Hot Spring Microbial Community Elemental Composition: Hot Spring and Soil Inputs,and the Transition from Biocumulus to Siliceous Sinter

3.1. Sample collection and analysis

Water samples were collected and analyzed, as previously described (Schuler et al., 2017; Havig and Hamilton, 2019a, 2019b). Microbial community samples (e.g., Supplementary Fig. S1) were collected with Teflon tweezers and placed in 5 mL cryovials. All sample sites were located by global positioning and marked on maps. Samples were flash frozen on dry ice in the field and kept frozen until they were processed. Rock, soil, and sinter samples were collected in either 50 mL centrifuge tubes or plastic bags, labeled, and photographed in place at the time of collection. Soils were collected from exposed soils, and all contained gravel to sand-sized pieces of obsidian and rhyolite/tuff. Rock samples were collected from the exposed outcrop of rhyolite/obsidian flows. Sinter samples were collected from the active precipitation region of hot springs, or from old sinter outcrops in hydrothermal areas. For images of representative biocumulus and sinter samples, see Supplementary Fig. S1. Contextual biota samples were collected and placed into either 5 mL cryovials or 15 mL centrifuge tubes, flash frozen in the field on dry ice, and kept frozen until processing.

Temperature and pH were measured onsite with a WTW 330i meter and probe (Xylem Analytics, Weilheim, Germany), and conductivity was measured with a YSI 30 conductivity meter and probe (YSI, Inc., Yellow Springs, OH). Water-dissolved silica values were estimated during sampling by using Hach DR1900 Portable Spectrometers (Hach Company, Loveland, CO) utilizing powder pillows for determining dissolved silica concentrations. Dissolved silica values should be treated as qualitative estimates, as higher temperatures and interferences from other aqueous species are known to affect the colorimetric chemical reaction involved with the Hach methods (unpublished data). Total ammonia [NH₄(T) = NH₃ + NH₄ ⁺] and nitrate concentrations were determined with DR1900 spectrometers via powder pillows on return to the lab to minimize intereferences from temperature and interfering chemical species (e.g., sulfide). Total dissolved inorganic nitrogen (TDIN) was calculated from the sum of NH₄(T) and nitrate.

Hot spring water was collected with a 140 mL syringe directly or drawn after collected with a long-handled polyethylene dipper (500 mL beakers attached to a pole). Water collectors were rinsed with sample water three times before filling. Water was filtered through 0.8/0.2 μm Supor^® syringe filters and dispensed into the appropriate sample bottles after a 10 mL flush to minimize contamination from the filter. Samples for ion chromatography (IC) analysis of major anions (Thermo Scientific Dionex ICS 5000+ IC system) were stored in 15 mL polypropylene centrifuge tubes. Samples for inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis (Thermo Scientific iCAP 6000 series ICP-OES) of major cations and for inductively coupled plasma mass spectrometry (ICP-MS) analysis (Thermo Scientific X Series 2 ICP-MS) for trace elements were stored in acid-washed (3 days soak in 10% TraceMetal Grade HNO₃ [Fisher Scientific, Hampton, NH] followed by triple rinsing with 18.2 MΩ/cm deionized water) 15 mL polypropylene centrifuge tubes and acidified with 400 μL of concentrated, OmniTrace Ultra^™ concentrated HNO₃ (EMD Millipore, Billerica, MA). Field blanks were taken daily by using 18.2 MΩ/cm deionized water transported to the field in 1 L acid-washed Nalgene bottles. IC, ICP-OES, and ICP-MS analyses were carried out by the Analytical Geochemistry Laboratory in the Department of Earth Sciences at the University of Minnesota.

Samples for dissolved inorganic carbon (DIC) analysis were filtered into a gas-tight syringe and then injected into Labco Exetainers^® (Labco Limited, Lampeter, United Kingdom) that was pre-flushed with He. Excess He was removed after the introduction of 4 mL of filtered sample with minimal agitation. Samples were stored inverted and on ice and refrigerated at 4°C until they were returned to the lab, where 1 mL of concentrated H₃PO₄ was added and the samples were shipped to the Stable Isotope Facility (SIF) at the University of California, Davis for analysis. The DIC analysis for concentration and ¹³C isotopic signal was acquired by a GasBench II system that was interfaced to a Delta V Plus isotope ratio mass spectrometer (IR-MS; Thermo Scientific, Bremen, Germany). Raw delta values were converted to final by using laboratory standards (lithium carbonate, δ¹³C = −46.6‰ and a deep seawater, δ¹³C = +0.8‰) that were calibrated against standards NBS-19 and L-SVEC. Samples for dissolved organic carbon (DOC) analysis were filtered through a 0.2 μm polyethersulfone syringe filter that had been flushed with ∼30 mL of sample, and then ∼40 mL was put into a 50 mL centrifuge tube and then immediately flash-frozen on dry ice. They were kept frozen and in the dark until analysis at the SIF. The DOC analysis for concentration and ¹³C isotopic signal was carried out by using O.I. Analytical Model 1030 TOC Analyzer (O.I. Analytical, College Station, TX).

Major biologically essential elements (BIO: carbon, nitrogen, and phosphorous) were determined as DIC and DOC (via IR-MS), as NH₄(T) and NO₃ ⁻ (via field spectrophotometer), and summed as TDIN and dissolved P in samples that were filtered through 0.2 μm syringe filters (via ICP-MS) (Fig. 7 and Supplementary Table S1). Trace elements that were analyzed included Li, Al, V, Mn, Fe, Co, Ni, Cu, Zn, Ga, As, and Mo (Figs. 6 and 8 and Supplementary Table S1). These were binned into elements that are relevant to the function of life (termed BIO-TRACE), which included V, Mn, Fe, Co, Ni, Cu, Zn, and Mo (as well as Mg, discussed above), and elements that are biologically non-essential (termed NON-BIO), including Li, Al, Ga, and As.

