Alkalo-Thermophilic Microbial Diversity of the Tecozautla Geyser, México

Microbial mats have been studied in many thermal systems; the most iconic is Yellowstone. In Mexico, the information on microbial mats is scarce and therefore novel. In this research, the thermophilic microbial composition of samples from four areas of the Tecozautla geyser, Hidalgo, Mexico, was studied: sediments (GD), salt deposits (GA), and microbial mats (GB and GC). The samples were taken at the outlet of the geyser (94 °C) and in storage pools with temperatures of 61.5-65 °C. Sequencing of the 16S rRNA gene amplicons was carried out, obtaining 1,425,506 readings, and was analyzed through the Quantitative Insights Into Microbial Ecology software package version 2 (qiime2). 32 phyla were identied in the four samples being the most representative for the GA sample: Armatimonadetes, Chloroexi, Cyanobacteria, and Thermi, with abundances of 46.35, 19.18, 3.27, and 1.82 %, respectively. For the GB sample, they were Proteobacteria, Bacteroidetes, Cyanobacteria, Spirochaetes, Thermi, and Firmicutes with abundances of 25.23, 22.04, 20.42, 12.31, 4.56, and 1.32 %, respectively. For the GC sample, abundances of 55.60, 9.85, 7.04, 7.01, and 6.15 % were observed for the phylum Chloroexi, Armatimonadetes, Proteobacteria, Cyanobacteria, and Acidobacteria, respectively. Finally, for the GD sample, the most abundant phyla were Chloroexi (36.10 %), Cyanobacteria (17.13 %), Armatimonadetes (15.59 %), Proteobacteria (5.45 %), and Nitrospirae with (3.21 %). The metabolic functionality of the microbial communities present in the samples was inferred using the 16S rRNA amplicons. This work represents the rst report of the microbial communities present in the Tecozautla geyser. The 4 samples sequenced from the Tecozautla geyser, resulted in a total of 5,660,646 raw paired-end (PE) readings, of which 2,830,823 were found in the forward direction, with these sequences it was carried out with the bioinformatic analysis. After removal of the primers with TagCleaner and quality ltering with Trimmomatic, 1,589,850 readings were obtained. DADA2 was used for removal of chimeras, borderline chimeras, and clustering of sequences de novo at 97 % similarity; obtaining a total of 1,425,506 readings of which 1,178,144 corresponded to GA, 1,668 to GB, 43,769 to GC, and 201,925 to GD. The OTUs obtained for each sample were 434, 75, 130, and 648, for GA, GB, GC, and GD, respectively. % GD 8.40 %, GA, GB, GC,

Introduction phenol solution (pH 8), 40 µL of SDS (20 %); followed by 3 cycles of shake-ice on a Tissue Lyses LT (QIAGEN) for 30 seconds at maximum speed (50 Hz). The samples were then centrifuged at 13,000 rpm in 1 minute. The supernatant was transferred to an HTP (hydroxyapatite) column by centrifuging at 1500 rpm for 2 minutes, followed by three continuous washes of the column with 500 µL of 120 mM sodium phosphate pH 7.2, each time centrifuging at 1500 rpm for 6 min. The DNA was eluted by adding 400 µL of a dipotassium phosphate solution (300 mM, pH 7.2), centrifuging at 1500 rpm for 6 minutes. Once eluted, the DNA solution was transferred to a G50 Sephadex column and centrifuged at 13,000 rpm for 1 minute. Subsequently, the DNA was precipitated overnight at -20°C with the addition of 40 µL of a sodium acetate solution (3 M) and 1 mL of absolute cold ethanol. The DNA was recovered by centrifugation at 13,000 rpm for 10 minutes. Subsequently, the pellet was washed with 70 % alcohol and resuspended in a volume of 20-50 µL of DNAase-free water.
For the extraction of metagenomic DNA from the GD sample, the following protocol was carried out. To obtain a homogeneous mixture, approximately 0.5 g of the sample were macerated with liquid nitrogen and extraction buffer (100 mM Tris-HCl, 20 mM NaCl, 100 mM EDTA pH 8, 1 % (p/v) PVP), with 1 % cetyltrimethylammonium bromide (CTAB) at 6 %, following three continuous freeze-thaw cycles with liquid nitrogen and 65°C. Subsequently, the samples were incubated for 30 min at 37°C with lysozyme (30 mg/mL). Then 0.2 mL of 10 % sodium dodecyl sulfate (SDS) and proteinase K (10 mg/mL) were added for 2 h at 65°C. The nucleic acids were extracted twice with a solution of phenol: chloroform: isoamyl alcohol (25:24:1) and once with chloroform: isoamyl alcohol (24:1), recovering the supernatant liquid after each centrifugation (10,000 x g for 10 min). The DNA was precipitated with 0.6 volumes of cold isopropanol, 3M sodium acetate (1/10 v), and centrifuged at 22700 x g for 15 minutes at 4°C. The DNA pellet obtained was washed with ethanol cold to 7 %, centrifuged, and the excess alcohol was allowed to evaporate. The DNA was resuspended in 30 µL of DNAase-free water. Finally, to purify the DNA, the commercial kit Zymo BIOMICS DNA (Zymo) was used. The DNA quality was veri ed by 0.8 % agarose gel electrophoresis and stained with EpiQuick DNA Stain (10 µL/100 mL). DNA concentrations were determined using a Nanodrop Lite spectrophotometer (Thermo Scienti c).

