Water column and sediment of crater El Chichón volcano were analyzed using high throughput next-generation sequencing to identify the microbial biodiversity and functional potential, the statistical and parameters are presented in Table 2.
Table 2
Data details of metagenome analysis of water column and sediment of El Chichón active volcano.
|
Water column
|
Sediment
|
Reads
|
10 767 992
|
32 227 870
|
Contigs
|
4 775
|
24 312
|
N50
|
26 634
|
9 633
|
Total assembly length (Mb)
|
6.13
|
47.3
|
Each metagenomic sample was sequenced in triplicate and the sequences were pooled for bioinformatics analysis.
|
Taxonomic classification environmental and lab culture
The environmental metagenomic analysis enable to identify members of the three domains of life, this information is a precedent for the rapid evolution that this extreme environment has had since its last eruption in 1982, after this geological event and studies in which the absence of life was reported. [44]. Unlike these reports, in this study, a set of methodologies with a multi-omic approach was used. Starting with a metagenomic study to achieve characterization in microbial cultures.
It is of great interest to deepen the study of microbial diversity in this environment, in order to have a better understanding of the microbiology from El Chichón volcano and its highly dynamic environment, sediment and water column samples from the crater lake were analyzed by metagenomics. It is exhibited in the natural environment that the hyperthermophilic water column was not very diverse (Shannon's index = 0.58), 80% corresponds to Archaea represented by the Candidatus Aramenus genus and 20% bacterium domain of the Hydrogenobaculum genus, both microorganisms are characteristic of hot springs and rich in sulfur. Candidatus Aramenus is a thermophilic and acidophilic candidate genus that has been identified only in the geothermal site Los Azufres, Michoacán [45], and which has a role in sulfur metabolism and Hydrogenobaculum a chemolytoautotrophic bacterium, which has been identified in solphatara pools and volcanic environments, and is characterized by obtaining ATP using H2 as an electron donor in the presence of a reduced sulfur source [46]. Regarding the mesophilic sediment, greater microbial diversity was considered (Shannon index = 1.41) finding the presence of the bacterial phylum Proteobacteria represented mainly by Acidithiobacillus and Desulfurella, the former, a microorganism normally found in acidic environments such as Rio Tinto [47] [48]) Copahue volcano [49] and Rio Agrío [50]; where they play a crucial role in the fixation of CO2 and production of H2SO4, it also obtains the energy from the oxidation of Fe2+ by means of an “upward potential gradient” [51,52]. On the other hand, Desulfurella has been found in acid mine residues [53], Rio Tinto sediments [54], among other thermophilic and acidic environments. In these ecological sites they are of great importance due to its role in the disproportionation of sulfur, given that it can use sulfide or thiosulfate as an electron acceptor, and as a donor acetate, pyruvate, propionate, among others. CO2 / H2 is presented as a product of the oxidation of these substrates. Another phylum identified in these samples is Firmicutes, highlighting the presence of the genera Thermoanaerobacterium, Ruminococcus, and Ignavibacterium. The dominant bacterial taxon in this sample is Thermotogae, which is represented by Athalassotoga, an anaerobic, heterotrophic, acidophilic and moderately thermophilic, which has been identified in similar environments as a hot-spring in Japan [55]; and in greater abundance Mesoaciditoga, this microorganism has also been reported in the Copahue-Caviahue volcanic system [56]. Regarding to the diversity of archaea, the presence of the phylum Euryarchaeota stands out, represented by Candidatus Methanofastidiosa, and the Thermoplasmatales: Ferroplasma, Thermoplasma, Cuniculiplasma and Acidiplasma. The Thermoplasmatales order has been identified at other volcanic sites, such as the Tenorio volcano, Costa Rica, where its main metabolic contribution is the oxidation of reduced sulfur compounds (for example, H2S) to sulfate [57]. It was also found the presence of the phylum Chlorophyta composed of green algae, this phylum is dominated by Trebouxiophyceae. Some microalgae belonging to this group have been identified in extreme conditions, for example, Endolithella mcmurdoensis in the McMurdo valleys, desert in Antarctica [58].
Another aspect of great relevance is the study of Extremophilic viruses. In this study, it was possible to identify the Mimiviridae and Pithoviridae families using the Lowest Common Ancestor (LCA) algorithm with a >80% bit-score identity [59] compared contigs to GenBank; Mimiviridae corresponds to a family of double-stranded DNA viruses, which is associated with protozoa. These viruses stand out for their participation in theories of the origin of life [60]. On the other hand, Phithoviridae is a single-stranded RNA virus, which also plays an important role in understanding evolutionary theory [61]. The Phytoviridae viruses rely on very diverse eukaryotic hosts, which include protists, algae, vertebrate animals, and insects[62].