3.2. Solid sample preparation and analysis

Solid samples (including biocumulus, sinter, rocks, and soils) were dried for 3 days in an oven set at ∼60°C. After drying, the samples were ground to a homogeneous powder with an agate mortar and pestle that was cleaned between samples by using an ethanol silica sand slurry followed by rinsing with 18.2 MΩ/cm deionized water and drying. The C and N analysis samples were weighed and placed into tin cups that were folded to seal the sample inside. The samples were then analyzed by using an Elementar Vario EL Cube or Micro Cube elemental analyzer (Elementar Analysensysteme GmbH, Hanau, Germany) that was interfaced to a PDZ Europa 20–20 IR-MS (Sercon Ltd., Cheshire, United Kingdom) at the SIF at the University of California, Davis. The samples were combusted at 1080°C in a reactor packed with copper oxide and tungsten (VI) oxide. After combustion, the oxides were removed in a reduction reactor (reduced copper at 650°C). The helium carrier then flowed through a water trap (magnesium perchlorate). N₂ and CO₂ were separated by using a molecular sieve adsorption trap before entering the IR-MS. During analysis, the samples were interspersed with several replicates of at least two different laboratory standards (bovine liver, glutamic acid, enriched alanine, nylon 6), which had been previously calibrated against NIST Standard Reference Materials (IAEA-600, USGS-40, USGS-41, USGS-42, USGS-43, USGS-61, USGS-64, and USGS-65).

Trace element analysis of biomass and soils were determined from ground and dried samples (∼0.050 g each) that were digested by using acid ratios adapted from the EPA method 3052 (Kingston and Walter, 1998) and carried out via an open-vessel digestion. Weighed samples were placed in Teflon beakers with 1 mL 30% H₂O₂ and 1 mL of trace element-clean concentrated HNO₃ and were heated at 30°C for 3 h to break down organics. The remaining 4 mL of the HNO₃ and 3 mL of trace element-clean concentrated HCl were added sequentially in 1 mL increments to avoid excess H₂O₂ from rapidly effervescing. The samples were heated for 1 h at 40°C before adding 1 mL of trace element-clean concentrated hydrofluoric acid (HF). The samples were left at 40–50°C until solutions became faint yellow or clear (approximately 3–4 h). The samples were then heated to just below boiling in a fume hood to drive off HF (reducing the sample to a volume of ∼0.5 mL), and then they were diluted with nanopore 18.2 MΩ/cm deionized water to 30 mL in a 50 mL centrifuge by using a 5 mL pipette. This gave a final concentration equivalent of ∼1 g sample per 0.6 L of water. Confirmation of digestion method element recovery was conducted with NIST SRM 278. The digested samples were analyzed with ICP-MS (Thermo Scientific X Series 2 ICP-MS) by the Analytical Geochemistry Laboratory in the Department of Earth Sciences at the University of Minnesota.

Elements measured in digested bulk biocumulus samples were binned into three categories: (i) major biologically essential elements (BIO, including C, N, and P); (2) trace biologically essential elements (BIO-TRACE, including Mg, V, Mn, Fe, Co, Ni, Cu, Zn, and Mo); and (3) biologically non-essential elements (NON-BIO, including Li, Al, Ga, and As) (Fig. 6 and Supplementary Table S2). Values are presented in units of mol/kg to facilitate comparison of concentration values that span over seven orders of magnitude, and these units will be used for sinter, rock, and soil digestion analysis results as well. Median values are used to represent data populations for each element, as mean values would be skewed by high concentration outliers.

All analytical results are reported in tables provided in the Supplementary Table S2, including biocumulus analytical results; biocumulus associated water sample site location, temperature, pH, conductivity, and analytical results (Supplementary Table S1); non-biocumulus water sample site location, temperature, pH, conductivity, and analytical results (Supplementary Table S1); and rock and biota sample locations and analytical results (Supplementary Table S2).

4.1. Aqueous geochemistry

Hot spring water samples had pH values that ranged from a low of 2.05 to a high of 9.49 (Fig. 5 and Supplementary Table S1). The MPS area sites (n = 46) had pH values, with one mode between 6.35 and 9.49 with an average of 8.4. Areas with phase separation sites (n = 55) ranged from 2.05 to 8.26 and exhibited a bimodal distribution between more acidic sites (n = 26) with an average pH of 3.0 and less acidic to circum-neutral sites (n = 29) with an average of 6.3. These pH distributions are consistent with previous work (e.g., Brock, 1971; Nordstrom et al., 2009; Holloway et al., 2011; Boyd et al., 2012; Inskeep et al., 2013). Temperatures ranged from 29.4°C to 92.8°C, with MPS sites ranging from 33.1°C to 92.8°C and phase separation sites ranging from 29.4°C to 91.6°C (Supplementary Table S1). All sites exhibited high concentrations of dissolved silica, with MPS sites ranging between 1.96 and 6.18 mM (average of 4.18 mM) and phase separation sites ranging between 1.18 and 6.82 mM (average of 3.07 mM) (Supplementary Table S1). In general, all aqueous geochemistry values and patterns observed are consistent with previous work done by the USGS in studying the geochemistry of hot springs in Yellowstone National Park (e.g., Ball et al., 2002, 2008, 2010; McCleskey et al., 2005, 2014).

