Bacterial Diversity in a Nonsaline Alkaline Environment
KeywordsBacterial Diversity Lost City Archaeal Population Methane Cycle Class Clostridia
Metagenome represents total genomes of all microbes inhabiting low-saline serpentinization-driven subterrestrial alkaline water ecosystems. Metagenomics will include all molecular biology techniques that were used to analyze metagenome, including sequencing 16S rRNA genes and specific functional genes, additionally to genome shotgun sequencing by new generation sequencing-NGS technology.
Naturally occurring continental alkaline environments comprise soda lakes, soda deserts (Grant 2006), and low-saline-alkaline environments. Low-saline-alkaline environments are generally related with a unique geochemical process known as serpentinization. Serpentinization occurs, on the deep sea floor or on the continental crust, when ultramafic rocks of Earth’s mantle (i.e., olivine and pyroxenes) are exposed to water, leading to the formation of new minerals like serpentine, magnetite, and brucite. During this process H2 and methane can be released and the abiogenic formation of low-molecular-weight organic compounds may occur (McCollom and Seewald 2007). Continental serpentinization is responsible for the formation of low-saline-alkaline groundwater that emerges on the continental surface in the form of seeps, pools, or springs. These extreme aquatic ecosystems are characterized for being extreme alkaline with low salt concentration and for its highly reducing power (negative Eh) where the availability of organic carbon is highly diminished. Anionic/cationic composition varies accordingly with the geological context, making unique environments. Such sites have been considered potential environments for the emergence of life on the early Earth (Sleep et al. 2011) and are discussed as Mars analogues (Blank et al. 2009). Although active serpentinization is occurring on all continents and comprise significant portions of the deep seafloor, such ecosystems are still poorly understood portions of the biosphere.
The first culture-dependent microbial investigation was performed on a spring water in the ophiolitic complex of Semail in Oman by Bath et al. (1987), followed by Tiago et al. (2004) studies on Cabeço de Vide aquifer in Southeast of Portugal, Pedersen et al. (2004) studies on the Maraquin site in Jordan, and Blank et al. (2009) on the Del Puerto Ophiolite in California.
Culture-independent studies based on total DNA extracted directly from the environment by Brazelton et al. (2012, 2013) at Tablelands Ophiolite alkaline springs in Canada, Tiago and Veríssimo (2013) in Cabeço de Vide aquifer in Portugal, Daae et al. (2013)in Leka ophiolite complex in Norway, and Suzuki et al. (2013) in The Cedars in United States of America, contributed to the characterization of the metagenome of these environments providing vital data to a better understanding of these ecosystems.
Cabeço de Vide Aquifer
Cabeço de Vide aquifer – CVA (located in Southeast Portugal) is constituted by a pristine borehole that gives access to the deep hyperalkaline groundwater (pH value around 11.4), with a distinct, highly stable chemical composition (Tiago et al. 2004; Tiago and Veríssimo 2013), and an Eh value of −215 mV. The alkaline groundwater is originated by serpentinization activity in an ophiolite-like context were infiltrated meteoric water interacts with deep mafic/ultramafic rocks that can be distributed vertically till 1 Km (Marques et al. 2008; Etiope et al. 2013). These particular serpentinization processes are very slow, occur at low temperature as a result of a 2,790 + 40 years BP recharge of the CVA system (Marques et al. 2008), and lead to the production of methane in significant amounts (Etiope et al. 2013).
Bacterial Diversity: Heterotrophic Aerobic Populations
The heterotrophic aerobic bacterial populations’ diversity from CVA was accessed by cultured-based approaches (Tiago et al. 2004). A total of 38 different populations were identified and summarily characterized phenotypically. Phylogenetic analyses identified populations belonging to families Dietziaceae, Microbacteriaceae, Dermacoccaceae, Intrasporangiaceae, Micrococcaceae, Actinomycetaceae, and Nocardiaceae of phylum Actinobacteria; families Staphylococcaceae and Bacillaceae of phylum Firmicutes; family Cyclobacteriaceae of phylum Bacteroidetes; and family Caulobacteraceae of class Alphaproteobacteria (Tiago et al. 2004). Despite the high diversity observed, the cultivable bacterial community was mainly constituted by three major populations: Dietzia natrolimnae, Microcella putealis/Microcella alkaliphila, and Microbacterium kitamiense all belonging to phylum Actinobacteria. The phenotypic characterization of the representative strains of each population determined that the majority of the populations were not alkaliphilic but rather alkalitolerant, despite the fact that they were isolated from a high-alkaline environment.
Microbial Diversity: Unculturable Populations
Cabeço de Vide aquifer microbial diversity was accessed by DGGE analyses, 16S rRNA clone libraries, and 16S-pyrotag sequencing analyses (Tiago and Veríssimo 2013).
A diverse bacterial community was identified in CVA by these methodologies. This community encompassed populations affiliated to phyla Bacteroidetes, Chloroflexi, and Nitrospira and to classes Acidobacteria, Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Clostridia, Actinobacteria, and Deinococci. Despite such high bacterial diversity, Clostridia and Betaproteobacteria were the predominant phylogenetic groups. In fact, the major populations detected were related to Hydrogenophaga sp., Dethiobacter alkaliphilus, and Candidatus “Desulforudis audaxviator” (Chivian et al. 2008), all capable of chemolithoautotrophy. These results suggested that the primary production in CVA was dependent on chemolithoautotrophic microorganisms. Other populations were associated with chemoorganotrophic organisms, including a great number of anaerobic populations (Tiago and Veríssimo 2013).
