Gaseous emissions from geothermal and volcanic areas: focus on methane and methanotrophs

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

Estimation of CO2 release from thermal springs to the atmosphere

Introduction Geodynamically active regions have long been recognized as areas of anomalous Earth degassing [Irwin and Barnes, 1980]. Areas found at plate boundaries are characterized by seismic, volcanic and geothermal activity as well as ore deposition. These processes are enhanced by the circulation of hydrothermal fluids in the crust, which transport volatiles from the deep crust or mantle to the surface [King, 1986]. Kerrick and Caldera, [1998], were the first to indicate the significant contribution of the CO2 degassing by extensional tectonic and hydrothermal activity in metamorphic belts during the Phanerozoic. Moreover, further studies concerning gas emissions from diffuse degassing tectonic structures on various geological regimes suggested in their majority elevated CO2 concentrations [Klusman, 1993]. In fact, it is worth noting that the estimated global hydrothermal CO2 flux from subaerial geothermal environments can be comparable to that of direct volcanic discharges [Kerrick et al., 1995; Seward and Kerrick, 1996]. Study Area The back-arc geothermal fields of Greece include, among others, the Tertiary sedimentary basins of both Sperchios Basin and north Euboea, which are located in central Greece. Their tectonic activity contributes in crust thinning [Papadakis at al., 2016 and references therein] and elevated heat flow values [Fytikas and Kolios, 1979]. These geothermal anomalies due to the tectonic activity and the geological and volcanic regime are expressed as hot springs (Ypatis, Psoroneria, Thermopyles and Kamena Vourla in Sperchios Basin and Edipsos and Ilion in north Euboea). Tectonics of central Greece seems to be of particular interest as major fault structures are found in the area. Sperchios Basin was formed through the activity of WNW-ESE trending faults [Georgalas and Papakis, 1966; Marinos et al., 1973], whilst the Sperchios tectonic graben itself is considered to be the extension of the North Anatolia strike-slip fault. Moreover, in the north Euboean Gulf, the major fault structures are those of the Atalanti Fault Zone (AFZ) that consist of several segments of normal faults, trending about NW-SE [Pavlides et al., 2004]. Materials and Methods Six groups of springs (Ypatis, Psoroneria, Thermopyles, Kamena Vourla, Edipsos and Ilion) were investigated in this study. Bubbling gases were sampled using an inverted funnel positioned above the bubbles and stored in glass flasks equipped with two stopcocks until analysis. Samples for dissolved gas analyses were collected in glass vials and were sealed underwater. In the laboratory, the concentrations of He, H2, H2S, O2, N2, CO2 and CH4, on the samples were analysed by an Agilent 7890B gas chromatograph with Ar as carrier. The total CO2 emitted through bubbling was measured at 6 different pools (Psoroneria, Psoroneria 2,Thermopyles, Leonidas, Kamena Vourla and Ilion), whereas at other springs (Koniavitis-Sperchios Basin, Edipsos-Damaria and Edipsos-Thermopotamos) an estimation of the release was made by visual inspection. The CO2 fluxes were measured using the floating chamber method [Mazot and Bernard, 2015] that was equipped with a portable fluxmeter (WEST Systems, Italy) based on the accumulation chamber method as suggested by Chiodini et al., [1998]. The flux data were processed with both the Graphical Statistical Approach (GSA) and the Stochastic Simulation Approach (SSA), with the latter being based on the algorithm of sequential Gaussian simulation [Deutsch and Journal 1998; Cardellini et al., 2003]. Zonal Statistics on the final CO2 flux maps was obtained using the ArcMap 10.3 (ESRI) Spatial Analyst tool and were used to estimate the total CO2 output to the atmosphere. Results and Conclusions Carbon dioxide is the prevailing gas species for the great majority of the under investigation sites, with only gases collected in the area of Kamena Vourla (Kamena Vourla and Koniavitis) being rich in N2. The total bubbling CO2 emission from the pools to the atmosphere ranged from 314 to 44,800 g/m2/day. At sites with greater surfaces, the CO2 release was estimated after performing direct measurements (28-Thermopyles, 74-Psoroneria) with the most elevated values being found in the areas of Thermopyles and Psoroneria (1 and 2 t/d, respectively) (Tab. 1); the maps were drawn following the SSA (Figure 1). The outgoing channels of the springs showed an elevated flow (> 250 l/s) of gas-charged water (> 15 mmol/l of dissolved CO2). Even though no bubbling was visible along the stream, the dissolved CO2 content sampled at different distances from springs of Psoroneria and Thermopyles, decreased up to an order of magnitude after few hundreds of metres, indicating an evident and intense, although not visible, CO2 degassing versus the atmosphere. Physico-chemical parameters (temperature and pH) along the outlet channels were also measured at the same sampling points showing correlations (negative in terms of temperature; T decreased from 33.1 to 30.3 and 40.8 to 39 °C, respectively and positive in terms of pH; pH increased from 6.11 to 7.05 and 6.05 to 7.70, respectively) with the distance. The CO2 output of the outgoing channels to the atmosphere was quantified considering thedifference between the initial and the final content of the dissolved CO2 as well as the water flow, obtaining values of > 10 t/d for Thermopyles and ~9 t/d for Psoroneria. Estimations were also made at Ypatis, Kamena Vourla, Koniavitis and Edipsos, where the mean values reached 1 t/d of CO2 for each spring. The obtained CO2 released from the bubbling pools to the atmosphere was directly compared with the one estimated from the outgoing channels (Tab. 1). The degassing along the outflow channel was almost always higher than the corresponding bubbling pool, sometimes even an order of magnitude, suggesting that most of the degassing is “hidden”. For each site the amount of CO2 released versus the atmosphere was calculated as (Figure 2): ΦtotCO2 = Φpool + Φstream The total CO2 released to the atmosphere as estimated for the study area is at ~ 30 t/d, with the major contribution deriving from the degassing along the outflow channels of the thermal springs. Such output is comparable and sometimes higher than that of each single active volcanic system along the South Aegean Volcanic Arc (15 - 38 t/d) and highlights the importance of “hidden” degassing along CO2 - oversaturated streams