The MPS sites had a relatively small range of SO₄ ²⁻ and Cl⁻ concentrations, falling between 0.14 and 0.37 mM with an average value of 0.19 mM for SO₄ ²⁻ and between 6.01 and 9.30 mM with an average value of 7.46 mM for Cl^- (Fig. 5 and Supplementary Table S1). Phase separation sites exhibited a wider range of concentrations, falling between 0.16 and 6.97 mM for SO₄ ²⁻ and between 0.01 and 20.29 mM Cl⁻, with a general bimodal distribution of high concentration of one constituent associated with a low concentration of the other, with Cl⁻ tracking with liquid phase input and SO₄ ²⁻ tracking with vapor phase input.

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FIG. 5.

SO₄ ²⁻ versus Cl⁻ plots for Yellowstone hot springs sampled for this study and in previous work. Top: samples broken down by hydrothermal area. Closed symbols for this work, open symbols for previous work. Bottom, left: samples binned by pH of hot springs sampled. Black outlined symbols for this work, gray outlined symbols for previous work. Bottom, right: hot springs that were sampled for biocumulus elemental composition determinations. GAP, The Gap Area; GGB, Gibbon Geyser Basin. Literature values from Havig (2009), Havig et al. (2011), Holloway et al. (2011), McCleskey et al. (2014), Schuler et al. (2017), Hamilton et al. (2019), Havig and Hamilton (2019a, 2019b), Gangidine et al. (2020).

Monovalent and divalent cations exhibited strong, though differing, correlation with phase input. Na and K concentrations were higher in MPS sites and phase separation sites with significant liquid phase input. Mg and Ca concentrations were higher in phase separation sites with significant vapor phase input (Supplementary Table S1). Similar to SO₄ ²⁻ and Cl⁻, MPS sites had Na and K concentrations that exhibited a relatively narrow range of values, with 9.85–16.41 mM and an average of 13.64 mM for Na and 0.23–0.55 mM and an average of 0.37 mM for K. Phase separation sites had a larger range of values for Mg and Ca, from below detection limits (<5 nM) to 0.617 mM and an average of 0.055 mM Mg and from 0.008 to 0.940 mM and an average of 0.169 mM Ca.

The DIC concentration ranged from 2.25 to 6.96 mM with an average of 4.09 mM DIC at sites with MPS, whereas the highest and lowest overall values were recovered from sites with phase separation inputs, ranging from 0.02 to 14.93 mM, with an average of 3.17 mM DIC. The DIC is present as primarily dissolved CO₂ and H₂CO₃ at acidic pH sites and predominantly as HCO₃ ⁻ at circum-neutral to alkaline sites. The DOC (measured as bulk values) was generally lower at MPS sites (20.45–161.5 μM with an average of 67.60 μM DOC) and higher at sites receiving predominantly phase separation inputs (38.22–492.3 μM with an average of 132.0 μM DOC). Inorganic fixed nitrogen, when above detection limits, was present as either a relatively low concentration of NO₃ ⁻ (typical for MPS sites) or a relatively high concentration of NH₄(T) (typical for input from vapor phase in phase separation sites). When above detection limits, NO₃ ⁻ concentration ranged from 9.50 to 71.42 μM, whereas NH₄(T) concentration ranged from 0.61 to >575 μM, which are consistent with previous observations (e.g., Holloway et al., 2011). Phosphorous concentration varied across sites, with a general pattern of lower concentration at MPS sites (range of 0.82–38.05 μM, average value of 8.31 μM) and higher concentration at phase separation sites (range of 1.04–134.73 μM, average value of 17.14 μM).

Element concentration ranges for each element were as follows—Li: 4.61–881 μM, Al: 1.20–1645 μM, V: 0.39–1600 nM, Mn: 3.71–10,867 nM, Fe: 7.07–174,722 nM, Co: below detection limits to 38.74 nM, Ni: below detection limits to 314.3 nM, Cu: below detection limits to 191.27 nM, Zn: 1.59–7046 nM, Ga: 0.057–523.1 nM, As: 28.68–54,470 nM, and Mo: 1.04–2139 nM. These elements fell into two patterns of behavior: either high concentration in MPS and phase separation sites with significant liquid phase input, or high concentration in sites with significant vapor phase input. Most elements had higher concentrations in the VPD sites, including Al, Mn, Fe, Co, Ni, Cu, and Zn. Elements with higher concentrations associated with liquid phase input included Li, As, and Mo.

4.2. Biocumulus

Biocumulus BIO element concentrations were similar for MPS samples and phase separation samples. The C concentrations ranged from 3.59 × 10⁻¹ to 2.35 × 10⁺¹ mol/kg, with median values of 6.07 × 10⁰ mol/kg for MPS samples and 4.22 × 10⁰ mol/kg for phase separation samples. The N concentrations ranged from 3.10 × 10⁻² to 2.74 × 10⁰ mol/kg, with median values of 5.45 × 10⁻¹ and 4.80 × 10⁻¹ for MPS and phase separation samples, respectively. The P concentrations ranged from 1.97 × 10⁻³ to 1.60 × 10⁻¹ mol/kg, with median values ranging of 1.44 × 10⁻² mol/kg for MPS samples and 1.85 × 10⁻² mol/kg for phase separation sites. The ratio of biocumulus C:N concentrations for MPS and phase separation sites fell along a 10.1 to 1 line (Fig. 10).