16S rRNA sequencing
The puri ed DNA samples were used as a template to amplify the 16S rRNA gene using primers from the V3-V5 regions, according to the Illumina MiSeq protocol. The rst universal 357-F (5´-CTCCTACGGGAGGCAGCAG − 3´) and CD-R (5´-CTTGTGCGGGCCCCCGTCAATTC-3´) [7] were used.

Analysis of amplicon readings
The readings obtained were processed by TagCleaner to remove the initiators and ambiguous bases, consecutively Trimmomatic was used to lter the sequences, conserving those that had a length greater than 100 bp ≤ Q20 in the Forward direction of the V3 region, due to their quality and abundance, in addition to the fact that they could not be paired because they were very distant from each other. QIIME2 v2019.1 software was used, the sequences were imported with Casava 1.8 single-end demultiplexed fastq, using a mapping le (.tsv) with the information of the samples. The analysis started with the DADA2 option, which consists of the puri cation and trimming of the sequences, carried out at 100 bp to preserve the greatest amount of information, as well as the elimination of errors in the sequencing. The identi cation and elimination of the chimeric sequences and the "borderline chimeras" were carried out using the uchime-de novo method, grouping them de novo in operative taxonomic units (OTU's) at 97% similarity threshold, using the VSEARCH algorithm for both cases. The taxonomic assignment of the sequences was carried out using a Bayesian classi er with the complement q2feature-classi er using the Greengenes database (gg-13-8-99-nb-classi er.qza) [8]. Additionally, graphs were made to describe the relative abundance at different taxonomic levels using ggplot2 in the R software (https://www.r-graph-gallery.com).
The diversity analyzes were carried out taking as a parameter the depth of the sampling (rarefaction) starting from the sample with the lowest readings obtained. The alpha diversity, the observed OTU's metrics, Chao 1, Shannon, and Simpson, were determined. In addition, Good coverage was determined (www.qiime2.org). Beta diversity was determined with qiime diversity and plotted by Emperor, through principal coordinate analysis (PCoA), using Bray-Curtis decrease distance matrices. The MetaCoMET web tool was used to determine the central microbiome using an OTU table in BIOM v2.1.0 format that contained the community abundance data, as well as its taxonomy (https://probes.pw.usda.gov/MetaCoMET).

Prediction of metabolic functions with PICRUSt-Galaxy
Prediction of metabolic functions was performed using PICRUSt. The analysis began with the singleend.qza le from where the chimeric sequences and the "borderline chimeras" were eliminated, using the uchime-de novo method, the sequences were grouped to closed-references in operative taxonomic units (OTU's) at 97 % identity. The generated le was exported to the BIOM v2.1.0 format and the taxonomic assignment to 97 % identity with Greengenes data was attached. It was then uploaded to the PICRUSt platform, (http://galaxy.morganlangille.com/), where the functional metabolic prediction was performed (https://forum.qiime2.org/t/how-to-create-a-feature-table-with-qiime2-for-picrust-with-the-taxonomicassignment/2526/6).

Description of the collection area
The location of the Tecozautla geyser is shown in Fig. 1A. The collection was carried out in June. In Figs. 1B and 1C, the sampling zones in the geyser are shown. The samples were collected in four areas of the Tecozautla geyser. The GA sample was located on the tube of the outlet water of the geyser, it had a composition with a high content of white salts with green areas, registering a temperature of 65°C ( Fig. 1B: GA). The GB sample corresponds to a microbial mat found in the soil directly receiving the water from the geyser, it had the characteristic of having white, brown, and green colors in addition to presenting a rigid and brous structure (65°C) (Fig. 1B: GB). The GC sample corresponded to a microbial mat of reddish, yellow, green, pink, white, and brown color, with a semisolid structure (muddy) with small brous sections, found in a heat ux of 61.5°C on a rock ( Fig. 1B: GC). Finally, the GD sample corresponded to the sediments and microbial mats submerged in the left outlet of the geyser, with constant turbulence due to the continuous outlet of water (62°C) (Fig. 1C: GD).