Microbial impact on the isotope composition of methane in both thermal and hyperalkaline waters of central Greece

Introduction The different origins of methane can be subdivided in biogenic (either directly produced by microbial activity or deriving by decay of organic matter at T > 150\ub0C) and abiogenic (from pure inorganic reactions). Among the latter, one of the most debated origins comes from serpentinization processes of ultramafic rocks in ophiolitic sequences at low temperatures (T < 80 \ub0C). Moreover, further secondary processes (diffusion, inorganic or microbial oxidation, etc.) may also contribute and thus mask the original chemical and/or isotope composition. Primary and secondary processes acting on CH4 can be recognised mainly through its isotope (d13C and d2H) composition and the ratio between CH4 and C2+C3 light hydrocarbons [Bernard et al. 1978; Schoell 1980]. Microorganisms may be involved in the methane cycle not only as active producers but also as consumers. Methane oxidizing bacteria (or methanotrophs) are microorganisms with the ability to use methane as the only source of carbon for energy and biomass production. Methanotrophs are ubiquitous and play an important role in the global carbon cycle, acting as a natural filter between the subsoil and the atmosphere. They were isolated from several environments such as soils, wetlands, freshwater, marine sediments, water columns, groundwater, rice paddies, and peat bogs [Murrell and Jetten, 2009]. Some species were adapted also at extreme environments characterized by high temperature (up to 81.6 \ub0C), extremely low or high pHs (1.5-11) or even anaerobic conditions. Due to the fact that methanotrophs metabolize preferentially light isotopes, biologic methane oxidation brings sometimes to extremely positive d13C and d2H values [Cadieux et al., 2016]. The Greek territory belongs to the geodynamically active Alpine-Himalayan orogenic belt. As such, it shows intense seismic activity, active volcanic systems and areas of enhanced geothermal fluxes. One of these areas is the Sperchios Basin and the northern part of Euboea Island in central Greece, where thermal manifestations are widespread [D\u2019Alessandro et al., 2014]. The complex geology of Greece includes also two important parallel running ophiolitic belts, with the Othrys Massif (central Greece) belonging to the westernmost of them. In and around this wide ophiolite outcrop, some cold hyperalkaline and some hypothermal (T < 30\ub0C) alkaline waters are present. In the present paper we discuss data about chemistry and methane isotope composition of bubbling or dissolved gases in both thermal springs and hyperalkaline springs of Central Greece. Sampling and Analytical Methods Free bubbling gas samples were taken using an inverted funnel. All free gas samples were stored in Pyrex bottles with two vacuum stopcocks. Samples for dissolved gas analyses were collected in glass vials sealed underwater. In the laboratory, the chemical analyses were carried out by gaschromatography (Agilent 7890B GC System) using Ar as the carrier gas. Dissolved gases were extracted after equilibrium was reached at constant temperature with a host-gas (high-purity argon) injected in the sample bottle. The measurement precision was better than \ub15% for common gases and \ub110% for trace gases such as the alkanes. The chemical composition of the dissolved gas phase was obtained from the gas-chromatographic analyses taking into account the solubility coefficients (Bunsen coefficient \u201c\u3b2\u201d, ccgas/mlwater STP) of each gas specie, the volume of gas extracted and the volume of the water sample (details in Capasso and Inguaggiato, [1998] and Liotta and Martelli, [2012]). Starting from the total amount of dissolved gases (ccSTP/L) we calculated the relative abundances for every single gas species in equilibrium with the dissolved gas phase and expressed the analytical results in \u3bcmol/mol of gas at atmospheric pressure, allowing the comparison of dissolved gases with free gases. Carbon and hydrogen isotope compositions of CH4 were measured using a Thermo TRACE GC and a Thermo GC/C III interfaced to a Delta Plus XP gas source mass spectrometer. 13C/12C ratios are reported here as d13C values (\ub10.1 \u2030) with respect to the V-PDB standard. 