Subsurface Archaea associated with rapid geobiological change in a model Yellowstone hot spring
Acidification of CPHistorical geochemical data suggest that the water chemistry of Cinder Pool (CP) has been relatively stable from the time of first reported geochemical data in 1947 until autumn 2018, followed by pronounced acidification between winter and spring 2019 (Supplementary Data 1, Fig. 1a, b). Images and documentation dating to even earlier (1927) reveal the presence of cinders covering ~50% of the spring surface at that time, a temperature near boiling (91.5 °C), and a description of having high sulfate and chloride levels (although data was not provided), suggesting that its chemistry has been generally stable since its discovery1. Spring pH ranged between ~3.6 and 4.5 in 22 yearly measurements spanning 71 years (1947–2018; multiple measurements in the same year were averaged to represent each year) (Fig. 1b), while the pH has been subsequently measured after 2018 as low as 2.5 (Fig. 1b). A single pH measurement of 2.5 was also recorded in a 2003 publication27, although other measurements in 2003, 2000, and 2001 were more consistent with the long-term average (i.e., pH 4.2–4.3; Supplementary Data 1). Scrutiny of chemical data accompanying the pH 2.5 measurement in 2003 indicates a SO42− concentration (~48 mg L−1) that is considerably lower than would be expected for CP, even when the pH is much higher (SO42− = 80 mg L−1; pH = 4.2–4.3). Considering that sulfuric acid is the predominant buffer of pH in these systems7,28, the pH 2.5 reading in 2003 is considered questionable. Nevertheless, the 2018 shift in pH towards more acidic conditions was accompanied by a notable change in the appearance of CP. Prior to autumn 2018, the spring waters were cloudy gray with the considerable suspension of kaolinite clay particles20 and black cinders10. However, between autumn 2018 and spring 2019, the spring waters visibly turned blue-green and contained colloidal S° particles that were also deposited along the pool shelves, while the pool also lacked its characteristic black cinders (Fig. 1a). The spring has maintained this appearance since spring 2019 until at least July 2022.Fig. 1: Historical geochemistry of Cinder Pool (CP).a Top panel shows the visual change in the appearance of CP in 2016 (left) and 2020 (right). Scale bars in the bottom right are ∼1 m. b Measurements of pH (n = 21; black line) and sulfate (SO42−) concentrations (n = 12; red line) in CP waters between 1947 and 2021. Years with multiple measurements were averaged to represent the entire year. c Paired measurements of SO42− and chloride (Cl−) concentrations (n = 12) between 1947 and 2021 in the context of the same measurements for 488 YNP springs derived from previous studies. Paired points for CP are colored based on the year they were recorded (averaged for multiple measurements/year as described above). End member fluid compositions as described in the manuscript text are indicated based on the abbreviations: MO meteoric only, HO hydrothermal only, MG meteoric plus gas, HB hydrothermal plus boiling, HBG hydrothermal plus boiling plus gas. Points for 2016, 2018, 2019, 2020, and 2021 are indicated by “16”, “18”, “19”, “20”, and “21”, respectively.Full size imageThe source of fluids in YNP hot springs can be broadly defined by concentrations of sulfate (SO42−) and chloride (Cl−)2,7. These indicators have been previously used to define the source of YNP springs as either (1) hydrothermal only (HO) waters that have moderate concentrations of SO42− (~30 mg L−1 depending on the depth of boiling; described below) but high concentrations of Cl− (~300 mg L−1), (2) meteoric-only (MO) waters containing lower concentrations of both solutes, or (3) MO waters infused with gas (MG) that have lower Cl− concentrations and higher SO42− concentrations (Fig. 1c). Subsequent boiling and/or evaporation of HO waters can concentrate Cl− and SO42− to higher concentrations (termed hydrothermal plus boiling; HB), while additional gas input into HO or HB waters can lead to particularly high concentrations of both Cl− and SO42− (hydrothermal + boiling + gas; HBG)7 (Fig. 1c). Geochemical data from surveys spanning 1947 to 2018 suggest that CP was largely sourced by hydrothermal (HO) waters that have undergone boiling and/or evaporation (HB) during this time frame (Fig. 1c).HO and HB waters are typically circumneutral7, while CP (which is also sourced by HB waters) has maintained a moderately acidic pH of ~4 until autumn 2018 (Fig. 