In addition to the taxonomic classification, the information generated from the metagenomic analysis allowed to identify the metabolic and functional potential of the microbiome, and with this information microbial culture strategies and metabolic and kinetic analyzes were developed to evaluate the participation of microorganisms in key biogeochemical processes such as carbon and sulfur metabolism. To understand these processes with greater precision, a culturomic stage was developed, in which it was possible the enrichment of Firmicutes (genera Ruminococcus, Thermoanaerobacter, Tepidanaerobacter) and archaea of the Euryarchaeota type (genera Methanobacterium, Methanothermobacter) (Fig 2b). Based on the sequences obtained from the analysis of the 16S rRNA gene, it was identified by prediction of genomic potential that the microorganisms present in laboratory cultures developed roles of vital importance in the preservation of the biogeochemical balance of sulfur and carbon (see Additional File Table 1 S1). Therefore, it is possible to use microbial cultures to understand the participation of microorganisms in the biogeochemical cycles of carbon and sulfur in El Chichón volcanic system.
Carbon metabolism
Based on the genomic potential of the natural environment, a metabolic map of the carbon cycle is proposed (Figure 3), considering the acquisition and intermediate metabolism of carbon in the extreme natural environment El Chichón.
The microbiome showed genomic characteristics that allow to carry out most of the metabolic pathways described for obtaining of a carbon source. The presence of the semi-SWEET system was identified that allow the internalization of sugars, as well as glycerol transporters and glycerol-3P molecules that can be used as carbon sources by the extremophilic microorganisms. The abundance of metabolic pathways for CO2 fixation was also observed, these results suggested that this is one of the most important mechanisms for obtaining carbon in this extreme environment; besides low concentration of sugars (or not) has been determined in the lake (data not show). On this regard, CO2 production was 5 ± 0.4a and 4 ± 0.1a mmol CO2 for mesophilic and thermophilic cultures, respectively. This type of metabolism has been reported in environments homologous to El Chichón volcano, such as Yellowstone National Park, where the presence of archaea from the Euryarcheota and Crenarchaota phyla has been reported, which at high temperatures carry out processes of methanogenesis and the metabolic cooperation of these microorganisms with bacteria has even been reported to facilitate the obtaining of CO and CO2, which they use as an electron donor and carbon source [63].
The dynamics of assimilation of carbon sources of the lab-culture showed that the mesophilic microorganisms consumed 54 ± 8a % of the substrates, while the hyperthermophilic culture consumed only 41± 3b % carbohydrates present in the growth media (Glc and maltose) were mainly consumed over the rest of substrates (glycerol, glyceryl triacetate, sodium pyruvate, sodium acetate and methanol) 90 and 35-60 % respectively of these substrates. Additionally methanogenesis was evaluated as it was the last step in the total degradation of organic matter, in addition to a mechanism for obtaining energy under anaerobic conditions [64]. Mesophilic cultures were observed to have higher methanogenic activity than hyperthermophiles (1.8 ± 0.3a and 0.5 ± 0.07b mmoles of CH4 after 15 days of culture). Results based on metabolic evidence show differences between mesophilic and hyperthermophilic cultures, and therefore allow an inference of the metabolic dynamics that takes place in this extreme natural environment, this suggests that glycolysis and CO2 fixation for methanogenesis are energy mechanisms most used by mesophilic consortia.
Sulfur metabolism
The functional analysis of the microbiome expressed in terms of functional orthologs using the KO database revealed that a high percent of the KOs was assigned to the KEGG metabolism pathway to sulfur metabolism, with this information the presence of potential to perform sulfate reduction and sulfur oxidation (SOX system) (Figure 4).
Volcanic systems are dominated by sulfur oxidizing microorganisms, while the presence of sulfate-reducing bacteria has not been described in detail [65–67]. It is important to consider that the active volcano El Chichón also presents acidic characteristics (pH 2-6), microorganisms have been described that have the capacity to carry out sulfate reduction processes under these conditions such as Desulfurella, Thermodesulfobium, Desulfurococcus, Desulfosarcinacetonica [68–70]. Regarding El Chichón volcano, there is the presence of Desulfurella sulfate reducing bacteria and Candidatus Aramenus, a sulfur oxidizing archeon (Fig 2a).
