Seasonal biogeochemical variations in a modern microbialite reef under early Earth-like conditions

Laguna Pozo Bravo presents physicochemical conditions reminiscent of early Earth

Laguna Pozo Bravo (Fig. 1a) is a small (500 m long, 52 m wide) shallow (~2.5 m deep) lagoon located at the foot of basaltic lava deposits in the northwest region of Salar de Antofalla (25° 30’ 49.82” S, 67° 34’ 38.92” W). Certain physicochemical conditions of this lagoon align with those of primitive Earth. Due to its high altitude (3330 m above sea level), surface pressure at Pozo Bravo remains consistently low (613.9–618.2 hPa), ~60% of average sea level pressure³⁹ (Supplementary Fig. S1). This reduced pressure leads to a partial pressure of oxygen between 128.9 and 129.8 hPa, considerably lower than the ~210 hPa at sea level, and comparable to oxygen levels estimated for the Late Paleozoic (Devonian–Carboniferous), when atmospheric oxygen may have transiently reached 60–100% Present Atmospheric Level (PAL) (~126–210 hPa)^2,40. Although the partial pressure of oxygen was much lower in the Precambrian after the Great Oxidation Event (1–10% PAL, ~2.1–21 hPa) and the Lomagundi Event (5–20% PAL, ~10.5–42 hPa), the oxygen regime at Pozo Bravo provides a useful modern analogue for studying microbial ecosystems under low atmospheric oxygen conditions. The UV index is persistently elevated, with high values (~6–9) in winter and extreme levels (~17–19) in summer (Supplementary Fig. S2). These values are among the highest measured on modern Earth, highlighting the extreme solar radiation of the region. While UV levels during the Archean (~4000–2500 Ma) would have been greater (>100) due to a negligible ozone layer⁴¹, Pozo Bravo still serves as a valuable modern analogue for studying microbial life under extreme UV conditions that may have prevailed on early Earth. Land surface temperatures at Pozo Bravo show substantial fluctuations between day and night (ΔT up to 49.8 °C in summer) and across seasons (Supplementary Fig. S3). Such pronounced diurnal and seasonal swings likely resemble those experienced on the early Earth due to a thinner atmosphere, lower greenhouse gas levels, and limited ocean coverage⁴². The lake water is alkaline (pH ~8), highly saline (over 35,000 mg L^–1), and rich in elements of volcanic origin, including sulphate (1920–1840 mg L^–1), boron (10.5–152 mg L^–1), lithium (21.3–101 mg L^–1), arsenic (0.07–0.86 mg L^–1), and manganese (0.05–0.60 mg L^–1) (Table 1). This water chemistry shares certain similarities with the metal-rich conditions of Precambrian Earth’s oceans, which likely held high concentrations of volcanic-derived elements^43,44,45,46. Overall, these extreme physicochemical conditions make Laguna Pozo Bravo a compelling model for ancient Earth, offering insights into adaptations, survival strategies, and geobiological mechanisms underlying Earth’s first life forms.

Laguna Pozo Bravo is a unique environment not only because it presents extreme physicochemical conditions, but also because these conditions vary between seasons (Table 1). In winter, when the water temperature is lower (9 °C) and the water level is higher (26 cm) due to reduced evaporation, the dissolved oxygen (DO) level is remarkably high at 171% air saturation. This high oxygen level corresponds with lower concentrations of ions (sodium, magnesium, chlorine, potassium, calcium) and minerals (e.g. calcite and aragonite), resulting in lower conductivity (150.4 mS cm^–1) and total hardness (8460 mg CaCO₃ L^–1). In contrast, during summer, the elevated water temperature (23 °C) and lower water level (7 cm), driven by high evaporation rates, result in reduced dissolved oxygen (27.5% air saturation). The increased concentration of ions and minerals under these conditions leads to a rise in both conductivity (175.2 mS cm^–1) and total hardness (10,250 mg CaCO₃ L^–1). Seasonal changes are also evident in biochemical oxygen demand (BOD₅) and chemical oxygen demand (COD). Both BOD₅ and COD values increase in summer to 230 mg L^–1 and 705 mg L^–1, respectively, compared to their winter levels of 161 mg L^–1 and 475 mg L^–1. This indicates a likely seasonal boost in microbial activity and biomass during the warmer months.

