3.3. Microbial Biodiversity
The biodiversity in the SZN sediment (
Table 4) was similar to that in other lake sediments worldwide (analyzed using the analogous sequencing depth) [
34,
35] and higher than values typically calculated for soil [
36,
37] or freshwater [
38,
39]. This confirms that the transitional environments of the sediments harbor much higher microbial diversity that the adjacent environments.
Microbial diversity was largely lost in the enrichments. The highest decline was noted between SZN and S1 of the cultures for all calculated indexes (
Table 4). It should be noted that the trophic structure of the microbial community in the S1 cultures was still influenced by the availability of organic matter originating from the basal sediment (dilution 2 × 10
−2). The second stage S2 was transitional, while at the third stage of cultivation when the availability of the basal material was 8 × 10
−6, the community was in fact dependent only on substrates delivered by the experimental medium. The decrease in biodiversity between the second and third stage of culturing was low, which suggests that the S3 community structures were determined mainly by the experimental conditions.
Subsequent transitions resulted in a decrease in unidentified sequences, which probably points to the disappearance of species that are not able to grow when deprived of the natural sediment and are unculturable to date. Furthermore, it was found that a majority of the genera that were lost during the successive stages of the experiment were aerobic (Gaiella, Gemmatimonas, Nitrospira, Conexibacter, Methylobacter, Thermoleophilum, Kofleria) or microaerobic (Sideroxydans, Magnetococcus, Anaeromyxobacter, and Sterolibacterium—threshold of 1% of the reads in SZN. The presence of aerobic microorganisms confirms the transitional character of the SZN sediment and the dualistic character of the microbial community (aerobic/anaerobic). The loss of aerobes was a consequence of the application of the culturing conditions.
The structure of the microbial communities was shaped by the available carbon sources (
Figure 4). Adaptation to the culturing conditions (available carbon sources) was also clearly visible for the methanogens.
Methanothrix, which is the most frequent
Archaea in the SZN sediment, disappeared gradually in a majority of the treatments. In the third stage of the experiment, it was outcompeted by other methanogenic genera (
Figure 3). The differentiation reflected the metabolic capabilities of
Archaea. The availability of the thermodynamically beneficial energy source (H
2) led to increased contribution of hydrogenotrophic
Methanobacterium (in H(+), H(−)CO
2/H
2, and H(+)CO
2/H
2) while the addition of acetate to the culture medium (H(+)acet and H(−)acet promoted
Methanosarcina. In this case, our experiment confirms the previously described fact that although both are acetotrophic,
Methanothrix and
Methanosarcina differ in the kinetics of enzymes involved in acetate assimilation. Hence, low acetate concentrations favour
Methanothrix, which is outcompeted by
Methanosarcina at high acetate levels [
40].
Methanothrix almost completely disappeared at S3 of the experiment. Only in the H(+) culture, at S3, did it still account for 13% of the methanogens (
Figure 3). If the outcompetition of
Methanothrix by
Methanosarcina can indeed be explained by competition for the substrate, the disappearance of
Methanothrix in H(+)acet and H(−)acet requires additional explanation. The composition of the basal medium (vitamins and trace element content) was the same in all cultures. Therefore, it seems that the primary cause of the changes in methanogen contribution would be the associated bacterial community (differentiated by the available carbon source).
Methanothrix may lack some key compounds, e.g., auxotrophic vitamins or amino acids. Recently, Hubalek et al. [
41] presented a comprehensive study showing that the proportion of genes encoding auxotrophy for vitamins and amino acids in the metagenome-assembled genomes of anaerobic, hydrocarbon-degrading communities is surprisingly high compared to those linked with energy conservation. This led them to the conclusion that metabolic interactions between obligate mutualistic microbial partners should be of central importance because beyond the canonical H
2-producing and syntrophic bacteria - methanogen partnership, a complex (although not fully defined) interactions play an important role in determination of the metabolism of the entire community.
Our study shows that the cooperation between acetotrophic methanogens (
Methanothrix) and acetogenic bacteria may be one of those relationships. It has already been proven that
Methanosarcina owe its physiological flexibility to
Clostridia—the most probable source of a unique (as for
Archaea) enzymatic system employing acetate kinase (AckA) and phosphoacetyl transferase (Pta) [
42]. Enzymes involved in methane production from acetate may not be the only ones “imported” via gene transfer. It cannot be excluded that other genes, not yet identified but enhancing survival in environmental conditions, were also incorporated by
Methanosarcina.