Biocumulus BIO-TRACE element concentrations were generally higher in biocumulus samples from phase separation sites compared with samples from MPS sites, consistent with higher concentrations of trace elements in phase separation hot spring water compared with MPS hot springs. The ranges of values recovered were Mg: 1.32 × 10⁻³ to 2.13 × 10⁻¹ mol/kg, V: 2.19 × 10⁻⁶ to 7.71 × 10⁻³ mol/kg, Mn: 1.59 × 10⁻⁵ to 2.35 × 10⁻² mol/kg, Fe: 8.11 × 10⁻⁴ to 4.07 × 10⁰ mol/kg, Co: 7.49 × 10⁻⁷ to 1.03 × 10⁻³ mol/kg, Ni: 1.32 × 10⁻⁶ to 3.65 × 10⁻⁴ mol/kg, Cu: 2.44 × 10⁻⁶ to 5.67 × 10⁻⁴ mol/kg, Zn: 2.47 × 10⁻⁵ to 1.67 × 10⁻² mol/kg, and Mo: 6.62 × 10⁻⁷ to 6.23 × 10⁻⁴ mol/kg. The median values for phase separation site biocumulus samples were approximately two to ten times the median values of MPS site biocumulus samples for all BIO-TRACE elements except Mn (which was nearly the same).

Biocumulus NON-BIO element concentrations did not follow a consistent pattern, with Al and As concentration ranges similar for biocumulus samples from MPS sites versus phase separation sites (1.88 × 10⁻¹ to 3.90 × 10⁰ mol/kg Al and 6.80 × 10⁻⁵ to 3.27 × 10⁻² mol/kg As), though median values for phase separation biocumulus samples were higher than for MPS biocumulus samples. Alternatively, Li and Ga concentrations were generally higher in MPS biocumulus samples (1.33 × 10⁻³ to 1.17 × 10⁻¹ mol/kg Li with a median of 1.13 × 10⁻² mol/kg Li and 4.85 × 10⁻⁵ to 7.36 × 10⁻³ mol/kg Ga with a median of 1.22 × 10⁻³ mol/kg Ga) compared with phase separation biocumulus samples (1.35 × 10⁻³ to 8.69 × 10⁻² mol/kg Li with a median of 5.65 × 10⁻³ mol/kg Li and 1.04 × 10⁻⁴ to 1.94 × 10⁻² mol/kg Ga with a median of 4.89 × 10⁻⁴ mol/kg Ga).

4.3. Contextual samples

The concentration of BIO, BIO-TRACE, and NON-BIO elements in rock and soil samples overlapped with biocumulus samples (Supplementary Table S2). The BIO element concentrations for rocks and soils were all at the low range of biocumulus concentration values. Soil C:N ratios had an average value of 21.7 to 1, which was more than double the C:N ratio of biocumulus samples (Fig. 10). The BIO-TRACE element concentrations for rocks and soils overlapped with either the mid-range values for biocumulus samples (e.g., Mg, V, Ni, Cu, and Mo) or the high-range values for biocumulus samples (e.g., Mn, Fe, Co, and Zn). NON-BIO element concentrations overlapped with the high end of biocumulus concentrations for Al, but with the low end of biocumulus concentrations for Li, Ga, and As. A comparison of median rock and soil values with median biocumulus concentrations from either MPS or phase separation sites shows that elements fall along, or close to, a 1:1 line (Fig. 11). The overall ranges of values for rock and soil samples for BIO, BIO-TRACE, and NON-BIO elements were as follows: C: 3.60 × 10⁻¹ to 1.48 × 10⁰ mol/kg, N: 1.61 × 10⁻² to 9.52 × 10⁻² mol/kg, P: 3.07 × 10⁻³ to 1.16 × 10⁻² mol/kg, Mg: 2.15 × 10⁻³ to 2.20 × 10⁻¹ mol/kg, V: 6.11 × 10⁻⁶ to 8.21 × 10⁻⁴ mol/kg, Mn: 5.04 × 10⁻⁴ to 4.09 × 10⁻² mol/kg, Fe: 9.71 × 10⁻² to 3.64 × 0⁻¹ mol/kg, Co: 4.48 × 10⁻⁶ to 7.73 × 10⁻⁴ mol/kg, Ni: 4.37 × 10⁻⁶ to 3.29 × 10⁻⁴ mol/kg, Cu: 3.82 × 10⁻⁵ to 9.22 × 10⁻⁴ mol/kg, Zn: 2.31 × 10⁻⁴ to 1.33 × 10⁻³ mol/kg, Mo: 3.00 × 10⁻⁵ to 8.38 × 10⁻⁵ mol/kg, Li: 5.32 × 10⁻⁴ to 7.06 × 10⁻³ mol/kg, Al: 1.79 × 10⁰ to 4.01 × 10⁰ mol/kg, Ga: 2.64 × 10⁻⁴ to 3.32 × 10⁻⁴ mol/kg, and As: 7.72 × 10⁻⁵ to 1.97 × 10⁻³ mol/kg.

Allochthonous biota biomass C concentrations fell in a small range of values (2.77 × 10⁺¹ to 4.09 × 10⁺¹ mol/kg) and a wide range of sources, including Lodgepole Pine needles, grasses, sedges, mosses, lichens, insects, bison feces, and bison fur. Biota N concentrations exhibited a larger range of values (4.58 × 10⁻¹ to 1.13 × 10⁺¹ mol/kg). Lichen, mosses, and Lodgepole Pine needles had the lowest values of N, whereas insects and bison fur had the highest values. The C:N concentration ratios ranged from a high of 70.7 to 1 for lichen to a low of 3.4 for bison fur, and all plant matter and bison feces values fell close to, or above, the soil C:N ratio, or below the biocumulus C:N ratio (Fig. 10).

