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

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

Antonina Lisa Gagliano, Sergio Calabrese, Kyriaki Daskalopoulou, Konstantinos Kyriakopoulos, Marcello Tagliavia, Walter D'Alessandro PII:

S0009-2541(20)30085-1

DOI:

https://doi.org/10.1016/j.chemgeo.2020.119546

Reference:

CHEMGE 119546

To appear in:

Chemical Geology

Received date:

13 August 2019

Revised date:

12 February 2020

Accepted date:

15 February 2020

Please cite this article as: A.L. Gagliano, S. Calabrese, K. Daskalopoulou, et al., Methanotrophy in geothermal soils, an overlooked process: The example of Nisyros island (Greece), Chemical Geology (2018), https://doi.org/10.1016/j.chemgeo.2020.119546

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© 2018 Published by Elsevier.

Journal Pre-proof

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

Antonina Lisa Gagliano1, Sergio Calabrese1-2, Kyriaki Daskalopoulou2,^, Konstantinos

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Kyriakopoulos3, Marcello Tagliavia4,#, Walter D’Alessandro1,*

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Istituto Nazionale di Geofisica e Vulcanologia, sezione di Palermo, via Ugo la Malfa 153, 90146, Palermo, Italy 2 Università degli Studi di Palermo, Department of Earth and Marine Sciences, via Archirafi, 36, 90123, Palermo, Italy 3 National and Kapodistrian University of Athens, Department of Geology and Geoenvironment, Panepistimioupolis, Ano Ilissia, 15784, Athens, Greece 4 Università degli Studi di Palermo, Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, Viale delle Scienze, Ed. 16, 90128 Palermo, Italy

* Corresponding author: [email protected] ^

now at: GFZ German Research Centre for Geosciences, Helmholtzstraße 6/7, Potsdam, Germany

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now at: Consiglio Nazionale delle Ricerche, IRIB, via Ugo la Malfa 153, 90146, Palermo, Italy

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Journal Pre-proof Abstract 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

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the highest CH4 output values (about 0.8 t a-1 from an area of about 30,000 m2) and Phlegeton the

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lowest (about 0.01 t a-1 from an area of about 2500 m2). The total output from the entire geothermal

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system of Nisyros should not exceed 2 t a-1. Previous indirect estimates of the CH4 output at

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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

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flux data because indirect methods totally disregard methanotrophic activity within the soil. Ten soil

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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

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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).

Keywords: methanotrophy, soil degassing, hydrothermal systems, methane output, greenhouse gases

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Journal Pre-proof 1. Introduction

Carbon degassing from soils consists of the release of C-compounds (eg CO, CO2, CH4, light hydrocarbons and many others) from the subsurface to the atmosphere. The different species emitted to the atmosphere reflect several sources, sinks and processes that are involved in their upflow as well as in their interactions with others matrices (solid and fluid). Knowledge on the amount of the compounds reaching the atmosphere is crucial in terms of both environmental impact

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and policy. In this respect, the last centuries have been and still are characterized by an increase in

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the amount of the C-gases (mainly CO2 and CH4) that escape from the subsurface and strongly

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affect the atmospheric chemistry and, as a result, have an impact on global warming. On one hand,

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anthropogenic sources are in some way known and estimable; on the other hand, natural sources of gaseous C-compounds are neither well studied nor well estimated. In the last years, several

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campaigns carried on geothermal and volcanic areas were devoted to the estimation of the amount

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of geogenic CO2 and its impact, and thus many areas were investigated (Chiodini et al., 1998; Cardellini et al., 2003a; Viveiros et al., 2010; Granieri et al., 2010; De Gregorio and Camarda,

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2016). On the contrary, only few areas were investigated for the amount of CH4 released from geothermal and volcanic sources (Cardellini et al., 2003b; D’Alessandro et al., 2009; 2011; Venturi et al., 2019).

At first sight, the estimation of CO2 might appear more relevant due to its higher amount in the atmosphere; however, going deeper into the matter, methane plays a key role in the abrupt climate change. In fact, a single CH4 molecule impacts the atmosphere radiative balance 25 times more than one CO2 molecule (IPCC, 2013). The total amount of CH4 released from geogenic sources is currently not well defined due to many factors that correspond to scarce dataset availability, difficulty in direct measurements of the CH4 flux and interaction with the biosphere that consumes CH4 in the soil (Hanson and Hanson 1996). The latter is the second most important sink of atmospheric methane (IPCC, 2013; Dutaur and Verchot, 2007) with its importance being recently 3

Journal Pre-proof demonstrated in the harsh environment of geothermal and volcanic soils (Castaldi and Tedesco, 2005, Pol et al., 2007, Gagliano et al., 2014, 2016, Venturi et al., 2019). For a long time, volcanic/geothermal soils have been considered inhospitable for methanotrophic microorganisms. However, the presence of microbially driven methane oxidation in different degassing areas (Pantelleria Island and Solfatara di Pozzuoli, Italy; Hell’s Gate, New Zealand; Kamchatka, Russia – Gagliano et al., 2014; 2016; Dunfield et al., 2008; Islam et al., 2007; Pol et al., 2007) has been confirmed by both direct methane consumption measurements and methanotrophs

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isolation from volcanic soils, as well as by soil DNA analyses. In particular, such investigations

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highlighted the presence of methanotrophs (Gagliano et al., 2014), including extremophile ones,

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whose metabolic activity could explain the anomalous behaviours in methane leakages of several

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geothermal/volcanic sites (D’Alessandro et al., 2009; 2011). In cases where CH4 upraise is highly sustained, microbial oxidation efficiency is a complex interplay between microbial communities,

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gas composition and soil characteristics (Gagliano et al., 2014; 2016). At some points, soil

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environmental parameters may not allow methanotrophic activity to take place and as such, methane fluxes to the atmosphere will be unchanged. On the contrary, at other places

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methanotrophy can contribute significantly in the reduction of the net emission of methane (D’Alessandro et al., 2009; 2011).

The Hellenic territory represents one of the most geodynamically active areas in the world, emitting geogenic methane from tectonically active sites, active and passive degassing volcanoes, sedimentary basins, etc. (Daskalopoulou et al., 2019). Studies based on direct methane measurements were recently carried out on diverse anomalously degassing areas of Greece improving our knowledge in the matter (D’Alessandro et al., 2011, Etiope et al., 2013a, 2013b, Daskalopoulou et al., 2018a). Nisyros Island, located in the southern part of the Aegean Sea, is a currently quiescent active volcano with intense fumarolic activity that is generated by the presence of a high enthalpy geothermal system (Marini et al., 1993).

