Metagenomic and Metabolic Analyses of Poly-Extreme Microbiome from an Active Crater Volcano Lake

Background: El Chichón volcano is one of the most active volcanoes in Mexico. Previous studies have described the poly-extreme conditions of the lake crater but its bacterial composition and the functional features of the complete microbiome have not been characterized yet. Methods: This study integrated two approaches to explain the microbiology diversity and abundance, one focused on the environmental genomic potential by metagenomics approach, and other culturomics of enrichment of bacteria and archaea. The microbial diversity of the anaerobic consortia cultivated in was carried out by metabarcoding analysis, the metabolic capacity by metabolomics uxes of carbon and enzymologic techniques for the analysis of sulfate reduction in laboratory-grown prokaryotic cells. Results: This work provides new information on the taxonomic and functional diversity of the Archea representative phyla Crenarchaeota and Euryarchaeota as well as the phyla Thermotogales and Aquicae for Bacteria. Through the analysis of microbial consortia cultivation and the genetic information collected from the natural environment sampling, metabolic interactions were identied between the microorganisms that support the life of the microbiome under multi-extreme conditions. A close relationship is proposed between the cycles of carbon and sulfur in an active volcano. Conclusions: This research contributes to the understanding of microbial metabolism under extreme conditions and potential knowledge of "microbial dark matter" that can be applied in biotechnological processes and evolutionary studies.

are sul de [7], which confers acidic conditions and a highly toxicity and hostility for the survival of microbial life.
Extreme natural environments are a source of microorganisms of geological and biotechnological interest; however, in "El Chichón" only the taxonomic diversity of thermoacidophilic bacteria has been examined so far, as fteen identi ed phyla were found in the sediment at 50 ° C such as Actinobacteria (33.1%), Proteobacteria (29.1%) and Acidobacteria ( 20.1%); nine phyla were found in the sediment at 92 °C where Firmicutes (52.7%, mostly Alicyclobacillus and Sulfobacillus) and Proteobacteria (44.8%, mostly Bradyrhizobium, Methylobacterium, Sediminibacterium) were the most abundant [8]. However, to our knowledge, there are no reports dealing with the diversity, culture and metabolism of poly-extreme archaea in crater-lake active volcano, presumably the predominant microorganisms in these environmental conditions, and which isolation and study remain elusive. Understanding the mechanisms used by extremophiles microorganisms to tolerate and thrive in multi-extreme conditions may provide useful information to assess habitability on earth, as well as its application in biotechnological processes.
In this work, by using complete metagenome analysis, enzyme activities and metabolic uxes determination, molecular and biochemical evidence related to the energy metabolism in this novel multiextreme environment is reported for thermoacidophilic bacteria and archaea for the rst time. This study provides new insights regarding the geobiochemical role of volcanic microorganisms.

Methods
The physicochemical environmental parameters have been previously described [6]. The samples were collected on November 8 th 2018 from El Chichón crater-lake, Chiapas, México.

