Integration of multiple metabolic pathways supports high rates of carbon precipitation in living microbialites

Domain: earth_science
Published: 2025-12-08
Editorial outcome: major_revision

Abstract
Microbialites are lithifying microbial mats that form multi-layered structures via biological carbon (C) uptake and carbonate precipitation. Here we relate C uptake and precipitation rates to taxonomic diversity and functional capacity of bacterial communities in supratidal freshwater microbialites. Diel assays and analysis of functional gene capacity reveal that photosynthesis is bolstered by light-independent, biological C uptake mechanisms, including biomineralization and chemoautotrophy. Through integration of these mechanisms, microbialites can capture inorganic C over a 24-hour cycle at a rate of 7-12 g C m-2 24h-1. Notably, up to 87% of the C taken up is precipitated as inorganic carbon, capturing 2.4−4.3 kg C m-2 year-1. Based on observed porosity and laboratory-based accretion rates, this equates to 13-23 mm of vertical calcium carbonate accumulation per year. Hence, contemporary microbialites provide a highly effective biological mechanism to precipitate dissolved CO2 as geologically stable carbonate mineral deposits.

Introduction
Microbialites are lithifying microbial mats formed through the biogenic precipitation of minerals, and their fossils represent the earliest microbial communities on Earth1,2. Microbialites with a distinctive layered structure, known as stromatolites, are well preserved in the fossil record and reflect an intimate connection between microbialites, the global carbon (C) cycle and Earth’s geological evolution3. However, the impact of specific geochemical and biological interactions between these microbial communities and their environment remains difficult to determine in their fossilized remains4. Living microbialites continue to thrive in both temperate and extreme environments (Fig. 1), and therefore provide important insight into fundamental biotic and chemical processes that may have occurred in ancient microbialite systems, and how they influenced the geochemical conditions of the environment4. C autotrophy is arguably the oldest metabolic process on Earth5. In most ancient and living microbialites, oxygenic photosynthesis appears to be the major source of bioavailable C and a key metabolic driver of microbialite formation6–8. While oxygenic photosynthesis is considered to have been the major biological C sequestration process taking place, evidence from Archean Strelley Pools formations indicates that non-oxygenic chemoautotrophic forms of C sequestration, such as the Wood-Ljungdahl (WL) pathway, are likely to have occurred in addition to the Calvin cycle9,10. These same pathways and other forms of non-oxygenic, chemoautotrophic C sequestration are proposed to still be active in modern microbialite formations. Functional gene abundances and sulfur isotope distribution patterns in the hypersaline, smooth mat formations of Hamelin Pools, Shark Bay, infer that non-RuBisCO-based forms of autotrophy, such as the 3-hydroxypropionate bicycle (3HP), the WL pathway, and sulfur cycling metabolisms, may be active11,12. Similarly, metagenomic analysis of Bahamian stromatolites suggests that sulfur cycling and methanogenesis may play an important role in the C cycle as well as in stromatolite formation in marine systems13–15. Unlike other microbial mats, microbialites precipitate carbonate through biological alkalinization, potentially aided by the trapping and release of cations by exopolymers to form C-containing minerals, such as calcite and aragonite2,16, that can sequester C for extended periods on the order of 106 years17. Notably, in addition to pH (Fig. 1), the chemical activities of carbonate (CO32-) and calcium (Ca2+) also strongly influence microbialite formation. Recent work shows that microbialites often develop in waters oversaturated with respect to metastable CaCO3 phases such as amorphous calcium carbonate or monohydrocalcite, highlighting that these and other permissive physicochemical conditions may also be required for biogenic precipitation to proceed18,19. Living microbialites are widely distributed along the southeastern coastline of South Africa20–22, southwestern coast of Australia and north coast of Northern Ireland23. These formations occur in the supratidal environment at the terrestrial-marine interface, where calcium-rich hard water from sand dune seeps mixes with tidal ocean waters23–26. In South Africa, individual microbialite systems may receive distinctly different freshwater nutrient inputs. For example, there is a six-fold difference in the average dissolved inorganic nitrogen (DIN) concentration at Cape Recife (76.6 ± 19.6 μM) and Schoenmakerskop (457.3 ± 333.4 μM), which are only 3 km apart24. Despite these differences, conserved functional guilds of bacteria are shared by both Cape Recife and Schoenmakerskop stromatolites27. The similarities between the functional capacities of these two systems lie in their potential to utilize and assimilate nitrogen, sulfur, and phosphorus, which likely impact their formation. In addition, the high proportion of metagenomic assembled genomes assigned as Cyanobacteria (40–70%) would suggest these systems have a substantial capacity for oxygenic photosynthesis27. In this study we relate the rates of C uptake to bacterial diversity and functional capacity in contemporary microbialites, quantifying their efficiency to remove dissolved CO2 from their freshwater environment.