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Journal Pre-proof Etiope et al (2007), using an indirect method, estimated the CH4 release from the hydrothermal system of the island to the atmosphere at 54 t a-1. This estimation was based on its whole on the literature data of the total CO2 output and the CO2/CH4 ratios measured in the main gaseous manifestations (Chiodini et al., 2005). The hydrothermal system of Nisyros though, presents a wide range in the fumarolic CO2/CH4 ratios (from 25 to 1805 in volume – Chiodini et al., 1993; Kavouridis et al., 1999; Brombach et al., 2003; Marini and Fiebig, 2005; Fiebig et al, 2013; 2015; Daskalopoulou et al., 2018a), and therefore, this method cannot be applicable as it introduces great

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uncertainty in the indirect estimation of the CH4 released into the atmosphere. Moreover, another

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important drawback of this method is that it disregards the possible microbial methane oxidation

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occurring within the soil. Thus, in order to overcome problems related to the indirect estimation of

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the CH4 output to the atmosphere we have performed measuring campaigns in the area of Lakki Plain, where the fumarolic activity of the volcanic/geothermal system of Nisyros is concentrated.

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In the present study, we present the results of flux measurements covering the main exhaling area of

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the island. Furthermore, a hydrothermal gas prospection was carried out at the main exhaling area focusing the attention on the soil CH4, CO2 and H2S content. To highlight methanotrophic activity

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in the soils of the studied area, ten top-soil samples were collected and laboratory consumption experiments were performed evidencing microbial oxidation activity in some of the soil samples. The presence of methanotrophic microorganisms has been confirmed by molecular analysis on microbial DNA extracted from soils. Additionally to this work, the total hydrothermal CH4 and CO2 output from Lakki Plain was estimated.

2. Geological setting

The volcano of Nisyros Island belongs to the easternmost volcanic group of the South Aegean Active Volcanic Arc (SAAVA) and is located in the Dodecanese. It was built up during the last 200 ka and is still considered active, though at present in a quiescent status (Vougioukalakis and Fytikas, 5

Journal Pre-proof 2005). Its volcanic activity has been characterized by an early submarine stage, a subaerial conebuilding stage culminating in the formation of a central caldera, and a post-caldera stage that took place when several dacitic-rhyolitic domes were extruded (Keller, 1982). No historical magmatic activity is known on Nisyros and the most recent activity was of hydrothermal character (Marini et al., 1993). Such activity has been concentrated within the caldera, in the southern Lakki Plain and in the southeastern flank of the Lofos dome and formed a series of hydrothermal craters, whose age decrease from southeast to northwest. The last events took place in 1871–1873 and 1887, when they

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partially destroyed the small Lofos dome. A large fumarolic field, the Lakki Plain, is now present in

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this area, mainly within the hydrothermal craters, and is strongly controlled by fracturing along the

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main NW- and NE-trending active fault systems (Papadopoulos et al., 1998, Fig. 1).

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The drilling of two deep wells for geothermal exploitation provided information regarding its high heat flow (Geotermica Italiana, 1983; 1984; Marini et al., 1993) and allowed to identify the

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hydrothermal system consisting of two separate permeable zones, the first at a depth > 1000 m and

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the other at ∼ 200 m. The deep permeable zone has been developed within the intrusive dioritic basement and is confined by an overlying carbonate and volcanic sequence. The deep permeable

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zone has a temperature range of 300–350 °C, and the heat is provided by magmatic fluids (Caliro et al., 2005; Lagios et al., 2007). The deep permeable zone provides vapour to the shallow permeable zone, which has a temperature range of 150–260 °C (Chiodini et al., 2002; Lagios et al., 2007). Finally, Lagios et al. (2007) proposed that a third still shallower reservoir exists at the periphery of the volcanic system and is fed by condensates. Fumaroles occur mainly at some of the phreatic craters such as Stefanos, Phlegeton, Mikros Polybotis, and at the eastern base of the Lofos dome. Temperature measurements at the phreatic craters have been conducted for decades, recording fumarole outlet temperatures of mostly 96–100 °C (Chiodini et al., 2002; Teschner et al., 2007). Smaller fumaroles occur along the flanks and on top of the Lofos dome and at the Kaminakia crater flank. Some degassing vents occur also along the southern and western internal flank of the caldera

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Journal Pre-proof following the NE–SW trends, which represents one of the main fault strikes recognized by Caliro et al. (2005), Lagios et al. (2005) and Tibaldi et al. (2008).

3. Sampling and analytical methods

Previous studies assessed a widespread CO2 degassing in both the fumarolic area and the nearby Lakki Plain (Caliro et al., 2005). The highest CO2 fluxes (> 300 g m2 day-1) were measured within

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the above described hydrothermal craters. Based on these results, we conducted our CH4 flux

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measurements in the craters with the highest CO2 output (Kaminakia, Stefanos, Phlegeton and

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Mikros Polybotis), whilst some additional measurements were added in the fumarolic fields of

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Lofos and Ramos (outside any crater) and in the low flux areas so as to get an insight into background values.

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Methane fluxes were measured with the accumulation chamber method (Livingston and Hutchinson,

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1995; Baciu et al. 2008; D’Alessandro et al. 2009). Measurements were made at 130 sites during three field campaigns (3-6 September 2009, 24 August – 4 September 2010 and 4-6 June 2013)

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with a flux chamber that had a cross-sectional area of 0.07 m2 and a height of 10 cm. The chamber top had also two fixed capillary tubes; one was for the collection of chamber gas samples, whereas the other balanced the inner and outer pressure. Three gas samples were drawn from the headspace in the chamber at fixed intervals after deployment (5, 10 and 15 min). The 20 mL samples were slowly collected (1 min) with the use of a syringe and were injected into a 12 mL pre-evacuated sampling vial (Exetainer

Labco Ltd.), through a three-way valve and a needle. The

overpressured vials were sent to the laboratory for CH4 and CO2 analysis. The flux of CH4 and CO2 from the soil was calculated as the rate of concentration increase in the chamber: = dC/dt V/A

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(1)

Journal Pre-proof where  is the flux of a gas, V is the volume of air in the chamber (m3), A is the area covered by the chamber (m2), C is the chamber concentration of a gas and dC/dt is the rate of concentration change in the chamber air for each gas. Gas concentrations were measured using the GC Perkin Elmer Clarus 500 equipped with Carboxen 1000 columns, Hot Wire and Flame Ionisation detectors with methanizer. The gas samples were injected through an automated injection valve with a 1000 µL loop. Calibration was made with certified gas mixtures. Analytical precision (±1) was always

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better than ±5%. The detection limit for CH4 was about 0.1 µmol mol-1. Volumetric concentrations were converted to mass concentrations accounting for atmospheric pressure and temperature. Flux

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values were expressed as mg m-2 d-1. Positive values indicate fluxes directed from the soil to the

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atmosphere and negative values indicate flow from the atmosphere into the soil or methane

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microbial oxidation.