Whole metagenome analysis
Samples of sediment and water column were taken for metagenomic analysis, samples were placed in three separate sterile tubes, immediately preserved by freezing in liquid nitrogen, and stored at −80 °C until DNA was extracted. In turn, metagenomic DNA was extracted by triplicate using the QIAGEN DNA isolation kit (Hilden, Germany) and the metagenomic DNA was sequenced using Illumina NextSeq platform with 2 X 150 pair-ends reads (Integrated Microbiome Resource, Halifax, Canada). The quality control of the reads was performed with Trimmomatic [9], functional and taxonomical analysis of metagenome was carried out using the co-assembly mode from SqueezeMeta v1.0.0 automatic pipeline [10], Assembly was done using Megahit [11]. Contig statistics were done using prinseq [12]. RNAs were predicted using Barrnap [13], 16S rRNA sequences were taxonomically classi ed using the RDP classi er [14], tRNA/tmRNA sequences were predicted using Aragorn [15]. ORFs were predicted using Prodigal [16]. Similarity searches for GenBank [17], eggNOG [18], KEGG [19], were done using Diamond [20]. HMM homology searches were done by HMMER3 [21] for the Pfam database [22]. Read mapping against contigs was performed using Bowtie2 [23]. Binning was done using MaxBin2 [24], Binning was done using Metabat2 [25], Combination of binning results was done using DAS Tool [26], Bin statistics were computed using CheckM [27].Pathway prediction for KEGG [19] and MetaCyc [28], databases was done using MinPath [29]. Gene abundance was estimated mapping individual reads to the reference coassembly contigs annotation. Finally, Squeezemeta manages the results and generates a table of genes (taxonomy, function, origin of contig and bin, abundance in samples and amino acid sequence), a table of contig, which gathers the data of the contigs (taxonomy, a liation of bin, abundance in samples and disparity), and a bin table with information related to the bins (taxonomy, integrity, contamination, abundance in samples and disparity). These three tables and the metadata were used to create a MySQL database which was analyzed using R (version 3.4.4, Inc., Boston, MA, U.S.A). The metagenomic data obtained were used to build metabolic schemes which were carried out using speci c genes involved in the metabolism of carbon and sulfur (see Additional le Table 1). Using an independent analysis system and using KEGG as a reference base [19] The identi cation of metabolic processes was carried out by comparing the genes of the metagenome with the KEEG database. Illustrations were made using CorelDrawn software (version 2016) [30].
Methanogenic and sulfate-reducing bacteria enrichment The culture system is called ´Lab-Lake´ which was carried out under strict anoxic conditions in the culture medium reported by Sowers et al. (1993) [31] modi ed by the addition of a mixture of carbon sources commonly used in the cultivation of Extremophiles under laboratory conditions: 10 mM glucose, 10 mM maltose, 10 mM glycerol, 20 mM glyceryl triacetate, 20 mM sodium pyruvate, 100 mM sodium acetate and 100 mM methanol [32] plus 100 µM FeSO 4 . The culture media were prepared in an anaerobic chamber (COY, Michigan, USA) that contained an atmosphere of 80 % N 2 , 15 % CO 2 , 5 % H 2 (v / v). The oxygen dissolved in the culture media was removed by gas exchange (constant bubbling with the gas mixture) for four hours and the addition of cysteine. Each experimental unit consisted of a 100 mL vial with 50 mL of operation, hermetically sealed and sterilized by humid heat for 40 min.
The rst stage of cultivation was carried out in situ the crater of the volcano and was started with the addition of approximately 50 mL of water column of the volcanic lake collected in various sections of the crater lake at different temperatures (30,35,40,45 and 82 ° C) injected using sterile syringes in sealed 100 mL bottles containing 10 mL of Lab-lake medium. These samples were kept at 60-70 ° C during their transport to the National Institute of Cardiology in Mexico City, which took 72 hours. After 8 days of incubation, 10 ml were transferred to 50 ml of mixture of fresh medium and sterile volcanic water and cultured at 37 ° C or 70 ° C without shaking. After repeating this procedure three times, 190 culture bottles resulted, from which methane production and CO 2 uxes were determined at different times during 24 days. From the continuous transfer to fresh media for 2 months and based on the growth curves obtained (production of CH 4 , CO 2 and protein content), enriched samples were obtained that were nally grouped into two representative sets: mesophilic and hyperthermophilic consortia and subsequently, the microbial growth parameters were each determined for 500 hours.

Cell harvesting
After 15 days of growth, cell cultures were harvested under anaerobic conditions at 5000 x g for 15 min at 4°C. Cell pellet (1 mL) was resuspended and washed in 10 volumes of a fresh solution containing 50 mM Tris, 20 mM MgCl 2 , 200 mM NaCl and 1 mM EGTA at pH 7.2 (TME-Na buffer) and further centrifuged at 5000 x g for 15 minutes at 4°C. After this, 5mM DTT, 1 mM PMS and 10% glycerol (v/v) were added to the cell pellet. Cells were frozen and kept at -80°C until use.

Metabarcoding analysis
DNA metagenomic was extracted of thawed washed cells of mesophilic and hyperthermopilic consortia after 15 days of growth. For bacterial identi cation the variable V4-V5 regions of the 16s rRNA were ampli ed with primers 515Fw (5'-GTGYCAGCMGCCGCGGTAA-3') and 926Rw (5'-CCGYCAATTYMTTTRAGTTT-3') (Walters et al., 2016), it was also analyzed the regions V6-V8, the same gene for archaea identi cation with primers 956Fw (5'-TYAATYGGANTCAACRCC-3') and 1401Rw (5'-CRGTGWGTRCAAGGRGCA-3') [33]. Barcoded DNA libraries were sequenced through Illumina MiSeq 2x300 platform. 16S rRNA reads were processed using MiSeq SOP Mothur [34], for reducing sequencing and PCR errors, sequencereads with < 50 nucleotides, > 1% and 7% of homopolymers and those with mitochondrial or chloroplast origins were discarded. Chimeric sequences were removed using default parameters in UCHIME [35]. Sequences were aligned with SILVA database version 132 [36] at 80% con dence threshold [37]. The aligned data sets were clustered into Operational Taxonomic Units (OTUs) at 98% sequence similarity using a pairwise distance matrix. The relative abundance of taxas was as a percentage of the number of sequences a liated with the speci c taxon against the total number of sequences obtained for that sample.

