The importance of methanotrophic activity in geothermal soils of Pantelleria island (Italy)

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 g-1 dry soil per hour while soils collected outside the geothermal area consume less than 6 ng g-1 h-1. The maximum consumption was measured in the shallowest part of the soil profile (1-3 cm) and high values (>100 ng g-1 h-1) 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 g-1 h-1) 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

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 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

Geochemical survey of Levante Bay, Vulcano Island (Italy), a natural laboratory for the study of ocean acidification

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Hydrogen and Carbon Monoxide-Utilizing Kyrpidia spormannii Species From Pantelleria Island, Italy

Volcanic and geothermal areas are hot and often acidic environments that emit geothermal gasses, including H2, CO and CO2. Geothermal gasses mix with air, creating conditions where thermoacidophilic aerobic H2- and CO-oxidizing microorganisms could thrive. Here, we describe the isolation of two Kyrpidia spormannii strains, which can grow autotrophically by oxidizing H2 and CO with oxygen. These strains, FAVT5 and COOX1, were isolated from the geothermal soils of the Favara Grande on Pantelleria Island, Italy. Extended physiology studies were performed with K. spormannii FAVT5, and showed that this strain grows optimally at 55\ub0C and pH 5.0. The highest growth rate is obtained using H2 as energy source (\u3bcmax 0.19 \ub1 0.02 h\u20131, doubling time 3.6 h). K. spormannii FAVT5 can additionally grow on a variety of organic substrates, including some alcohols, volatile fatty acids and amino acids. The genome of each strain encodes for two O2-tolerant hydrogenases belonging to [NiFe] group 2a hydrogenases and transcriptome studies using K. spormannii FAVT5 showed that both hydrogenases are expressed under H2 limiting conditions. So far no Firmicutes except K. spormannii FAVT5 have been reported to exhibit a high affinity for H2, with a Ks of 327 \ub1 24 nM. The genomes of each strain encode for one putative CO dehydrogenase, belonging to Form II aerobic CO dehydrogenases. The genomic potential and physiological properties of these Kyrpidia strains seem to be quite well adapted to thrive in the harsh environmental volcanic conditions

Methylacidimicrobium thermophilum AP8, a Novel Methane- and Hydrogen-Oxidizing Bacterium Isolated From Volcanic Soil on Pantelleria Island, Italy

The Favara Grande is a geothermal area located on Pantelleria Island, Italy. The area is characterized high temperatures in the top layer of the soil (60\ub0C), low pH (3\u20135) and hydrothermal gas emissions mainly composed of carbon dioxide (CO2), methane (CH4), and hydrogen (H2). These geothermal features may provide a suitable niche for the growth of chemolithotrophic thermoacidophiles, including the lanthanide-dependent methanotrophs of the phylum Verrucomicrobia. In this study, we started enrichment cultures inoculated with soil of the Favara Grande at 50 and 60\ub0C with CH4 as energy source and medium containing sufficient lanthanides at pH 3 and 5. From these cultures, a verrucomicrobial methanotroph could be isolated via serial dilution and floating filters techniques. The genome of strain AP8 was sequenced and based on phylogenetic analysis we propose to name this new species Methylacidimicrobium thermophilum AP8. The transcriptome data at \u3bcmax (0.051 \ub1 0.001 h 121, doubling time ~14 h) of the new strain showed a high expression of the pmoCAB2 operon encoding the membrane-bound methane monooxygenase and of the gene xoxF1, encoding the lanthanide-dependent methanol dehydrogenase. A second pmoCAB operon and xoxF2 gene were not expressed. The physiology of strain AP8 was further investigated and revealed an optimal growth in a pH range of 3\u20135 at 50\ub0C, representing the first thermophilic strain of the genus Methylacidimicrobium. Moreover, strain AP8 had a KS(app) for methane of 8 \ub1 1 \u3bcM. Beside methane, a type 1b [NiFe] hydrogenase enabled hydrogen oxidation at oxygen concentrations up to 1%. Taken together, our results expand the knowledge on the characteristics and adaptations of verrucomicrobial methanotrophs in hydrothermal environments and add a new thermophilic strain to the genus Methylacidimicrobium

Estimation of the geogenic carbon degassing of Greece

Greece belongs to the most geodynamically active regions of the world and as such it has to be considered an area of intense geogenic degassing. Here we review all the papers already published in the scientific literature on both the geochemistry of gas manifestations and the CO2 and CH4 release, in an attempt to obtain the first nationwide inventory of the natural output of these carbon gases in Greece. The best studied and most exhaling area is the South Aegean Active Volcanic Arc (SAAVA), which releases more than 1.3 × 105 tons of CO2 per year. Continental Greece, on the contrary, is much less studied but may release CO2 in the same order of magnitude in its eastern-central and northern parts. The western and south-western parts of Greece are conversely the main areas in which methane and higher hydrocarbons degas. Methane output of Greece is much less constrained, but the presence of one of the biggest thermogenic gas seepages of Europe, which releases about 200 tons of CH4 per year to the atmosphere, underscores its potentially high contribution. © 2019 Elsevier Lt

The importance of methanotrophic activity in geothermal soils of Pantelleria island (Italy)

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 g-1 dry soil per hour while soils collected outside the geothermal area consume less than 6 ng g-1 h-1. The maximum consumption was measured in the shallowest part of the soil profile (1-3 cm) and high values (>100 ng g-1 h-1) 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 g-1 h-1) 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.PublishedVienna, Austria4.5. Studi sul degassamento naturale e sui gas petroliferiope

