Chemical reactions driven by geology, for example the water-rock interaction known as serpentinization, have been shown to support microbial life in several environments including the subsurface. However, many questions remain about how life exists under serpentinizing conditions including: what specific chemical species support life’s processes, how diverse are the types of microbial activities (or metabolisms) that operate in these environments, and how do the endemic microbes deal with the chemical stresses that result from living in a water-rock driven ecosystem. In this work, we leverage studying a natural serpentinizing spring in Northern California, Ney’s spring, which is chemically and microbially similar to marine serpentinzing systems. This makes it unique across the known terrestrial serpentinized springs that have previously been studied, providing distinct insight into the microbial activities and physiologies that are present in these systems.

Our research started by geochemically and microbial characterizing the aqueous environment at Ney springs. (Reported in Trutschel et al. Science of the Total Environment. 2022) Using water isotopes, we demonstrated that the fluids collected at Ney springs are separated from the meteoric water cycle, providing evidence that these fluids are minimally altered by either ground water or surface water (Figure 1). As such, the geochemical and microbial data observed in this work is largely influenced by the water-rock interactions. Geochemically, the site at Ney’s is distinct from other terrestrial systems due to the high sulfide concentrations (~1 mM) and ammonia concentrations. The gas composition at Ney’s has shifted from hydrogen (<1% gas composition) to methane (>80%). Based on the geochemical composition of the spring and thermodynamic calculations, we predicted that metabolisms that either used or produced methane in this environment could be present—with anaerobic methane oxidation being particularly thermodynamically favorable. However, no evidence of Archaea, or methanogenic (or anaerobic methane oxidizing) archaea were found in either the microbial community analysis or metagenome. Methane isotopes supported the potential for methane to be thermogenic in origin. Similarly, no phylogenies or genetic support for aerobic methane oxidation were observed. Metabolisms capable of simple fermentation reactions, and sulfur oxidation were observed in the metagenomes and we were later able to support these potential metabolisms through cultivation of different species, including heterotrophic sulfur-oxidizing Halomonas and Rosinatronobacter species (Figure 2).

Because of the ease of access to Ney springs, we were able to perform seasonal analyses of the aqueous geochemistry and microbial community (Reported in Trutschel et al. Frontiers in Microbiology, 2022). Though many taxa were often observed at Ney springs (occasionally > 5,000 ASVs), only 96 ASVs were observed consistently at every sampling trip. Of these 96 resident ASVs only 16 were observed in abundance of 1% of the total community. We were able to obtain high quality metagenomes assembled genomes of these 16 core taxa and investigate correlations between their seasonal abundance and site geochemistry (Figure 3). For example, Tindalia sp., which were consistently the most abundant ASVs at the site, were strongly correlated with ammonia levels in the spring (Figure 3F). Genomically these organisms were shown to be capable of amino acid fermentation reactions that could generate ammonia as a byproduct. As such, we hypothesize that fluctuations in ammonia levels observed at Ney springs are driven by the activities of this group which is important because ammonia is a potentially potent inhibitor of various microbial activities.

The origin of sulfide, and its potential to support microbial activities have remained an outstanding question at Ney springs. Minimal evidence for biological generation of sulfide (through sulfate reduction) was observed in the genetic/metagenomic data from the site. Our attempts to culture organisms capable of sulfate/sulfur reduction were unsuccessful (data not shown). To further investigate the potential for sulfate reduction we investigated sulfide and sulfate isotopes from Ney springs. The similarity in isotope values (δ³⁴S_VCDT 14.34 vs 16.67 & Δ³³S or 0.024 and 0.056 for Zn-S and Sulfate [Thode] samples respectively) suggest that there is little evidence of biological sulfate reduction in this system, but some potential for sulfur oxidation. As mentioned previously, sulfur oxidation was supported by our genetic/metagenomic investigations and is present in three of the 16 core ASVs consistently abundant at Ney springs.

The major findings of this work support a dynamic community is supported at Ney springs that is largely the result of water-rock interactions. We were able to successfully link various metabolic activities to important taxonomic groups, providing insight into how microbes are surviving in these environments. Notably we see evidence for some of the metabolisms we predicted would thrive in this ecosystem (i.e., sulfur oxidation) and not others (i.e., methanogenesis, methane oxidation) and this might be due to the presence of other stressors (i.e., ammonia) or the extreme pH of this system (> pH 12). Our future work will focus on understanding how the microbes we cultured from this environment, including notably the autotrophic Thiomicrospira.

Last Modified: 12/31/2024
Modified by: Annette R Rowe