Lithological characteristics of sediments
In the upper interval (from the surface to 50 cm) of the St.5 GC. 3 core, sediments are represented by reduced diatomaceous aleurite-pelitic ooze with oil inclusions; in the middle interval (from 50 to 65 cm) – by a watered and oil-saturated aleurite layer; in the lower one (from 120 to 151 cm) – grey clay with many oil inclusions and massive stratified gas hydrates. The sediments of the St.5, GC.3 core were highly saturated with gas; the methane concentration varied from 4 to 18 mM/L along the core depth (Fig. 2а). The highest methane concentrations were recorded at depths of 20 and 100 cm (13 and 18 mM/L, respectively). Methane homologues in the gas were mainly represented by ethane (from 1 to 10 µM/L).
Pore waters of sediments in all sedimentary layers were of bicarbonate-calcium-sodium type. Salinity was higher than background one [32]; total ions varied from 136.6 mg/L to 278.8 mg/L along the depth of the core. Pore waters were enriched with bicarbonate ions (up to 3.3 mM/L at a depth of 80 cm) (Fig. 2b). Nitrate (0.7–1.4 µM/L), and nitrite ions (0.7–4.5 µM/L) were present along the entire profile of the core. The concentration of sulfate ions was lower along the entire core depth than at the reference sites and in the Baikal water [27, 32], accounting for 2.9 to 8.3 µM/L (Fig. 2c).
Degradation of hydrocarbons under methanogenic and sulfate-reducing conditions
Cultivation of microbial communities in the cultures enriched with bicarbonate ions and sulfate ions for one year revealed a different degree of conversion for n-alkanes and PAHs. In the GUI sample, the greatest loss of n-alkanes (28%) was determined during the cultivation of the microbial community under sulfate-reducing conditions where the Σalk content in the sample decreased to 5000 μg in comparison with control enrichment cultures (7000 μg). Under metagenomic conditions, a decrease in the number of alkane fraction of oil was insignificant (6600 μg), accounting for 6%. The ΣPAH concentration in the presence of sulfate and bicarbonate ions in the GUI_SO4 and GUI_HСO3 enrichment cultures decreased by 20 to 37%, respectively (Fig. 3). Conversion of oil hydrocarbons in GUI_HСO3 was accompanied by the generation of methane. A native sample of sediments was initially saturated with gas; the methane concentration in the enrichment cultures at the beginning of the experiment was 13 mM/L. After three months of cultivation, the methane concentration increased to 27 mM/L. Its highest concentration (32 mM/L) was recorded after six months of cultivation in the enrichment cultures containing the surface sample of sediments. This level of methane concentration was maintained until the end of the experiment (31 mM/L).
In the cultures with deep sediments (GUII) enriched with bicarbonate ion, the conversion of n-alkanes was 20%, and in those enriched with sulfate ion – less than 1.5% (Fig. 3). The degree of PAH conversion was 45–46% irrespective of the present electron acceptors. In the GUII enrichment cultures, the methane concentrations during the entire experiment remained almost the same (3.0 to 3.8 mM/L) because the values were comparable to those determined at the beginning of the experiment (3.2 mM/L). The loss of sulfate ions in the enrichment cultures containing both surface and deep samples was 25% of the initial concentration.
Bacterial and archaeal community composition in enrichment culture under methanogenic conditions
Analysis of the 16S rRNA gene clone libraries of bacterial communities revealed the members of 12 phyla in the GUI_HCO3 and GUII_HCO3 enrichment cultures. Bacteria assigned to Firmicutes, Chloroflexi, Proteobacteria (δ), and Armatimonadetes (OP10) were common to two samples. The members of the phylum Bacteroidetes and Ca. Aminicenantes (OP8) were found only in the enrichment culture of the surface sample; Proteobacteria (α), Caldiserica (OP5), Ca. Atribacteria (OP9), Ca. division (AC1), Ca. division (OP11), and Ca. Parcubacteria (OD1) – only in the enrichment culture of the deep sample (Fig. 4).
In the GUI_HCO3 clone library, the bulk of the Firmicutes sequences (20 clones) belonged to uncultured bacteria from the sediments of freshwater lakes, Antarctic cold seeps and oil sands tailings (Supplementary Table S1). In the 16S rRNA gene library of the GUII_HCO3 sample, five sequences showed the highest similarity to the members of Peptococcaceae (Fig. 5).