Microbial mats in Pozo Bravo exhibit diel vertical geochemical gradients

In Laguna Pozo Bravo, the combination of minimal physical disturbance, abundant volcanic-derived element inputs, strong light availability, and low sedimentation rates, provides ideal conditions for the growth of microbial mats, especially along the lagoon’s southwest margin (Fig. 1a, d, e), where they exhibit cerebroid- or snake-like morphologies due to biogenic gas accumulation (Fig. 2a). In the absence of considerable physical disruption, microbial mats organise into layers, each containing microorganisms with distinct metabolic activities. The microbial mats in Pozo Bravo display three main layers: a green surface layer, a reddish-pink middle layer, and a dark brown underlying layer (Fig. 2b).

a Overview of Pozo Bravo soft microbial mats displaying cerebroid- or snake-like structures. b Close-up look at a cross-section of a soft microbial mat exhibiting a green surface layer (G), a reddish-pink middle layer (R), and a dark brown underlying layer (D). c Spatial distribution of elements (deconvoluted counts) in a freeze-dried soft microbial mat sample determined by µXRF spectrometry. The area of µXRF scanning (4.2 mm × 4.3 mm) is marked with a black frame, and the dotted lines highlight the green, reddish-pink, and dark brown layers. d Oxygen and hydrogen sulphide profiles (mean values, n = 3) of a soft microbial mat, measured in situ during the middle of the day (from 13:00 to 16:00; yellow symbols) and night (from 20:00 to 01:00; blue symbols).

Microbial mat layered structures are shaped by steep vertical geochemical gradients generated by environmental factors and the metabolic activities of mat organisms. In the Pozo Bravo microbial mats, oxygen is detectable in the green surface layer, where its concentration rises during the day (370.69 µM at 0.1 cm depth) and drops at night (24.00 µM at 0.1 cm depth) (Fig. 2d), suggesting oxygenic phototrophic activity. With oxygen available mainly in this layer, aerobic respiration may be confined to it. A small increase in oxygen concentration is also detected during the day (16.05 µM at 0.45 cm depth) at the interface between the reddish-pink and dark brown layers (Fig. 2d), possibly reflecting oxygenic phototrophic activity driven by far-red light. Manganese is mainly localised between the green and reddish-pink layers (Fig. 2c and Supplementary Fig. S4), which may suggest manganese oxidoreduction in this zone. Sulphur is concentrated within the reddish-pink and dark brown layers (Fig. 2c and Supplementary Fig. S4), which correlate with the observed hydrogen sulphide profiles (Fig. 2d). In the reddish-pink layer, hydrogen sulphide levels decrease during the day (4.80 µM at 0.3 cm depth) but increase again at night (11.88 µM at 0.3 cm depth), possibly reflecting anoxygenic phototrophic activity driven by sulphide oxidation. Nevertheless, since pH could not be simultaneously measured during hydrogen sulphide profiling, day-night variations in hydrogen sulphide concentrations due to pH-dependent speciation cannot be excluded. The fact that sulphur concentrates in the dark brown layer may further suggest sulphur oxidoreduction activity in this area (Fig. 2c and Supplementary Fig. S4). Arsenic also accumulates in the dark brown layer, implying potential arsenic oxidoreduction activity within this anaerobic region (Fig. 2c). The evaluation of elemental composition in the mapped area of a soft microbial mat sample can be found in Supplementary Table S1.

The observed spatial and temporal variations in chemical composition suggest distinct microbial communities within each mat layer (Fig. 2). The green surface layer is likely dominated by oxygenic phototrophs, which couple inorganic carbon assimilation to light energy, oxidising water and producing oxygen as a byproduct. This layer thereby would provide an ideal micro-environment for aerobic and facultative aerobic heterotrophs. Aerobic heterotrophs gain energy by oxidising organic carbon exudates through oxygen respiration, while facultative aerobic heterotrophs can switch to using other terminal electron acceptors when oxygen is limited. The reddish-pink layer is potentially dominated by anoxygenic phototrophs, which use sulphide as an electron donor for photosynthesis and release elemental sulphur as a byproduct. The dark brown layer is likely dominated by anaerobic heterotrophs and fermenters. Anaerobic heterotrophs, mainly sulphate reducing bacteria (SRB), oxidise organic carbon using a range of terminal electron acceptors, while fermenters use organic carbon as both electron donor and acceptor. Sulphide-oxidising bacteria, which oxidise reduced sulphur compounds with oxygen or nitrate, might also be abundant in the dark brown layer.

Both eukaryotes and prokaryotes may participate in the organomineralisation process

In Laguna Pozo Bravo, the formation of lithified mats and microbialites (Fig. 1b–e) is driven by the constantly elevated calcium carbonate saturation index (aragonite SI is 2.78 in winter and 3.36 in summer; calcite SI is 2.94 in winter and 3.54 in summer), which indicates supersaturation of this mineral throughout the year, as well as the presence of abundant EPS, which may serve as a template for carbonate nucleation (Fig. 3). The elevated calcium carbonate saturation index (Supplementary Tables S2 and S3) likely results from environmental conditions like water evaporation, reflected in the seasonal variations in water column height (Table 1). Nevertheless, the spatial and temporal separation of metabolic activities observed in the microbial mats (Fig. 2) may also be responsible for local differences in the saturation index which promote carbonate precipitation. Consequently, mineralisation in Pozo Bravo may be both biologically induced and influenced. Chemical analyses conducted on the microbial mats (Table 2) revealed increased concentrations of arsenic, calcium, magnesium, iron, boron, and manganese compared to their levels in the water (Table 1). This suggests that the EPS matrix of the mats acts as a chelator for cations. Therefore, the precipitation of calcium carbonate is likely enhanced by mechanisms that weaken the capacity of the EPS matrix to bind cations. For instance, extreme UV radiation (Supplementary Fig. S2) may contribute to partial EPS destruction through Maillard browning reactions (involving the reaction of sugars and aminoacids)⁴⁷, whereas the activity of heterotrophic microorganisms may lead to microbial decomposition of the EPS. Additionally, the notably higher concentrations of calcium and magnesium in the mats (Table 2) compared to the water (Table 1) suggest that saturation of the EPS cation-binding capacity might also facilitate calcium carbonate precipitation¹².