1H/2H ratios are reported here as d2H values (\ub12 \u2030) with respect to the V-SMOW standard. The oxygen and hydrogen isotopic compositions of water were analysed on unfiltered samples with the use of Analytical Precision AP 2003 and FinniganMAT Delta Plus IRMS devices, respectively. The isotope ratios are expressed as the deviation per mil (\u3b4\u2030) from the reference V-SMOW. The uncertainties (\ub11\u2030 were \ub10.1% for \u3b418O and \ub11% for \u3b42H. Results Five thermal springs, with temperatures from 33 to 80\ub0C, were sampled in the study area. All show elevated fluxes of bubbling gases whose prevailing species are either CO2 or N2. Methane concentrations range from 27 to 4000 \u3bcmol/mol, whilst the isotope composition of CH4 covers a wide range with d13C values ranging from -21.7 to +16.9\u2030 and d2H values ranging from -124 to +370\u2030. Seven alkaline hypothermal waters were collected in five areas (Amplas, Platystomo, Kaitsa, Smokovo and Soulanta) while 10 hyperalkaline waters in two areas (Archani and Ekkara); all samples were collected from different springs and wells and some of the sites presented bubbling. All samples present low concentrations of H2 (from <2 to 2500 \u3bcmol/mol), CO2 (up to 26,000 but generally below 1000 \u3bcmol/mol) and O2 (up to 16,000 but generally below 3000 \u3bcmol/mol). Gases in alkaline waters (pH <10) are in their majority dominated by CH4 (from 128,000 to 915,000 \u3bcmol/mol). Hyperalkaline (pH > 11) waters are N2 dominated (from 727,000 to 977,000 \u3bcmol/mol) and have CH4 concentrations from 11,500 to 279,000 \u3bcmol/mol. Also all these samples display a wide range of isotope compositions of CH4 (d13C from -74.5 to -14.5 \u2030 and d2H from -343 to -62 \u2030). Discussion Thermal springs Methane in most of the bubbling gases found in the thermal waters of Greece display a small range in isotope composition close to -21\u2030 for carbon and to -130\u2030 for hydrogen [Daskalopoulou et al., 2018] and plot in the middle of the field of volcanic and geothermal systems (Figure 1). In the study area, only the hottest (Edipsos) of the thermal manifestations displays similar values. All the remaining samples fit a methane oxidation trend reaching extremely positive values (Figure 1). If we consider the lowest values as the deep hydrothermal marker the obtained \u394H/\u394C values range between 5 and 13 which are close to those typical of microbially driven oxidation [Coleman et al., 1981]. Although the outlet temperature of the hottest manifestations is at the upper limit for methanotrophic microrganisms [Sharp et al., 2014], we can hypothesize that environmental conditions are not favourable for their survival at this site. On the contrary, methanotrophs can thrive in the sites characterized by lower temperatures (33-65 \ub0C), strongly consuming methane. The most positive values were measured at Psoroneria and indicate a very high consumption fraction. Considering again the values of Edipsos as the deep hydrothermal marker, a Rayleigh fractionation modelling in a closed system and kinetic fractionation factors for microbial oxidation [Coleman et al., 1981] we estimate a consumption of more than the 75% of the initial CH4. Alkaline and hyperalkaline waters Alkaline waters present mostly isotope values for CH4 compatible with a biogenic origin (d13C from - 62.0 to -37.5 \u2030 and d2H from -247 to -154 \u2030). Only the sample of Kaitsa falls above the biogenic field, indicating possible fractionation due to CH4 oxidation (Figure 2). Most of the hyperalkaline waters have CH4 isotope values compatible with an abiogenic origin through serpentinization processes (Figure 2). But some of the CH4 collected in the hyperalkaline waters show values falling in the biogenic field, with at points, very negative d13C values (< -70\u2030). Methanogens were found also in other hyperalkaline waters taking advantage of the presence of sometimes very high hydrogen concentrations [Woycheese et al., 2015; Miller et al., 2018]. Also methanotrophs were rarely found in hyperalkaline waters [Woycheese et al., 2015; Miller et al., 2018] and their presence may justify the most positive values found in the study area (Figure 2)

