In recent years, the tree of life has expanded substantially. Despite this, many abundant yet uncultivated microbial groups remain to be explored.

The distribution and diversity of sulfate/sulfite reduction prokaryotic (SRP) communities in hot springs from the Quzhuomu and Daggyai Geothermal Zone of Tibetan, China, was reported for the first time. In hot springs that are naturally hyperthermal and anoxic, the sulfur cycle is one of the most active cycles of the elements. The distribution of SRP in response to temperature is of great importance to the understanding of biogeochemical cycling of sulfur in geothermal features. Little is known about the SRP in geothermal zone. In this study, the diversity of SRP was investigated in the sediments from the Daggyai and Quzhuomu geothermal zone using PCR amplification, cloning and sequencing of the dissimilatory sulfite reductase beta subunit gene (dsrB). The abundance of dsrB and 16S rRNA genes, were determined by quantitative polymerase chain reactions. In addition, correlations of the SRP assemblages with environmental factors were analyzed by the aggregated boosted tree (ABT) statistical analysis. The results showed that SRP populations were diverse, but were mainly composed of Desulfobacterales, Desulfovibrionales, Syntrophobacterales, Clostridia and Nitrospirales, and large fraction (25%) of novel sequences have branched groups in the dsrB phylogenetic tree. In Quzhuomu geothermal zone, sulfate-rich hot springs are characterized by thick bacterial mats that are green or red and the SRP populations mainly appear at mid-temperature (50 °C to 70 °C). In low-sulfate hot springs in the Daggyai geothermal zone, although gray or pink streamers are widely formed at 60 °C to 80 °C, they prefer to inhabit in green mat at lower temperature (30 °C to 50 °C). With increasing temperature, the diversity of the dsrB gene at the OTU level (cutoff 97%) decreased, while its relative abundance increased. This result suggests that temperature played an important role in affecting dsrB gene distribution.

Anoxygenic phototrophic sulfur bacteria flourish in contemporary and ancient euxinic environments, driving the biogeochemical cycles of carbon and sulfur. However, it is unclear how these strict anaerobes meet their high demand for iron in iron-depleted environments. Here, we report that pyrite, a widespread and highly stable iron sulfide mineral in anoxic, low-temperature environments, can support the growth and metabolic activity of anoxygenic phototrophic sulfur bacteria by serving as the sole iron source under iron-depleted conditions. Transcriptomic and proteomic analyses revealed that pyrite addition substantially up-regulated genes and protein expression involved in photosynthesis, sulfur metabolism, and biosynthesis of organics. Anoxic microbial oxidation of pyritic sulfur and consequent destabilization of the pyrite structure were postulated to facilitate microbial iron acquisition. These findings advance our understanding of the survival strategies of anaerobes in iron-depleted environments and are important for revealing the previously underappreciated bioavailability of pyritic iron in anoxic environments and anoxic weathering of pyrite.

Investigating the distribution of hydrogen-producing bacteria (HPB) is of great significance to understanding the source of biological hydrogen production in geothermal environments. Here, we explored the compositions of HPB populations in the sediments of hot springs from the Daggyai, Quzhuomu, Quseyongba, and Moluojiang geothermal zones on the Tibetan Plateau, with the use of Illumina MiSeq high-throughput sequencing of 16S rRNA genes and hydA genes. In the present study, the hydA genes were successfully amplified from the hot springs with a temperature of 46–87°C. The hydA gene phylogenetic analysis showed that the top three phyla of the HPB populations were Bacteroidetes (14.48%), Spirochaetes (14.12%), and Thermotogae (10.45%), while Proteobacteria were absent in the top 10 of the HPB populations, although Proteobacteria were dominant in the 16S rRNA gene sequences. Canonical correspondence analysis results indicate that the HPB community structure in the studied Tibetan hot springs was correlated with various environmental factors, such as temperature, pH, and elevation. The HPB community structure also showed a spatial distribution pattern; samples from the same area showed similar community structures. Furthermore, one HPB isolate affiliated with Firmicutes was obtained and demonstrated the capacity of hydrogen production. These results are important for us to understand the distribution and function of HPB in hot springs.

The microorganisms are main drivers of biogeochemical processes in geothermal ecosystems. The dissimilatory nitrate-to-ammonium reduction pathway (DNRA) could act as an alternative source of ammonium and provide an important nitrogen supply for the maintenance of geothermal ecosystems. Investigating the distribution of DNRA-functional bacteria is of great significance to understanding the source of biological nitrogen production in geothermal environments. In this study, we characterized the community distribution of microorganisms harboring nrfA genes in the sediments of hot springs from the Tibet-Yunnan geothermal zone, with the use of Illumina MiSeq high-throughput sequencing of nrfA genes and R language software for statistical analysis. In the present study, the nrfA genes were successfully amplified from the hot springs with a temperature of 38°C–80°C. The nrfA-based phylogenetic analysis showed that the DNRA pathway is widespread within the geothermal ecosystems, with microorganisms harboring nrfA genes predominantly belonging to phyla Chloroflexi, Proteobacteria, Deinococcus-Thermus (top 10), etc. Genus-level analysis revealed Thermoflexus (Chloroflexi) as the dominant taxon in the DGQ, while Geothrix (Acidobacteria) showed peak abundance in weakly acidic sites. The DNRA-functional community structure and nrfA gene abundance also showed a sample variability, even among samples from the same region, there were differences in dominant populations and overall nrfA gene abundance between them. Statistical analysis results indicate that the distribution of nrfA type microorganisms was mainly influenced by physicochemical factors, including pH, SO₄²⁻, and NO₂⁻ concentrations. These findings deepen our understanding of the nitrogen cycle in extreme environments and provide valuable perspectives on the role of nitrogen metabolism in both contemporary and ancient geothermal systems.