a–p Scanning electron microscopy images from microbial mat samples. Different recognized structures are marked with an arrow: Orange, EPS serving as a template for mineral precipitation; Red, mineral structure; Green, filamentous cyanobacterium; Yellow, bacillus; Purple, spirochete; Blue, diatom (genera identified morphologically: Navicula in a and b; Halamphora in c, d and k; Nitzschia in e).

The organomineralisation in Pozo Bravo has been further analysed through SEM of a microbial mat sample (Fig. 3). The SEM images obtained revealed the presence of diatoms (Bacillariophyceae), filamentous cyanobacteria, and other prokaryotic cells (cocci, bacilli, and spirilla) forming aggregates embedded in the EPS matrix, where abundant mineral precipitation takes place. These observations suggest the potential contributions from both prokaryotic and eukaryotic microorganisms in the mineralisation processes that lead to the formation of a lithified microbial mat or microbialite. Nevertheless, as outlined by Petryshyn et al.⁴⁸, it is important to acknowledge that the microorganisms observed could play different roles, ranging from builders (e.g. diatoms and cyanobacteria) that are responsible for the construction of the structure, trapping and binding particles or driving mineral precipitation via their metabolism, to tenants that reside within the structure but are not directly responsible for its accretion or lithification, or squatters that are passively incorporated to the structure post-lithification. Based on morphological characteristics, we could identify at least three genera of diatoms: Navicula (Fig. 3a, b), Halamphora (Fig. 3c, d, and k), and Nitzschia (Fig. 3e), previously reported to compose microbial mats and microbialites in the central Andes region⁴.

Environmental conditions and microbial activity shape microbialite structure and composition

In Laguna Pozo Bravo, modern microbialites extend along the entire margin, forming a reef analogue for early Earth biostructures (Fig. 1b–e). These microbialites vary in height from 0.05 to 0.65 m and display a wide range of macrostructures, including domical, discoidal, and tabular shapes (Fig. 4a). However, the most distinctive feature of these microbialites is their internal organization, which transitions gradually from a thrombolite core to a dendrolite middle layer and, finally, to a stromatolite surface³⁸ (Fig. 4b). Thrombolite and dendrolite mesostructures are characterized by lighter colouration, whereas stromatolite mesostructures appear darker (Fig. 4b, c). Consequently, we have divided the internal organization of the microbialites into two distinct zones: (I) an internal light area and (II) an external black rim. Notably, these two zones are also present in the lithified microbial mats, suggesting that both mats and microbialites underwent similar mineralisation processes.

a Overview of Pozo Bravo bioherms displaying diverse external morphologies (macrostructure). DM, domical shape; DS, discoidal shape; TB, tabular shape. b Cross-section of domical bioherm, showing an external dark rim followed by an internal light area. The division between the two areas is indicated by a green dotted line. c On the left, a polished hand specimen of a domical bioherm showing the dark external rim (above) and the light internal area (below). On the top-right, a photomicrograph of the dark external rim (plain-polarised light) with thin layers of dark reddish oxides, corresponding to manganese oxides intergrown with calcite. On the bottom-right, a photomicrograph of the light internal area (cross polars) dominated by calcite with detrital grains of plagioclase (Pl) and volcanic rock lithic fragments (Lv). d Representative Raman spectra in the dark external rim characterised by ramsdellite and calcite peaks. e Representative Raman spectra in the light internal area characterised by calcite bands.