Determination of the physicochemical parameters of the geyser water and total sulfur
The results obtained from the chemical composition of the Tecozautla geyser are presented in Table 1. It was observed that the water presented a conductivity of 1215 µmoh/cm and 82 mg/L of Total Dissolved Solids. The pH was between 9.24 and 10 indicating alkaline waters. The presence of total sulfur (47 ± 2.51 mg/L) sodium (195 mg/L) and chlorides (156.33 mg/L) predominated. Elements such as Al, As, Ba, Cd, Cr, Mn, Pb, Zn, and Hg were not detected. The water temperature at the time of sampling was 94°C at the source. Finally, sulfates (28.6 mg/L), carbonates (16.29 mg/L) and a minimal concentration of nitrates (0.2 mg/L) were detected. Thermophilic diversity in the Tecozautla geyser The SJA-176 class was present in GC (0.05 %) and GD (0.10 %), which is a bacterium not yet cultivated, and nally the Fimbriimonadia class was found in the GC (~ 0.02 %) and GD (< 0.01 %), having as the only cultivated species Fimbriimonas ginsengisoli. Furthermore, at the class level, a relative abundance of unclassi ed sequences was obtained in the GC (0.29 %) and GD (1,44 %) samples.
The phylum Cyanobacteria is present in all samples of the Tecozautla geyser, with relative abundances of 3.27 %; 20.24 %; 7.01 %, and 17.13 % for GA, GB, GC, and GD respectively.
The class Gloeobacterophycideae is present only in the GA sample with 0.10 %, Nostocophycideae with 1.78 % in the GC sample, as well as Oscillatoriophycideae in the GA, GB, and GC samples with abundances of 5.94x10-3 %, 12.61 %, and 0.03 %, respectively. Finally, the Synechococcophycideae class was presented with abundances of 0.01 % for GA; 1.44 % for GB; 4.80 % for GC, and 1.54 % for GD.
The organisms found in the Tecozautla geyser at the genus level within the Cyanobacteria phylum were: Gloeobacter (GA), Leptolyngbya (GA and GB), and Pseudanabaena (GA, GC, and GD).
Another abundant group in the Tecozautla geyser samples was the phylum Chloro exi with abundances of 19.28 % for GA, 1.26 % for GB, 55.61 % for GC, and 36.10 % for GD.
Several classes of microorganisms belonging to the phylum Chloro exi were found in the Tecozautla geyser, among them Anaerolineae with a single cultured bacterium: Anaerolinea thermolimosa. This bacterium presented relative abundances of 18.53 %, 1.02 %, 1.75 %, and 2.84 % for GA, GB, GC, and GD samples, respectively. Chloro exia presented abundances of 0.02, 0.24, 53.66, and 32.18 % for GA, GB, GC, and GD respectively. The class Thermomicrobia has an isolated and identi ed strain; Thermomicrobium roseum was found in GD (0.78 %); Two little-known classes were identi ed Elin6529 in GD and TK17 in GC and GD. Unidenti ed microorganisms were found at the class level with 0.62 %, 0.14 %, and 0.06 % for GA, GC, and GD, respectively.
The phylum Deinococcus-Thermus was identi ed in the Tecozautla geyser with abundances of 1.82 % for GA, 4.56 % for GB, 0.23% for GC, and 1.52 % for GD. Belonging to this phylum, the Truepera genus was identi ed in the GA sample and the Trueperaceae family in the GA, GC, and GD samples. The Proteobacteria were among the most abundant members with the presence of 0.19 %, 25.10 %, 7.04 %, and 5.45 % for the GA, GB, GC, and GD samples respectively. In the Tecozautla geyser all classes of Proteobacteria were identi ed: Alpha, Beta, Delta, Epsilon, and Gamma, which shows the great diversity in the microenvironments analyzed.

Archeas
The archeal phyla found in the Tecozautla geyser were Crenarchaeota and Euryarchaeota in the GA and GD samples, respectively, with relative abundances < 0.001% in both samples, and with 0.002 % of unclassi ed sequences in the GD sample.