Results
Microbialite systems and environmental factors
Microbialite formations from four distinct sites along ~105 km of the southeastern coastline of South Africa were selected for this study (Supplementary Fig. 1). In the east, near Gqeberha (formerly Port Elizabeth), are the Cape Recife (CR) and Schoenmakerskop (SK) microbialite sites that have been confirmed as stromatolites22,24,25. Thyspunt (TP), near Oyster Bay, is the most westerly site, while OV745 is located ~10 km to the east of TP. At CR, SK, and TP, freshwater flows over a series of fluvial formations along shallow barrage pools in the intertidal zone (Fig. 2A–C). The OV745 site feeds from a small concrete water tank, that overflows to create a ~3 m vertical waterfall microbialite formation (Fig. 2D) that terminates in the intertidal zone. The formations at TP and OV745 also show a distinctive layered structure (Fig. 2C, D, inserts), but whether or not they can be classified as stromatolites remains to be confirmed. The formations sampled in this study are distinct from most lake and marine formations in that they are continually fed by undiluted, fresh groundwater inflows. The water flowing over the surface is a few mm to a few cm in depth depending on the location. High flow rates (up to 28 L min-1) and relatively consistent pH (~8.5) across season and location (freshwater source to marine influenced end member) highlights the consistent and well mixed nature of these sites26.

The four microbialite systems in this study (including the two stromatolites) are distinguished by the different nutrient properties of their freshwater inflows (Fig. 2E), notably with respect to dissolved nitrogen levels. Most of the total dissolved nitrogen in the four systems occurs in the form of NO3- (69–85%), with SK having the highest NO3- concentration (328.01 µM), followed by CR (69.71 µM), OV745 (31.49 µM) and TP with the lowest concentration (12.28 µM). While excessively high for most natural water bodies, NO3- levels between 200 µM−300 µM are relatively common for groundwater in the dune fields around Gqeberha28. Concentrations of NH4+ and PO43- were generally low, but varied between the systems, while SK and OV745 received substantially more dissolved organic C (DOC) than the other two systems (Fig. 2E). The nutrient concentrations at the four sites were found to be chemically distinct from one another (Supplementary Fig. 2), confirming that they receive separate and unique inflows and highlighting the diversity of geochemical environments in which these microbialites thrive.

Microbial community diversity analysis
Non-metric Multi-Dimensional Scaling (NMDS) plots of 16S rRNA gene Operational Taxonomic Units (OTU0.03) showed distinct bacterial communities at the four sites, with two major clusters: SK together with CR in one cluster and OV745 in another (Fig. 3A; Supplementary Table 1). The samples from TP were separated from the other clusters, but there were too few replicates to consider them a definitive group. Consistent with a previous study27, the clustering of SK with CR bacterial communities infers that nutrient availability may not be the principal determinant of diversity at these formations. Overall, taxa belonging to 44 Eubacteria phyla were detected, with the majority of dominant OTUs classified as Cyanobacteria, Bacteroidetes, or Proteobacteria (Fig. 3B). Cyanobacteria were the most abundant OTUs at all sites, with several shared between CR and SK. OTU 1, of the family Phormidiaceae, was present in nearly all samples and was dominant at OV745, comprising up to 63% of the total reads (Fig. 3B).

Carbon uptake
Photosynthesis is accepted to be the major factor driving microbialite formation6. In addition, the transformation of dissolved inorganic C (DIC) into carbonate minerals through alkalinization also contributes to particularly high rates of C uptake in these systems. The DIC pool at the study sites is dominated (98%) by bicarbonate (HCO3-)26 making it an ideal source for tracking C uptake29,30. Daytime bicarbonate (H13CO3-) 13C stable isotope tracer experiments were used to measure the C uptake of microbialite communities at CR, SK, TP and OV745, representing four contrasting nutrient regimes. H13CO3- uptake was observed in samples from all the sites (Fig. 4A). The highest specific uptake rates (the measurement of uptake of 13C into particulate C (PC) over time) were detected at CR (0.0132 ± 0.0019 h−1), followed by OV745 (0.0097 ± 0.0013 h−1), SK (0.0065 ± 0.0012 h−1) and TP (0.0012 ± 0.0001 h−1) (Fig. 4A, and Supplementary Table 2). There was no significant difference in C uptake rates between OV745 and SK, however, all other site combinations were significant with the largest difference observed between TP and CR (p = 0.00002; Supplementary Table 3). With the exception of ammonium, none of the nutrients measured correlated with the specific 13C uptake rates (Supplementary Fig. 3, and Supplementary Table 4). While CO2 uptake and release were not directly measured in this study, the use of 13C stable isotope tracer methods is a well-established method for assessing C uptake in aquatic systems29.