A Raman spectroscopy analysis of a microbialite sample (Fig. 4d, e) revealed that both distinct internal zones are primarily composed of calcite (characteristic Raman modes at 282, 711, and 1085 cm⁻¹). However, the dark external rim also appears to contain ramsdellite (bands at 573–577 and 647–654 cm⁻¹)⁴⁹, a black Mn(IV) oxide mineral that probably explains the colour difference between zones and imparts the distinctive outer colouration of lithified mats and microbialites. The presence of ramsdellite in the outermost zone could be attributed to microbial activity, as microorganisms are well-known for mediating the precipitation and transformation of Mn(IV) oxide minerals^50,51,52. The spatial association of manganese accumulation at the oxic–anoxic transition between the green and reddish-pink mat layers (Fig. 2c and Supplementary Fig. S4) supports a biotic origin for ramsdellite in Pozo Bravo. In this region of the mats, microbial oxidation of dissolved Mn(II) may proceed by oxygen-dependent multi‑copper oxidases, superoxide-mediated pathways, sulphur-coupled oxidation, nitrate reduction, ferric iron-mediated oxidation, or anoxygenic photosynthesis, leading to Mn(IV) oxide precipitation⁵³. Abiotic oxidation of Mn(II) may also occur via molecular O₂ or UV photooxidation in Pozo Bravo⁵³. However, because abiotic Mn(II) oxidation by O₂ is far slower than biotic pathways⁵⁴ and manganese accumulates poorly in the mat’s photic surface zone (Fig. 2c and Supplementary Fig. S4), a purely abiotic origin for the ramsdellite is unlikely. In the Archean oceans, low concentrations of Mn(II) oxidants (like oxidised sulphur, nitrate, and iron oxides) and high concentrations of labile Mn reductants (like ferrous iron) would have limited Mn(II) oxidation in the absence of O₂, making Mn(II) oxidation via molecular O₂ or superoxide produced through photosynthesis the most plausible mechanisms⁵³. Consequently, ramsdellite could prove useful as a biosignature of oxygenic photosynthesis in Earth’s earliest biostructures, but interpretations must remain cautious, since abiotic oxidation of Mn(II) could have also been catalysed by UV photooxidation.

Powder X-Ray diffraction (XRD) analyses of microbial mat (soft and lithified) and microbialite samples (Supplementary Table S4 and Supplementary Figs. S5 and S6) provided further insights into their mineralogy, and the environmental and microbial processes shaping it. Both types of microbial mats are predominantly composed of magnesium calcite, with minor contributions from silicate minerals such as quartz (SiO₂) and plagioclase feldspars. In the soft mats, the plagioclase component consists of andesine ((Ca,Na)(Al,Si)₄O₈) and albite (NaAlSi₃O₈), while the lithified mats contain albite and anorthite (CaAl₂Si₂O₈). The differential abundance of magnesium calcite in the soft microbial mat layers (Supplementary Table S4) suggests that carbonate precipitation mainly occurs in the green surface and reddish-pink middle layers, where oxygenic photosynthesis and anoxygenic photosynthesis coupled with sulphate reduction, respectively, increase carbonate alkalinity, favouring precipitation. This pattern is consistent with the slight calcium accumulation observed principally in the reddish-pink middle layer of the soft microbial mat sample (Fig. 2c). Together, these observations support the idea that net carbonate precipitation depends on the balance between different metabolic activities as well as their temporal and spatial variations. Contrary to microbial mats, microbialites are primarily composed of calcite throughout their structure, with minor contributions from hydrotalcite (Mg₆Al₂CO₃(OH)₁₆·4H₂O), illite ((K,H₃O)(Al,Mg,Fe)₂(Si,Al)₄O₁₀[(OH)₂,(H₂O)]), and silicate minerals, including quartz, albite, andesine, and labradorite ((Ca,Na)(Al,Si)₄O₈). Microscopic examination of the microbialite samples (Fig. 4c) suggests that quartz and plagioclase are detrital clasts, likely sourced from adjacent volcanic rocks. This volcanic input may also account for the presence of hydrotalcite, which is commonly formed as an alteration product of basalts in alkaline water environments⁵⁵. The presence of low-magnesium calcite in the microbialites may also be the result of post-depositional alteration, where magnesium ions in magnesium calcite are replaced by calcium ions, leading to the formation of a more stable mineral. All these findings suggest that the mineralogical composition of microbial mats and microbialites reflects a combination of biogenic and detrital inputs, with microbialites further shaped by diagenetic transformations.

The carbon and nitrogen systematics of soft microbial mat and microbialite samples (Supplementary Table 5) revealed notable differences in the content of total organic carbon (TOC) and total nitrogen (TN). The soft microbial mat sample exhibited higher levels of TOC (2.1% of dry weight) and TN (0.42%), compared to the microbialite sample, which had only 0.35% TOC and no detectable TN. While this pattern could reflect greater microbial activity and organic matter inputs in the microbial mats, it may also arise from post-depositional processes in the microbialites, such as remineralisation of organic matter, diagenetic loss of nitrogen, and a decrease in the proportion of organics due to mineral precipitation during lithification. The isotopic composition of organic carbon (δ¹³C_org) measured in the soft microbial mat (−18.2‰) and microbialite (−23.4‰) samples primarily suggests carbon fixation via the reductive pentose phosphate (Calvin−Benson−Bassham) cycle, employed by oxygenic (e.g. diatoms and cyanobacteria) and anoxygenic (e.g. purple sulphur bacteria) phototrophs^56,57,58,59. Nevertheless, the notable δ¹³C_org variation (~5‰) observed between samples may reflect additional contributions from alternative carbon fixation pathways and/or post-depositional alteration, including heterotrophic remineralisation and selective preservation processes. Minor contributions from the reductive acetyl-CoA (Wood–Ljungdahl) pathway would have shifted δ¹³C_org towards more negative values, whereas small inputs from the reductive tricarboxylic acid, 3-hydroxypropionate, 3-hydroxypropionate-4-hydroxybutyrate, or dicarboxylate-4-hydroxybutyrate cycles would have yielded less negative δ¹³C values, collectively modifying the overall isotopic signature of the bulk organic carbon pool^59,60,61. The bulk nitrogen isotopic composition (δ¹⁵N) of the soft microbial mat sample (0.4‰) suggests nitrogen fixation, likely mediated by cyanobacteria, through nitrogenase enzymes⁶². These results are consistent with the phototrophic activities inferred from the microelectrode measurements (Fig. 2d) and the organisms identified through SEM imaging (Fig. 3) of the microbial mats.