1b). Several other low pH HB waters have been previously observed within the NGB7. The moderately acidic pH in CP (prior to 2018) has been attributed to the hydrolysis of molten S° that occurs at depths of >18 m that leads to the formation of S2O32– 11. Oxygen (O2)-dependent oxidation of S2O32−, catalyzed by trace iron sulfide in the cinders, forms SxO62− that can then react with sulfide to yield S2O32− and S° 11. Alternatively, SxO62− can be disproportionated to form S2O32− and SO42− 11. The relative rates of these reactions in CP prior to 2018 are not known although similar concentrations of S2O32− measured between 1995 and 1997 suggest that rates of S° hydrolysis and rates of S2O32− formation have been relatively constant over yearly time scales11. The consumption of O2 by reaction with S2O32− and the consumption of sulfide involving reactions with SxO62− would limit the amount of sulfuric acid that could be formed, thereby maintaining a less acidic pH than other sulfuric acid buffered acidic springs in YNP7.Between November 2018 and March 2019, the pH of CP markedly decreased to 2.8 in 2019, 2.7 in 2020, and 2.6 in 2021. This coincided with a marked increase in SO42− concentrations of ~3–5 fold above historical ranges (Fig. 1b), while Cl− concentrations fluctuated without clear trends during this time (Supplementary Fig. 1c). Thus, CP transitioned from an HB water type to an HBG water type between autumn 2018 and spring 2019 and has remained this way since (Fig. 1c). This is interpreted to reflect a substantial increase in H2S/S° oxidation that results in the formation of SO42− and H+ (sulfuric acid). Several observations suggest a fundamental restructuring of CP’s unique sulfur cycling due to dramatic physical and chemical changes at this time. As described in more detail below, the molten S° layer was detected at a depth of 18 m in 2016. However, in 2020 and 2021 there was no evidence of molten S° at ~18 to 20 m depth as previously documented, and sampling equipment could be freely dropped to a depth of 22 m (length of the cable) without interruption. In the absence of the molten S° at depth, the S° hydrolysis product S2O32−, and the cinders that catalyze SxO62− formation from S2O32− and H2S, it is possible that such reactions that previously competed for H2S or O2 (i.e., those involving S2O32− and SxO62−) are no longer taking place in CP. This in turn would allow for sulfur compounds (H2S and S°) to now be oxidized, thereby contributing to spring acidification.Alternative scenarios underlying the dramatic changes in CP waters also warrant consideration, and the three most logical are presented below. First, it is possible that the waters sourcing CP may have shifted either via replacement of the primary source or by altered mixing of multiple water sources. Water isotope values (δ2H and δ18O) can be used to further deconvolute the sources of hydrothermal waters because distinctive isotope values are associated with distinct water sources and the various influences upon them including meteoric water recharge, boiling (and/or evaporation), and water–rock interactions7,29. The water isotope values measured among the measured depths in CP in 2020 were near the range of water isotope values observed in CP across multiple months in 201613 (depth-resolved water isotope measurements were not made in 2016). The 2020 CP water isotope values were slightly right-shifted relative to those of 2016, suggesting a minor increase in the evaporation and concentration of CP water isotopes between 2016 and 20207 (Supplementary Fig. 2). These data thus do not support the hypothesis that the source of waters in CP dramatically shifted between 2016 and 2020, consistent with the SO42− and Cl− measurements indicating that the primary change to CP waters was increased input or availability of H2S for oxidation.A second alternative explanation is that a change in the water level of CP could potentially alter residence times which could allow for more oxidation of sulfur compounds in the spring and increased acidification. Such a scenario would also likely result in increased evaporation and concentration of solutes. However, the minimal increase in water isotope values (Supplementary Fig. 2) and similar Cl− concentrations (Supplementary Fig. 1c) accompanying a ~3–5 fold increase in SO42− concentration pre- and post-acidification (Fig. 1b) argue that increased residence time was of minimal importance in acidification.A third possible explanation is that a change in the plumbing system of CP