None of the archaeal populations determined by the methodologies used had a 16S rRNA gene similarity value higher than 90 % with any archaeal isolate (Tiago and Veríssimo 2013). Major populations were phylogenetically related to phylum Euryarchaeota, mainly to the South Africa gold mines-SAGMEG lineage, although some sequences were related to the anaerobic methanotroph group 1 (ANME-1).
PCR surveys using specific sets of primers towards genes encoding key enzymes of autotrophic CO2 fixation pathways, and sulfur and methane cycles were performed in CVA (Tiago and Veríssimo 2013).
From all autotrophic CO2 fixation pathways screened, only CBB cycle was detected. All sequences belonged to form I RuBisCO (cbbL gene), and the phylogenetic analyses grouped the translated sequences on a monophyletic cluster comprising highly similar sequences, showing very low diversity. Sequences were affiliated with translated cbbL sequences of bacteria belonging to Betaproteobacteria.
The presence of sulfate-reducing prokaryotes (SRP) and sulfur-oxidizing prokaryotes (SOP) in CVA was determined by the detection of aprA. Phylogeny analyses identified two clusters for SRP-related sequences, both affiliated with translated aprA sequences belonging to populations of class Clostridia, namely, Candidatus Desulforudis audaxviator (one of the major populations detected in CVA) and Desulfotomaculum spp. SOP-related sequences were distributed by three small clusters affiliated to translated sequences of aprA belonging to Proteobacteria.
One gene encoding a key enzyme involved on methane cycle was detected in CVA, namely, methyl coenzyme M reductase gene (mcrA). Translated sequences formed a single cluster phylogenetically affiliated with several mcrA sequences belonging to ANME-1 (domain Archaea), a phylogenetic group that has been associated to anaerobic oxidation of methane (AOM).
Metagenomic of CVA: A Case Study
The use of culturing (heterotrophic aerobic bacteria) and non-culturing methodologies to access CVA microbial diversity provided complementary data, leading to a better understanding of the structural and functional diversity of this unique environment.
Microbial populations inhabiting CVA are most likely autotrophic or anaerobic or microaerophilic, as determined by 16S rRNA analyses. This assumption was reinforced by the fact that none of the aerobic heterotrophic populations isolated in CVA were detected by clone libraries or DGGE analyses, and only 16S-pyrotag detected some of the isolated populations. Therefore the aerobic heterotrophic isolates constitute most likely seed banks and are present in very low quantity in the environment but may represent an important factor to the ecosystem resilience (Pedrós-Alió 2006). Archaeal populations detected show low diversity, with the dominance of populations belonging to phylum Euryarchaeota, namely, euryarchaeotal SAGMEG lineage (of unknown metabolic role). Nevertheless, populations belonging to ANME-1 (usually associated to anaerobic oxidation of methane) were also detected.
The low diversity observed on functional genes may be indicative of a high level of selective pressure ongoing on CVA due to the existing physicochemical characteristics, leading to the selection of organisms with enzyme forms more suitable to perform under such specific conditions.
The CVA metagenomic overview allows to depict a comprehensive picture of this unique ecosystem. The CBB carbon fixation pathway could be putatively assign to the major population belonging to Betaproteobacteria that was phylogenetically affiliated with species Hydrogenophaga flava a facultative chemolithoautotrophic.
It was also possible to envision a straight relation between detected populations with their functional role regarding the sulfur and methane cycles. Indeed, it was possible to envision that the anaerobic oxidation of methane (AOM) may be occurring in CVA. In fact, microbial populations were identified as SRP and ANME-1 and may mediate a syntrophic consortium responsible for AOM. Additionally, during this process methane is oxidized with sulfate as the terminal electron acceptor leading to the release of hydrogen sulfide, and some populations identified as SOP may be taking advantage of the produced HS- contributing to the energy cycling in CVA.
The overall functional role of the majority of the populations detected in CVA is still unclear. Despite the fact that some metabolic pathways could be, with some degree of certainty, putatively assigned to some specific groups, the fact is that much is still a grey area. The use of the shotgun metagenome sequencing and the metatranscriptome sequencing can be next step. These techniques will yield good data that, most probably, will answer those pertinent questions.
Other Continental Serpentinization-Driven Alkaline Environments
Microbial studies of two terrestrial serpentinizing sites, Tablelands in Newfoundland, Canada (Brazelton et al. 2012), and The Cedars in north of San Francisco, United States of America (Suzuki et al. 2013), revealed identical microbial structure to the one described in CVA. The majority of populations detected were affiliated to classes Clostridia and Betaproteobacteria, showing that these microorganisms may indeed represent ubiquitous populations on these environments. Additionally, in The Cedars site a new kind of “deep groundwater-fed spring” was described, with highest pH and lowest redox potential at The Cedars. Its microbial community structure was clearly distinct from the latter sites; it was constituted mostly by candidate division members that clustered into a clade with phylotypes reported from the oceanic serpentinizing site, Lost City (Schrenk et al. 2004; Brazelton et al. 2006; Brazelton et al. 2010; Suzuki et al. 2013).
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