The microbialite reef in Pozo Bravo shows seasonal variations in microbial community composition

A molecular diversity analysis based on the small subunit rRNA gene of Bacteria and Archaea was conducted over an annual cycle to study the prokaryotic composition of the water, microbial mats, and microbialites (Fig. 5). The results revealed significant differences in community composition (PERMANOVA, F = 6.9934, R² = 0.60847, p ≤ 0.001, n_obs = 11) between the water and the benthic microbial ecosystems (Fig. 5a, c). The microbial community in the water is dominated by two phyla, Bacteroidota (60.91–88.31%) and Pseudomonadota (11.10–37.69%), largely represented by the classes Flavobacteriia (60.49–88.12%) and Gammaproteobacteria (10.40–35.83%). These classes are composed mainly of heterotrophic and halophilic bacteria from the genera Psychroflexus, Owenweeksia, and Halomonas. The prevalence of these taxa may reflect organic matter processing in the water and suggests adaptations to the hypersaline conditions and elevated organic content of the environment. In contrast, microbial mats and microbialites support more diverse and complex microbial communities (Fig. 5b). These communities are dominated by several phyla, including Pseudomonadota (24.34–63.67%), Bacteroidota (3.42–38.35%), Bacillota (0.18–23.64%), Cyanobacteriota (1.02–14.31%), Spirochaetota (0.02–12.21%), Chloroflexota (0.53–8.29%), Deinococcota (0.25–5.49%), Planctomycetota (1.44–4.46%), and Verrucomicrobiota (0.08–2.93%). Pseudomonadota is mainly represented by the classes Gammaproteobacteria (4.52–56.02%), Alphaproteobacteria (6.64–20.70%), and Deltaproteobacteria (0.64–10.87%). These groups include previously reported anoxygenic phototrophs such as purple sulphur bacteria (Thiocapsa, Thiococcus, Thiocystis, Thioflavicoccus, Thiohalocapsa, Thiorhodococcus, Thiorhodovibrio, Halochromatium, Ectothiorhodospira, and Halorhodospira) and purple non-sulphur bacteria (Rhodomicrobium, Roseospira, Rhodospira, and Rhodovibrio), heterotrophic sulphur oxidisers (Sulfurimonas, Sulfurifustis, Sedimenticola, Desulfobacula, Desulfobacter, Desulfovibrio, and Desulfuromonas), sulphur reducers (Desulfobacter, Desulfobacula, Desulfovibrio, Desulfuromonas, Desulfuromusa, Shewanella, and Geobacter), and metal/metalloid reducers potentially capable of reducing iron, arsenic, manganese, cobalt, selenium, or chromium (Desulfuromonas, Desulfuromusa, Desulfovibrio, Geobacter, and Shewanella). The phyla Bacteroidota, Bacillota, Spirochaetota, Deinococcota, Planctomycetota, and Verrucomicrobiota are primarily represented by the classes Bacteroidia (0.11–10.61%), Cytophagia (0.48–8.41%), Flavobacteriia (0.62–18.84%), Saprospiria (0.20–8.17%), Deinococci (0.25–5.48), Bacilli (0.00–23.08%), Clostridia (0.12–12.19%), Phycisphaerae (0.30–2.68%), Spirochaetia (0.02–12.19%), and Opitutae (0.00–1.24%). These groups are predominantly composed of heterotrophic bacteria, with some likely capable of sulphur reduction (Bacteroides, Marinifilum, Anaerophaga, Clostridium, Desulfotomaculum, Halanaerobium, Spirochaeta, and Sediminispirochaeta), sulphur oxidation (Cytophaga, Clostridium, Flavobacterium, and Spirochaeta), ammonification (Bacteroides and Anaerophaga), nitrite reduction (Flavobacterium and Maribacter), and denitrification or nitrogen fixation (Clostridium, Desulfotomaculum, Halanaerobium, and Bacillus). Cyanobacteriota is represented by oxygenic phototrophs from different genera, such as Halothece, Coleofasciculus, Halomicronema, and Synechococcus. Among these genera, Coleofasciculus and Synechococcus include some species potentially capable of nitrogen fixation. Meanwhile, Chloroflexota, primarily represented by Anaerolineae (0.33–5.29%), includes green non-sulphur bacteria (Candidatus Chlorothrix) which may be capable of anoxygenic photosynthesis. The prokaryotic composition observed in the microbial mats and microbialites aligns with the microbial activities inferred from the microelectrode measurements (Fig. 2d) and the bulk carbon and nitrogen isotopic compositions (Supplementary Table S5), including oxygenic and anoxygenic photosynthesis, carbon and nitrogen fixation, and sulphur, arsenic, and manganese oxidoreduction. The diversity of bacterial genera likely capable of these activities highlights the complex metabolic interactions driving the biogeochemical cycles within the benthic ecosystems. This diversity contrasts with that of the water column, which is dominated by a few potential heterotrophic taxa, reflecting the distinct ecological roles of these habitats. The functional potential of the genera identified in Laguna Pozo Bravo was inferred from the BacDive database⁶³.