Taxonomic assignments at the genus level
For the relative abundances at the genus level, lower abundance percentages were observed because some of the sequences could not be classi ed at a lower taxonomic level. This situation suggests di culties at DNA extraction due to the high content of exopolysaccharides and salts. Figure 2C shows the relative abundances of the microorganisms with the highest incidence.
In the GA sample, the genus Caldilinea showed an abundance of 0

Analysis of beta diversity and the central microbiome
The beta diversity analysis was carried out using the Bray-Curtis principal coordinate analysis (PcoA), presented in 3D, which determines the fraction of minimum abundance per sample of shared taxa. In Fig. 4A it is observed that the 4 samples are separated from each other, that is, that none of the samples is superimposed. The Venn diagram of the central microbiome (Fig. 4B)

Prediction of functional pro les
The prediction of the functional pro les of the microbiome based on the 16S rRNA gene was carried out using the OTU table of assigned taxa and their relative distribution, in order to generate the relative abundances of the functional categories based on sequenced genomes through analysis with PICRUSt, using the Kyoto Encyclopedia of Genes and Genomes (KEGG). The results obtained from the four samples of the Tecozautla geyser presented similar functions to each other, for level 2 (Fig. 5A). The highest abundances were the pathways related to membrane transport, carbohydrate metabolism, amino acid metabolism, energy metabolism, replication and repair, cofactor, and vitamin metabolism. These results show some of the main routes of microorganisms in the Tecozautla geyser, but at a not so speci c level.
The most abundant metabolic pathways obtained for level 3 shown in Fig. 5B, were similar to each other, predominating ABC membrane transport, DNA repair and protein recombination, prediction of general functions, membrane transport, metabolisms of the methane, and purine metabolism. It is observed that oxidative phosphorylation stands out from energy metabolism, followed by routes that intervene in carbon xation and methanogenic metabolism (Fig. 5C).

Chemical composition of thermal water
According to the results obtained, the existence of sulfate (SO4 2− ) may favor the presence of microorganisms that use sulfur in their metabolic pathways. Sulfate is considered one of the most important sources of energy in thermal ecosystems. There is a wide variety of microbial mats that thrive in geothermal hot springs and form unique ecosystems made up of physiologically and phylogenetically diverse microorganisms. In fact, hot springs that contain sulfur, such as the Tecozautla geyser, form microbial mats of various colors (white, gray, yellow, red, pink, green, etc.), which harbor different types of microorganisms such as lithotrophic oxidizing bacteria of sulfur, anoxygenic phototropic bacteria, cyanobacteria, and heterotrophic bacteria. Some of the major members of the microbial mats have been isolated, but most of the existing microbial diversity remains uncultivated and therefore unidenti ed, thus its physiological and ecological functions remain not fully understood [9].
Arsenic was not detected in the waters of the geyser, unlike that described by Núñez-Benítez [10] who reported 43,772 mg/L. Likewise, this author reported the presence of silice with a concentration of 112.65 mg/L. Although it is water from the same area, the composition of the water can be highly variable since it depends on the path you take from the depth of the geyser and the depletion of the minerals. The chemical composition of the water has a strong impact on the microbial communities since several of the minerals present are a source of energy for them, such as Arsenic, which favors the proliferation of species such as Thermus aquaticus and Thermus thermophilus who use it as a source of energy [11].