High diversity of methanotrophic bacteria in geothermal soils affected by high methane fluxes

Volcanic and geothermal systems emit endogenous gases by widespread degassing from soils, including CH4, a greenhouse gas 25 times as potent as CO2. Recently, it has been demonstrated that volcanic/geothermal soils act as source, but also as biological filter for methane release to the atmosphere. For long time, volcanic/geothermal soils has been considered inhospitable for methanotrophic microorganisms, but new extremophile methanotrophs belonging to Verrucomicrobia were identified in three different areas (Pozzuoli, Italy; Hell’s Gate, New Zealand; Kamchatka, Russia), explaining anomalous behaviours in methane leakages of several geothermal/volcanic sites. Our aim was to increase the knowledge of the relationship between methane emissions from volcanic/geothermal areas and biological methane oxidation, by investigating a geothermal site of Pantelleria island (Italy). Pantelleria Island hosts a high enthalpy geothermal system characterized by high temperature, high CH4 and very low H2S fluxes. Such characteristics are reflected in potentially great supply of methane for methanotrophs and scarce presence of inhibitors of their activity (H2S and NH3) in the Pantelleria soils. Potential methanotrophic activity within these soils was already evidenced by the CH4/CO2 ratio of the flux measurements which was lower than that of the respective fumarolic manifestations indicating a loss of CH4 during the gas travel towards the earth’s surface. In this study laboratory incubation experiments using soils sampled at Favara Grande, the main hydrothermal area of Pantelleria, showed very high methane consumption rates (up to 9500 ng CH4 h1 g1). Furthermore, microbiological and culture-independent molecular analyses allowed to detect the presence of methanotrophs affiliated to Gamma- and Alpha-Proteobacteria and to the newly discovered acidothermophilic methanotrophs Verrucomicrobia. Culturable methanotrophic Alpha-proteobacteria of the genus Methylocystis were isolated by enrichment cultures. The isolates showed a wide range of tolerance to pH (3.5 – 8) and temperatures (18 – 45 C), and an average methane oxidation rate of 450 ppm/h. A larger diversity of proteobacterial and verrucomicrobial methanotrophs was detected by the amplification of the methane mono-oxygenase gene pmoA. This study demonstrates the coexistence of both the methanotrophic phyla Verrucomicrobia and Proteobacteria in the same geothermal site. The presence of proteobacterial methanotrophs was quite unexpected because they are generally considered not adapted to live in such harsh environments. Their presence at Favara Grande could be explained by not so low soil pH values (> 5) of this specific geothermal site and by the high methane availability. Such species could have found their niches in the shallowest part of the soils, were the temperatures are not so high, thriving 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