The members of the phylum Chloroflexi were the second most common bacteria in gene libraries. Their largest number (10 clones) was identified in the GUII_HCO3 library. In the phylogenetic tree, they did not form a single cluster but separate branches with the sequences of uncultured bacteria retrieved in the sediments of the cold methane seep in the Sea of Okhotsk, mud volcanic sediments and oil sands tailings during аnaerobic biodegradation of longer-chain n-alkanes (Fig. 5). The sequences from the surface sample (three clones) were homologous to the sequences of uncultured bacteria from riverine sediments contaminated with nitrobenzene as well as to consortium of microorganisms involved in anaerobic digestion of sludge.
The members of the phylum Proteobacteria (δ) and Armatimonadetes (OP10) were minor (one clone in each library). The uncultured Syntrophaceae bacteria and bacterial sequences of the genera Syntrophus sp. and Smithella sp., whose cultured homologues were obtained from syntrophic associations of methanogenic archaea and propionate-, benzoate- and alkane-oxidizing microorganisms, represented the phylum Proteobacteria (δ).
The bacterial sequences assigned to the phylum Bacteroidetes (two clones), which were detected only in the surface sample, were homologous to the uncultured bacteria from chemolithotrophic denitrification reactor and sediments of low-sulfate Lake Pavin. Three clones were assigned to Сandidate division OP8. These clones were closely related to uncultured bacteria from water-flooded petroleum reservoirs, wastewater and freshwater ecosystems.
The 16S rRNA gene library of bacteria from the deep sample was very diverse. There were six sequences assigned to the uncultured candidate division AC1 bacterium, poorly known taxa detected in the deeper layers of lakes [33], forming two subclusters in the phylogenetic tree. One sequence cluster was homologous to the uncultured bacteria from the PAH degrading bacterial community of contaminated soil; another – to the sequences of the uncultured bacteria from the sediments of Lake Biwa and phreatic limestone sinkholes, Mexico. The phylum Caldiserica was the next most represented in the gene library (three clones). The sequences were 96 to 99% homologous to the sequences of the uncultured bacteria from boreal oligotrophic peat wetlands and subalpine stream sediments and 94% − to the uncultured bacteria from thermal vents in Yellowstone Lake (Supplementary Table 1S).
Minor sequences (one clone each) were identified as the members of the phyla Proteobacteria (α) and Ca. Atribacteria (OP9). The Caldovatus sediminis and Crenalkalicoccus roseus thermophils isolated from hot springs were the closest homologues of the MW595807 (Proteobacteria α) sequence. The MW595808 clone was identified as uncultured bacteria closely related to microorganisms from the candidate phylum Atribacteria (OP9) found in the methanogenic reactor and boreal oligotrophic peat wetlands (Fig. 5). Two sequences from the gene library of the deep sample had low similarity (82 to 93%) with uncultured bacteria from the candidate phylum, OD1 (also referred to as Parcubacteria), and Ca. division (OP11) (not shown in the phylogenetic tree).
The members of the phylum Euryarchaeota and TACK group archaeon were detected in the 16S rRNA clone library of archaeal genes from the GUI_HCO3 enrichment culture. The phylum Euryarchaeota (20 clones) was represented by the orders Thermoplasmata and Methanomicrobia, whose closest homologues had been identified in Canadian oil sands reservoir, gas-hydrate potential area, freshwater, and sea floor sediments (Supplementary Table 2S) (Fig. 6). TACK group archaeon consisted of 10 sequences, the closest homologues of which were detected in groundwater of the deep-well injection site, Tomsk-7 (Russia) and in sediments with different geographical locations. The MW617261 clone was related (98%) to uncultured "Aigarchaeota" archaeon from the microbial community of thermal vents in Yellowstone Lake. Currently "Aigarchaeota" is a proposed archaeal phylum combining features of hyperthermophilic and mesophilic life during the evolution of its lineage [34].
Notably, the sequences from the 16S rRNA gene library of bacteria from the microbial community of thermal vents in Yellowstone Lake already appeared in this study during the analysis of the gene library of bacteria from the GUII_HCO3 enrichment culture. Thus, the MW595804 clone showed a similarity of 94% to uncultured Candidate Division OP5 from Yellowstone Lake.