Analysis of shotgun metagenome sequenced data from all four sites revealed the presence of genes involved in photosynthesis, as well as genes that may represent alternative routes for C uptake. The relative abundance of genes associated with oxygenic photosynthesis was higher at CR, SK and TP compared to OV745 (Fig. 4B, and Supplementary Table 5). Contrary to expectations, the relative abundances of photosystem and photosynthetic pigment genes at OV745 were lower than that observed for SK, which has similar, but on average lower, C-specific uptake rates. However, we noted that carbonic anhydrases, specifically gene cynT (K01673), were abundant in all sampled sites. Taxonomic classification of bacterial contigs showed that genes encoding CAs were primarily from Cyanobacteria (Coleofasciculaceae, Oscillatoriaceae and Chamaesiphonaceae), Proteobacteria (Desulfobulbaceae) and Bacteriodetes (Cytophagales) phyla (Supplementary Table 5). There was a similar high abundance of a gene predicted to encode trimethylamine-corrinoid protein Co-methyltransferase (K14083) assigned to the Proteobacteria (Rhodobacteraceae and Desulfovibrionaceae) and Chloroflexi in all formations except for CR that could be indicative of the potential for some methanogenic capacity. The presence of genes associated with both branches of the WL pathway, mainly from Desulfobulbaceae, Cytophagales, the cyanobacterial Chamaesiphonaceae and Leptolyngbyaceae families as well as unclassified Chloroflexi (Supplementary Table 5) highlight the capacity for chemoautotrophic C fixation in all formations and, in particular, at OV745 (Fig. 4B).

To further investigate the contribution of non-photosynthetic mechanisms to C uptake, we focused on OV745, which had a lower abundance of genes associated with photosynthesis, but a relatively high C uptake rate. To assess the contribution of non-photosynthetic C assimilatory pathways, we conducted diel H13CO3- tracer experiments using standardized samples (17 mm diameter and 10 mm depth), allowing us to calculate absolute C uptake rates. Mercuric chloride (HgCl2) was used in parallel control treatments to halt biological (induced) incorporation to determine the fraction of observed uptake that was due to the presence of extracellular organic molecules (influenced) and abiotic precipitation. These experiments confirmed the temporal reproducibility and homogeneity of both C uptake and the metagenomic profile of samples from the OV745 formation. There was no significant difference between uptake rates observed from OV745 samples collected in 2018 and 2019 (p = 0.3025). Daytime C uptake rates were consistent and statistically similar in all four OV745 experiments spanning both years (Fig. 5A) and the patterns of C uptake genes remained similar, with an abundance of genes encoding CAs present as well as several copies of genes involved in proton generation (Fig. 5C).

Night-time rates (Fig. 5B) represented on average 80% of C uptake observed during the day, with average daytime and night-time rates of 0.0112 ± 0.0030 h−1 and 0.0090 ± 0.0036 h−1, respectively. Furthermore, HgCl2-treated control experiments placed abiotic incorporation/precipitation at only 8 to 12% of the daytime rates and 11 to 15% of the night-time rates (Fig. 5A, B), significantly lower (p < 0.00506) than all 13C uptake measurements, indicating that the rates observed are biologically driven. Since the amount of microbialite sample used in each incubation was known for the 2019 study, we were able to calculate the absolute uptake rate in terms of the surface area of stromatolite formation. In the OV745 system, absolute C uptake was calculated to range from 4.20 to 5.18 g C m−2 12h−1 during the day and 1.75 to 6.70 g C m−2 12h−1 at night. When combined, the rates ranged from 6.68 to 11.88 g C m−2 24h−1 (Fig. 6), corresponding to annual uptakes of 2.44 to 4.34 Kg C m−2 yr−1, or 8.94 to 15.90 Kg CO2 m−2 yr−1.