The impact of methanotrophic activity on methane emissions through the soils of geothermal areas

Methane plays an important role in the Earth’s atmospheric chemistry and radiative balance being the most important greenhouse gas after carbon dioxide. It has recently been established that geogenic gases contribute significantly to the natural CH4 flux to the atmosphere (Etiope et al., 2008). Volcanic/geothermal areas contribute to this flux, being the site of widespread diffuse degassing of endogenous gases (Chiodini et al., 2005). In such an environment soils are a source rather than a sink for atmospheric CH4 (Cardellini et al., 2003; Castaldi and Tedesco, 2005; D’Alessandro et al., 2009; 2011; 2013). Due to the fact that methane soil flux measurements are laboratory intensive, very few data have been collected until now in these areas. Preliminary studies (Etiope et al., 2007) 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. Such methods, although acceptable to obtain order-of-magnitude estimates, completely disregard possible methanotrophic activity within the soil. 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 oxidise 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 been considered inadequate for methanotrophic microrganisms. 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 di Pozzuoli (Pol et al., 2007), in New Zealand at Hell’s Gate (Dunfield et al., 2007) and in Kamchatka, Russia (Islam et al., 2008). Both the Italian and the Hellenic territories are geodynamically very active with many active volcanic and geothermal areas. Here we report on methane flux measurements made at Pantelleria (Italy) and at Sousaki and Nisyros (Greece). The total methane output of these three systems is about 10, 19 and 1 t a-1, respectively (D’Alessandro et al., 2009; 2011; 2013). The total emissions obtained from methane flux measurements are up to one order of magnitude lower than those obtained through indirect estimations. Clues of methanotrophic activity within the soils of these areas can be 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. Furthermore laboratory methane consumption experiments made on soils collected at Pantelleria and Sousaki revealed, for most samples, CH4 consumption rates up to 9.50 μg h-1 and 0.52 μg h-1 respectively for each gram of soil (dry weight). Only few soil samples displayed no methane 2 consumption activity. Finally, microbiological and molecular investigations allowed us to identify the presence of methanotrophic bacteria belonging to the Verrucomicrobia and to the Alpha- and Gamma-Proteobacteria in the soils of the geothermal area of Favara Grande at Pantelleria. While the presence of the former was not unexpected due to the fact that they include acidophilic and thermophilic organisms that were previously found in other geothermal environments, the latter are generally considered not adapted to live in harsh geothermal environments. Their presence in the soils of Pantelleria could be explained by the fact that these soils do not have extremely low pH values (>5). Indeed thermotollerant methanotrophic Gamma-proteobacteria, have been previously found in the sediments of thermal springs in Kamchatka (Kizilova et al., 2012). Such species could find their niches in the shallowest part of the soils of Favara Grande were the temperatures are not so high and they thrive on the abundant upraising hydrothermal methane. References: Cardellini C., Chiodini G., Frondini F., Granieri D., Lewicki J., Peruzzi L., 2003. Accumulation chamber measurements of methane fluxes: application to volcanic–geothermal areas and landfills. Appl. Geochem. 18, 45–54. Castaldi S., Tedesco D., 2005. Methane production and consumption in an active volcanic environment of Southern Italy. Chemosphere 58, 131–139. Chiodini G., Granieri D., Avino R., Caliro S., Costa A., 2005. Carbon dioxide diffuse degassing and estimation of heat release from volcanic and hydrothermal systems. J. Geophys. Res. 110, B08204. D’Alessandro W., Bellomo S., Brusca L., Fiebig J., Longo M., Martelli M., Pecoraino G., Salerno F., 2009. Hydrothermal methane fluxes from the soil at Pantelleria island (Italy). J. Volcanol. Geotherm. Res. 187, 147–157. D’Alessandro W., Brusca L., Kyriakopoulos K., Martelli M., Michas G., Papadakis G., Salerno F., 2011. Diffuse hydrothermal methane output and evidence of methanotrophic activity within the soils at Sousaki (Greece). Geofluids 11, 97–107 D’Alessandro W., Gagliano A.L., Kyriakopoulos K., Parello F., 2013. Hydrothermal methane fluxes from the soil at Lakki plain (Nisyros island, Greece). Bull. Geol. Soc. Greece, vol. XLVII Proc. of the 13th International Congress, Chania, Sept. 2013 Dunfield P.F., Yuryev A., Senin P., Smirnova A.V., Stott M.B., Hou S., Ly B., Saw J.H., Zhou Z., Ren Y, Wang J., Mountain B.W., Crowe M.A., Weatherby T.M., Bodelier P.L.E., Liesack W., Feng L., Wang L., Alam M., 2007. Methane oxidation by an extremely acidophilic bacterium of the phylum Verrucomicrobia. Nature, 450, 879–882. Etiope G., Fridriksson T., Italiano F., Winiwarter W., Theloke J., 2007. Natural emissions of methane from geothermal and volcanic sources in Europe. J. Volcanol. Geotherm. Res. 165, 76–86. Etiope G., Lassey K.R., Klusman R.W., Boschi E., 2008. Reappraisal of the fossil methane budget and related emission from geologic sources. Geophys. Res. Lett. 35, L09307. Islam T., Jensen S., Reigstad L.J., Larsen Ø., Birkeland N.K., 2008. Methane oxidation at 55°C and pH 2 by a thermoacidophilic bacterium belonging to the Verrucomicrobia phylum. Proc. Natl. Acad. Sci. 105, 300–304. Kizilova A.K., Dvoryanchikova E.N., Sukhacheva M.V., Kravchenko I.K., Gal’chenko V.F., 2012. Investigation of the communities of the Hot Springs of the Uzon Caldera, Kamchatka, by Molecular Ecological Techniques. Microbiology, 81, 606-613. Pol A., Heijmans K., Harhangi H.R., Tedesco D., Jetten M.S.M., Op den Camp H.J.M., 2007. Methanotrophy below pH 1 by a new Verrucomicrobia species. Nature, 450, 874–878