Thermophilic Diversity Analysis
The analysis of the sequences of the Tecozautla geyser allowed the identi cation of 30 bacterial phyla and only 2 archaea, 70 classes, 79 orders, 72 families, and 44 genera. An important observation is that bacteria without taxonomic assignment with high percentages in abundance were found in the 4 samples analyzed, some authors have described similar results; the lack of taxonomic assignment can be attributed to the fact that their identi cation has not been possible due to the di culty of their isolation preventing identi cation and characterization. It was observed that the phylum Armatimonadetes was the most abundant in the GD sample. The Chthonomonadetes class is part of this phylum which is present in the GD sample with ~ 0.02 % of the total sequences, the only cultivated strain is Chthonomonas calidirosea, which can grow in an interval of 50-73°C, pH 4.5-5.8 and 2 % NaCl [12]. The OS-L class, which corresponds to unidenti ed microorganisms, was also found in samples GA, GC, and GD, this class was found in Octopus Springs in Yellowstone National Park [13]. The species Fimbriimonas ginsengisoli, which belongs to the class Fimbriimonadia, can indirectly provide information about the class. It is a strict anaerobic mesophilic microorganism (https://www.genome.jp/Tools-bin/taxsummary).
The phylum Armatimonadetes is a moderately abundant and phylogenetically diverse bacterial group, little studied and phylogenetically associated with Chloro exi [12]. Three cultured individuals are known, two previously mentioned and Armatimonas rosea. The candidate phylum OP10 (now Armatimonadetes) was rst described in the ecology study of Obsidian Pool, a geothermal hot spring in Yellowstone National Park [12].  [19]. In the same way, they have been detected in the thermal areas of the Pakistanis Himalayas in conditions of 60-95°C with a pH of 6.2-9.4 [20], and in El Coquito, located in the Colombian Andes that present temperatures of 29°C in the source and pH of 2.7 [21]. The Cyanobacteria class was found in Ghats, India, with abundances of 96.42 % and 87.35 % of the total classes for the AT (58°C, pH 8.56) and TP (48°C, pH 8.76) samples [22].
From the analysis of the phylum Cyanobacteria that was detected in the samples from the Tecozautla geyser, the most studied genera are Gloeobacter, which contains two species that are Gloeobacter violaceus and Gloeobacter kilaueensis, the latter isolated in a cave near the lava caldera of the volcano Kilauea in Hawaii. Both are non-thylakoidal and carry out oxygenic photosynthesis [23].
Leptolyngbya is one of the most common and frequent lamentous cyanobacterial genera in thermal environments, occurring in a wide range of terrestrial, aquatic, and extreme environments and equally distributed in Mexico [24]. Finally, the genus Pseudanabaena is anoxygenic photoautotrophic thermophilic cyanobacterium, also found in Yellowstone National Park, and is one of the main responsible for the formation of microbial mats in Chocolate Pool to 52°C, in addition to having been found in geysers in areas that exceed 60°C, as well as in the Amazon River at ~ 30°C, which shows that it can grow at different temperature ranges and habitats [23]. An important point to note about lamentous cyanobacteria is that they are encapsulated by exopolysaccharides, making them di cult to lyse and nucleic acids can be trapped, making them inaccessible for polymerase chain reaction and sequencing [25].
The phylum Chloro exi has been found in the Garga hot spring microbial mats, where the incidence of Chloro exi and Chlorobi did not represent > 10 % of the total number of sequences in the Ga2-verh and Ga3-sred samples [26]. The phylum Chloro exi is one of the most found in thermal environments in the world, the presence of Chloro exi predominates in microbial mats with temperatures of 53-65°C and slightly alkaline pH (7.75-7.91), similar to the samples of the Tecozautla geyser [27]. They have also been identi ed in microbial mats (66°C, pH ∼ 6.5), with relative abundances of ~ 18 to 50 % [28], which are similar to the percentages found in the geyser understudy for Cyanobacteria and Chloro exi. Chloro exi was identi ed in microbial mats from hot springs in Costa Rica, constituting 93 % of all readings, the conditions of the sample area were 37-60°C and pH 6.1-7.5 [29]. Likewise, it is known that the phylum Chloro exi is very widespread in microbial mats of hot springs in Japan [30], Yellowstone (USA) [31], Kamchatka, Thailand, Tibet [23], and in the Andes [32].
The abundances of the two most common chlorophotrophs of Chloro exi (Rosei exus castenholzii and Chloro exus spp.) vary with temperature. At temperatures lower than 60°C, Rosei exus spp. are more abundant, but when temperatures are higher, Chloro exus spp. they are predominant at ~ 70°C [30]. In the Tecozautla geyser, Hidalgo, the genera with the highest relative abundance were Rosei exus and Chloro exus. The rst is a photosynthetic, lamentous, thermophilic bacterium lacking chlorosomes, with a single cultured species Rosei exus castenholzii with optimal growth temperature of 45-55°C and pH 7.5-8 [30]. The second genus contains only two species: Chloro exus aurantiacus and Chloro exus aggregans, both with optimal growth temperatures of 55°C, the rst with a growth limit of 70°C and the second of 60°C, they are generally found in neutral alkaline hot springs [35]. Chloro exus are photoheterotrophic and chemoheterotrophic, they inhabit microbial mats of thermal waters together with cyanobacteria, their growth can occur at temperatures of 30 to 70°C. Furthermore, they are facultative bacteria capable of growing photoheterotrophically under anaerobic and chemoheterotrophic conditions under aerobic conditions [33,35].
The phylum Deinococcus-Thermus is considered a group of microorganisms abundant in hot springs [21,36]. With a presence in hot springs and microbial mats in Tibet (China), Sichuan (China), Mushroom Spring and Calcite Springs, YNP (USA), Siloam (South Africa), Bulgaria (Eastern Europe), Chile (South America) [14,28]. The phylum Deinococcus-Thermus includes the class Deinococci and this is currently divided into the orders Deinococcales and Thermales. The rst is made up of the Deinococcus, Deinobacterium, and Truepera genera. All members of Deinococcus are radioresistant, with two known thermophiles Deinococcus geothermalis and Deinococcus murrayi [36]. In the case of Truepera, the only cultivated species is Truepera radiovictrix resistant to ionizing radiation with optimal growth at pH 7.5-9.5 up to pH 11.2, with the ability to grow in multiple extreme conditions in alkaline, moderately saline, and high-temperature habitats [37]. The Thermales order encompasses ve genera (Thermus, Meiothermus, Marinithermus, Oceanithermus, and Vulcanithermus). Cultured representatives of Thermus are thermophilic and hyperthermophilic. The members of Thermales have recovered from a large set of natural and man-made thermal environments. These bacteria and their cellular components are of biotechnological interest with possible applications in bioremediation or molecular biology, for example, thermostable enzymes [38]. Their ecological importance stands out in that they play an important role in the carbon, nitrogen, and sulfur cycles, such as Thermus oshimai, Thermus thermophilus [39], or as Thermus scotoductus with oxidizing mixotrophic characteristics of sulfur [40]. In the case of the Tecozautla geyser, only the genera Thermus and Meiothermus were found.
Regarding the phylum Proteobacteria, its presence has been reported in habitats with temperatures of 29-35°C and pH 3.5-6.5, with relative abundances of 60 % which decreases signi cantly at 68°C (pH 6.9) with < 10 % [32]. They have also been found in microbial mats, sediments, and hot springs in Eritrea (Africa) with relative abundances of 6.2 to 82.3 %, with conditions of temperatures of 44 to 110°C and pH of 6.97 to 7.54 [41], which allows elucidating that Proteobacteria inhabit a wide spectrum of environmental conditions. The diversity of Proteobacteria is based on the classes, that is, Alpha, Beta, Epsilon, and Gamma, a habitat with the presence of all four classes is an indication of the high diversity of this phylum, as was the case of the Tecozautla geyser that shares the same diversity with the thermal devil's eye [40]. The phylum Proteobacteria is home to the largest variety of bacteria in all environments in the world, including phototrophic and anoxygenic bacteria that predominate in many geothermal environments. They are an important part of ecosystems because they participate in the sulfur and carbon cycle such as purple sulfur bacteria (PSB), type I methanotrophs, which belong to Gammaproteobacteria, purple sulfur-free bacteria (PNSB) that belong to Betaproteobacteria and Alphaproteobacteria, in addition to type II methanotrophs. Sulfate-reducing bacteria (SRB) belong to Deltaproteobacteria, being the main components in environments related to geothermal, alkaline lakes, and saline environments [20,25].
The PSBs belong to the Chromatiales order in the Gammaroteobacteria and within the Chromatiales the PSBs are separated into the Chromatiaceae and Ectothiorhodospiraceae families. All PSB species and their families can perform anoxygenic photosynthesis under anoxic conditions and x CO 2 by the Rubisco enzyme and the Calvin-Benson-Bassham cycle. PSBs are mesophilic organisms that can also photo-assimilate small organic molecules or grow heterotrophically in the dark. Under favorable conditions, they have the ability to reduce N 2 to ammonia. In addition to converting sulfur into less toxic compounds such as sulfate [33]. Nitrogen xation is widely distributed among PNSB. Many of them can use sulfur as an electron donor, but can generally only tolerate low sulfur concentrations < 0.5 mM. Like PSBs, PNSBs are ubiquitously found in mesophilic, circumferential neutral aquatic, or terrestrial environments. Exceptions occur, some prefer acidic, alkaline, or hypersaline conditions. Isolates of thermophilic microorganisms with growth > 50°C are unknown; only mildly thermophilic PNSB species have been isolated (Blastochloris sp. and Rhodocista sp.) which grow up to 47°C, these bacteria were identi ed in slightly alkaline hot springs [33]. The third physiological group of chlorophotrophic proteobacteria is the aerobic anoxygenic purple bacteria (AAPB), with species belonging to the α, β, and γ proteobacteria. Unlike the other two groups, AAPBs require oxygen and organic molecules for their growth. They lack the ability to use CO 2 as their primary carbon source. However, they can obtain up to ~ 15 % of their cellular carbon by anaplerotic CO 2 xation reactions [33]. AAPBs are found in freshwater and marine aquatic environments, as well as in soil crusts and microbial mats from hot springs. They have recently been found in the microbial mats of Octopus Springs and Mushroom springs as well as in the Tecozautla geyser.
Another phylum found in the Tecozautla geyser was the Bacteroidetes phylum. This phylum is a large group of anaerobic and gram-negative chemoganotrophic organisms that do not form endospores and are not mobile by sliding, with wide distribution in the environment [42], with presence in water samples, wet sediments, and microbial mats from hot springs from Eritrea, Ethiopia with abundances of 2.7 to 8.4 % and with growth conditions of 49.5°C to 100°C and pH of 6.97 to 7.54 [41]. They have also been detected in Himalayan hot springs with relative abundances of 74.28 % at temperatures of 60-80°C and pH 8.0-8.5 [43]. Which suggests high adaptability to alkalo-thermophilic conditions.

