Microbiological evidences of methanotriphic activity in the soils of the geothermal area of Pantelleria island (Italy)

Methane plays an important role in the Earth\u2019s atmospheric chemistry and radiative balance being the second most important greenhouse gas after carbon dioxide. Methane is released to the atmosphere by a wide number of sources, both natural and anthropogenic, with the latter being twice as large as the former. It has recently been established that significant amounts of geological methane, produced within the Earth\u2019s crust, are currently released naturally into the atmosphere. Active or recent volcanic/geothermal areas represent one of these sources of geological methane. Microbial oxidation in soils contributes for about 3-9% to the total removal of CH4 from the atmosphere. Recent studies evidenced methanotrophic activity also in soils of volcanic/geothermal areas notwithstanding their harsh environmental conditions (high temperatures, low pH and high concentrations of H2S and NH3). The purpose of our study was to verify the methanotrophic potential and the bacterial diversity of the soils of the main geothermal area of Pantelleria island (Italy). Laboratory incubation experiments with soil samples collected at the main exhalative area showed methane consumption values of up to 9500 ng per g of dry soil per hour while soils collected outside the geothermal area less than 6 ng/g/h. Geothermal soils showed their maximum methane consumption in the shallowest part of the soil profile (0-3 cm) mantaining high values (>100 ng/g/h) at least up to dephts of 15 cm. Furthermore they showed the maximum consumption at about 37\ub0C, showing a still recognizable consumption (>20 ng/g/h) at 80\ub0C, and a positive correlation with the methane concentration in the incubation atmosphere. These results can be considered a clear evidence of the presence of methanotrophs. In order to evaluate the bacterial diversity, soil metagenomic DNA was extracted from Le Favare and analysed using a Temporal Temperature Gradient Electrophoresis (TTGE) analysis of the amplified Bacterial 16S rRNA gene. The amplification of metagenomic DNA with primers targeting Proteobacterial and Verrucomicrobial MMO (methane monooxygenase) genes is in progress. Enrichment cultures on a mineral medium in a CH4-enriched (25%) atmosphere allowed to isolate different strains that are under characterization

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

A modified culture medium for improved isolation of marine vibrios

Marine Vibrio members are of great interest for both ecological and biotechnological research, which often relies on their isolation. Whereas many efforts have been made for the detection of food-borne pathogenic species, much less is known about the performances of standard culture media toward environmental vibrios. We show that the isolation/enumeration of marine vibrios using thiosulfate-citrate-bile salts-sucrose agar (TCBS) as selective medium may be hampered by the variable adaptability of different taxa to the medium, which may result even in isolation failure and/or in substantial total count underestimation. We propose a modified TCBS as isolation medium, adjusted for marine vibrios requirements, which greatly improved their recovery in dilution plate counts, compared with the standard medium. The modified medium offers substantial advantages over TCBS, providing more accurate and likely estimations of the actual presence of vibrios. Modified TCBS allowed the recovery of otherwise undetected vibrios, some of which producing biotechnologically valuable enzymes, thus expanding the isolation power toward potentially new enzyme-producers Vibrio taxa. Moreover, we report a newly designed Vibrio-specific PCR primers pair, targeting a unique rpoD sequence, useful for rapid confirmation of isolates as Vibrio members and subsequent genetic analyses

Methanotrophic activity and diversity of methanotrophs in volcanic geothermal soils at Pantelleria (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 or geothermal soils are not only a source of methane, but are 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 (Italy), Favara Grande, whose total methane emission was previously estimated at about 2.5Mga−1 (t a−1). Laboratory incubation experiments with three top-soil samples from Favara Grande indicated methane consumption values of 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, the maximum methane consumption was measured in the topsoil layer, and values greater than 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 DNA extracted from the three samples was probed by Polymerase Chain Reaction (PCR) using standard proteobacterial primers and newly designed verrucomicrobial primers, targeting the unique methane monooxygenase gene pmoA; the presence of methanotrophs was detected at sites FAV2 and FAV3, but not at FAV1, where harsher chemical–physical conditions and negligible methane oxidation were detected. The pmoA gene libraries from the most active site (FAV2) pointed to a high diversity of gammaproteobacterial methanotrophs, distantly related to Methylocaldum-Metylococcus genera, and the presence of the newly discovered acido-thermophilic Verrucomicrobia methanotrophs. 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 of 3.5 to 8 and temperatures of 18–45 C, and consume 160 nmol of CH4 h−1 mL−1 of culture. Soils from Favara Grande showed the largest diversity of methanotrophic bacteria detected until now in a geothermal soil. While methanotrophic Verrucomicrobia are reported as dominating 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

The occurrence of mycorrhizal fungi in Betula aetnensis Raf. roots: from ecological role to conservation strategies