4.4. Siliceous sinter

Concentrations of BIO and BIO-TRACE elements in siliceous sinter samples were consistently at, or below, values of associated biocumulus samples for both MPS sites and phase separation sites, with siliceous sinter median values all lower than associated biocumulus median values (Fig. 12 and Supplementary Table S2). Sinter C:N ratios were more scattered than biocumulus values, but they still fell along a 10.1 to 1 line (Fig. 10). For NON-BIO elements, Li and Al concentrations in siliceous sinter samples were at, or below, values for associated biocumulus samples, and median sinter values were lower than biocumulus median values. Ga and As concentrations were similar to biocumulus values, and median values varied (MPS sites had sinter median values lower than biocumulus median values, and phase separation sites had sinter median values similar to biocumulus median values). Analyses for Mo in all sinter samples, for Co in MPS sinter samples, and for P in phase separation sinter samples were at or below detection limits; therefore, these values were excluded from this study. The overall ranges of values for siliceous sinters for BIO, BIO-TRACE, and NON-BIO elements were as follows: C: 6.73 × 10⁻² to 1.40 × 10⁰ mol/kg, N: 9.86 × 10⁻³ to 1.48 × 10⁻¹ mol/kg, P: 5.09 × 10⁻⁴ to 3.23 × 10⁻³ mol/kg, Mg: 1.86 × 10⁻⁴ to 1.94 × 10⁻² mol/kg, V: 7.24 × 10⁻⁷ to 1.16 × 10⁻⁴ mol/kg, Mn: 3.30 × 10⁻⁶ to 1.38 × 10⁻² mol/kg, Fe: 1.46 × 10⁻⁴ to 5.18 × 10⁻¹ mol/kg, Co: 1.19 × 10⁻⁶ to 2.73 × 10⁻⁴ mol/kg, Ni: 1.35 × 10⁻⁶ to 5.43 × 10⁻⁵ mol/kg, Cu: 2.57 × 10⁻⁶ to 1.81 × 10⁻⁴ mol/kg, Zn: 5.21 × 10⁻⁶ to 1.23 × 10⁻³ mol/kg, Mo: at or below detection limits, Li: 2.32 × 10⁻⁴ to 1.90 × 10⁻² mol/kg, Al: 1.65 × 10⁻² to 2.49 × 10⁰ mol/kg, Ga: 2.67 × 10⁻⁵ to 2.04 × 10⁻² mol/kg, and As: 9.06 × 10⁻⁵ to 2.17 × 10⁻¹ mol/kg.

In summary, whether a hot spring and endemic biocumulus are in hydrothermal areas that exhibit either MPS or phase separation influences the elemental composition of the water (aqueous geochemistry), biocumulus, and siliceous sinter.

5.1. Characterizing hot spring water

A total of 102 hot spring water samples were collected from 8 hydrothermal areas across Yellowstone National Park from 2015 to 2018 (Fig. 2) and were analyzed for major and trace elements (Figs. 6–8). Coupled to this, 618 hot spring samples reported in select literatures sources (Havig, 2009; Havig et al., 2011; Holloway et al., 2011; McCleskey et al., 2014) were added for context. Elements of interest were placed into three separate bins as follows: (1) biologically essential elements that are major components of cellular material, including C, N, and P (BIO); (2) biologically essential elements that are minor or trace components of cellular material, including Mg, V, Mn, Fe, Co, Ni, Cu, Zn, and Mo (BIO-TRACE); and (3) elements that are biologically non-essential, including Li, Al, Ga, and As (NON-BIO).

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FIG. 6.

SO₄ ²⁻ versus Cl⁻ plots with hot spring biologically non-essential (NON-BIO) element concentration bins overlain, including (in increasing atomic weight) Li, Al, Ga, and As. Element ranges for bins given for each element.

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FIG. 7.

SO₄ ²⁻ versus Cl^- plots with hot spring major biologically relevant (BIO) element concentration bins overlain, including (in increasing elemental atomic weight) DIC, DOC, TDIN [TDIN = NH₄(T) + NO₃ ⁻ + NO₂ ⁻], and total dissolved phosphorous (P). Element ranges for bins given for each element. DIC, dissolved inorganic carbon; DOC, dissolved organic carbon; TDIN, total dissolved inorganic nitrogen.

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FIG. 8.

SO₄ ²⁻ versus Cl⁻ plots with hot spring trace biologically relevant (BIO-TRACE) element concentration bins overlain, including (in increasing elemental atomic weight) Mg, V, Mn, Fe, Co, Ni, Cu, Zn, and Mo. Element ranges for bins given for each element.

BIO elements—the major components of life—were measured as DIC, DOC, TDIN [the sum of NO₃ ⁻, NO₂ ⁻, and NH₄(T)], and total dissolved phosphorous (P) in hydrothermal waters, with the concentrations binned and plotted within the SO₄ ²⁻ versus Cl⁻ field (Fig. 7). The DIC concentrations generally increased with increasing LPD input, with the majority of the highest concentrations measured (>6000 μM) found in the MPS area and lower DIC values in VPD hot springs due to low pH driving DIC to CO_2(g), which is readily lost. Conversely, the highest DOC concentrations were observed in hot springs with vapor phase input, and most of the lowest DOC values (<50 μM) were found in the MPS area. This indicates that DOC is picked up from surface sources and concentrated in VPD hot springs, but it is not as readily concentrated in LPD systems, potentially due to the continuous turnover of hot spring water and/or heterotrophic breakdown, which is consistent with DOC δ¹³C values compared with allochthonous sources in previous work (Havig, 2009; Havig et al., 2011).

The TDIN concentrations were the lowest in the MPS area and greatest in the VPD area. A spread of concentrations was observed in remixed to LPD areas, which is consistent with volatilization as NH_3(g) during subsurface phase separation and concentrated as NH₄(T) with vapor phase condensate. Holloway et al. (2011) presented a more in-depth discussion about TDIN and its connection to subsurface processes and input.

High P concentrations were found across both MPS and phase separation hot springs, indicating that input of P from allochthonous and autochthonous sources may act as sources of P for microbial communities present, without a consistent pattern. Most of the lowest P sites plot in the MPS and LPD space, suggesting that the autochthonous hot spring source of P is less significant than that of allochthonous sources. This is consistent with the accumulation of P due to the breakdown of allochthonous organic material and/or dissolution of primary minerals in low pH phase separation dominant hot springs.