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The CH4 and CO2 flux datasets were processed following the Sinclair's portioning method, to extract the main data populations and define the threshold values (Sinclair, 1974). Each dataset was

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plotted in a probability plot and the single populations were identified in correspondence of either the inflection points (main populations) or the changes in direction (secondary populations) of the

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curvature on the probability plot, by visual analysis (Table 1 and 2). CH4 and CO2 flux maps were drawn following the stochastic simulation approach. Each dataset was initially converted by normal score transformation to follow a Gaussian distribution. These normal score transformed data were used to compute omnidirectional variograms, and were interpolated with the sequential Gaussian simulations (sGs) method by using the executable “sgsim” of GSLIB (Deutsch and Journel, 1998) and performing 100 equiprobable realizations for each area. Grid resolution for each map and statistics on the realizations are listed in Table 3 and 4. The most probable map for each area was produced averaging the results of the 100 realizations, using the Etype post-processes method. Zonal Statistics on each map, performed by ArcMap 10.3 (ESRI) Spatial Analyst tool, was used to estimate the CH4 and CO2 output above the background threshold value for each area. 8

Journal Pre-proof The percentage relative error (Er%) associated to the CH4 and CO2 flux maps was calculated over the 100 realizations as, (2)

where Ea and Xm represent the absolute error, and the CH4 and CO2 flux mean value was obtained from the post-processing of the 100 realizations, respectively.

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During the three campaigns, the soil gas samples were collected through a Teflon tube of 5 mm ID

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using a tight plastic syringe to avoid air gases contamination and were injected into a 12 mL preevacuated sampling vial (Exetainer Labco Ltd.) with the same method used for the accumulation

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chamber. The overpressured vials were analysed for CH4, H2S and CO2 by using Micro Gas

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Chromatography, with methane detection limit being 10 µmol mol-1 per each module. In cases

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where methane was below this limit, samples were measured again with by using a Perkin Elmer Clarus 500 GC equipped with Carboxen 1000 columns and Flame Ionisation detector as described

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above. Calibration was made with certified gas mixtures. Analytical precision (±1) was always

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better than ±3%. Data on soil gases species were processed by graphical approach to ascertain threshold values and the main data population, and plotted by using GIS platform. At each sampling site the soil temperature was also measured at 20 and 50 cm depth using thermal probes and a digital thermometer. Reading was recorded 10–15 min after the insertion of the thermal probe into the soil in order to achieve thermal equilibrium. Ten top-soil samples (0-3 cm) were collected in June 2013 from Kaminakia and Stefanos craters, the selection of which was based on the preliminary results of the previous campaigns. Soils were sampled using a sterile hand shovel and stored in sterile plastic bags. At each sampling site, temperatures were measured as described above. Soil pH was determined using a pH meter in a mixture of 1/2.5 of soil and distilled deionised water.

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Journal Pre-proof Methane oxidation potential of the soil was analysed as described by Gagliano et al. (2014), transferring 15 g of each air-dried soil sample in a 160-mL glass serum bottle and enriching the bottle headspace with 25% of methane. Bottles were incubated at controlled room temperature (2325°C) and CH4 concentration was measured at the beginning of the experiment and at about 24h intervals for 5 days. Methane concentration inside the vials was measured using GC as above. All incubation experiments were made in duplicate and the results were expressed as average value in ng of CH4 per g of soil (dry weight) per h (ng g-1 h-1). Taking into consideration the above

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experimental conditions, only values above 10 ng g-1 h-1 can indicate significant microbial oxidation

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activity. Lower values are doubtful because they can sometimes be obtained also in sterilised soil

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samples (D’Alessandro et al., 2011).

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Total bacterial DNA was extracted using the FastDNA Spin for Soil Kit (MP Biomedicals, Solon, OH, USA) from 0.5 g of air-dried soil, following the manufacturer’s instructions. The DNA quality

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and concentration was assessed on agarose gel (1%) electrophoresis and by spectrophotometric

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analysis using Nanodrop (NanoDrop ND-1000, Celbio SPA, Milan, Italy) and used as template for the amplification of the 16S rRNA gene with the universal primers pair 27F/1492R (Lane, 1991)

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and of pmoA as detailed below.

In order to enrich for methanotrophic bacteria, 15 g of soil were placed in 125-mL sealed serum bottles in atmosphere supplemented with methane (25%) and incubated at 37° for 2 weeks. Two g of enriched soil crumbles were transferred in 125-mL serum bottles containing 20 mL of low salt mineral medium M3. After 2 weeks incubation at 37°, genomic DNA was extracted from 10 mL M3-CH4 broth culture of each sample by the method described by Sambrook et al. (1989) and was used as template for the amplification of 16S rRNA and pmoA genes. The gene encoding the key methane oxidation enzyme pMMO was detected by amplification of total soil DNA using the primers A189f and A682r (Holmes et al., 1995), targeting the  subunit of the proteobacterial pmoA gene. Polymerase Chain Reaction (PCR) was carried out in a final volume of 50 μL, containing 100 ng of total DNA, 200nM of each oligonucleotide primer, 0.20mM dNTPs, 10

Journal Pre-proof and one unit of recombinant Taq polymerase, (Invitrogen, USA). The PCR program consisted of an initial denaturation step at 95 °C for 4 min, followed by 28 cycles consisting of a denaturation step at 95 °C for 45 s, annealing at 56 °C for 45 s, an extension at 72 °C for 45 s, and a final extension at 72 °C for 5 min.

4. Results

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4.1. Soil flux measurements

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Results of the flux measurements are summarized in Table 1 and Table 2. Values range from –3.4 to 1419 mg m-2 d-1 for CH4 and from 0.1 to 383 g m-2 d-1 for CO2. To get an insight in the methane

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output of the Lakki Plain, we focalised our measurements in restricted exhaling areas: Kaminakia,

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Stefanos, Phlegeton and Mikros Polybotis craters, the southeastern flank of the Lofos dome, the

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base of the caldera rim north of Kaminakia and the Ramos fumarolic area. Some measurements were also made in areas of lower hydrothermal output. The latter sites display the lowest CH 4 flux

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values that have never exceeded 2.6 mg m-2 d-1, whilst frequently present negative values. Of the investigated exhaling areas, those where the most recent activity occurred show the lowest CH4 flux values (Lofos, Mikros Polybotis micros and Phlegeton ~ 0-100 mg m-2 d-1), while fluxes to the older craters reach progressively higher values (Stefanos up to 714 mg m-2 d-1 and Kaminakia up to 1419 m-2 d-1). High CH4 fluxes are sometimes measured along the base of the caldera rim with values reaching 570 mg m-2 d-1 at Ramos. The probability plot of all data indicates a polymodal distribution of the data. The portioning method of Sinclair (1974; 1991) was applied to extract data populations. Three populations (A, B and C, Table 1) were identified from the CH4 flux dataset: “A” comprised values from -33,5 to 30 mg m-2 d-1, “B” from 30 to 100 mg m-2 d-1 and “C” values >100 mg m-2 d-1. Table 1 summarizes the percentage contribution of each population to the whole dataset and the statistical parameters. Two 11

Journal Pre-proof main populations were detected from the CO2 dataset and a third population of very high value was found in the Kaminakia area (Table 2). sGs simulation was applied as described by Cardellini et al. (2003a), by producing E-type maps with the mean expected value for each cell. Methane total output was calculated by the simulation maps at each area (Table 3 and Table 4). Total estimated methane output is at the order of 1.57 t a-1 with the main contribution being that of Kaminakia crater (0.89 t a-1) and the lowest of Phlegeton

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4.2. Soil incubation experiments and molecular analyses

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(0.02 t a-1).

