleria island (Italy). Close to the fumarolic area (Le Favare) the mean detected temperature is about 90 °C at 30 cm of depth and the mean measured pH of the soil is about 4.8. Laboratory incubation experiments with soil samples collected at the main exhalative area showed methane consumption values of up to 9500 ng/g of dry soil per hour while soils collected outside the geothermal area consumed less than 6 ng/g/h. Geothermal soils showed their maximum methane consumption in the shallowest part of the soil profile (0-3 cm). Furthermore they showed the maximum consumption at about 37°C, showing a still recognizable consumption (>20 ng/g/h) at 80°C. These results can be considered a clear evidence of the presence of methanotrophs. In order to evaluate the bacterial diversity, soil metagenomic DNA was extracted from Le Favare and analysed using a Temporal Temperature Gradient Electrophoresis (TTGE) analysis of the amplified Bacterial 16S rRNA gene. The amplification of metagenomic DNA with primers targeting Proteobacterial and Verrucomicrobial MMO (methane monooxygenase) genes is in progress. Enrichment cultures on a mineral medium in a CH4-enriched (25%) atmosphere led to the isolation of different strains that are under characterization.

Greece, a country characterised by intense seismic and volcanic activity, has a complex geodynamic and geological setting that favours the occurrence of many gas manifestations. In this study, we address the origin of CH 4 and light hydrocarbons in cold and thermal emissions discharging along the Hellenic territory. Also, we investigate their possible relationship with the main geochemical composition of the gases and the different geological settings of the sampling sites. For this purpose we collected 101 new samples that were analysed for their chemical (O 2 , N 2 , CH 4 , CO 2 , He, Ne, Ar, H 2 , H 2 S and C 2 -C 6 hydrocarbons) and isotopic (R/R A , δ 13 C-CO 2 , δ 13 C-CH 4 and δ 2 H-CH 4 ) composition. Results show that CH 4 presents a wide range of concentrations (from 13 C-CH 4 from −79.8 to +45.0‰ vs. V-PDB; δ 2 H-CH 4 from −311 to +301‰ vs. V-SMOW). Greece was subdivided in four geologic units (External [EH] and Internal [IH] Hellenides, Hellenic Hinterland [HH] and active Volcanic Arc [VA]) and a decreasing CH 4 concentration from EH to HH was recognized, whereas CH 4 showed intermediate concentrations in VA. The CH 4 /(C 2 H 6 + C 3 H 8 ) ratios (from 1.5 to 93,200), coupled with CH 4 isotopic features, suggest that the light alkanes derive from different primary sources and are affected by secondary processes. An almost exclusive biotic, mainly microbial, origin of CH 4 can be attributed to EH gases. Cold gases at IH have mainly a thermogenic origin, although some gases connected to continental serpentinization may have an abiogenic origin. Methane in gases bubbling in thermal waters of IH, HH and VA and fumarolic gases of the VA seem to have an abiogenic origin, although their chemical and isotopic characteristics may have been produced by secondary oxidation of thermogenic CH 4 , a process that in some of the sampled gases causes extremely positive isotopic values (δ 13 C-CH 4 up to +45.0‰ vs. V-PDB and δ 2 H-CH 4 up to +301‰ vs. V-SMOW).