The 16S rRNA library of archaeal genes of the GUII_HCO3 enrichment culture was less diverse in the composition than the GUI_HCO3 enrichment culture. The gene library was 100% composed of the sequences, the closest homologues of which were identified in peatland ecosystems (Supplementary Table 2S). Of them, 87% were the sequences of the uncultured Methanomicrobiales archaeon, and 13% − the sequences of uncultured bacteria that formed a branch in TACK group archaeon (Fig. 6).
Bacterial and archaeal community composition in enrichment culture under sulfate-reducing conditions
In the gene libraries of both samples, more than 30% of the detected sequences belonged to microorganisms of the phylum Caldiserica, аn anaerobic, thermophilic and thiosulfate-reducing bacterium [35] (Fig. 4). Sequences of the uncultured bacteria detected in the hydrocarbon-contaminated aquifer and pristine subalpine stream sediments were the closest homologues (Fig. 4). The members of the phylum Firmicutes, the families Thermoactinomycetaceae and Gracilibacteraceae, as well as unclassified Clostridia, were the second most common microorganisms. The members of the phyla Proteobacteria (δ), Ca. Atribacteria (OP9) and Chloroflexi were common for both samples.
The sequences assigned to the phylum Proteobacteria (δ) had high similarity with uncultured deltaproteobacteria from Zacaton (volcanically controlled hypogenic karst, Tamaulipas, Mexico) and with Syntrophus sp. previously detected in the enrichment cultures of this study under methanogenic conditions. The phylum Chloroflexi was also represented by the sequences that were previously detected in the enrichment cultures enriched with bicarbonate ion.
Sequences of the phyla Ca. Latescibacteria, Bacteroidetes, Actinobacteria, and Planctomycetes were detected only in the surface sample, and the phyla Acidobacteria and Ca. division (AC1) – only in the deep sample (Fig. 7). The sequences assigned to the phylum Planctomycetes had a low similarity (90 to 92%) with the closest homologues from methane hydrate-bearing deep marine sediments in the Pacific Ocean and deep-sea mud volcanoes in Eastern Mediterranean. One of the sequences was assigned to Ca. division WS3 (Latescibacteria). Metabolic reconstruction suggests a prevalent saprophytic lifestyle in all “Latescibacteria” orders, with marked capacities for the degradation of proteins, lipids and polysaccharides predominant in the plant, bacterial, fungal/crustacean, and eukaryotic algal cell walls [36]. Uncultured eubacterium clone from industrial and mining acid sulfate wastewaters was the only closest homologue (98%) of this sequence.
Two clones related to uncultured Actinobacteria from boreal oligotrophic peat wetlands and an ammonium-rich aquifer-aquitard system in the Pearl River Delta (China) had a low similarity (Supplementary Table 3S). In the phylogenetic tree, two clones from the gene library of the deep sample (MW617252) formed a branch in a separate cluster and were similar to uncultured candidate division AC1 bacterium from deepest phreatic sinkhole and sediment of a freshwater Lake Biwa. Two other sequences with unclear phylogenetic position formed another branch in this cluster (Fig. 7).
The clone library of the 16S rRNA archaeal genes from the GUI_SO4 enrichment culture consisted of 100% members of Euryarchaeota. In the phylogenetic tree, sequences of the order Methanomicrobia formed three branches (Fig. 8). The group with the greatest number of sequences (22 clones) clustered with uncultured euryarchaeota from sinkhole ecosystems, which had been previously identified in the archaeal gene library of the cultures enriched with bicarbonate ions and of Methanoregula formicica, methane-producing archaeon isolated from methanogenic sludge. Five sequences showed the highest similarity with the members of Methanosaeta sp. from the microbial community of anaerobic methanotrophic archaea of the ANME-2d cluster in freshwater sediments of Lake Ørn. The sequences homologous to the archaeal sequences from the microbial community of freshwater sediments of Lake Ørn was already identified in the gene library of archaea from the surface sample, which had been cultivated under methanogenic conditions (Supplementary Table 4S). Three clones were related (98%) to uncultured euryarchaeote from the microbial community of thermal vents in Yellowstone Lake (Supplementary Table 4S).
In the 16S rRNA library of archaeal genes from the GUII_SO4 enrichment culture, as in GUI_SO4, sequences of the order Methanomicrobia from sinkhole ecosystems dominated (87%). Five sequences showed the highest similarity to uncultured Methanomicrobiales archaeon from the Canadian oil sands reservoir. TACK group archaeon was represented by two sequences with 97–96% identity with the sequences of uncultured archaea from sediments of various ecosystems (Supplementary Table 4S).