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Specific laboratory assays were set up in order to assess the actual potential of methane consumption by soil microorganisms. In particular, soil incubation experiments were carried out in

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a methane-enriched atmosphere, where methane concentration was measured over time.

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Methane consumption values ranged from 4.3 (KAM1) to 39.7 ng g-1 h-1 (KAM2) (Table 5). Only five soil samples showed consumption values significantly above the value of 10 ng g-1 h-1; four of

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them were from the Kaminakia crater (KAM2 to KAM5) and one from Stefanos (STE4). Three soil samples showed values close to 10 ng g-1 h-1 (STE1, STE2 and STE5), indicating a very low methanotrophic activity, which was negligible in other two samples, namely STE3 and KAM1. The highest temperature, measured in the soil at 20 cm, was recorded in the site STE1 (70 °C) and the lowest in STE4 (27 °C). Soil pH was very low in all sampled sites, varying from 1.37 in STE4 to 3.67 in KAM2. No obvious relationship between methane consumption rates and soil temperature or pH was found, as the highest values referred to the sites STE4 and KAM2 showed the lowest and the highest pH values, respectively, and intermediate temperatures. Conversely, the very low consumption values recorded in STE1-2 were characterized by the highest temperature and very low pH. The same behaviour was found also in KAM1, whose pH and temperature though, were not as extreme. 12

Journal Pre-proof In order to get further indications about the actual presence of methane-consuming bacteria, molecular assays based on the detection of functional genes involved in methane oxidation were carried out. The most striking evidence was expected from analyses on soil DNA. Unfortunately, total DNA samples purified from volcanic soils failed to amplify targeted genes (including the 16S gene, used as control - data not shown), presumably because of the peculiar soil chemical composition, which favours the carry-over of inhibitory compounds in DNA preparations (Yamanouchi et al., 2019 and references therein). Further efforts aiming to improve DNA

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amplificability were unsuccessful.

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In order to overcome such limitation, an alternative experimental strategy was contrived.

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Enrichment cultures were set up in liquid media and DNA was extracted from cultivated cells.

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Cultures were enriched in methanotrophic bacteria, which yielded high quality DNA, useful for downstream analyses. After PCR amplification, we were able to detect pmoA sequences in all

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samples except for STE3, STE5 and KAM1 (Tab. 5). These results matched with previously

4.3. Soil gases

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among various soils.

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observed methane consumption rates, thus confirming the differentiated methanotrophic activity

The portioning method of Sinclair (1974; 1991) was applied to extract data populations from the CH4, CO2 and H2S soil gas concentrations dataset (Table 6). Three main populations have been identified according to the CO2 content; values close to the atmosphere (A), moderate/high content of CO2 (B, up to 47%) and very high (C, up to 75%) content of CO2. The H2S probability plot indicated three different populations, A and B included samples with very low content or absence of H2S, whilst C presented H2S concentrations arriving up to the 17%. Like for CO2 and H2S species, also for CH4 data three populations were identified: A, B, C included very low value, moderate and high value of methane, respectively (details are provided in Table 6). The hydrothermal component 13

Journal Pre-proof in the soil gases is more abundant in Mikros Polybotis, Phlegeton, Stefanos and Kaminakia, but some of the samples from Kaminakia and Stefanos recorded also high contents of atmospheric air (Fig. 2a). In the CO2-CH4-H2S triangular diagram the distribution of these hydrothermal components in the soil gases was evaluated (Fig. 2b). Elevated concentrations of H2S were found at Phlegeton and Mikros Polybotis, close to the fumarolic compositions. Lofos dome and Stefanos showed variable concentrations of H2S going from high concentrations close to the composition of the relative fumaroles, down to concentrations sometimes below the detection limit. Finally,

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samples from Ramos and Kaminakia showed low to very low H2S concentrations. CO2 is the main

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gas in all the samples with the most soluble of the three gases (H2S) being the most depleted and the

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least soluble (CH4) being the most enriched with respect to the fumarolic composition. All

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remaining gases plot almost along the CH4 – CO2 axis. These gases were sampled in a site with lower fumarolic gas flux, where air diffusion from the atmosphere to the ground is less hampered

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than other sites. Moreover, in some sites the CH4 and CO2 relationship is affected by the microbial

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CH4 oxidation. Carbon dioxide and H2S are correlated in the binary diagram in Fig. 2c, and as a general trend, samples with higher CO2 values (> 30%) present elevated H2S concentrations (up to

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18%). On the same graph the main secondary processes affecting the soil gas composition were evidenced: i) dilution by atmospheric air, which does not affect the original CO2/H2S ratio; ii) interaction with groundwater or condensed hydrothermal vapour, which increases the CO2/H2S ratio due to solubility differences of the two gases; iii) oxidation of H2S by atmospheric O2.

4.4. Temperatures

Temperature measurements provide a track of the hydrothermal gases upraise. Higher temperature indicates higher gas flux and an enrichment of the soil gases in the hydrothermal component. Temperature distribution maps at 20 cm depth suggest that temperatures are above 30°C at all the investigated sites with the exception of some points along the western flank of Kamankia crater. 14

Journal Pre-proof Higher temperatures, in the range from 50 to 100 °C were recorded at the Southern part of Stefanos crater, at Phlegeton and Mikros Polybotis. At Kaminakia, Lophos and Ramos, the measured temperatures at 20 cm depth were in the range of 30 to 50 °C (Fig. 3a). Higher temperatures were measured at 50 cm depth, reaching values close to 100 °C almost at all the investigated points of Stefanos, Mikros Polybotis, Phlegeton and Lophos areas (Fig. 3b). Temperature distribution seems to follow a fracturing pattern along the main NW- and NE-trending active fault systems, with the higher temperatures being recorded in correspondence of the main

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fumarolic manifestations; Stefanos, Mikros Polybotis and Phlegeton.