Ogni anno, 22 Tg di CH4 vengono rilasciati in atmosfera da numerose sorgenti sia naturali che antropiche. Il metano riveste un ruolo molto importante nella chimica dell’atmosfera terrestre e nel bilancio dell’energia radiante assorbita, essendo il secondo gas serra più potente dopo la CO2. Le aree vulcaniche e geotermali contribuiscono al flusso di metano in atmosfera, essendo vaste aree di degassamento. Studi preliminari hanno stimato che le emissioni globali di metano dai sistemi geotermali e vulcanici europei sono nel range di 4-16 kt a-1. Questa stima è stata ottenuta indirettamente dai dati delle emissioni di CO2 o H2O e dal rapporto del flusso CO2/CH4 oppure H2O/CH4 misurati nelle principali fumarole. La stima del metano emesso globalmente dalle aree vulcaniche e geotermali non è ancora ben definita in quanto il bilancio tra le emissioni per degassamento dai suoli e il consumo di metano per ossidazione microbica è ancora poco noto. Inoltre, le misure di flusso di metano sono molto difficili da eseguire e si hanno a disposizioni pochi dati. Alcuni metodi, seppur accettabili al fine di ottenere stime sul flusso di metano, escludono completamente la possibilità che il metano venga rimosso per via microbica dai batteri metanotrofi. A scala globale, l’ossidazione microbica del metano contribuisce alla rimozione di circa il 3-9% del metano dall’atmosfera. Ma l’importanza dei batteri metanotrofi è ancora maggiore in quanto questi ossidano la maggior parte del metano prodotto nel suolo e nel sottosuolo prima che questo raggiunga l’atmosfera. Le condizioni ambientali dei suoli vulcanici e geotermali (ad esempio scarso contenuto in ossigeno, alta temperature, attività protonica, ect.) sono stati da sempre considerati inospitali per i batteri metanotrofi. Tuttavia, di recente è stata dimostrata la presenza di batteri acidofili e termofili appartenenti al phylum dei Verrucomicrobia. Questi organismi sono stati individuati alla Solfatara di Pozzuoli (Italia), ad Hell’s gate (Nuova Zelanda) ed in Kamchatka (Russia). Qui riportiamo l’attività metanotrofa riscontrata nei suoli dell’Isola di Pantelleria (Italia), dell’Isola di Vulcano (Italia), di Sousaki (Grecia), di Nea Kameni- Santorini (Grecia), e dell’Isola di Nisyros (Grecia). Evidenze di rimozione microbica del metano in questi suoli era già stata riscontrata nel rapporto dei flussi di CO2/CH4, che risultava sempre inferiore rispetto a quello atteso, indicando una perdita di CH4 durante il suo movimento verso la superficie. Esperimenti per la misura del consumo di metano sono stati eseguiti usando i suoli di Pantelleria, Vulcano, Nea kameni, Nisyros e Sousaki. Questi esperimenti hanno rivelato tassi di consumo fino a 950, 48, 15, 39 e 520 ng CH4 h-1 per ogni grammo di suolo (peso secco), rispettivamente. Solo pochi campioni non hanno indicato consumo di metano. L’analisi dei gas del suolo e le caratteristiche chimico-fisiche del suolo ci hanno permesso di discriminare i fattori principali che influenzano la presenza dei metanotrofi e il tasso dei consumo del metano. La composizione del gas dal suoli, e in particolare il contenuto di CH4 e di H2S rappresentano il fattore discriminate per i metanotrofi. infatti, l’isola d Vulcano e di Nisyros, il cui contenuto in H2S raggiunge circa 250000 ppm, mostrano i consumi più bassi. In aggiunta nei suoli geotermali e vulcanici l’H2S contribuisce all’abbassamento del pH dei suoli. I valori di consuma maggiori sono stati misurati nell’isola di Pantelleria dove l’H 2S è meno di 20 ppm e il pH è vicino alla neutralità. Analisi microbiologiche e molecolari hanno permesso di riscontrare nei suoli di Pantelleria la presenza di batteri metanotrofi affiliati ai Gamma ed agli Alfa-Proteobatteri ed agli acido-termofili Verrucomicrobia. Il metanotrofo coltivabile appartenete al genere Methylocystis (Alfaproteobatterio) e il Gammaproteobatterio Methylobacterium sono stati isolati attraverso colture di arricchimento. Gli isolati mostrano ampi range di tolleranza di pH e temperatura e un tasso di ossidazione fino a 450 ppm/h. Attraverso l’amplificazione del gene pmoA, basandosi sui metodi coltura-indipendenti è stata rivelata un’ampia diversità di batteri metanotrofi appartenenti ai Proteobatteri (α- e γ-) ed ai Verrucomicrobia. Questo è il primo report in cui si dimostra la coesistenza di entrambi i phyla di metanotrofi in un sito geotermale/vulcanico. La presenza dei metanotrofi Proteobatteri era inaspettata perché le condizioni di sito sono state considerate inadeguate e può essere spiegata del pH non eccessivamente basso (>5) di questo specifico sito geotermale. Queste specie possono aver trovato la loro nicchia negli strati più superficiali dei suoli di Favara Grande a Pantelleria dove le temperature non sono così alte ed è presente una forte risalita di metano. capire l’ecologia dei metanotrofi nei siti geotermali e vulcanici aumenterà le conoscenze nel loro ruolo nelle emissioni di metano in atmosfera. Yearly, 