To confirm that the C taken up by the microbialites was contributing to accretion (defined as the precipitation of mineral inorganic C), additional studies were conducted at OV745 in 2022. Replicate uptake samples collected from the same incubations were acidified and the loss of inorganic C and isotopic enrichment were compared to their non-acidified counterparts. Inorganic C represented 82 ± 6% of the total C, and 87 ± 3% of the total isotope enrichment observed in bicarbonate uptake was lost with the removal of the inorganic fraction (Supplementary Fig. 4). This means that the majority of the C taken up by the microbialite communities is precipitated as carbonate, which has a higher potential for long term storage.

The accretion of C in mineralized parts of microbialite formations has been estimated in a previous study from the relative proportions of porosity, detrital silicate, and biogenic phosphatic and calcium carbonate precipitates in material from CR22. We have since analysed carbonate material from Cape Recife, Schoenmakerskop and Cape St Francis (OV745) using powder X-ray diffraction spectroscopy (XRD; Supplementary Fig. 4). All spectra indicate that the precipitating phase is calcite rather than another CaCO3 polymorph (aragonite or vaterite).

The amount of C stored in the stromatolites depends on the proportions of calcite, mineral detritus washed in with the freshwater supply, phosphatic microbialite precipitates, and pore space, which we estimated from the evaluation of scanning electron microscope (SEM) imagery (Fig. 7A) and mass balance calculations (see “Methods” section). The C concentration in the analyzed microbialite samples ranged between 5.3 and 9.5 wt% (Fig. 7B). The pore space varied significantly (between 24 and 55.6 vol%). Detrital silicates were typically rare (0–2.3 vol%) and where biogenic phosphate (up to 30 vol% in one selected area) was abundant22, the stored C content was lowest. There is no textural evidence for the authigenic growth of silicate minerals. Typically, the mineralized parts of the stromatolites from CR did not show significant amounts of silicate detritus and phosphate, and in such domains the C contents ranged between 8.2 and 9.5 wt% (average: 8.7 wt% C, corresponding to 32 wt% CO2) with a (water-filled) porosity between 42.0 and 55.6 vol% (average 50.2 vol%). At an average density of 1.85 t m−3, this corresponds to an accretion of 161 kg C m−3 or the equivalent of 592 kg CO2 m−3 in mineralized stromatolite material at CR (Fig. 7B).

The accretion of 8.94 to 15.90 kg CO2 m−2 yr−1 measured at OV745 equates to the precipitation of 20.3 to 36.2 kg calcite m−2 yr−1. At an observed porosity of ~50 vol% (Fig. 7B) this corresponds to a deposition of 13 to 23 mm thick porous calcite precipitate per year. Under controlled laboratory settings, nascent microbialites cultured from field samples can grow at a rate of ~1 mm in diameter per week (Fig. 8), suggesting that the rate of deposition observed in the field could be even higher under conditions where macro- and micro-nutrients are non-limiting and the water supply is consistent.

Methods
Study site description
All four microbialite systems that are the focus of this study are entirely and directly fed by constant groundwater seep inflows and the water flows along a vertical gradient of microbialite (rock) at flow rates up to 28 L min-1 26. The depth of the water over the microbialites varies from millimeters to centimetres. The Cape Recife (CR) and Schoenmakerskop (SK) sites are within 3 km of each other. Microbialite formations at both sites begin to form at the freshwater inflows in the supratidal zone (Supplementary Fig. 1). At CR these formations follow a long ~8 m stream before branching off into two barrage pool structures, while at SK and Thyspunt (TP), the freshwater inflows run into larger, shallow barrage pools where encrusting microbialites form. The OV745 microbialites at Cape St Francis form below a shallow man-made reservoir (Supplementary Fig. 1D). Freshwater in this system is fast-flowing and the OV745 structure forms a large ~3 m tall waterfall microbialite that ends abruptly just above the subtidal zone (Fig. 2B, Supplementary Fig 1D). All microbialites sampled for this study s show a thin green surface layer (~1-2 mm think) indicative of cyanobacterial abundance (Fig. 2A-D, insets).