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5.1. Hydrothermal gases prospection

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5. Discussion

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The major hydrothermal activity at Nisyros Island is located at the craters of Stefanos, Kaminakia, Mikros Polybotis, and Phlegeton and is expressed as fumarolic vents. Exhalation of hydrothermal

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gas occurs diffusively along the whole Lakki Plain area though. The fumarolic manifestations are dominated by water vapour that accounts for 91 to 99%. For the remaining gases the composition generally follows the order CO2 > H2S > H2 ≈ N2 ≈ CH4 ≫ He > O2 ≈ CO (Marini and Fiebig, 2005). Methane displays a wider range in composition with respect to the other gases, which is reflected in a wide range in CO2/CH4 ratios (from 25 to 1805 by volume – Daskalopoulou et al., 2019). Processing these literature CO2/CH4 data of fumaroles at Nisyros following the portioning method of Sinclair (1974), at least three statistically different populations can be recognised (Fig 4). The main difference between the fumarolic areas can be summarised in a lower content in H2O and H2S and a higher in CO2 and CH4 in the fumaroles of Kaminakia that, as explained by Marini and Fiebig (2005), was the consequence of the condensation of water vapour close to the surface. Dissolution in the liquid phase can change the relative concentrations of the remaining gases depending on their 15

Journal Pre-proof solubility. This results in depletion of the most soluble species (H2S), a relative increase of CO2 and a strong increase of the less soluble gases such as CH4. Hydrothermal gases originate from a unique geological parental liquid at 350°C in the deeper permeable zone (>1000 m of depth) of the hydrothermal system within the caldera. At shallower depths (200 m), hydrothermal gases intercept a second permeable zone at 150 - 260°C and close to the surface, mainly in the Kaminakia area, shallow groundwater (Pantaleo and Walter, 2014). The interaction with the different aquifers further influences the final composition of the released gases.

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Soil gases were sampled from the sub-surface and were not affected by the air dilution occurring in

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the shallower layer (15 - 0 cm of depth, depending on the permeability). Hydrothermal gas

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composition combined with data on (high) soil temperatures reflects the energy of the hydrothermal

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system and the potential of the hydrothermal gas in shaping of the soil properties. Soil gas samples from the sub-surface showed that the less soluble gases (CH4 and H2) were more abundant in

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Kaminakia and NE part of Stefanos, where, instead, H2S and to a lesser degree also CO2, were

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solubilized with the lowering of the temperature as confirmed by measurements in the soils. On the contrary, enrichment in hydrothermal gases such as CH4, CO2, H2S is found mainly at Phlegeton,

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Mikros Polybotis and in the SW part of the Stefanos Crater. These areas showed the highest temperatures (close to the boiling point) both at 20 and 50 cm depth, whilst in the areas where the fumarolic manifestations take place, native sulphur, sulphate crystals and high level of soil alteration are present. Hydrothermal gases are indicators of the exhaling activity and the amount that escapes through the surface strictly depends on the sites’ characteristics. These include among other: i) soil permeability and porosity that both favour the dilution of the gases from atmospheric air, ii) water content of the soil, iii) chemistry of the soil that depends, but also interacts, with the gases and iv) microbial activity that may have a drastic impact on the hydrothermal gases (Gagliano et al., 2014; 2016). At Lakki Plain the soil chemistry strictly depends on the gases upraise. Daskalopoulou et al. (2014), Venturi et al. (2018) and Gagliano et al. (2019) after analysing the soil properties demonstrated that the chemistry of the soils at Lakki Plain is ruled by both the pH and the 16

Journal Pre-proof sulfur content. They identified a good inverse correlation between S concentration and pH hypothesizing that both parameters strongly depend on the H2S degassing and oxidation at the soil surface. The soil gas prospection was carried on in the same field campaign of Daskalopoulou et al (2014) and Gagliano et al. (2019) and had indicated high values of H2S in the soils gases that were sampled at Stefanos. The highest soil alteration and an assemblage that mostly comprises of hydrothermal alteration minerals (Quartz, Sulfur, Wollastonite, Gypsum, Alunogen and Gismondine) was also found there. The dataset reported in this work confirms the correlation

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between the pH, the S content in the soil and the hydrothermal flux from the soil.

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5.2. Methane output estimation

Methane flux distribution maps were used to estimate the CH4 output of the investigated areas Fig.

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5). The areas presented very different flux values evidencing also very different CH4 outputs.

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Phlegeton shows an output of about 0.01 t a-1 from an area of approximately 3000 m2. Additionally Stefanos releases about 0.42 t a-1 and covers an area of about 35,000 m2, whilst Kaminakia exhales

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about 0.8 t a-1 from an area of approximately 30,000 m2. The flux measurements presented in this work do not cover the whole exhalative area but most likely, the remaining areas would not have added significant amounts of CH4 to the entire output of the geothermal system. In fact, the remaining areas that present the highest hydrothermal fluxes (Mikros Polybotis, Megalos Polybotis, Lofos) and have strong fumarolic emissions, present also characteristics very similar to Phlegeton, whose CH4 flux is low, and thus, their contribution can potentially be of the same order of magnitude. A more substantial contribution could probably derive from the area northeast and southwest of Kaminakia along the caldera border, where soil gases seem to be enriched in CH4 in the same way as at Kaminakia. Nevertheless, previous studies on CO2 soil degassing (Caliro et al., 2005) indicated that in these areas the fluxes tend to decrease rapidly away from the Kaminakia

17

Journal Pre-proof crater, especially in the southwest direction where their possible contribution to the total output is trivial. Summing up the contributions of all the areas where measurements were made we obtain a CH 4 output of 1.39 t a-1 (Table 3). The remaining areas not covered by measurements will not add significantly to the output. Our best estimation of the total CH4 output of the geothermal system of Nisyros is therefore less then 2 t a-1, which is more than one order of magnitude lower than the previous estimation (54 t a-1 - Etiope et al., 2007). The latter was made simply by multiplying the

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estimated average CH4/CO2 ratio of the fumarolic emissions with the total CO2 output as obtained

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by Chiodini et al. (2005). As previously evidenced (D’Alessandro et al., 2009; 2011), part of the

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difference could be attributed to the disregarding of methanotrophic activity within the soils.

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Another source of error in the estimation of Etiope et al. (2007) derives from the great variability in space of the CO2/CH4 ratios of the fumarolic emissions at Nisyros as also evidenced by Marini and

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Fiebig (2005). Such great variability can most possibly introduce a great error in the CO2/CH4 ratio

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used to obtain the total CH4 output. The CO2/CH4 ratio used by Etiope et al. (2007) is indeed low (167 by volume), close to the mean value of the Kaminakia crater, which is by no means

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representative of the whole area. Other strongly degassing areas show all considerably higher mean values accounting for a significant part of the difference in output estimation. A strong temporal variation to explain the great difference in CH4 output estimate cannot even be invoked. Literature data of the CO2 output (Cardellini et al., 2003a; Caliro et al., 2005; Bini et al., 2019) point towards a long time stability of the geothermal system of Nisyros. Repeated CO2 flux measurement campaigns that were made both before (Cardellini et al., 2003a; Caliro et al., 2005) and after (Bini et al., 2019) our CH4 flux measurements, gave values within 10% difference. Furthermore, fumarolic CO2/CH4 ratios do not show seasonal variations (Marini and Fiebig, 2005) and instead showed a fourfold increase induced by a seismic crisis in 1996-1997 that occurred close to the island (Chiodini, 2009). Both the CH4 flux measurements presented in the current study and

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Journal Pre-proof the indirect estimation of Etiope et al. (2007) refer to a period far from the variations caused by the seismic activity and in particular when the CO2/CH4 ratios returned to pre-crisis values.