Betula aetnensis Raf. is an endemic tree species of particular conservation value. It only thrives in the north-eastern slopes of Mount Etna (Sicily), from 1200 to 2100 m a.s.l. This pioneer plant is able to begin primary succession on nutrient-poor and water-limited soils (C = 0.17%; N = 0.05 \u2030; P2O5 = 4.1 ppm), where beneficial mycorrhizal fungi (MF) may play a crucial role. In order to investigate MF role in B. aetnensis, plant roots from natural sites and nursery grown specimens were analyzed for both ectomycorrhizal and endomycorrhizal structures. Typical structures of both symbiosis were detected by root staining and morphological observations. Ectomycorrhizae (EM) were more abundant in natural sites ( 4888%) than in nursery ( 4877%). Clear morphological differences in the EM root tips suggest the occurrence of different fungal species. About 50% of roots had arbuscular structures, both in natural habitats and nursery. The community structure of EM and AM fungal symbionts was characterized by DGGE analysis. Mycorrhizal dependence trials are in progress to elucidate the relative importance of ectoand endomycorrhizal symbionts for this endemism, whose conservation could be strongly linked to mutualistic associations established at root level

Blue biotechnology: enhancement of bioremediation using bacterial biofilms on biodegradable scaffolds

Petroleum hydrocarbons are still the most threatening environmental pollutants. A promising non invasive and low-cost technology for the treatment of contaminated sites is based on bioremediation by biodegrading microorganism endowed with catabolic ability towards oil and derivatives. New methods are needed to enhance and optimize natural biodegradation, such as the immobilization of hydrocarbons degraders in many types of supports. We developed a scaffold-bacteria bioremediation system to clean up oil contamination based on degradable 3D scaffolds. The polycaprolactone component is biodegradable, produced in the melt, i.e. at low cost and without the use of toxic solvents. The biofilm is made of highly performing HC-degrading bacteria such as the marine hydrocarbonoclastic bacteria (HCB) (1) or solid n-alkane degrading Actinobacteria (2, 3). The bacterial biofilm is observed within the whole structure of scaffold using scanning electron microscopy. The bioremediation efficiency of such systems was tested on crude oil by GC-FID analysis and compared whit planktonic cells. The biofilms formation was a promoting factor for biodegradation showing hydrocarbon removal up to 70% and 15% more in respect to the planktonic cells. Increasing availability of the contaminants and a better interaction between the hydrophobic substrate and the bacterial cells resulted in developing the degradation rate. Biofilm-mediated bioremediation is a new tool to be developed for bioremediation of acquatic system

The gut microbiota structure of the terrestrial isopod Porcellionides pruinosus (Isopoda: Oniscidea)

We used a combination of culture-dependent and independent approaches to study in depth the microbial community associated with the digestive tract of the terrestrial isopod Porcellionides pruinosus (Brandt, 1833). Specimens from different sampling sites in Tunisia harbored distinct microbiota profiles indicating the impact of both host origin and environmental factors on shaping the microbial flora within P. pruinosus. Our results revealed unexpected bacterial diversity especially via metagenomic analysis; a total of 819 operational taxonomic units (OTUs) assigned to two major bacterial phyla; Proteobacteria and Bacteroidetes. We used Nutrient Agar to isolate the cultivable fraction of bacteria associated with the gut of three geographically distant populations of P. pruinosus. The isolated bacteria belong to Actinobacteria, Firmicutes and Proteobacteria. Enrichment cultures on carboxymethylcellulose (CMC) medium gave evidence that the gut of this Oniscidea harbors cellulolytic Firmicutes and Proteobacteria probably involved in the lignocellulose degradation and then in mediating the functional role of terrestrial isopods as litter decomposers and regulators of nutrient cycling in soil ecosystems

Cover crop impact on soil organic carbon, nitrogen dynamics and microbial diversity in a mediterranean semiarid vineyard

Cover crop (CC) management in vineyards increases sustainability by improving soil chemical and biological fertility, but knowledge on its effects in semiarid soils is lacking. This study evaluated the effect of leguminous CC management on soil organic carbon (SOC) sequestration, soil nitrate content and microbial diversity in a semiarid vineyard, in comparison to conventional tillage (CT). SOC and nitrate were monitored during vine-growing season; soil respiration, determined by incubation experiments, microbial biomass and diversity was analyzed after CC burial. The microbial diversity was evaluated by bacterial and fungal automated ribosomal intergenic spacer analysis (ARISA) and high-throughput sequencing of 16SrDNA. CC increased nitrate content and, although it had no relevant effect on SOC, almost doubled its active microbial component, which contributes to SOC stabilization. An unexpected stability of the microbial communities under different soil managements was assessed, fungal diversity being slightly enhanced under CT while bacterial diversity increased under CC. The complete nitrifying genus Nitrospira and plant growth-promoting genera were increased under CC, while desiccation-tolerant genera were abundant in CT. Findings showed that temporary CC applied in semiarid vineyards does not optimize the provided ecosystem services, hence a proper management protocol for dry environments should be set up