is now delivering more vapor phase gas that contributes H2S and acidity when oxidized. Such a scenario could be consistent with increased surface deformation, subsurface gas accumulation, and seismic activity that has been taking place near NGB just prior to these changes21, and the transition from HB-type to HBG-type waters in CP. Sulfur species isotope analyses would help deconvolute the sources of SO42− in CP, but samples for sulfur isotopic analyses were not collected prior to acidification. Thus, it is unclear if this process may also be contributing to the acidification of CP. Regardless, the disappearance of the molten S° cap either by consumption or displacement would in effect make H2S more available for oxidation, similar to increased vapor phase input. The acidification of hot springs involves the oxidation of H2S by O230. More specifically, partial oxidation of H2S at acidic pH (90% amino acid identity to other homologs from UYS MAGs), but that was only present on unbinned contig sequences. Proteins are grouped based on their functionalities and associations in complexes. TetH (tetrathionate hydrolase), SQO sulfide:quinone oxidoreductase, SOR sulfur oxygenase reductase, SoxABCD Sulfolobus oxidase, SoxM Sulfolobus oxidase, CbsAB cytochrome b 558/566, SoxLN cytochrome ba complex, DoxBCE Desulfurolobus oxidase, DoxAD/TQOab Desulfurolobus oxidase/thiosulfate-quinone oxidoreductase, HdrAB1C1B2C2 (heterodisulfide reductase), DsrE3 DsrE3 sulfurtransferase, Dld dihydrolipoamide dehydrogenase, LplA lipoate-protein ligase A, LbpA lipoate binding protein A/glycine cleavage system H protein, TusA tRNA 2-thiouridine synthesizing protein A, SreABC sulfur reductase, SAOR sulfite:acceptor oxidoreductase, HcaLS [NiFe]-hydrogenase group 1 g. SoxEFGHI and FoxABCDEFGH (ferrous iron oxidation) gene sets were also investigated, but not identified in any of the MAGs and not shown here for brevity. A complete description of the enzymes/proteins found in individual UYS MAGs is provided in Supplementary Data 4.Full size imageTo assess the potential role of the UYS in sulfur biogeochemical cycling, the metabolic functional potentials of these populations were evaluated in greater detail based on their reconstructed genomes (Fig. 5, Supplementary Data 3). The UYS encoded the capacity for autotrophy via full complements of enzymes involved in the 3-hydroxypropionate/4-hydroxybutyrate cycle (3HP-4HB) (Supplementary Data 4), consistent with the general potential for autotrophy in most other Sulfolobales36. Consistently, the SoxM subunit that has been suggested as a marker for (facultatively) heterotrophic growth of Sulfolobales37 was absent in all UYS MAGs (Fig. 5, Supplementary Data 4). Given that all known Acidilobus and Vulcanisaeta spp. are characterized heterotrophs without known autotrophic capacity38,39, the UYS are likely the sole primary producers in the CP surface and subsurface waters, consistent with their considerable dominance in CP water communities over time.Also consistent with almost all other Sulfolobales36, the UYS universally encode the ability to reduce O2 via terminal cytochrome oxidases, although not via Sulfolobus oxidase (SoxABCD) complexes that are common among many Sulfolobales36 but rather via Desulfurolobus oxidase complexes (DoxBCE) (Fig. 5, Supplementary Data 4). An additional terminal oxidase complex (CbsAB-SoxLN) was encoded in the 2020 CP MAGs along with several other UYS MAGs from other YNP springs, although homologs of CbsAB-SoxLN were not present in the 2016 CP MAGs or several others recovered from sediments of other hot springs (Fig. 5). Thus, a potentially important metabolic difference between the pre- and post-acidification (2016 and 2020, respectively) CP Sulfolobales was the ability to use different terminal cytochrome oxidase compliments for aerobic respiration. The capacity to use multiple terminal oxidases has been suggested as an adaptation to varying oxygen tensions/availabilities37,40 that likely substantively differed between the low ORP 2016 CP waters and the high ORP 2020 CP waters (Fig. 2c). Consequently, these data point to the ecological succession of UYS strains within CP that are, at least in part, related to strain-level differences in aerobic respiration capacities.A defining feature of most cultured Sulfolobales is the ability to grow chemolithoautotrophically by coupling the oxidation of sulfur compounds (e.g., S0) to aerobic respiration37. The slow kinetics associated with abiotic oxidation of S0 with O2 at temperatures More