a Relative abundance of the twenty major prokaryotic phyla in the different seasons. A, Autumn; W, Winter; Sp, Spring; S, Summer. b Comparison of alpha diversity metrics (Shannon index) between samples throughout the different seasons. c Principal coordinate analysis (PCoA) based on Bray-Curtis dissimilarity between samples throughout the different seasons.

The molecular diversity analysis also revealed seasonal variations in the prokaryotic composition of microbial mats and microbialites. Cyanobacteria increase in abundance during periods of higher light availability and decrease when light levels are lower (Fig. 5 and Supplementary Fig. S7). In the microbial mats, they reach their highest relative abundances in summer, whereas in the microbialites, the peaks occur in autumn and spring. These differences may be related to the greater susceptibility of microbialites to desiccation, making summer conditions more extreme and thus less favourable for cyanobacterial proliferation. Green non-sulphur bacteria (GNSB) show a similar pattern, peaking in summer and spring, reflecting their strong dependence on light availability as well. Purple sulphur bacteria (PSB) also increase during months with higher light, but reach their maximum abundances primarily in autumn in both microbial mats and microbialites (Supplementary Fig. S7). This deviation from a mid-summer peak, as seen in cyanobacteria and GNSB, may be linked to the activity of SRB. These bacteria show greater abundances in spring and summer in the mats, and in autumn in the microbialites (Supplementary Fig. S7). Elevated levels of sulphide produced by SRB could promote the autumnal bloom of PSB. In contrast, cyanobacteria and GNSB do not depend on sulphide, which could explain why their seasonal trends align more directly with light intensity rather than the sulphur cycle. These findings suggest that multiple environmental factors, including light availability and the presence of redox-active species, drive the seasonal compositional shifts and metabolic interactions within these microbial ecosystems.

Symbiotic microorganisms regulate biogeochemical cycles that shape the structure and mineralisation of mats

A metagenomic analysis was performed on a soft microbial mat to explore the metabolic diversity of their microorganisms and to elucidate their roles in the biogeochemical cycles (Supplementary Table S6). The high relative abundance of genes encoding subunits of Photosystems I and II (psa and psb, respectively), along with the presence of puf genes associated with bacterial phototrophic reaction centres, suggests co-occurring oxygenic and anoxygenic photosynthesis within the microbial mat (Fig. 6a), as confirmed by microelectrode measurements. The presence of nif genes may indicate that the microorganisms in the mat fix atmospheric nitrogen through nitrogenase enzymes, as reflected by the bulk nitrogen isotopic composition (δ¹⁵N = 0.4‰) (Supplementary Table S5). Since vnf genes encoding vanadium-dependent nitrogenases were not identified, nitrogen fixation is likely carried out solely by molybdenum-dependent nitrogenases (nif). Regarding carbon fixation, the high relative abundance of cbb and prk genes suggests that the mat’s primary CO₂ assimilation occurs via the reductive pentose phosphate cycle, in line with the isotopic composition of organic carbon (δ¹³C_org = −18.2‰) (Supplementary Table S5), 16S rRNA data revealing abundant oxygenic phototrophs (cyanobacteria) in the mat community (Fig. 5a), and microelectrode measurements demonstrating strong oxygen production in the surface layer (Fig. 2d) consistent with active CBB-based photosynthesis. However, the presence of acl genes associated with the reductive tricarboxylic acid cycle, acs genes involved in the reductive acetyl-CoA pathway, and mcl and mcr bacterial genes related to the 3-hydroxypropionate bicycle suggests the potential operation of alternative carbon fixation routes, which could contribute to the observed isotopic range. Genes involved in the oxidation of hydrogen (hya), sulphur (sqr, fcc, sox, dsr, hdr, sor, soe, apr, sat, and tcdh), nitrogen (hao and nxr), arsenic (aio), and manganese (mnxG) compounds were also identified, indicating the capacity of the microbial mat community to engage in these processes. Some of them may even be obtaining energy through the oxidation of these compounds. Finally, the identification of genes involved in the reduction of sulphur (sat, apr, and dsr), nitrogen (narG, napA, nir, norB, and nosZ), iron (mtr) and arsenic (arr) compounds suggests that some microorganisms in the mat could use alternative electron acceptors to oxygen for respiration. In fact, the mtr system has been implicated not only in the reduction of iron but also in the reduction of manganese, cobalt, and chromium⁶⁴, expanding the possibilities for anaerobic respiration in Pozo Bravo. These findings align with the microelectrode measurements, which revealed a large anoxic section in the microbial mat where anaerobic microorganisms flourish.