22 Tg of CH4 are released in to the atmosphere from several natural and anthropogenic sources. Methane plays an important role in the Earth’s atmospheric chemistry and radiative balance being the most important greenhouse gas after carbon dioxide. Volcanic/geothermal areas contribute to the methane flux, being the site of widespread diffuse degassing of endogenous gases. Preliminary studies estimated a total CH4 emission from European geothermal and volcanic systems in the range 4-16 kt a-1. This estimate was obtained indirectly from CO2 or H2O output data and from CO2/CH4 or H2O/CH4 values measured in the main gaseous manifestations. The total estimated CH4 emission from geothermal/volcanic areas is still not well defined since the balance between emission through degassing and consumption through soil microbial oxidation is poorly known. Moreover, methane soil flux measurements are laboratory intensive and very few data have been collected until now in these areas. Such methods, although acceptable to obtain order-of-magnitude estimates, completely disregards possible methane microbial oxidation within the soil carried on by the methanotrophs. At the global scale, microbial oxidation in soils contributes for about 3-9% to the total removal of methane from the atmosphere. But the importance of methanotrophic organisms is even larger because they oxidize the greatest part of the methane produced in the soil and in the subsoil before its emission to the atmosphere. Environmental conditions in the soils of volcanic/geothermal areas (i.e. low oxygen content, high temperature and proton activity, etc.) have long been considered inadequate for methanotrophic microorganisms. But recently, it has been demonstrated that methanotrophic consumption in soils occurs also under such harsh conditions due to the presence of acidophilic and thermophilic Verrucomicrobia. These organisms were found in Italy at the Solfatara at Pozzuol (Italy), at Hell’s Gate (New Zealand) and in Kamchatka (Russia), pointing to a worldwide distribution. Here we report on methane oxidation rate measured in Pantelleria Island (Italy), Vulcano Island (Italy), Sousaki (Greece), Nea Kameni (Santorini) and Nisyros (Greece) soils. Clues of methane microbial oxidation in soils of these areas can be already found in the CH4/CO2 ratio of the flux measurements which is always lower than that of the respective fumarolic manifestations indicating a loss of CH4 during the travel of the gases towards earth’s surface. Laboratory methane consumption experiments made on soils collected at Pantelleria, Vulcano, Nea Kameni, Nysiros and Sousaki revealed for most samples consumption rates up to 950, 48, 15, 39 and 520 ng CH4 h-1 for each gram of soil (dry weight), respectively. Only few soil samples displayed no methane consumption activity. Analysis on soil gases and chemical-physical characteristics of the soils allowed us to discriminate the main factors that influenced the methanotrophs presence and the methane consumption rate. Soil gases composition, and in particular the amount of the CH4 and H2S, represent the main discriminating factor for methanotrophs. In fact, Vulcano and Nisyros Island, whose soil gas contained up to 250000 ppm of H2S, showed the lowest consumption rate. Moreover, in geothermal/volcanic soils H2S contribute to the soil pH lowering; highest methane consumption were recorded in Pantelleria island were H2S is less than 20 ppm and pH close to the neutrality were measured. Microbiological and molecular analyses allowed to detect the presence of methanotrophs affiliated to Gamma and Alpha-Proteobacteria and to the newly discovered acido-thermophilic methanotrophs belong to the Verrucomicrobia phylum in soils from Pantelleria. Culturable methanotrophic Alphaproteobacteria of the genus Methylocystis and the Gammaproteobacteria Methylobacterium were isolated by enrichment cultures. The isolates show a wide range of tolerance to pH and temperatures and an average methane oxidation rate up to 450 ppm/h. A larger diversity of (α- and γ-) proteobacterial and verrucomicrobial methanotrophs was detected by a culture-independent approach based on the amplification of the methane mono-oxygenase gene pmoA. This is the first report describing coexistence of both the methanotrophic phyla (Verrucomicrobia and Protebacteria) in the same geothermal site. The presence of proteobacterial methanoptrophs, in fact, was quite unexpected because they are generally considered not adapted to live in such harsh environments and could be explained by not really low pH values (> 5) of this specific geothermal site. Such species could have found their niches in the shallowest part of the soils of Favara Grande were the temperatures are not so high and thrive on the abundant upraising methane. Understanding the ecology of methanotrophy in geothermal sites will increase our knowledge of their role in methane emissions to the atmosphere.