Methanothrix seems to lack such benefits. In this work, we found a relationship between the contribution of
Methanothrix and acetogenic bacteria (the latter presented as KEGG-revealed expected abundances of genes responsible for the synthesis of enzymes involved in the acetate-generating Wood–Ljungdahl pathway - carbon monoxide dehydrogenase [EC 1.2.7.4]/acetyl-CoA synthase [EC 2.3.1.169]). We hypothesize that
Methanothrix gains more benefits from cooperation with acetogenic bacteria than from substrate delivery and interspecies electron transfer. Examples of such a relationship have already been demonstrated in co-cultures (alanine transfer between
Methanococcus maripaludis and
Desulfovibrio vulgaris [
43]).
A support for the deduced relationships seems to be the disappearance of
Methanothrix in treatments containing acetate, where the growth of acetogenic bacteria was inhibited by excess product concentration (
via a mechanism described previously by Wang and Wang [
44] (
Figure 6). Full confirmation of the necessity of
Methanothrix – acetogen cooperation requires comprehensive studies. The use of model co-cultures subjected to transcriptional, proteomic and metabolic analyses or shotgun metagenome/metatranscriptome analyses of environmental samples would explain the exact nature of the deduced cooperation between
Methanothrix and acetogenic bacteria.
Interestingly, the proportion of methanogens was not in line with the methane production rate detected in vivo (
Figure 1). The treatment that exhibited the highest methane production (growing with each transfer and characterized by the shortest lag phase) was also characterized by a very low proportion of the identified methanogens (4.3% of the reads). Instead, unexpectedly, high contribution of
Caldiserica was found in H(+)CO
2/H
2. These bacteria are one of the most intriguing elements of the SZN consortium.
Caldiserica, formerly known as OP5, was first described based on environmental 16S rRNA fragments isolated from Obsidian Pool (Yellowstone) [
45]. The first culturable species of the phylum, i.e.,
Caldisericum exile, was isolated by Mori and co-workers a decade later [
46] from a hot spring in Japan. In the present experiment,
Caldiserica was found in almost all of the treatments and was particularly abundant in H(+)CO
2/H
2. The culturing conditions in which CO
2/H
2, yeast extract, and tryptone were used were highly suitable for these bacteria, whose participation in the community structure grew successfully with the consecutive culture stages reaching over 30% of the sequence reads in S3. In the other variants containing H
2/CO
2, yeast extract, and tryptone separately, they accounted for 0.17% and 3.2% of the sequences in the final stage of the experiment. Almost no
Caldiserica representatives were detected in the treatments containing acetate (even in the presence of yeast extract (H(+)acet)).
C. exile, the only known culturable representative of the phylum to date, was described as anaerobic, thermophilic, and thiosulfate-reducing bacterium. Therefore, it could be expected that the presence of
Caldiserica would hinder methane production by competition for hydrogen with methanogens. In this work, we found that the presence of huge numbers of
Caldiserica did not reduce methanogenesis but seemed to even stimulate it. Similar observations have been reported by Ma and co-workers [
47], who investigated degradation of hexadecane to methane as a function of sulphate concentration. In that research, the most effective culture (containing 0.5 mM sulphate) contained a high proportion of
Caldiserica. These authors did not emphasize the role of
Caldiserica but their analysis of the whole community suggested that there is a possibility of cooperation between incomplete-oxidizing sulphate reducers and methanogens, as incomplete oxidation of organic intermediates may generate H
2 through sulphate reduction. In fact, sulphur disproportionation may be carried out with protons being either a substrate or a product of the reaction, as described below (Equations (1), (2)) [
48,
49]:
The ecology of
Caldiserica is currently being discovered and described. The first reports on representatives of the phylum were associated with extreme thermophilic environments, e.g., the hot springs mentioned above [
50] and hydrothermal vents [
51]. Further studies provided growing evidence that representatives of the phylum can occupy other environmental niches as well. Interestingly, their high contribution (reaching as much as 60% of the total community) has been confirmed in permafrost [
52,
53], which denies their exclusively thermophilic character. Additionally, a negative effect of the increased temperature has been observed in this specific location [
52].