Concentrations of BIO-TRACE elements (Mg, V, Mn, Fe, Co, Ni, Cu, Zn, and Mo) were binned and plotted within the SO₄ ²⁻ versus Cl⁻ field and are shown in increasing atomic weight (Fig. 8). In general, for Mg, V, Mn, Fe, Co, Ni, Cu, and Zn, there was a clear difference between the phase separation and MPS areas, with the lowest concentrations found in MPS samples, and elevated concentrations found in phase separation dominant sites. Mo had the highest concentrations in the LPD phase separation sites, indicating input from concentrated liquid phase, and minimal dissolution in acidic hot springs.

NON-BIO elements can act as tracers of fluid interactions and uptake into biofilms that are not directly linked to biologic function. Binning and plotting concentrations of these and other NON-BIO elements in SO₄ ²⁻ versus Cl⁻ space help to elucidate the effects of phase inputs on concentrations of these elements in hot springs (Fig. 6). Li and As are elevated in phase separation hot springs with dominant LPD input (typically >600 μM Li and >25 μM As), indicating that they are accumulated while the source water is circulating at depth and then transported to the surface via the deep hydrothermal source and concentrated in the liquid phase during subsurface boiling. Al is sparingly soluble in circum-neutral water, especially in silica-enriched hydrothermal waters, and is thus transported via the liquid phase in relatively low concentrations (typically <40 μM). Conversely, relatively high concentrations of Al are observed in VPD hot springs (>200 μM) due to leaching from local rock and soil by sulfuric acid generated from oxidation of vapor phase accrued H₂S. Though there is a relative paucity of Ga concentration analyses, Ga exhibits higher concentrations in the MPS sites. There are, however, indications that elevated Ga concentrations are also found in phase separation sites. The lowest Ga concentrations were observed in remixed springs. This may be explained by a solubility minimum for α-GaOOH around pH 4, with higher pH water concentrating Ga as Ga(OH)₄ ⁻ in the liquid phase, and dissolution of Ga by acidic water concentrating Ga as Ga(OH)₂ ⁺, Ga(OH)²⁺, and Ga³⁺ (Benézéth et al., 1997).

Overall, LPD and MPS hot springs can be characterized as relatively enriched in elements that are transported by the liquid phase (including Li, DIC, Cl, As, and Mo). Conversely, VPD and remixed hot springs are relatively enriched in (1) elements that are transported with the vapor phase [TDIN specifically as NH_3(g), and SO₄ ²⁻ from the oxidation of H₂S_(g)]; (2) elements that are mobilized through acid leaching from subsurface and surface soil and/or rock by H₂SO₄ (including Mg, Al, V, Mn, Fe, Co, Ni, Cu, and Zn); and (3) elements that are introduced from breakdown of allochthonous organic material (including DOC, TDIN, and P). From this, we predict that microbial communities that live in these hot spring water types sequester elements differentially, which are, in turn, reflected in their elemental compositions.

5.2. Characterizing biocumulus elemental compositions

Element concentrations in biocumulus samples span one to >3 orders of magnitude (Fig. 9). This is likely due to multiple factors discussed in detail later, but to facilitate a comparison of biocumulus between MPS and phase separation dominant sites and with associated siliceous sinter deposits, the median values have been determined and used to highlight general trends. Average values were found to be influenced by small numbers of high concentration samples, whereas median values better represented the middle of the population of data points. Further, no difference was observed between chemotrophic and phototrophic biocumulus for either MPS or phase separation dominant sites, and so they will be treated together in the following discussion.

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FIG. 9.

Biocumulus concentration and median values by increasing atomic weight for elements that are grouped into one of three bins, including biologically non-essential (NON-BIO, including Li, Al, Ga, and As), major biologically essential elements (BIO, including C, N, and P), and trace biologically essential elements (BIO-TRACE, including Mg, V, Mn, Fe, Co, Ni, Zn, Mo). Biocumulus samples grouped as either no phase separation sites (royal blue left-justified open circles and closed bar, n = 74) or phase separation sites (raspberry red right-justified open circles and closed bar, n = 39). Data from this study and literature (Havig, 2009; Havig et al., 2011; Schuler et al., 2017; Hamilton et al., 2019; Havig and Hamilton, 2019a, 2019b).

Most biocumulus samples are predominantly Si as the most abundant element (by dry weight), followed by C, Al, N, Ca, Fe, Mg, and P, indicating that inorganic components (represented by the high concentrations of rock-forming elements such as Si, Al, Ca, Fe, and Mg) as well as organic components (represented by C, N, and P) represent significant fractions of biocumulus (Havig, 2009). Thus, biocumuli are taken as primarily a mixture of three components as follows: (1) biomass and EPS represented by C, N, and P; (2) allochthonous rock and soil particulates broken down from two mineralogical components; and (3) autochthonous silica precipitated from the supersaturated hot spring water (Figs. 3 and 4) (Havig, 2009). The two mineralogical components of allochthonous rock/soil (represented by soil and rock samples overlying literature values for Yellowstone rhyolites) and autochthonous silica precipitate (represented by siliceous sinter samples approaching 100% SiO₂) can be taken as two end members of a mixing line, with biocumulus falling on the line and moving toward the 100% silica end member with increasing silica precipitation over time (SOM) (Fig. 2) (Havig, 2009).

