Unravelling extremophiles diversity of volcanic habitats

Introduction Acidic habitats of natural origin are broadly distributed on the Earth. Nevertheless little is known about the microbial diversity of these extreme ecosystems, generally originated by volcanic and hydrothermal activities. Since their values of temperature, pH and concentration of chemicals are close to known limits for life, they represent challenging habitats for those microorganisms that normally colonize conventional environments and only a small fraction of Bacteria are expected to thrive in such a harsh type of ecosystem. Extremophiles inhabiting volcanic habitats, including crater lakes and hot springs, are of interest to microbiologists for their potential biotechnological application. In this study the composition of the microbiome inhabiting acidic water samples collected from six crater lakes located in Costa Rica (Río Cuarto, Hule, Irazú, Botos and Poás) and Mexico (El Chichón) was investigated. Furthermore the bacterial community able to colonize water collected from three different sites of an acidic Icelandic hot-spring was analyzed. Main purposes of the present work were (i) to explore the phylogenetic diversity of Bacteria adapted to the peculiar physio-chemical conditions and (ii) the understanding of the microbiome’s composition shaping forces in natural acidic waters. In order to overcome the low level of cultivability of environmental bacteria, the Denaturing Gradient Gel Electrophoresis (DGGE) fingerprinting technique was applied on the metagenome extracted from the lake waters. Moreover, cultivation dependent methods were applied to investigate the physiological capabilities of the microorganisms that colonize thermal spring field related to the El Chichón volcanic system, where pioneering vascular plants were retrieved. Establishment of tight interplay of microbes and plants are well known to occour in many different type of extreme environments (Puente et al., 2004; Siddikee et al., 2010) For this reason a special attention has been dedicated to plant growth promoting (PGP) rhizobacteria, with the aim of discovering bacteria able to aid the plant to cope with the inhospitable environmental conditions. Materials and Methods Water samples were collected in March 2010 at different depths along a vertical profile of the water column from 1 m to the maximum depth for Laguna Hule (H), Río Cuarto (RC) and Botos (B) lakes. Water samples were collected from different sites at the surface layer in the case of Poás, Irazú (LN09, LE09, LE10) and El Chichón (LE, SP, AS8, ASgas) volcanic lakes (December 2009 and March 2010) and from the Icelandic hot-spring (P). Immediately after collection the water samples (0.1 to 0.5 l) have been filtered through Sterivex filters with 0.2-m pore size (Millipore). After adding RNA Later the filters were stored at –20°C until DNA extraction. The rhizosphere of a single individual of vascular plants observed in the acidic (pH 4.1) SO4-type steam-heated pools (T 42°C) within the Soap Pool field (SP) of El Chichón system was collected, stored in a sterile bag and transferred to the laboratory for cultivation experiments. Temperature, pH, conductivity and redox potential were measured in-situ using a CTD profiler. For details about the applied molecular and cultivation methods please refer to the manuscript in preparation (Mapelli et al.) Results and discussion The geochemistry of some of the Laguna Húle, Río Cuarto, Botos and Poás lakes was deeply studied in the last decades (Tassi et al., 2009), nevertheless few data are available about the microbiome composition of these extreme habitats. DGGE fingerprinting results showed the highest number of bands in the samples collected along the depth profiles of Laguna Húle and Río Cuarto lake, where the water was characterized by pH ranging from neutrality to slightly acidic value and temperature was comprise between 20 and 26 °C. A low bacterial diversity, revealed by the lowest number of DGGE bands, was observed in the extremely acidic water (pH 0.83) of the Poás lake in accordance to what previously observed in a similar environment (Löhr et al., 2006). In the case of Botos, Río Quarto and Húle lakes the analyses of the partial 16S rRNA sequences obtained from the excised DGGE bands rarely allowed to find a match higher than 97% of similarity with described species. Chlorobium limicola, a green phototrophic anaerobic bacterium, was identified from samples H15m and H21m, where the redox potential of the water shifted to negative value. The richness of undescribed species mainly belonging to Proteobacteria and Bacteroidetes classes, or which classification is unknown, indicate that novel bacterial species adapted to the peculiar geochemical setting of these crater lakes may inhabit them. On the other hand most of the partial 16S rRNA sequences detected from the Irazù lake, in addition to those recovered from the Icelandic hot-spring and El Chichón lake, showed higher percentage of similarity to described species previously isolated from analogous environments. The occurrence of bacterial species such as Hydrogenobaculum acidophilum or Acidicaldus organivorans from the acidic high-temperature geothermal waters collected in Iceland is tightly related to the physiochemical condition of these ecosystems. Similar considerations may be done in the case of the El Chichón crater system, where the water in the different investigated sites is characterized by high sulfate concentration. According to DGGE data the microbiome colonizing the El Chichón lake was dominated by the species Acidithiobacillus thiooxidans and Thiomonas intermedia which play a crucial role in the sulphur biogeochemical cycle. To estimate the overall similarity among the samples a Principal Component Analysis (PCA) has been applied after the conversion of DGGE pattern into line plot profiles. This approach allowed to compare the water samples taking into account the whole bacterial community, including those bands that was not possible to excide from the gels to obtain the 16SrRNA sequences. As stated by the distribution observed for the Botos lake samples (Fig. 1a) and in the cases of the Poás lake sample (Fig. 1b), pH value represent one of the most important factors determining the relationship among the bacterial communities composition. Indeed the role of pH have been widely demonstrated in biogeography of soil (Fierer et al., 2006; Griffiths et al., 2011 ) and it is possible to hypothesize its importance also in the prediction of the microbial community structure of freshwater lake systems. However the results of the multivariate analysis indicated that samples characterized by similar pH value can be distinguished according to the lake of provenance, a fact that likely reflect the microbiome adaption to the specific environmental parameters shaping overall each ecosystem. In correspondence of the SP site located in the El Chichón volcanic system, pioneer vascular plants were observed. These extremophile plants living at high T (42°C) and low pH (4.1) were associated with PGP rhizobacteria, some of them known for their capability to grow in extreme conditions. In vitro tests evidenced that 13% of the 44 selected PGP strains were effectively able to survive at in situ conditions. Moreover metabolic properties of the selected strains were explored, especially concerning their potential PGP activities. Several strains showed 1-aminocyclopropane-1-carboxylate (ACC)-deaminase activity, one of the best studied PGP activity, with a role in protecting the plant from environmental stress. Noteworthy percentage of the isolates showed also other PGP activities such as phosphate solubilisation (25%), siderophores production (16%) and the ability to affect phytohormones balance producing indole-3-acetic acid (20%). Conclusions The analyzed natural acidic ecosystems hosted a bacterial community which composition and richness degree appeared to be strongly related to the extreme geochemical parameters of the water. The use of DGGE fingerprinting showed the presence of 16S rRNA sequences poorly related to any known bacteria in the Botos, Río Cuarto and Húle lakes and indicated that volcanic systems might be considered as a source of novel extremophiles, possibly characterized by interesting biotechnological properties. By the way cultivation experiments allowed to identify rhizobacteria displaying multiple PGP activities. These isolates are able to help pioneer plants to cope with the harsh condition of thermal spring sites of the El Chichón lake. In its entirety the data set obtained in the present study indicate that acidic habitats of volcanic and hydrothermal origin can represent a resource for sustainable agriculture in acidic soils. References 1. Siddikee M.A., Chauhan P.S., Anandham R., Han G., Sa T. (2010). J. Microbiol. Biotechnol. 20(11): 1577–1584 2. Puente M.E., Li C.Y., Bashan Y., (2004). Plant Biol. 6: 643-650. 3. 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