5.3 Methanotrophic activity

Methane oxidizing bacteria (or methanotrophs) are microorganisms with the ability to use methane as the only source of carbon for energy and biomass production. They represent a subgroup of

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Methylotrophic bacteria, aerobic bacteria that use single-carbon compounds (methane, methanol,

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methylated amines, halothanes, and methylated compounds containing sulfur) as a major source of

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cellular carbon (Hanson and Hanson 1996). Distribution, ecology and activity of methanotrophs are

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important to understand the global methane cycle, as well as to implement management strategies aiming to reduce methane emissions in the atmosphere. Methanotrophs are ubiquitous and play an

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important role in the global carbon cycle, acting as a natural filter between the subsoil and the

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atmosphere. They were isolated from several environments such as soils, wetlands, freshwater, marine sediments, water columns, groundwater, rice paddies, and peat bogs (Kolb and Horn 2012,

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McDonald et al., 2005, Cebron et al., 2007). Methanotrophic bacteria have been overall grouped in three different phyla: Proteobacteria, Verrucomicrobia and NC10 (Hanson and Hanson 1996; Ettwig et al., 2009).

Microbial activity has the potential to oxidize great quantities of CH4 also within the soils of geothermal areas (Castaldi and Tedesco, 2005; Pol et al., 2007). Geochemical clues of this oxidizing activity have been evidenced in many geothermal areas by: i) CH4 consumption in soil incubation experiments (Castaldi and Tedesco, 2005; D’Alessandro et al., 2011; Gagliano et al., 2014), ii) CH4/CO2 ratios lower than the corresponding CH4/CO2 ratios in the gases of the main fumarolic vents (D’Alessandro et al., 2009; 2011; Tassi et al., 2013) and iii) C-isotope fractionation of CO2 and CH4 in the soil gases (Tassi et al., 2015; Venturi et al., 2019). Examples have been

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Journal Pre-proof found in many volcanic/geothermal areas of Italy (Solfatara di Pozzuoli, Pantelleria and Vulcano islands, Vicano–Cimino hydrothermal system, Larderello geothermal field) and Greece (Sousaki). Similar clues can be evidenced also for Nisyros. The ratios between the fluxes of CH4 and of CO2, measured in the different fumarolic areas were always within the range of the CH4/CO2 ratios of the fumaroles of the corresponding area or lower (Fig. 6). The lower values indicate substantial CH4 consumption and are mostly related to sites where the CH4 flux is lower (Fig. 6a) and the soil temperature is decreased (Fig. 6b) favouring methanotrophic activity (Hanson and Hanson, 1996).

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Another indication is that five out of ten soil samples show clear CH4 consumption in the

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incubation experiments (Table 5) while for other three samples consumption is doubtful. Samples of

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the former group and two of the latter were positive for pMMO (particulate Methane Mono-

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Oxygenase), thus confirming the actual presence of methanotrophic bacteria. In particular, the bacterial functional gene pmoA encodes the alpha subunit of pMMO, a membrane (almost entirely

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transmembrane), multimeric, copper-based metalloprotein (~300 kDa α3β3γ3 trimer), which

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represents the key enzyme involved in the first step of biochemical methane oxidation. In methanotrophs, two genetically unrelated MMOs have been reported to occur: soluble MMO

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(sMMO) expressed by a subset of methanotrophs, characterized as iron-based and membrane-bound, and the particulate MMO (pMMO), expressed by nearly all methanotrophs (Ross and Rosenzweig, 2017; Semrau et al., 2010). Moreover, a distantly related pMMO has been described in Verrucomicrobia, as well (Pol et al., 2007), and a molecular method for its detection has been reported (Gagliano et al., 2016). Unfortunately, we couldn’t get evidences about the presence of Verrucomicrobia, as our DNA analyses were successful only for enrichment cultures, but not for soils. As the pmoA presence is a unique, distinctive property of methanotrophic microorganisms, its detection is a reliable and effective system in order to unequivocally unveil their presence. Even if we weren’t be able to carry out direct analyses on soil DNA, pmoA gene was reliably detected in enrichment cultures, thus confirming the presence of methanotrophic bacteria. These 20

Journal Pre-proof results were fully consistent with previous observations; however, further assessment of the bacterial methane consumption might allow a more precise estimation of the actual extent of overall methane fluxes. The CH4 consumption activity has been compared to many environmental parameters of the sampled soils (Table 5). The observed relationships are not so straightforward but greater consumption activities are generally related to higher pH (Fig. 7a) and lower temperature (Fig. 7b), lower H2S (Fig. 7c) and H2 (Fig. 7d) concentrations as well as lower CH4 values (Fig. 7e). Soil pH

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and temperature are important parameters in regulating the methanotrophic activity. The latter

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seems to be restricted to soil samples with pH values higher than 2.5 (Fig. 7a). It is worth noting

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that Sharp et al. (2014) indicates this value as the lower limit for proteobacteria (both Alpha and

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Gamma). Verrucomicrobia can also thrive at lower values (<2.0), but their presence in the soils of Nisyros has not been evaluated. Even though some methanotrophs may be active at temperatures up

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to about 80 °C (Sharp et al., 2014), at Nisyros it seems that microbial oxidation activity seems to be

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limited in temperatures below 50 °C (Fig. 7b).

As for H2S and H2, high concentrations of such electron donors have been hypothesized to favour

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more chemolithotrophic bacterial communities than methane oxidizing ones (Gagliano et al., 2016), especially in aerobic conditions. Moreover, H2S, similarly to other sulfur compounds, has been reported as strong MMO inhibitor (Lee et al., 2015), even if the potential for sulphur oxidation in a methanotroph was recently described in a Methylococcaceae genome (Anantharaman et al., 2016), and a methanotroph was isolated from an H2S-rich environment (Zhang et al., 2016). Taken together, microbial competition between chemolithotrophs and methanotrophs, as well as the possible inhibition of the latter group by toxic compounds, might further account for the inverse correlation observed between presence of methanotrophic bacteria and H2S concentrations.