Discussion
In subsurface and deep sediments of the Gorevoy Utes natural oil seep, under methanogenic and sulfate-reducing conditions, we recorded the loss of n-alkanes and PAHs accompanied by the methane formation. In the enrichment cultures containing surface sediments, the n-alkane conversion was the most intense in the presence of sulfate ions, and in those containing deep ones – of bicarbonate ions, which can be due to the composition of microbial communities developing under various conditions. In deep sediments, the microbial community is more oriented to the anaerobic oxidation of PAHs, to which a high degree of their biodegradation (up to 46%) testifies, regardless of the present electron acceptors.
Cultivation of the surface sediment under methanogenic conditions led to the dominance of the members of the phylum Firmicutes in enrichment cultures, whose closest homologues are uncultured bacteria from sediments of freshwater bodies with unknown metabolism that is likely not associated with anaerobic oxidation of hydrocarbons. The members of the phylum Chloroflexi and Ca. Aminicinantes (OP8) can provide the loss of n-alkanes in the GUI_HCO3 enrichment culture. The members of the phylum Chloroflexi are regarded as microorganisms with a high level of hydrolytic enzymes indicating their involvement in the decomposition of complex organic matters [37]. The reconstructed central metabolic pathways suggested that Aminicenantes bacterium is an anaerobic organotroph capable of fermenting carbohydrates and proteinaceous substrates and of performing anaerobic respiration with nitrite [38]. At the same time, the members of the phylum Chloroflexi and Ca. Aminicinantes (OP8) are increasingly found in ecosystems associated with hydrocarbons. Ca. Aminicenantes are often found associated with fossil fuels and hydrocarbon-impacted environments; Chloroflexi harbouring genes for anaerobic hydrocarbon degradation have been found in hydrothermal vent sediments [12, 39, 40]. Perhaps, archaea assigned to the TACK group and the order Thermoplasmata, comprising 33 and 43%, respectively, of the archaeal gene library of the GUI_HCO3 sample, participate in anaerobic alkane oxidation. Phylogenetic reconstructions, protein homologue modelling and functional profiling of metagenomes and genomes revealed that among Archaea, in addition to Archaeoglobi previously shown to have this capability, genomes of Ca. Bathyarchaeota, Heimdallarchaeota, Lokiarchaeota, Thorarchaeota, and Thermoplasmata also suggest fermentative hydrocarbon degradation using archaea-type FAE [12, 41]. The ability to degrade oil in oil-contaminated soils was shown for methanogenic archaea of the families Methanomicrobiaceae, Methanosarcinaceae, and Ca. Methanofastidiosa as well as for the order Thermoplasmatales [42].
In the cultures enriched with bicarbonate ions, methane generation accompanied degradation of hydrocarbons. Methane generation rates in Lake Baikal vary significantly depending on the geological structure of the lake sites [43, 44]. The methane concentration (32.54 mM/L) identified during the cultivation of the surface sample after six months of the experiment significantly exceeded the values that had been previously determined (up to 11.2 mM/L) under conditions of laboratory modelling during the cultivation of microbial communities from the methane seep and mud volcanoes [45, 46].
In the deep sample, under methanogenic conditions, bacteria from the phylum Chloroflexi (proportion in the gene library – 33%) and Firmicutes (17%) represented by the order Peptococcaceae can play the main role in the alkane degradation. Microorganisms from the order Peptococcaceae are most often detected in anoxic environments associated with the anaerobic degradation of aromatic hydrocarbons [47] and in methanogenic short-chain alkane-degrading culture together with methanogenic Archaea (Methanosaetaceae and Methanomicrobiaceae) [48]. In the GUII_HCO3 enrichment cultures, the bulk of the archaeal sequences (83%) was the members of the class Methanomicrobia. Despite the presence of sequences of syntrophic bacteria and methanogenic archaea in the gene libraries of the deep sample, there was no significant methane generation. In some cases, the absence of methane generation in deep sedimentary strata was previously shown both in native natural sediments and in the experimental conditions [44, 46], despite the presence of methanogenic archaea in the composition of microbial communities.