Analysis of other identi ed taxonomic groups
Other phyla with less abundance but no less important were identi ed, all of them with the capacity to grow in environments with high temperatures and in alkaline waters, characteristics of the Tecozautla geyser. The phylum Acidobacteria, Actinobacteria, Aqui cae, Chlorobi, Dictyoglomi, Firmicutes, Gemmatimonadetes, Nitrospirae, Planctomycetes, Spirochaetes, Synergistetes, Tenericutes, Thermotogae, and Verrucomicrobia were identi ed, which have been reported for their ability to grow in extreme temperature and alkaline environments [44].
Bacteria of the phylum Arquea were also detected, including Euyarchaeota and Crenarchaeota. Euryarchaeota members include halophiles, methanogens, thermoacidophils, and hyperthermophiles. Methanogens are a relatively diverse group that produce methane from CO 2 through hydrogenotrophic methanogenesis and use H 2 as an electron donor [45]. In the case of Crenarchaeota, they have been cultivated at an optimum temperature of 80°C and in the case of extreme thermophiles, at a growth temperature of 65 to 70°C. These bacteria can be acidophilic, predominantly aerobic, or facultative anaerobic, as well as sulfur-dependent thermophilic and ammonium-oxidizing bacteria [46]. These bacteria have been found in the area of the Lake Baikal rupture, where archaea, mainly Crenarchaeota, constituted 19.8 % of the total number of sequences in areas with a temperature of 74°C, while for the Ga3-sred sample their abundance was 0.04 % at a temperature of 55°C. These results agree with the results obtained in this work, it was observed that the elevated temperature favors the growth of Crenarchaeota because in the sampling area the temperature was between 62 and 65°C, very close to the optimal growth temperature reported by other authors [18].