Conclusions

21

Journal Pre-proof Flux measurements confirm that Nisyros, like other active volcanic/geothermal systems, releases significant amounts of CH4 (~1.4 t a-1) through diffuse soil degassing. The previous estimate (54 t a1

), made at Nisyros by cross-correlating CO2 output data with the average CO2/CH4 ratio of its

gaseous manifestations (Etiope et al., 2007), is more than one order of magnitude larger. Although volcanic/geothermal areas are significant sources of GHG to the atmosphere, this study further confirms that their contribution has been previously overestimated as possibly also other natural sources (Ferré et al., 2020) and underscores the need of CH4 flux measurements in the field. Two

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major drawbacks affect the indirect estimation of the CH4 output. The first one is the large range in

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CO2/CH4 ratios that characterise the gases released the fumaroles of the island, which introduces a

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great uncertainty in calculating indirectly the total CH4 output. The second one, probably the most

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important, is that methanotrophic activity in the soil is totally disregarded. The incubation experiments made on soils collected in the studied area evidenced for most of the samples a small

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but significant CH4 consumption activity (4.3 – 39.7 ng g-1 h-1). Considering a bulk soil density of

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1000 kg m-3 and a depth of 1 cm for the most elevated methanotrophic activity in the soil (e.g. Gagliano et al. 2014), these values were expressed in kg m-2 a-1 (from 0.38 to 3.48) for a better

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comparison with annual CH4 output data. In fact, even the lowest value, extended to the whole studied area, may easily account for an order of magnitude difference in CH4 output, which seems to be annually lacking with respect to the indirect estimate of Etiope et al. (2007). Molecular analyses aiming to detect a methanotrophs-specific gene (pmoA) confirmed the presence of such a functional gene involved in the methanotrophic activity in the soils with the highest CH4 consumption activity. Soils in geothermal areas can be considered extreme environments and many factors may result limiting for methanotrophic activity. Although our dataset is limited, it indicates that microbial CH4 oxidation generally occurs in soils with less than 50°C temperature, pH higher than 2.5 and low H2S and H2 concentrations (at most tens of ppm) in the soil gas. Until now we have no data on possible species thriving in the soils of Nisyros but they have to be considered extremophiles. 22

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Acknowledgements We are grateful to Jens Fiebig, Mika Gousgouni, Artemis Kontomichalou and Silvia Milazzo for their help in the field. We kindly acknowledge the owner of the Volcano Café, Mr. Sideris Kontogiannis, for his logistical support to all volcanologists working in the Nisyros Caldera (and also for many beers spent for free and for the delightful music of his Cretan Lyra at dusk). All chemical analyses were made at the laboratories of the INGV of Palermo and we are grateful to all

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the laboratories’ responsible and technicians: F. Grassa, M. Martelli and F. Salerno.

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Microbiological analyses were made at the University of Palermo (STEBICEF). This research did

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not receive any specific grant from funding agencies in the public, commercial, or not-for-profit

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sectors.

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probability plots revisited. J. Geochem. Explor. 41, 1-22. Tassi, F., Nisi, B., Cardellini, C., Capecchiacci, F., Donnini, M., Vaselli, O., Avino, R., Chiodini, G., 2013. Diffuse soil emission of hydrothermal gases (CO2, CH4, and C6H6) at Solfatara crater (Campi Flegrei, southern Italy). Appl. Geoc. 35, 142-153. Tassi, F., Venturi, S., Cabassi, J., Vaselli, O., Gelli, I., Cinti, D., Capecchiacci, F., 2015. Biodegradation of CO2, CH4 and volatile organic compounds (VOCs) in soil gas from the Vicano–Cimino hydrothermal system (central Italy). Org. Geochem. 86, 81–93 Yamanouchi, K., Takeuchi, M., Arima, H., Tsujiguchi, T., 2019. Development of a method to extract protozoan DNA from black soil, Parasite Epidemiology and Control, e00081, https://doi.org/10.1016/j.parepi.2018.e00081. Venturi, S., Tassi, F., Vaselli, O., Vougioukalakis, G.E., Rashed, H., Kanellopoulos, C., Caponi, C., Capecchiacci, F., Cabassi, J., Ricci, A., Giannini, L., 2018. Active hydrothermal fluids circulation triggering smallscale collapse events: the case of the 2001–2002 fissure in the Lakki Plain (Nisyros Island, Aegean Sea, Greece). Nat. Hazards 93, 601–626 27

Journal Pre-proof Venturi, S., Tassi, F., Magi, F., Cabassi, J., Ricci, A., Capecchiacci, F., Caponi, C., Nisi, B., Vaselli, O., 2019. Carbon isotopic signature of interstitial soil gases reveals the potential role of ecosystems in mitigating geogenic greenhouse gas emissions: Case studies from hydrothermal systems in Italy. Sci. Total Environ. 655, 887-898 Viveiros, F., Cardellini, C., Ferreira, T., Caliro, S., Chiodini, G., Silva, C., 2010. Soil CO2 emissions at Furnas volcano, São Miguel Island, Azores archipelago: volcano monitoring perspectives, geomorphologic studies, and land use planning application J. Geophys. Res. 115, B12208 Volentik, A.C.M., Principe, C., Vanderkluysen, L., Hunziker, J.C., 2005. Explanatory notes on the

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“Geological map of Nisyros Volcano (Greece)”. In: Hunziker, J.C., Marini, L. (eds.) The geology, geochemistry and evolution of Nisyros volcano. Implications for the volcanic hazard.

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Mém. Géol. (Lausanne) 44, 7-25.

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Vougioukalakis, G.E., Fytikas, M., 2005. Volcanic hazards in the Aegean area, relative risk evaluation, monitoring and present state of the active volcanic centers. In: Fytikas, M.,

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Vougioukalakis, G.E. (Eds.), The South Aegean Active Volcanic Arc. Developments in Volcanology, 7, 161–183.

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Zhang, W., Ge, X., Li, Y.-F., Yu, Z., Li, Y., 2016. Isolation of a methanotroph from a hydrogen

Captions

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838-844.

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sulfide-rich anaerobic digester for methanol production from biogas. Process Biochem. 51(7),

Figure 1 – a) Map of the Aegean area with the position of the island of Nisyros; b) simplified geological map of the island of Nisyros (modified after Volentik et al., 2005); c) view from the NE of the Lakki Plain [line X-Y in (b)] within the caldera of Nisyros with the main fumarolic areas evidenced.

Figure 2 – a) Binary diagram of the main dry fumarolic gases (CO2, CH4, H2 and H2S) vs. the main atmospheric gases (N2 and O2) evidencing a mixing line between these two end-members in the soil gases of the study area; b) CO2 - CH4 - H2S triangular diagram showing strong differences in the fumarolic component of the soil gases of the study area; c) CO2 vs. H2S binary diagram evidencing 28

Journal Pre-proof the main secondary processes affecting soil gases starting from the original fumarolic gas composition on the top right of the graph. Blue line: dilution by atmospheric air; Green line: interaction with liquid water and consequent chemical fractionation due to solubility differences; Red line: H2S oxidation.

Figure 3 – Distribution maps of the soil temperatures measured at 20 cm (a) and 50 cm depth (b) in

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the Lakki Plain area.

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Figure 4 – Probability plot of the logarithm of the CO2/CH4 ratio measured in the fumarolic gases at

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Nisyros (data from Daskalopoulou et al., 2019). A wide range of values and three main statistically

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different populations (evidenced by the lines A, B and C) can be recognised. The populations have

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been defined following the method of Sinclair (1991).