a Relative abundance in the metagenome (presented as reads per kilobase per million mapped reads, RPKM) of genes associated with hydrogen, carbon, nitrogen, oxygen, sulphur, manganese, iron, arsenic, and selenium cycling. b Metabolic potential of the metagenome-assembled genomes. Grey squares indicate the presence of a gene.

The reconstruction of thirteen metagenome-assembled genomes (MAGs) from the metagenomic contigs further expanded our understanding of the biogeochemical cycles in the microbial mats (Fig. 6b and Supplementary Tables S7 and S8). Of these thirteen, four are nearly complete and nine are of medium quality. However, they are estimated to have very low contamination, with seven MAGs having contamination levels less than or equal to 1% (see “Methods”). Four of these MAGs belong to the phylum Cyanobacteriota, with two (MAGs 7 and 8) identified as members of the family Geitlerinemaceae and the other two (MAGs 9 and 10) classified under the genus Halothece (Supplementary Table S8). MAGs 7 and 10 contain nif, napA, and norB genes, suggesting that cyanobacteria would play a key role in the nitrogen cycle, particularly in nitrogen fixation and denitrification. All four cyanobacterial MAGs possess genes for carbon fixation (cbb and prk) and sulphide oxidation (sqr). On the one hand, the presence of cbb and prk genes indicates that cyanobacteria drive carbon fixation through the reductive pentose phosphate cycle, which appears to be the primary carbon fixation process in the mat. On the other hand, the detection of the sqr gene suggests that cyanobacteria may use sulphide oxidation as an alternative electron source for photosynthesis, resembling some anoxygenic phototrophs. The ability of cyanobacteria to perform anoxygenic photosynthesis via sulphide oxidation is supported by the microelectrode measurements (Fig. 2d), which showed a decrease in the hydrogen sulphide levels during the day in the green surface layer, where cyanobacteria are found (Fig. 2b). Our results suggest the possibility of photosynthetic competition for sulphide between cyanobacteria and PSB. Such competition could further explain why PSB thrive in autumn, whereas cyanobacteria and GNSB are more abundant in summer. Interestingly, MAG 11 appears to be a phototroph capable of anoxygenic photosynthesis through sulphide oxidation. Its genetic potential also suggests that it fixes nitrogen and carbon via the reductive pentose phosphate cycle, complementing the activities of cyanobacteria. This MAG does not belong to the PSB but is instead classified within the Thiohalospiraceae family (Supplementary Table S8). Members of Thiohalospiraceae have been described exclusively as chemolithoautotrophs that obtain energy by oxidising inorganic sulphur compounds⁶⁵. Thus, the discovery of photosynthesis-related genes in this MAG may broaden the metabolic potential known for this family. Future isolation of an anoxygenic phototroph from Thiohalospiraceae would confirm this finding. MAGs 12 and 13, belonging to the order Desulfobacterales, are predicted to participate in both the reduction and oxidation parts of the sulphur cycle. MAG 13 also contains acs genes for carbon fixation via the reductive acetyl-CoA pathway, suggesting that non-phototrophic groups may contribute to primary production. Links between each gene and its associated metabolic pathway, EC number, and HMM profile are provided in Supplementary Table S9.

The integration of the metagenomic analysis with the biogeochemical data enabled us to construct a conceptual model of the Pozo Bravo microbial mats, detailing microbial stratification, predicted metabolic functions, and their roles in key biogeochemical cycles (Fig. 7). During the day, cyanobacteria and diatoms in the green surface layer perform oxygenic photosynthesis and assimilate inorganic carbon through the reductive pentose phosphate cycle. Certain cyanobacteria also fix atmospheric nitrogen and perform anoxygenic photosynthesis, releasing elemental sulphur instead of oxygen. The oxygen generated in the green surface layer supports aerobic and facultative aerobic organisms, including chemoorganotrophic eukaryotes (Supplementary Table S10), which consume the organic exudates. Some of these organisms oxidise reduced compounds of hydrogen, sulphur, nitrogen, arsenic, and manganese. Anoxygenic phototrophs in the deeper reddish-pink layer contribute to primary production by assimilating inorganic carbon via the reductive pentose phosphate cycle (PSB), the reductive tricarboxylic acid cycle (purple non-sulphur bacteria, PNSB), and the 3-hydroxypropionate bicycle (GNSB and PNSB). During anoxygenic photosynthesis, PSB use thiosulphate, sulphide, or elemental sulphur as electron donors, whereas GNSB and PNSB use organic compounds or hydrogen. In the bottom dark brown layer of the mat (Fig. 2b), anaerobic heterotrophs oxidise the organic exudates using iron, manganese, cobalt, chromium, or other oxidised compounds as terminal electron acceptors, while fermenters use organic carbon as both electron donor and acceptor. Once night falls, photosynthesis ceases, and the residual oxygen is rapidly depleted by respiration. As a result, the anoxic zone expands, creating favourable conditions for anaerobic heterotrophs. Among them, SRB reduce sulphate or other oxidised sulphur compounds, contributing to the regeneration of sulphide levels. Certain SRB assimilate inorganic carbon via the acetyl-CoA pathway, further linking the carbon and sulphur cycles in the mat. The metabolic activities observed in the mat play a crucial role in shaping its mineralisation. The spatial and temporal separation of these metabolisms, as observed in the mat, may create localised differences in the saturation index that influence where and when carbonate precipitation occurs. Seasonal shifts in community composition also alter the balance of microbial metabolisms, potentially leading to cyclical patterns of organomineralisation. Therefore, the dynamic interplay between microbial processes and environmental conditions ultimately determines the lithification of the mat.