5.3. Allochthonous materials as potential inputs to biocumulus

Given that hot springs are open systems, it is unsurprising to find evidence for input of allochthonous material, ranging from observing dead insects (e.g., dragonflies, beetles, crickets, grasshoppers, etc.), animals (e.g., voles, newts, mice, bison, deer), animal excrement (e.g., bison feces), and plant matter (e.g., grasses, sedges, Lodgepole Pine needles and wood, etc.) from the local endemic biota in hot springs and hot spring runoff channels to noting transport of soil particulates from wind, rain and hail impact, and precipitation runoff. Thus, it is important to attempt to identify any impact of inputs into hot springs on element uptake in biocumuli.

The impact of potential input of C and N as biomass from allochthonous sources was examined by using C:N ratios (Fig. 10). Biocumulus from both MPS and phase separation sites fell along a 10.1 C to 1 N line, suggesting that this is a signal of hot spring microbial community biomass akin to the Redfield Ratio (6.625 C to 1 N) used to describe oceanic phytoplankton (Redfield, 1934, 1958). Silica sinter samples that are presumed to have derived from biocumulus also fell along this 10.1:1 line, suggesting the biomass preserved was sourced from biocumulus. Local soils clustered along a 21.7 C to 1 N line, suggesting significant depletion in N relative to biocumulus. Further, most soils had C and N concentrations that were below most biocumuli, making it unlikely that soils were a major contributor of those elements. Biota C:N ratios were either much lower (e.g., average C:N values ≤6.4 for insects and bison fur) or much higher (e.g., average C:N values ≥20.0 for plants and bison feces), and they exhibited a wide range of N concentration versus a relatively narrow range of C concentration. This evidence supports the hypothesis that most of the C and N in biocumulus samples is accrued from inorganic sources in the hydrothermal water [e.g., DIC, NH₄(T), NO₃ ⁻] or atmosphere (e.g., biological fixation of N₂). This is supported by previous work that has shown that hot spring biocumulus samples had different δ¹³C and δ¹⁵N values compared with potential sources of allochthonous input (Havig, 2009; Havig et al., 2011).

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FIG. 10.

Carbon and nitrogen in biocumulus, soils, and contextual samples. Top: Nitrogen concentration plotted against carbon concentration for samples from this study and literature for MPS sites (MPS, n = 75), phase separation sites (PhaseSep, n = 47), no phase separation site sinter samples (n = 8), phase separation site sinter samples (n = 10), local soil samples (n = 8), plant samples (mosses, lodgepole pine needles, grasses and sedges, n = 7), bison feces samples (n = 3), bison fur samples (n = 2), and an insect sample (n = 1). MPS, minimal phase separation. Literature values taken from Havig (2009), Havig et al. (2011), Schuler et al. (2017), Hamilton et al. (2019), and Havig and Hamilton (2019a, 2019b).

Mineralogical allochthonous inputs are assumed to be represented by local volcanic rocks and the local soils derived from them, which can be transported as particulates into hot springs from wind, precipitation impact, or fluvial transport. Median rock and soil element concentrations plotted against median values for MPS or phase separation site biocumuli median values fall close to a 1:1 line, suggesting a systematic input of these elements from a mineralogical source (Fig. 11). The C and N fall above the 1:1 line, representing the uptake of those elements from hot spring water sources (described above). Al is sparingly soluble in circum-neutral to alkaline water and is not essential to biological functions; Al is, however, a major component of the local volcanic rocks and thus an ideal tracer for mineralogical soil particulate input into biofilms in MPS sites. A line that intercepts the Al median value and is parallel to the 1:1 line is taken to represent where elements would fall if their input into biofilms was solely from allochthonous mineralogical particulate input (dashed line, Fig. 11). Elements that fall on, or close to, the Al line include Fe, Mn, Zn, Cu, Mo, and Co, with Ni, V, and Mg not as close. This proximity suggests that a significant portion of these elements are taken up by biocumuli from allochthonous particulates. Elements that fall above this line, besides C and N, include P, Li, Ga, and As. P is an essential element for life, so focused uptake is not unexpected. However, though they are not known to be essential components for life, enrichment of Li, Ga, and As (which are relatively enriched in MPS water) (Fig. 6) suggests that uptake from the hot spring water is a significant contribution to biocumuli compositions. These patterns of element uptake from autochthonous water and allochthonous particulate sources for MPS biocumuli are consistent with what has been previously reported (Havig, 2009).

Phase separation sites tend to have lower pH values (Fig. 5), where Al is more soluble (Fig. 6) and potentially drives the dissolution of mineralogical particulates. Thus, disentangling uptake of dissolved constituents versus input from allochthonous particulate material is a greater challenge for phase separation site biocumulus. The Al line for phase separation biocumulus falls closer to the 1:1 line, with the elements Fe, Li, Mn, Zn, and Co falling on or close to the Al line (Fig. 11). Elements that fall above the line, besides C and N, include Mg, P, Ga, As, V, Cu, Mo, and Ni. Elements in phase separation biocumuli that exhibit similar characteristics to those from MPS include BIO elements C, N, and P and BIO-TRACE elements Mn, Fe, Co, and Zn. Relatively greater enrichment in Mg, V, Ni, Co, and lower enrichment in Li may be indicators of the influence of lower pH/higher H⁺ activity on the ability of biocumuli to uptake these elements coupled to elevated concentrations, but further study is needed to disambiguate these results.

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FIG. 11.

Biocumulus median element concentration values plotted against rock and soil median concentration values for MPS sites (left) and phase separation sites (right). 1:1 Line (solid) and a parallel line centered on Al (dashed) given for each plot.