Sample collection and laboratory cultures
Field expeditions were conducted during the austral summer (January 16–18, 2018, and February 20, 2019) and austral winter (July 9-10, 2022). Daytime temperatures in the freshwater microbialite pools typically average between 23°C and 24°C during the summer months20 and 16°C to 18 °C in July. In 2018 microbialite biomass and water samples were collected from the shallow flowing region as the seep water enters each of the four different formations at TP (34˚11'24“S 24˚42'13“E) in the west, followed by OV745 (34˚11'47“S 24˚46'37“E), CR (34˚02'42“S 25˚34'07“E), and SK (34˚02'28“S 25˚32'18“E) (Fig. 2). Additional samples were collected from SK, CR and OV745 in August 2019 for 16S rRNA metabarcoding analysis and from OV745 in July 2022 for C uptake assessments. Samples were removed from the microbialite face, placed in Ziploc plastic bags and stored in a cooler. Ten liters of inflow water were collected in acid-washed plastic bottles. The microbialite and water samples were transported to the field laboratories (~2 h), where water for use in the tracer experiments was filtered through 0.22 µm nylon membrane filters (Pall) under vacuum. Water for nutrient analysis was filtered through 0.3 µm pre-combusted (450°C for 4 hr) GF-75 filters (Advantec). Biomass samples for DNA analysis were preserved in RNALater (Thermo Fisher Scientific) and stored at −20 °C. The remaining biomass was pulverized in filtered site water, and granular particles were removed with a metal sieve before use in 13C tracer experiments. During 2019, OV745 samples were collected with an auger generating samples 17 mm in diameter and 10 mm in depth. Half of the sample was used for 13C tracer experiments, while the remainder was preserved in RNALater for metagenomics analysis and stored at −20 °C.

Laboratory cultures of nascent microbialites were generated from a dilute slurry of pulverized samples resuspended in the freshwater inflow collected from the Schoenmakerskop site in November 2022. The slurry was inoculated into ninety-six well microtiter plates and fed with filtered source water from the site under standard grow lights in a 12-h light-dark cycle. The water was exchanged weekly, and representative microbialites were collected every seven days over the six-week period of incubation.

Uptake measurements
2018: Due to their rock-like structure, sampled microbialite biomass were broken up and strained through a 1.5 mm stainless steel sieve to remove large particles and create a slurry of the live material. Water collected from the freshwater inflows was filtered through a nylon membrane (0.22 µm) and used as a substrate for the microbialite incubations. To ensure that the microbial community did not run out of bicarbonate during the incubation, 1 mL of the slurry (100-500 µg microbialite carbon) was added to 500 mL of filtered station water in acid washed (10% HCl) 500 mL PETG incubation bottles. Due to the remoteness of the sampling sites, samples had to be transported for ~1–2 hr before inoculation into incubation bottles. Each treatment bottle was inoculated with 400 µM 13C-labeled bicarbonate (H13CO3; 99%; Cambridge Isotope Laboratories, Andover, MA). Tracer additions (10% of ambient concentrations) were estimated based on literature values of 4 mM bicarbonate in these systems26. Incubations were set up between 12:30 and 14:50 and terminated before dusk.

Samples were incubated in a microbialite barrage pool or water baths at ambient light and temperature levels for 4 hr. Since reported experiments took place during the austral summer, all daytime incubation periods occurred when ample light was available for photosynthesis. Incubations were terminated by filtering the samples onto pre-combusted (450°C for 4 hr) 0.3 µm GF-75 filters (Advantec) under gentle (5 psi) vacuum filtration using acid washed (hereafter meaning submerged in 10% HCl for ~24 h followed by at least six rinses with type 1 ultrapure water) 25 mm polysulfone filtration funnels (Pall Corporation). Once on the filter, samples were rinsed with unamended, filtered site water (under vacuum) to wash away unincorporated isotope. Separate dedicated funnels and filtration systems were used to produce filtered site water and to collect samples to determine the natural proportion of 13C in the microbialites at each site. Filters were stored at −20 °C in sterile microcentrifuge tubes until they were dried at 40 °C overnight and analyzed on a Sercon Integra2 isotope ratio mass spectrometer.

The atom percent (atom %) enrichment, the proportion of the total carbon mass that is 13C, of the microbialite mass used in the incubations is 1.0975 for CR, 1.0975 for SK, 1.0970 for OV 745 and 1.0981 for TP. For reference, 13C exists in nature at 1.11%, therefore all samples showed clear and consistent concentrations similar to the expected natural abundance. Any bicarbonate released from the microbialite slurry would have diluted our additions, deflating the actual rates and thus making our estimates even more conservative. This is shown through the use of no-addition controls, prepared from the same slurry, incubated for the same amount of time in the same water but did not receive any H13CO3. The average specific uptake rate for control treatments was a negligible -0.0001 hr-1.