The sulfate reduction is a process of biogeochemical relevance associated with volcanic environments, this metabolism can follow two routes: i) the assimilatory pathway that is carried out mainly for the synthesis of biomolecules [71] and ii) the non-assimilatory pathway that is the anaerobic process in which sulfate is used as a terminal electron acceptor, allowing the oxidation of organic and inorganic compounds, in this process a large amount of sulfide is produced, which is re-oxidized to sulfate by microbial activity [72,73]. These processes are abundantly distributed in El Chichón volcano microbiome.
Several sulfur reducing extremophilic microorganisms contain in their genome a set of genes involved in sulfate reduction: sat, aprBA, dsrABCMK [72]. These genes were identified in the metagenome of the mesophilic and hyperthermophilic environmental samples from El Chichón volcano. In addition to the biogeochemical relevance of sulfate reduction, reducing sulfate bacteria use this process to obtain energy [74]. The bioenergetic mechanism of this process has not been fully elucidated. Recently, it has been proposed that the QrcABCD complex of Desulfovibrio vulgaris is electrogenic, and the mechanism is due to the balance of electrons and protons on opposite sides of the membrane, it is suggested that it is not an H+ pump but a proton channel instead [74]. It has been evidenced as a new respiratory system in prokaryotes mediated by an electrogenic complex that involves a redox loop where the menaquinone (a low redox potential quinone) and the substrate sites are on the same side of the membrane, defining a new type of prokaryotic respiratory system [74]. The lab-culture microorganisms from El Chichón volcano showed evidence of carrying out processes of reduction of FeSO4 and formation of sulfide, which can be excreted from the cell interior (see Additional Table 2). Hyperthermophilic microbial consortia showed 1.6 times more ability to reduce sulfate to sulfide in microbial cultures (Supplemental Table S3). This is reproducible when evaluating the flux of the non-assimilatory sulfate reduction metabolic pathway in mesophilic and hyperthermophilic microbial consortia in cytosolic and membrane fractions (Table 3).
Table 3
Enzymatic activity of the non-assimilatory sulfate reduction pathway in El Chichón volcano culture.
Activity in
|
Mesophilic microorganisms
(nmoles of H2S / min mg protein)
|
Hyperthermophilic microorganisms
(nmoles of H2S / min mg protein)
|
Enriched cytosolic fraction
(ATP + GSH + NADPH)
|
20.5 ± 2.5 bB
|
399.6 ± 36.4 bA
|
Enriched membrane fraction
(ATP + GSH + NADPH)
|
168 ± 30.2 aB
|
886.7 ± 31.6 aA
|
Enriched cytosolic fraction
(-ATP -GSH -NADPH)
|
10 ± 2 bA
|
< 1 ± 0.3 cB
|
Enriched membrane fraction
(-ATP -GSH -NADPH)
|
128 ± 42.3 aA
|
< 1 ± 0.4 cB
|
Mean values ± standard deviation (n = 3). Significant differences (p ≤ 0.01) using ANOVA/Fisher’s least significant difference (LSD) test, are indicated in column by different lowercase letters (a, b, c) between cell fractions, and capital letters (A, B) indicated significant differences (p ≤ 0.01) in row between treatments.
|
The results suggested that the highest activity of the non-assimilatory reduction sulfate pathway in the microbial consortia of El Chichón volcano is found in the membrane fraction; in the case of mesophilic consortia there is 8.2 times more than in the cytosolic fraction. Similar to that determined in hyperthermophilic cultures where there is 2.2 times more activity in the membranes than in the cytosol, which suggest that hyperthermophilic consortia use the non-assimilatory pathway for sulfate reduction as an alternative mechanism for obtaining energy.
It is necessary to consider that, energetically, sulfate is a poor electron acceptor for microorganisms since the sulfate-sulfite redox pair is E 0 '-516 mV, which is too negative to allow reduction by NADH or Fedred, which are the main intracellular electronic mediators. To overcome this problem, the sulfate is first converted to APS by the enzyme ATP sulfurylase (Sat), at the cost of a single ATP molecule. The APS-sulfite redox pair has an E 0 'of -60 mV, which allows APS to be reduced by NADH or reduced ferrodoxin using the enzyme adenylyl sulfate reductase (Apr), which requires the input of 2 electrons. In the final step, the sulfite is reduced by dissimilatory sulfate reductase (Dsr) to form sulfide, requiring the input of 6 electrons. However, it has recently been shown that the conservation of energy through sulfate reduction processes are due to the transfer of protons from cytochrome c3 to the menaquinone group, through the Qrc complex. Additionally, the presence of this respiratory system in the prokaryotic membrane has been reported [74].