Methanotrophic activity and diversity of methanotrophs in volcanic-geothermal soils at Pantelleria island (Italy)

Volcanic and geothermal systems emit endogenous gases by widespread degassing from soils, including CH4, a greenhouse gas twenty-five times as potent as CO2. Recently, it has been demonstrated that volcanic/geothermal soils are not only a source of methane, but also sites of methanotrophic activity. Methanotrophs are able to consume 10-40 Tg of CH4 a-1 and to trap more than 50% of the methane degassing through the soils. We report on methane microbial oxidation in the geothermally most active site of Pantelleria island (Italy), Favara Grande, whose total methane emission was previously estimated in about 2.5 Mg a-1 (t a-1). Laboratory incubation experiments with three top-soil samples from Favara Grande indicated methane consumption values up to 59.2 nmol g-1 soil d.w. h-1. One of the three sites, FAV2, where the highest oxidation rate was detected, was further analysed on a vertical soil profile and the maximum methane consumption was measured in the top-soil layer and values >6.23 nmol g-1 h-1 were still detected up to a depth of 13 cm. The highest consumption rate was measured at 37°C, but a still detectable consumption at 80°C (>1.25 nmol g -1 h-1) was recorded. The soil total DNAs extracted from the three samples were probed by PCR using standard proteobacterial primers and newly designed verrucomicrobial primers, targeting the unique methane monooxygenase gene pmoA; the presence of methanotrophs was detected in sites FAV2 and FAV3, but not in FAV1, where harsher chemical-physical conditions and negligible methane oxidation were detected. The pmoA gene libraries from the most active site FAV2 pointed out a high diversity of gammaproteobacterial methanotrophs, distantly related to Methylococcus/Methylothermus genera and the presence of the newly discovered acido-thermophilic methanotrophs Verrucomicrobia. Alphaproteobacteria of the genus Methylocystis were isolated from enrichment cultures, under a methane containing atmosphere at 37°C. The isolates grow at a pH range from 3.5 to 8, temperatures of 18 – 45 °C and consume 160 nmol of CH 4 h-1 ml-1 of culture. Soils from Favara Grande showed the largest diversity of methanotrophic bacteria until now detected in a geothermal soil. While methanotrophic Verrucomicrobia are reported to dominate highly acidic geothermal sites, our results suggest that slightly acidic soils, in high enthalpy geothermal systems, host a more diverse group of both culturable and uncultivated methanotrophs

Duvalo (North Macedonia): A "volcano" without volcanic activity

T he Duvalo locality is located in the SW of the Republic of North Macedonia, in the Ohrid region, near the village of Kosel. It is an area of strong soil degassing, called “volcano” by the local people despite volcanic activity has never been documented in the recent geologic history of the area [1]. A large area (thousands of sqm) shows signs of strong alteration and is devoid of vegetation. Until the 19thcentury sulphur was mined from this area [1]. In August 2019, a campaign of soil CO2 flux measurements and soil gas sampling was made. Duvalo is sometimes referred to as an active geothermal feature but no signs of enhanced geothermal gradient were found and the soil temperatures at 50 cm depth in this campaign were always within the range of local mean air temperatures. Soil CO2 flux values ranged from 1.3 to 59,000 g/m2/d and can be modelled with the overlapping of 3 or 4 flux populations. A possible biological background is estimated in 6.8±1.8 g/m2/d while the other populations are characterized by an anomalous average flux ranging from 180 to 33,000 g/m2/d. The CO2 total emission, estimated both with a statistical and geostatistical approach, provided similar values in the order of 50 t/d. This has to be considered as a minimum value because only areas with evident signs of alteration have been investigated. Nevertheless, the estimated output is quite high for an area unrelated with recent volcanism or geothermal activity. The chemical composition of soil gases shows: CO2 (96.6%), N2 (1.8%), H2S (0.6%) and CH4 (0.3%) as the main gases. The present composition is almost indistinguishable from previous analyses made in 1957 and 1977 [1] pointing to a stability of the system in last decades. The isotope compositions indicate for CO2 (δ13C -0.2 ‰) a pure carbonate rock origin, for CH4 (δ13C -34.4 ‰ and δ2H -166 ‰) a thermogenic origin and for He (R/RA 0.10) a pure crustal origin. The H2S released at Duvalo may be produced by either microbial or thermochemical sulphate reduction favoured by hydrocarbons whose presence can be inferred by the uprise of thermogenic methane. Partial oxidation of H2S during its upflow, producing sulphuric acid, may be responsible of the production of abundant CO2 through dissolution of carbonate rocks. Similar processes have been evidenced also in other parts of North Macedonia [2]. These gases rise up through the N–S trending normal faults bordering the seismically active Ohrid basin graben [3] being released to the atmosphere through the soils of Duvalo “volcano”

Degassing and Cycling of Mercury at Nisyros Volcano (Greece)