Alpha diversity analysis
Studies based on the sequencing of 16S rRNA genes have facilitated the understanding of microbial diversity, knowing its composition, function, and dynamics. Although there is no general agreement on which diversity index is the best, in general, it is necessary to use a series of them for greater accuracy In the 4 samples of the geyser differences are observed between them, the GD sample of light blue color, presented the greatest diversity, because it does not show a de ned asymptote. In the remaining samples a similar behavior is observed among themselves and they follow an asymptotic trend, which suggests that the greatest amount of diversity possible was identi ed and the sampling effort was adequate as described [48]. The Good coverange graph (Fig. 3B) measures how well the sampling was carried out in an environment and indicates the percentage of individuals sampled in a microbial community, where values greater than 0.9%, evidence that the sequencing effort is su cient to represent the largest number of species [49]. The result observed in Fig. 3B therefore showed that the sequencing effort was su cient to represent the largest number of species.
The Chao1 index (richness) (Fig. 3C) estimates the abundance of individuals in a sample, in the so-called species accumulation curve, where the x-axis is the number of individuals sampled or the sampling units examined, which in this case is the sequencing depth, and the y-axis is the number of species observed.
The Chao1 index is calculated taking into account the total number of species, as well as the number of rare species and those found twice in the sample.
The results for the four samples of the geyser show different behaviors among themselves, this is due to the fact that in the GD sample (microbial mat-sediments) a greater number of individuals is estimated, followed by the GA sample (salts), and the two Microbial mats from the GB and GC samples, which have similar behavior to each other, with a smaller number of individuals.
The Shannon-Weiner index (diversity) (Fig. 3D) is de ned as the estimator of species richness and uniformity. Typical values are generally between 1.5 and 3.5 in most ecological studies, and the index is rarely greater than 4 [47,50]. The results re ect that the GD (microbial mat-sediments) and GC (microbial mat II) samples have greater diversity because both exceed the value of 3.5, but the GD sample is even more abundant because it is ~ 5, in addition to that values greater than 4 have greater diversity and are not so common. In the case of the GA and GB samples, they have values around 3 which indicates that they have an average diversity.
The Simpson metric also called dominance index (Fig. 3E), which derives from the probability theory, allows quantifying the diversity of habitat, re ects the probability of nding two individuals belonging to the same species in two successive extractions, that is, the closer the value of this index is to 1, there is a greater possibility of the dominance of a species, and the closer it is to the value of zero, the greater the biodiversity of a habitat. This index gives a higher weight to common or similar species and underestimates rare species [51]. Therefore, in the GB sample, there is a greater probability of predominance of a particular species, followed by the GD sample and nally, the GA and GC samples presented similar percentages.