Biocumulus in silica saturated hot springs continuously takes up silica due to precipitation, increasing the potential for biocumuli to be preserved as siliceous sinter in the rock record. Comparing biocumuli with associated siliceous sinter deposits would provide evidence for the question of which elements sequestered in biocumuli are retained through the transition to siliceous sinter. Specifically, retention of elements may provide evidence for an element concentration biosignature that could be preserved in the rock record even without the presence of associated microfossils. Whether or not trace elements taken up as allochthonous particulate material are sequestered/associated with biomass/EPS or remain as separate particulates trapped in silica could be tested by measuring whether there was loss of the elements of the same magnitude as Al. Similar elemental mass loss as that of Al would suggest that all that was lost was associated with soil particulates that were not incorporated in the silica precipitate. This would also suggest that all elements retained were associated with preservation of biomass within the silica sinter matrix. Also, retention of the soil particulate signal and hot spring water signal would give information on the environment at the time of the biocumulus formation, which would potentially be useful for teasing apart what the water chemistry and local soil compositions were. Less loss of Al might mean there was more Al sequestered into the silica from the dissolved component in phase separation biocumulus to sinter transition. All of this would serve as a first step toward potentially developing a novel biosignature that could be used for looking for signs of life in the ancient rock record as well as on other planets.

5.4. Biocumulus comparisons to sinter

BIO, BIO-TRACE, and NON-BIO element concentrations for biocumulus and sinter samples from MPS and phase separation sites are shown in Fig. 12. Biocumulus and sinter sample concentrations and median values are displayed for each element, with the overall element retention from biocumulus to sinter calculated from median values.

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FIG. 12.

Elemental compositions and median values for top: no phase separation area biocumulus and sinter, and bottom: phase separation area biocumulus and sinter, both plotted against element in increasing atomic weight. Elements binned as described in Section 3.2. Data from this study and literature (Havig, 2009; Havig et al., 2011; Schuler et al., 2017; Hamilton et al., 2019; Havig and Hamilton, 2019a, 2019b). Percent retention between biocumulus median and sinter median given below each element column.

5.4.3. Conceptual model

This work paints with an admitted broad stroke, comparing biocumulus from a wide range of environmental conditions and a likely equally wide range of microbial community compositions. However, patterns of sequestration and retention through the initial phases of diagenesis indicate a general pattern suggesting fidelity in the preservation of signals from biomass/metabolic processes (C, N, P, BIO-TRACE elements, As), allochthonous mineralogical particulate inputs (Al, BIO-TRACE elements), and precipitation of primary minerals (silica) from the hydrothermal water (Ga). Although recent work suggests that the elemental compositions of cellular components of biocumuli may be similar to the biomass of other microbial communities (Neveu et al., 2016), separating and removing the input and impact of soil particulates as “contamination” neglects a significant fraction of major and trace element sources for the biocumulus samples, and it ignores the potential for biocumulus to act as an effective recorder of the geochemical environment driven by the intimate interactions and relationships between cellular biomass, EPS, primary mineral precipitates, and allochthonous particulates.

To represent the patterns observed in Yellowstone sites in this study, a conceptual model highlighting the sources of elements, sequestration of these elements, and then retention through earliest diagenesis is shown in Fig. 13. Breakdown of local rock and input of C, N, and P from local biota contributes to forming soils (Fig. 13, Step 1). Soils and hot spring water then serve as primary sources of elements, with hot spring water continuously bathing growing biocumulus with elements and physical transport mechanisms (wind, precipitation impact, runoff transport) delivering soil particulates (as well as direct and indirect biota inputs) to adhere to the inherently sticky biocumulus EPS (Figs. 4 and 13, Step 2). Biocumulus accumulate/sequester elements while building structures (e.g., mats, filaments, stromatolites), becoming increasingly silica enriched with time (Fig. 13, Step 3). Complete infilling with silica (often associated with loss of hydrothermal input due to avulsion of outflow channels and/or changes in hot spring water output) drives the transition from biocumulus to siliceous sinter, initiating the loss of BIO and BIO-TRACE elements associated with the biomass/EPS (Fig. 13, Step 4). Total loss of hot spring water input completes breakdown and loss of dead hydrothermal microbial community, further driving loss of BIO and BIO-TRACE elements, with retention of Ga and As incorporated within the silica precipitate (Fig. 13, Step 5). Silica that co-precipitated with microbial structures potentially preserve physical features (Fig. 13, insets for Step 5). Better constraining and connecting co-preservation of geochemical signatures with microbially produced micro- and macroscopic morphological features in modern settings will help to better interpret the rock record on Earth. If geochemical signatures can be fully characterized and quantified, there is greater potential for determining biogenicity in rocks where morphological features may have been lost through diagenetic processes.

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FIG. 13.

Conceptual model for flow of elements into hot spring biocumulus systems and retention/loss through diagenesis to silica sinter. (1) Weathering of local rock and detritus from local biota contribute to local soil formation. (2) Primary inputs of elements into biocumulus from deposition of soil particulates via aeolian transport and hot spring water delivering elements for precipitation (Si as silica), fixation (C and N), and uptake/sequestration (N, P, trace elements). (3) Microbial communities grow and build structures from autochthonous accumulation of biomass, EPS, and precipitation of silica and allochthonous accumulation of soil particulates. Accumulation of BIO, BIO-TRACE, and NON-BIO elements occurs. Silica content increases with time, acting to dilute other elemental constituents. (4) Buildup of biocumulus leads to eventual outflow path avulsion, decreasing hot spring input/temperature and causing death of thermophilic microbial community; decomposition/breakdown drives loss of BIO and BIO-TRACE elements accumulated with biomass/EPS. (5) Complete outflow avulsion leads to desiccation of biocumulus and diagenesis from biocumulus to siliceous sinter. Retention of silica, systematic loss of BIO and BIO-TRACE elements, NON-BIO element loss (Li, Al), and retention (Ga, As). Biocumulus textures, BIO, and BIO-TRACE elements preserved through diagenesis by biocumulus co-precipitated silica and later infill of silica (lower right).