Nisyros Island (Greece) is an active volcano hosting a high-enthalpy geothermal system. During June 2013, an extensive survey on Hg concentrations in different matrices (fumarolic fluids, atmosphere, soils and plants) was carried out at Lakki Plain, an intra-caldera area affected by widespread soil and fumarolic degassing. Concentrations of gaseous elemental mercury (GEM), H2S and CO2, were simultaneously measured in both the fumarolic emissions and the atmosphere around them. At the same time, 130 samples of top soils and 31 samples of plants (Cistus Creticus and Salvifolius and Erica Arborea and Manipuliflora) were collected for Hg analysis. Mercury concentrations in fumarolic gases ranged from 10,500 to 46,300 ng/m3, while Hg concentrations in the air ranged from high background values in the Lakki Plain caldera (10-36 ng/m3) up to 7100 ng/m3 in the fumarolic areas. Outside the caldera, the concentrations were relatively low (2-5 ng/m3). The positive correlation with both CO2 and H2S in air highlighted the importance of hydrothermal gases as carrier for GEM. On the other hand, soil Hg concentrations (0.023-13.7 µg/g) showed no significant correlations with CO2 and H2S in the soil gases, whereas it showed a positive correlation with total S content and an inverse one with the soil-pH, evidencing the complexity of the processes involving Hg carried by hydrothermal gases while passing through the soil. Total Hg concentrations in plant leaves (0.010-0.112 μg/g) had no direct correlation with soil Hg, with Cistus leaves containing higher values of Hg respect to Erica. Even though GEM concentrations in air within the caldera are sometimes orders of magnitude above the global background, they should not be considered dangerous to human health. Values exceeding the WHO guideline value of 1000 ng/m3 are very rare (<0.1%) and only found very close to the main fumarolic vents, where the access to tourists is prohibited.PublishedID 47835146A. Geochimica per l'ambiente e geologia medicaJCR Journa

Rapporto tecnico sulle attività di campionamento della “Campagna Oceanografica CISAS_1” Augusta-Priolo 19-23 ottobre 2017

Le attività di campionamento ed acquisizione dati svolte durante la campagna CISAS_1 si inseriscono in seno al progetto “Centro internazionale di studi avanzati su ambiente ed impatti su ecosistema e salute umana (CISAS)” del CNR. Tra gli obiettivi principali del progetto, lo sviluppo di una complessa e decisa azione di ricerca scientifica volta ad una profonda comprensione dei fenomeni di inquinamento ambientale e dei loro risvolti sull’ecosistema e la salute umana. Le aree di indagine del progetto sono rappresentate dai Siti di Interesse Nazionale di Priolo, Milazzo-Pace del Mela e Crotone che, per specificità e modalità di impatto antropogenico sull’ambiente, l’ecosistema e la salute umana, coprono un ampio spettro di tipologie di interesse

Rapporto tecnico sulle attività di campionamento della “Campagna Oceanografica CISAS_2” Crotone 07-12 dicembre 2017

Le attività di campionamento ed acquisizione dati svolte durante la campagna CISAS_2 si inseriscono in seno al progetto “Centro internazionale di studi avanzati su ambiente ed impatti su ecosistema e salute umana (CISAS)” del CNR. L’obiettivo principale del progetto CISAS è la comprensione dei processi e dei meccanismi di trasferimento di alcuni contaminanti convenzionali (metalli pesanti, POPs, radionuclidi, ecc.) e di alcuni contaminanti emergenti (PDBE, composti farmaceutici di nuova generazione, ecc.) dall’ambiente inteso come l’insieme di atmosfera-suoli-acque sotterranee-matrici marine (acque e sedimento) all’ecosistema e all’uomo. Le aree di indagine del progetto sono i Siti di Interesse Nazionale (SIN) di Priolo, Milazzo-Pace del Mela e Crotone che, per specificità e modalità di impatto antropogenico sull’ambiente, l’ecosistema e la salute umana, coprono un ampio spettro di tipologie di interesse. La campagna oceanografica CISAS_ 2 è stata dedicata alla caratterizzazione ambientale del SIN di Crotone, nonché all’identificazione delle sorgenti dei contaminanti la cui distribuzione si ritiene di interesse (per i valori di concentrazione riscontrati nelle diverse matrici ambientali e per livello di tossicità associata agli effetti degli stessi sulla salute dell’ecosistema e dell’uomo) e i pathways di deposizione nelle aree di interesse