Beta diversity and the central microbiome
The results obtained from the beta diversity indicated a great microbial diversity in the four samples analyzed. Most of the OTU's were not shared which suggests a great microbial diversity. This result can be explained from the point of view of temperature, the GA sample was taken from the geyser source where the temperature was 94 ºC, while for the GB, GC, and GD samples the temperatures were 65, 61.5, and 62 ºC respectively. It is important to point out that the GD sample was the one that showed the main physical and chemical differences, in it the presence of exopolysaccharides was observed and it was the one that presented the greatest di culty in DNA extraction. It follows, therefore, that the temperature that determines the microbial diversity in systems similar to the one studied. Similar results have been obtained from other hot spring areas, where it has been observed that microbial diversity depends on the sampling area [52]. Central microbiome analysis is used to understand stable and consistent components across complex microbial ensembles. The results were obtained according to the "a liation" technique that is based on the presence and absence of OTUs between the different microbiomes that are compared, taking into account 1623 bp for the normalization of the data (rarefaction). It was observed that the 4 samples are very diverse due to the temperature of the sampling, which differentiates the populations. This result shows that the samples have a lower relationship with each other. By detecting different pro les it is deduced that each sample presents a typical and unique diversity.

Functional pro les
The prediction of the functional pro les yielded relevant information on the possible metabolic functions of the studied communities. The most abundant metabolic pro le was membrane transport that is related to ABC transporters, phosphotransferase system, and bacterial secretion system (https://probes.pw.usda.gov/MetaCoMET). Based on the results obtained, the importance of ATPdependent transporters or ABC transporters that belong to the class of primary transporters is deduced, being very important routes in archaea and bacteria. In the case of the Extremophiles, these transporters are used as a survival mechanism to regulate osmotic pressure, generation of exopolysaccharides and are part of one of the main transport routes at high temperatures. These routes are classi ed into two groups; carbohydrate and di/oligopeptide absorption transporters. In archaea they can accumulate substrates at much higher concentrations within cells. Hyperthermophilic archaea show important metabolic adaptations for carbohydrate growth under hostile conditions. So far, for carbohydrate absorption, only ABC-type transporters have been described, which are equipped with exceptionally high a nity compared to mesophilic bacterial systems allowing these organisms to e ciently remove all available carbohydrates from the extreme environment [53].
DNA repair, as well as the maintenance of the genome and its expression, is an established mechanism due to growth at high temperatures, where DNA decomposition is accelerated and where some repair proteins are widely conserved, being observed mainly in hyperthermophilic archaea. In addition, under conditions with high levels of radiation including ionizing radiation and UV radiation, repair of massive DNA damage is mediated by energy and protein metabolism, occurring mainly in thermophilic organisms [54]. Regarding energy metabolism, the results agree with the relative abundances found for some bacterial groups where aerobic respiration is the main mechanism for energy generation. In this case, oxidative phosphorylation indicates that electron transfer to a terminal electron receptor, such as oxygen, nitrate, or sulfate, generates ATP. Similar results have been reported in hot springs in Finland [55]. This shows that thermal environments follow a trend in their prediction in metabolic functions, although some of them showed results associated with activities in thermal environments, in others the metabolic capacities of the microbial community were not fully re ected, as is the case of Sulfur metabolism which is very reduced, this can be attributed to a low quantity and quality of the genomes annotated in the databases that are related to the species observed in the thermal water samples [56].
The functional predictions of the PiCRUSt-KEGG pro les indicated metabolic pathways related to membrane transport, carbohydrate metabolism, amino acid metabolism, energy metabolism, replication and repair, cofactor, and vitamin metabolism. Membrane transport is widely related to the ABC transporters, the phosphotransferase system, and the bacterial secretion system, which contribute substantially to the adaptability of these microorganisms to thermal environments, to name a few. The results of this research provided new prospects for the industrial, scienti c and biotechnological potential that can be derived from alkalo-thermophilic microorganisms.

Declarations
-Funding: This work was supported by the National Council for Science and Development (CONACyT-México).
-Con ict of interest: The authors declare that they have no con ict of interest.
-Consent to participe: Not applicable.
-Consent for publication: Not applicable.
-Availability of data and material: Not applicable -Code availability: Not applicable