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Figure 5 – Distribution maps of soil CO2 (a) and CH4 (b) flux measurements at Lakki Plain.

and Ramos (5).

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Enlarged views are shown for the areas of Stefanos (1), Kaminakia (2), Lophos (3), Phlegeton (4)

Figure 6 – Binary diagrams of CH4/CO2 ratios vs. CH4 (a) and vs. soil temperature at 20 cm (b). The three coloured bands refer to the range of CH4/CO2 ratios measured in three of the fumarolic areas: Kaminakia (yellow), Stefanos (blue) and Phlegeton (red).

Figure 7 – Binary diagrams of CH4 consumption vs. soil pH (a), soil temperature at 20 cm depth (b), H2S (c) and H2 (d) soil gas concentrations at 50 cm depth and CH4 fluxes (e). Blue dots refer to soil samples where PCR analysis resulted positive for the pmoA bacterial functional gene, while red dots to negative samples.

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Journal Pre-proof Declaration of interests

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☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Table 1 - Estimated parameters of partitioned populations (POP) for methane flux measurements at Lakki Plain Study area

POP.

n° data

%

area

min

m2

max

mean

std dev.

mg m-2 day-1

A

12

100

3192

0.0

28.1

9.93

9.77

A

5

100

1320

-2.38

32.8

16.9

13.8

A

14

70

-1.48

19.50

6.49

6.91

B

6

30

40.4

931

230.25

345

A

9

43

0.30

8.50

4.19

2.74

B

7

33

20.0

85.0

51.1

23.6

C

5

24

110

714

277

259

A

28

55

B

15

29

C

8

16

A

5

50

Stefanos

Kaminakia

10996

34864

30904

-4.33

20.4

2.44

6.77

42.5

238

139

69.7

247

1420

851

459

-33.5

4.31

-5.33

18.2

12.6

570

221

243

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Lofos

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Phlegeton Mikros Polybotis

2028 B

5

50

83304

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Tot.

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Ramos

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Positive flux values indicate a release of CH4 into the atmosphere, while negative values an uptake of CH4 from the atmosphere.

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Table 2 – Estimated parameters of partitioned populations (POP) for carbon dioxide flux measurements at Lakki Plain Study area

POP.

n° data

%

area

min

max

2

m 67

B

4

33

Mikros Polybotis

A

5

100

Lofos

A

15

75

B

5

25

A

11

52

B

10

48

A

10

25

B

23

58

C

7

17

A

5

50

B

5

50

Stefanos

Kaminakia

Ramos

3192

0.10

23.3

8.69

10.3

35.0

93.3

64.7

26.4

1320

0.57

43.6

21.7

19.2

10996

0.10

23.5

8.52

8.07

27.8

70.6

48.9

16.7

0.51

23.33

8.03

6.96

383

88.7

108

34864

30.3 30904

2028

83304

21.03

9.92

5.88

27.6

81.7

48.6

17.8

112

261

185

57.9

0.10

19.3

7.00

7.56

33.3

156

80.2

52.1

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Tot.

0.10

of

8

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A

std dev.

-1

g m day

-p

Phlegeton

mean -2

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Journal Pre-proof Table 3 - Methane output obtained from simulations. area

2m x 2m

m2

Stefanos

8716

34864

0.30

714

32.9

28.8

0.42

±0.012

Ramos

507

2028

-25.8

558

108

104

0.08

±0.007

Phlegeton

798

3192

0.00

28.1

9.22

8.11

0.01

±0.003

Kaminakia

7726

30904

-3.55

1419

71.2

144

0.80

±0.147

10996

-1.48

931

18.8

31.0

0.08

±0.002

1.39

±0.171

mean

st dev

total output

mg m-2 d-1

t a-1

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Tot

2749

max

na

Lophos

min

Er

n° of cells

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Area

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Table 4 – carbon dioxide output obtained from simulations. Area

Stefanos

n° of cells

area

min

max

2

mean -2

st dev

total output

-1

t a-1

2m x 2m

m

gm d

8716

34864

1.30

235

23.0

15.8

269

153

46.8

25.8

34.7

±0.9

Ramos

507

2028

2.06

Phlegeton

798

3192

0.20

91.9

23.2

24.2

27.0

±7.6

Kaminakia

7726

30904

4.60

184

46.1

20.3

520

±26

Lophos

2749

10996

0.10

69.1

16.9

13.6

68.1

±12

920

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81984

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Tot

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Table 5 – Geographic position of the soil samples collected for CH4 consumption experiments and microbiological analysis and the relative environmental parameters. X UTM

Y UTM

Zone 35S 515848 515074 515103 515036 515051 515472 515505 515441 515081 515470

4048154 4048077 4048158 4048126 4048103 4048026 4048072 4048080 4048120 4048107

ng g-1 h-1

°C

4.3 8.7 9.5 9.8 10.9 15.5 22.6 32.8 37.7 39.7

39 45 44 70 70 37 36 42 27 37

pH

2.72 1.65 1.89 1.40 1.66 3.26 2.81 3.07 2.73 3.67

H2S

H2

CH4 flux

ppm

ppm

mg m-2 d-1

89 5100 147 962 <10 <10 <10 <10 <10 <10

4 2277 295 1360 137 7 5 41 71 8

3.09 5.10 119 34.0 1.70 2.18 1.47 -1.27 714 0.90

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KAM1 STE3 STE5 STE1 STE2 KAM4 KAM3 KAM5 STE4 KAM2

CH4 Temp. consumption

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Sample

pmoA

+ + + + + + +

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Samples ordered by increasing CH4 consumption. Temperature measured on site at 20 cm depth.

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pmoA gene tests were performed on soil enrichments.

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Table 6 – Chemical composition of the soil gas samples Populations

n data

min

max

Mean

dev st

All Data CH4

102

0.000

1.914

0.171

0.285

A

35

0.000

0.002

0.001

0.001

B

53

0.006

0.304

0.128

0.968

C

14

0.463

1.914

0.737

0.402

All Data H2S

102

0.000

17.83

2.924

5.260

A

36

0.000

0.000

0.000

0.000

B

35

0.000

0.719

0.069

0.167

C

31

1.150

17.83

9.464

5.442

All data CO2

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%

102

0.280

75.27

24.08

A

9

0.280

1.600

0.801

23.22

B

75

2.069

47.35

16.41

11.73

C

18

53.84

75.27

67.65

6.938

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0.495

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Highlights The total geothermal CH4 output of Nisyros, based on flux measurements, is < 2 t/d Indirect methods, disregarding methanotrophic activity, overestimate CH4 output Microbial CH4 oxidation in the geothermal soils of Nisyros has been accertained Methanotrophic activity is highest when soil temperature is < 50°C and pH is > 2.5 Methanotropy is effective in limitig CH4 emissions also in this extreme environment

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7