Caldiserica was also identified in lake waters both in deep anoxic parts [
54] and, surprisingly, the upper layers [
55]. The results presented in this study pointing to the presence of these bacteria in the sediments of the shallow subsidence reservoir support the recent discoveries of
Caldiserica capability to live in mesophilic conditions or even temperatures close to zero (such as those occurring in shallow lake sediments in winter) and to cope with oxidative stress. Bearing in mind the variety of environments occupied by
Caldiserica (evidence of genomic diversity), it may be expected that the methodological progress in the field of environmental genomics will soon facilitate description of other species belonging to this phylum colonising various environments and eluding culturing attempts.
Other representatives of microbial “dark matter” that have recently come into the limelight and were found in the Szczecin reservoir sediments were those of
Methanomassiliicoccus. This methanogenic methylotrophic
Archaea was first described in human faeces [
56]. Further, its relatives were found in the intestinal tracts of other organisms [
57] or faeces-affected sludges (e.g., from wastewater treatment plants) [
58,
59]. Most research of these microorganisms focuses on their interaction with human health [
60,
61,
62] or the unique methylotrophic but H
2-dependent metabolism [
63].
Methanogenic Thermoplasmata (including
Methanomassilicoccales) use a reduced methanogenic pathway, in which methanol and other methylated compounds are reduced to methane in the presence of H
2 [
56]. This metabolic pathway has long been considered to have minor environmental importance, as it was reported to be used by only two methanogenic species. Recently, the visibility of
Thermoplasmata-related sequences has been enhanced by the description of culturable species and
M. luminensis genome sequence deposition in public databases [
64]. The ecology of this newly described archaeal phylum is currently being recognized. Comparative phylogenetic studies performed by Paul et al. [
65] have implied that
Methanomassiliicoccales may be a part of the microbiome occurring in various environments. To date, these assumptions have been confirmed for extreme environments such as hot springs [
66], formation waters connected with oil reservoirs [
67], wetland soils [
68], lake sediments [
69], and deep subsurface (coal) [
70]. The present study, indicating that the Szczecin reservoir sediment (SZN) is occupied by
Methanomassiliicoccaceae, is in line with the aforementioned discoveries and is the second report, after Fan and Xing [
69], on their presence in lake sediments. Surprisingly, the enrichment cultures in the presented experiment revealed that this group of methanogens is especially enriched in the presence of acetate. All currently published enrichment cultures and a sole
M. luminyensis isolate were obtained on methanol or methylamines as a carbon source and H
2 [
68]. Hence, it could be expected that
Methanomassiliicoccus would find the best growth conditions in medium H(+)CO
2/H
2, where organic substrates and hydrogen were added. Surprisingly, only 0.17% of the sequences in this treatment were affiliated to this genus, vs. nearly 2.5% in H(+)acet, which means that it was 27 times more abundant in these conditions than in the original sediment. These results contrast with previous studies of
Methanomassiliicoccus-containing enrichment cultures. In experiments presented by Lv et al. [
67], addition of acetate to the culture medium resulted in replacement of
Methanomassiliicococcacae by
Methanosaeta and
Methanosarcina (both known for acetotrophic metabolism). In the present study,
Methanosarcina was also dominant in all acetate-amended cultures (which is not a surprise) but in H(+)acet
Methanomassiliicoccus accounted for nearly 12% of all methanogens. The difference between these two experiments may result from the different origin of the inoculates. Lv and co-workers [
67] investigated communities derived from oil production waters, while our study was developed based on a community retrieved from a shallow lake sediment; therefore, they may represent distinct species. The utilization of acetate by
Methanomassiliicoccaceae in lake sediments may be a result of adaptation to in situ conditions. Shallow lake littoral zones are often overgrown by aquatic macrophytes. The roots of these plants are known to exude organic acids. Acetate is thus an abundant substrate in the sediment and, provided anaerobiosis is maintained, can be used by methanogens. Our hypothesis pointing to stimulation of
Methanomassiliicoccaceae by acetate exudates seems to be confirmed also by Fan and Xing [
69], who reported that representatives of the genus are more abundant in littoral- macrophyte overgrown sediments than in deeper parts of the lake (dominated by algae). Also, the sequence retrieved from the SZN metagenome exhibited high similarity with 16SrDNA fragments isolated from environmental littoral samples overgrown by reed (AB896665.1) and rice (KU522088.1; GU134476.1).