Biochemical characterization of microbial cultures
Carbon metabolism was determined by measuring CO 2 and CH 4 uxes in a gas chromatography equipment (Shimadzu GC2010, Japan) as described previously by Santiago-Martínez et al (2015, 2016) [38,39], while carbon sources consumption was determined by spectrophotometry, gas chromatography (see Additional le Text 1).
Sulfate-reduction pathway was determined throughout the growth curve by measuring the synthesis of sul de by the cell consortia. All steps were carried out under strict anaerobiosis: cell-free supernatant and washed cells were used to determine the extra and intracellular sul de and mixed with a solution containing 23.7 mM zinc acetate, 60 mM NaOH, 0.18 mM N,N-dimethyl-p-phenylenediamine (DMPD) dissolved in 5 N HCl and 0.1 mL of sample, or different amounts of sul de and 2.8 mM FeCl 3 . Sul de was quanti ed by the methylene blue formation [40] Limit of sensitivity was 3 nmol and linear up to 350 nmol [41].
Enzymatic determination of the dissimilatory sulfate reduction pathway Enriched cytosolic and membranal fractions of cells among its logarithmic growth phase were collected afterwards its lysate by sonication with ve 20 seconds intervals with a minute rest in which anaerobiosis was preserved by a constant ux of N 2 (10 mL/min). Enzymatic activity was carried out in anaerobic conditions in a reaction mixture containing 0.1 mg of protein of each cellular extract, 5mM ATP and 1mM GSH and 100 μM NADPH as electron donors. Reaction was started with the addition of 50 μM FeSO 4 in a nal volume of 1 mL of HKE-Na buffer. The assay was attained at 37°C for 30 min (reaction was lineal until 60 min). Sul de produced was determined by the described spectrophotometric method for methylene blue.

Statistical Analysis
Statistical analyzes and graphs of metabolism were performed in Origin 5.0 (www.OriginLab.com). For metabarcoding diversity based on Operational Taxonomic Units (OTUs) were done in STAMP [42], the bar plot visualization was constructed with a relative abundance of the genus or phyla level > 0.05% of abundance count table and rest summarized in others. Venn diagram and metagenomic analysis were performed with Rstudio software [43].

Environmental general features of El Chichón volcano
The physicochemical characteristics of water column and sediment along the crater-lake were variable depending on the location. A priori perimetric exploration allowed sampling of sediment and water column at different temperatures. The sites analyzed and parameters determined in this study are shown in Table 1 and Figure 1.

Metagenome analysis
Water column and sediment of crater El Chichón volcano were analyzed using high throughput nextgeneration sequencing to identify the microbial biodiversity and functional potential, the statistical and parameters are presented in Table 2. Each metagenomic sample was sequenced in triplicate and the sequences were pooled for bioinformatics analysis.
Taxonomic classi cation 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 identi ed 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 identi ed in solphatara pools and volcanic environments, and is characterized by obtaining ATP using H 2 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) nding 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 xation of CO 2 and production of H 2 SO 4 , it also obtains the energy from the oxidation of Fe 2+ 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 sul de or thiosulfate as an electron acceptor, and as a donor acetate, pyruvate, propionate, among others. CO 2 / H 2 is presented as a product of the oxidation of these substrates. Another phylum identi ed 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 identi ed 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 identi ed 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, H 2 S) 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 identi ed 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 classi cation, 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 identi ed 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 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 identi ed 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 CO 2 xation 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, CO 2 production was 5 ± 0.4 a and 4 ± 0.1 a mmol CO 2 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 CO 2 , 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 ± 8 a % of the substrates, while the hyperthermophilic culture consumed only 41± 3 b % 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 ± 0.3 a and 0.5 ± 0.07 b mmoles of CH 4 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 CO 2 xation 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 sulfatereducing bacteria has not been described in detail [65][66][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][69][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 sul de 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 identi ed 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, de ning 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 FeSO 4 and formation of sul de, 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 sul de in microbial cultures (Supplemental Table S3). This is reproducible when evaluating the ux of the non-assimilatory sulfate reduction metabolic pathway in mesophilic and hyperthermophilic microbial consortia in cytosolic and membrane fractions (Table 3). 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-sul te redox pair is E 0 '-516 mV, which is too negative to allow reduction by NADH or Fed red , which are the main intracellular electronic mediators. To overcome this problem, the sulfate is rst converted to APS by the enzyme ATP sulfurylase (Sat), at the cost of a single ATP molecule. The APSsul te 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 nal step, the sul te is reduced by dissimilatory sulfate reductase (Dsr) to form sul de, 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].

Conclusion
Our results demonstrated the presence of a complex microbiome, composed of archaea, viruses, bacteria, and microalgae. The phyla Crenarchaeota, Aqui cae and Termotogae dominated the microbiome of the crater lake of the El Chichón volcano, while the lab-cultures are dominated by the phyla Firmicutes and Euryarchaeota. Reducing and methanogenic sulfate populations were enriched in the lab-culture system, poorly represented in the natural environment, with which we detected important differences in the functions of mesophilic and hyperthermophilic microbial cultures, these differences are related to carbon assimilation and energy metabolism of the extremophiles from El Chichón volcano. Future experiments should, therefore, aim to determine regulatory mechanisms in energy metabolic pathways to make proper correlations with the volcanic system.