The illustration depicts the major microbial guilds and biogeochemical cycles in the different layers of the microbial mat.

Distinct prokaryotic compositions define modern microbialites around the world

To understand whether microbialites found in different locations around the world exhibit a uniform prokaryotic composition or whether their composition is influenced by spatial and environmental factors, we performed a geographically broad comparative analysis of modern microbialites from North America, South America, and Australia (Fig. 8). Our study reveals that these microbial structures do not share a similar prokaryotic composition; instead, their composition varies according to geographic location and environmental characteristics, a pattern also recently observed in the eukaryotic communities of microbialites worldwide⁶⁶. For instance, the freshwater microbialites from Pavilion Lake³⁰ contain 140 distinct prokaryotic genera characteristic of freshwater environments (Fig. 8b). Some members of these genera contribute to carbonate precipitation processes, including freshwater cyanobacteria (Aphanocapsa, Chroococcus, Oscillatoria, Cyanomargarita, and Tolypothrix) and sulphate reducers (Sulfuricaulis, Sulfurisoma, Sulfurivirga, Desulforhabdus, and Bilophila). The microbialites from Clifton Lake²⁸, which develop under poikilosaline conditions (i.e. widely fluctuating salinity), host 24 distinct prokaryotic genera, whose members are adapted to salinity variation. These include halophilic or halotolerant cyanobacteria (Gloeocapsa, Scytonema, and Chondrocystis), anoxygenic phototrophs (Chloracidobacterium), and sulphate reducers (Candidatus Allobeggiato, Desulfocella, Desulfospira, and Dissulfuribacter) that drive carbonate precipitation. Microbialites from Pozo Bravo and Socompa⁶⁷ lakes, which are exposed to similar conditions of hypersalinity, high radiation, low oxygen pressure, and slight alkalinity, share 8 distinct prokaryotic genera adapted to these extreme conditions (Fig. 8b). These genera include polyextremophilic anoxygenic phototrophs (Thiorhodovibrio) and sulphate reducers (Dethiosulfatibacter), which modify carbonate alkalinity and drive mineralisation. Despite the shared extreme conditions, the microbialites from Pozo Bravo host 47 distinct prokaryotic genera not found in Socompa with the data currently available. These include polyextremophilic anoxygenic phototrophs (Thiococcus, Thioflavicoccus, and Thiorhodococcus) and sulphate reducers (Pseudodesulfovibrio) involved in carbonate precipitation, as well as genera involved in organic matter decomposition or fermentation (Acetohalobium, Halanaerobaculum, Natronorubrum, Salinirubrum, Salimicrobium, Sporohalobacter, Anaerostipes, Faecalibacterium, Holdemanella, Intestinimonas, Succiniclasticum, and Agathobacter). While the absence of these genera in Socompa reflects the influence of geographic location in shaping the prokaryotic community composition, interestingly, 14 prokaryotic genera are shared by all modern microbialites. These include genera involved in photosynthesis (Thiohalocapsa and Candidatus Alysiosphaera), sulphur reduction (Desulfonema and Desulfovibrio), and organic matter decomposition (Sedimenticola, Sediminispirochaeta, and Peredibacter), reflecting core ecological processes critical for microbialite formation and stability. The variability in prokaryotic compositions among modern microbialites demonstrates that the process of organomineralisation depends not on the taxonomy of the organisms within the community but rather on their metabolic potential and the prevailing environmental conditions.

a Worldwide distribution of modern microbialites. b UpSet plot depicting common prokaryotic genera among modern microbialites around the globe. Vertical bars show the number of prokaryotic genera in each intersection, while horizontal bars display the number of prokaryotic genera in each set. The taxonomic classification of genera found only in Pozo Bravo microbialites and genera common to all microbialites is shown in the form of a pie chart.