In marine sediments, sulfate ion is the most preferable electron acceptor, and the degradation rate of petroleum hydrocarbons gradually decreases under sulfate-reducing - methanogenic - nitrate-reducing conditions [49]. The content of sulfate ions in the fresh waters of Lake Baikal is not high (55 µM/L) [50]. In Baikal areas associated with hydrocarbon seepages, the concentrations of some ions in pore waters from sediments were abnormally high [27, 32]. No elevated concentrations of sulfate and nitrate ions were in the investigated core. The increased salinity was mainly due to the concentration of bicarbonate ions that do not prevent the development of microorganisms with different types of metabolism. The addition of sulfate ions into the experimental vials containing surface sedimentary layer led to the formation of a more diverse bacterial community and greater loss of n-alkanes in comparison with methanogenic conditions. The members of the phyla Caldiserica, Firmicutes and Chloroflexi, as well as of the class Deltaproteobacteria, occupied the dominant position there. Notably, in the 16S rRNA gene libraries of bacteria from GUI_HCO3 and GUI_SO4, we identified the same closest homologues assigned to Deltaproteobacteria, Chloroflexi and Methanomicrobia, despite the difference in cultivation conditions. The ability to adapt to sulfate stress was shown for bacteria of the genus Smithella and archaea of the genus Methanoculleus, the key alkane degraders and methane producers. In conditions of mixed electron acceptors, in the medium, depending on the sulfate concentration, there is a competition and coexistence of sulfate-reducing and methanogenic populations during the anaerobic decomposition of hexadecane [51], which, probably, also takes place in the Baikal sediments where the same microorganisms participate in the hydrocarbon degradation irrespective of the present electron acceptors.
Microorganisms present in enrichment cultures can be involved not only in the degradation of n-alkanes but also PAHs. Dong X. et al. [12] revealed that aromatic compounds can be anaerobically degraded by bacteria related to Dehalococcoidia, Anaerolineae, Deltaproteobacteria, Aminicenantes, and TA06, as well as by archaea (Thermoplasmata and Ca. Bathyarchaeota), via channelling into the central benzoyl-CoA degradation pathway.
A decrease in the PAH concentration in the sediments of the oil seepage site near Gorevoy Utes Cape has been observed over the past ten years since the discovery of natural oil seepage in 2005. Thus, in 2006, ΣPAH (24 compounds) in the sediments varied from 0.9 to 70 ng/g, and in 2016 – from 1.6 to 16 ng/g [21, 52]. In contrast to PAHs in oil sampled from the lake and the water surface, the proportion of PAHs in oil from the sediments was relatively low [21]. The low PAH concentration is likely due to the impact of the microbial communities in both surface and deep sediments under anaerobic conditions regardless of the present electron acceptors where PAHs primarily undergo oxidation, and n-alkanes are mainly oxidized in the water column under aerobic conditions and sub-surface sediments in case of their enrichment with sulfates. This experiment indicates that the presence of sulfate ion affects the n-alkane degradation that occurs only in enrichment cultures containing surface sediments and less significant for the processes occurring in enrichment cultures containing deep sediments, which corresponds to the results of determining the activity of the sulfate reduction in the sediments of Lake Baikal. The activity of sulfate reduction (from 0.3 to 1200 nM/(dm3day)) is reliably recorded in the upper 15 to 20 cm [44] of sediments and up to 60 cm near the Posolsk Bank methane seep [53]. In deep sediments, the maximum rates of sulfate reduction did not exceed 7 nM/(dm3day) [44].
Therefore, the conducted experiments have revealed a wide range of microorganisms that are potential participants in oil biodegradation under anaerobic conditions of Lake Baikal. The detailed analysis of phylogenetic diversity in petroleum reservoirs on the global scale determined the core of the microbiome that includes three classes of bacteria (γ-Proteobacteria, Clostridia and Bacteroidia) and one class of archaea (Methanomicrobia), which are widespread in petroleum reservoirs and underlie the functioning of the ecosystem in petroleum reservoirs [11]. Grey and co-authors [54] presented similar results that the members of four phyla (Firmicutes, Proteobacteria, Bacteroidetes, and Methanomicrobia) are mostly found in petroleum reservoirs and environments contaminated with hydrocarbons (aquifers, sediments and soils). The results of comparing the structure of microbial communities from sediments associated with oil and from petroleum reservoirs confirm the general idea that the identified main composition of microbial communities participates in complex syntrophic interactions responsible for the complete degradation of alkanes and other hydrocarbon components [55]. Syntrophy is a key mechanism of anaerobic biodegradation of hydrocarbons not only under methanogenic conditions but also in the presence of sulfate ion, ferric iron or nitrate ion [56]. We also do not exclude syntrophic interactions for microbial communities in the Baikal sediments because, in all investigated samples, there were microbial communities that carry out interdependent sequential reactions in the general metabolic process that one member of the community cannot carry out [8].