Methane efflux from the soil and methanotrophic activity in volcanic-geothermal areas: Examples from Italy and Greece

Methane plays an important role in the Earth’s atmospheric chemistry and radiative balance being the most important greenhouse gas after carbon dioxide. It 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. Due to the fact that methane flux measurements are laboratory intensive, very few data have been collected until now and the contribution of this source has been often indirectly estimated. Both the Italian and the Hellenic territories are geodynamically very active with many volcanic and geothermal areas. Here we report on methane flux measurements made at Pantelleria (Italy), Sousaki and Nisyros (Greece). The total outputs of these three systems are about 10, 19 and 2 t a-1 respectively. These figures are up to one order of magnitude lower than those obtained through indirect estimations. At the global scale, microbial oxidation in soils contributes to the total removal of methane from the atmosphere. Environmental conditions in the soils of volcanic/geothermal areas (i.e. low pH, high temperature, 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 thermo-acidophilic Verrucomicrobia. Here we present the results of laboratory incubation experiments on soil samples collected at the main exhalative areas that highlighted methanotrophic activity also at Pantelleria and Sousaki. Soil metagenomic DNA was extracted from some of the Pantelleria samples and analysed using Temporal Temperature Gradient Electrophoresis (TTGE) of the amplified Bacterial 16S rRNA gene in order to evaluate the total bacterial diversity. Soil DNA amplification with primers targeting Proteobacterial and Verrucomicrobial methane monooxygenase genes (pmmo) revealed the presence of methanotrophs affiliated to both phyla up to a depth of 11 cm and a temperature of 80°C. The diversity of proteobacterial methanotrophs was investigated by creating a clone library of the amplified methane mono-oxygenase encoding gene, pmmoA. The clone sequences are close to those of uncultured type I methanotrophic proteobacteria. An attempt to isolate methanotrophs was carried out on soils from Pantelleria, sampled at different depths, by enrichment cultures on a mineral medium in a methane-enriched atmosphere. No isolates were obtained from enrichments carried out at 65°C while incubation at 37°C allowed to isolate a few methanothropic strains that were identified as Methylocystis spp

Methanotrophy in geothermal soils, an overlooked process: The example of Nisyros island (Greece)

A multidisciplinary field campaign was carried out at Nisyros Island (Greece). Hydrothermal gases were sampled and analysed, and CH4 and CO2 fluxes from the soils were measured with the accumulation chamber method. The sampling area (Lakki plain) covers an area of about 0.08 km2, and includes the main fumarolic areas of Kaminakia, Stefanos, Ramos, Lofos and Phlegeton. Flux values measured at 130 sites range from −3.4 to 1420 mg m−2 d−1 for CH4 and from 0.1 to 383 g m−2 d−1 for CO2. The fumarolic areas show very different CH4 degassing patterns, Kaminakia showing the highest CH4 output values (about 0.8 t a−1 from an area of about 30,000 m2) and Phlegeton the lowest (about 0.01 t a−1 from an area of about 2500 m2). The total output from the entire geothermal system of Nisyros should not exceed 2 t a−1. Previous indirect estimates of the CH4 output at Nisyros, based on soil CO2 output and CH4/CO2 ratios in fumarolic gases, were more than one order of magnitude higher. The present work further underscores the utmost importance of direct CH4 flux data because indirect methods totally disregard methanotrophic activity within the soil. Ten soil samples were collected for CH4 consumption experiments and for metagenomic analysis. Seven of the soil samples showed small but significant CH4 consumption (up to 39.7 ng g−1 h−1) and were positive for the methanotrophs-specific gene (pmoA) confirming microbial CH4 oxidation in the soil, notwithstanding the harsh environmental conditions (high temperature and H2S concentrations and low pH). © 2020 Elsevier B.V

Gas geochemistry and CO2 output estimation at the island of Milos, Greece

Twenty gas samples have been collected from the natural gas manifestations of Milos Island, the majority of which is found underwater along its coast. Furthermore, three anomalous degassing fumarolic areas (Kalamos, Paleochori and Adamas) have been recognized on-land. Almost all the gases are CO2-dominated with CO2 ranging from 88 to 99% vol for the samples taken underwater, while the on-land manifestations show a wider range (15–98%) due to air contamination. Methane reaches up to 1.0% vol, H2 up to 3.2% vol and H2S up to 3.5% vol indicating a hydrothermal origin of the gases. The isotope composition of He points out to mantle contributions up to 45%, while the C-isotope composition of CO2 (from −1.9 to +1.3‰ vs. V-PDB with most of the values around −0.5‰) suggests a prevailing limestone origin. Isotope composition of CH4, ranging from −18.4 to −5.0‰ vs. V-PDB for C and from −295 to +7‰ vs. V-SMOW for H, points to a geothermal origin with sometimes evident secondary oxidation processes. Additionally, CO2-flux measurements showed high values in the three fumarolic areas (up to 1100, 1500 and 8000 g/m2/d at Kalamos, Paleochori and Adamas respectively) with the highest CO2-flux values (up to about 23,000 g/m2/d) being measured in the sea at Kanavas with a floating chamber. The south-western part of the island was covered with a lower density prospection revealing only few anomalous CO2 flux values (up to 650 g/m2/d). The total output of the island (30.5 t/d) is typical of quiescent closed-conduit volcanoes and comparable to the other volcanic/geothermal systems of the south Aegean active volcanic arc (Nisyros, Kos, Nea Kameni, Methana and Sousaki). © 2018 Elsevier B.V