The phylogenetic diversity revealed in methanogenic and sulfate-reducing microcosms that were obtained in this experiment mostly coincides with the composition of microorganisms included in the “microbiome core” of petroleum reservoirs. The members of the phyla Firmicutes, Chloroflexi, Caldiserica (OP5), as well as of the class Deltaproteobacteria, predominated in the bacterial 16S rRNA gene libraries. Archaea in the clone libraries of the 16S rRNA genes were mainly represented by the sequences of the class Methanomicrobia. In the experiment, there were no members of the class γ-Proteobacteria; the class Bacteroidia was present only in the surface sediments. At the same time, γ-Proteobacteria and Betaproteobacteria ranged from 0.3 to 9 and 0.5 to 15%, respectively, in the surface sediments of the oil seeps according to the analysis of the structure of microbial communities in sediments from the Gorevoy Utes oil seep areas using high-throughput sequencing [57]. In general, from 46 to 80% of microbial communities from the different sedimentary layers of oil seeps consisted of unique OTUs, and only 1 to 2% were shared [57, 58]. Sequences of Actinobacteria, Cyanobacteria, Proteobacteria, Thaumarchaeota, and Euryarchaeota dominated the communities in sediments [57, 58]. The members of the phyla Chlorobi, Gemmatimonadetes, Nitrospirae, Planctomycetes, Armatimonadetes, Ca. Saccharibacteria Ca. Aminicenantes, Ca. Parcubacteria, and TM6 were minor in the 16S rRNA gene libraries [57].
The dominant taxa detected in the experimental enrichment cultures are more typical of microbial communities from deep methane hydrate-bearing sediments of the St. Petersburg methane seep (Lake Baikal) where Chloroflexi (38%), Armatimonadetes (previously OP10/JS1 group) (19%) and Caldiserica (OP5) (8%) were the major components [59]. The investigated sediments, in particular the deep sample, included not only oil but also gas hydrates. Therefore, the influence of gas-saturated fluids may be responsible for the composition of microbial communities close to hydrate-bearing sediments and for the presence in sediments and enrichment cultures of the members of “rare taxa”: Planctomycetes, Ca. Atribacteria (OP9), Ca. Armatimonadetes (OP10), Ca. division (OP11), Ca. Latescibacteria (WS3), Ca. division (AC1), and Ca. Parcubacteria (OD1), which can be involved in hydrocarbon oxidation.
Microbial communities of enrichment cultures are mainly represented by uncultured microorganisms, whose closer homologues were identified in thermal habitats, sediments of mud volcanoes and environments contaminated with hydrocarbons, which are rather distant geographically from Lake Baikal. The detection of the same phylotypes of anaerobic bacteria phylogenetically similar to microorganisms from marine oil strata and high-temperature sediments in cold sediments of distant geographical locations is owing to their distribution by ocean currents [60]. Ocean currents play a key role in the passive spread of spores of thermophiles to distant locations from their origins. The transfer of cells from underground habitats to the overlying ocean also contributes to the marine microbial biodiversity, including representatives of the “rare biosphere” [61].
Lake Baikal located in the central part of the Baikal Rift Zone is not connected with the World Ocean by ocean currents. The identification of sequences of microorganisms having the closest homologues from mud volcanoes, oil and gas basin of the Sea of Okhotsk and Canada, as well as from thermal vents of Yellowstone Park, in the sediments of Lake Baikal associated with the discharge of hydrocarbons may be due to the activity of hydrothermal vents located at a depth of 5–6 km, the generation of which was the most intense at the beginning of the Neogene [62]. In the same period, a modern system of mid-ocean ridges was formed in the World Ocean at the boundary between the Miocene and Pliocene. The entry of thermophiles from terrestrial hot springs located in the area of the Baikal rift may be another probable source of their occurrence in surface sediments. Thanks to the complex system of gradient and convection currents that determine the general circulation of water masses covering all three basins of Lake Baikal [63], thermophilic prokaryotes from terrestrial thermal vents could be brought in and buried in the surface sediments. All these hypotheses require study and will be the subject of further research.