2019: For the tracer experiment, half-core sections were broken up and strained through a 1.5 mm stainless steel sieve to remove large particles and create a slurry of the live material. To confirm that the use of a slurry did not artificially inflate or deflate rate evaluations, we compared the rates of whole (intact) versus slurry microbialite preparations. This effort revealed no significant difference in specific uptake rates between crushed-slurry and whole microbialite source material (p = 0.4; Supplementary Fig. 5).

Water collected from the freshwater inflow was filtered (0.2 µm) and used as a substrate for the microbialite incubations. The slurry was divided between nine incubation bottles for nighttime experiments and eight incubation bottles for daytime experiments that included two non-labeled controls, one HgCl2-killed control (taken only at night) and three dual labeled algal amino acid treatments (not reported here). Thus, the core area per bottle ranged from 6.3 mm2 to 9.5 mm2 depending upon the number of bottles per experiment (Supplementary Data 1). The portions of samples used in night-time experiments were incubated in filtered site water without tracer additions during the day and then divided between night-time assays before the addition of tracer. Abiotic controls were supplemented with 200 µl of a HgCl2 saturated solution to cease microbial growth allowing for the determination of chemical carbonate precipitation. Each treatment was inoculated with 13C labeled H13CO3 as described above (99%; Cambridge Isotope Laboratories, Andover, MA). All daytime experiments were started by 11:25 am. Night-time experiments started at midnight and incubated in a barrage pool at ambient light and temperature levels for 4 hr. Incubations were terminated by filtering the samples onto pre-combusted 0.3 µm GF-75 filters (Advantec) to collect the tracer enriched biomass. Night-time assays were filtered in the dark before dawn. Temperatures fluctuated in the barrage pool by ~5 °C over the full diel cycle. Filters were stored at -20 °C in sterile microcentrifuge tubes until they were dried at 40 °C overnight and analyzed on a Sercon Integra2 isotope ratio mass spectrometer.

Experiments in 2022 were conducted to assess the proportion of bicarbonate uptake that is incorporated into the inorganic vs organic fraction to assess accretion rates. All methods were the same as those used in 2018 except that these samples were incubated for a full 24 h to obtain net C incorporation.

Rate calculations
Bicarbonate uptake rates were calculated following standard stable isotope tracer methods described in Hama et al. 43 and refined by Collos and Slawyk (1985)44. An extensive study of groundwater chemistry for the region had been conducted by Dodd et al. 26, recording HCO3- concentrations ranging from 4096 µM to 4597 µM for stromatolite inflows. The lowest reported averages (4 mM) of freshwater DIC for these systems26 were used in our calculations to ensure that we did not overestimate C uptake, therefore, the presented rates should be considered a realistic but potential underestimation. The ratio of microbialite mass to water volume (<1 µg stromatolite C per ml site water) was intentionally low to ensure that bicarbonate would not become depleted. For this study, two different rates were calculated, specific and absolute carbon uptake. For clarity, specific uptake rates are expressed in units of time-1 and are comparable to growth rates. Absolute uptake rates should be interpreted as the amount of time for a specific substrate (HCO3-) to be taken up by a target community in units of carbon per volume (here, converted to mass or area) per unit of time. Specific and absolute uptake rates were calculated as follows:

v = PCat%xs / (DICat%xs * Time) (1)

ρ = (PCat%xs * [PC]) / (DICat%xs * Time) (2)

where v is specific uptake rate (h−1), ρ is absolute uptake rate (g C m−2 h−1), and atom percent (at%) is the percentage of carbon atoms that are 13C.

13C at% = (13C / (12C + 13C)) * 100 (3)

Atom% excess (at% xs) refers to the percentage of 13C atoms in excess of the natural abundance in the particulate carbon (PC) sample (atom % of PC after incubation minus the atom % of the initial/ambient PC). DIC (dissolved inorganic carbon) at% xs is the percentage of 13C atoms added with the addition of the 13C labeled HCO3- substrate, the 13C in excess of the natural abundance (atom % of DIC source pool after addition–atom % of the initial/ ambient PC). PC stands for particulate carbon but in the case of [PC] this refers to the mass of particulate carbon (Supplementary Data 1). In 2019, 17 mm × 10 mm cores, with a surface area of 227 mm−2, were divided among replicate bottles, averaging 7.5 mm−2, allowing absolute uptake rates to be calculated based on the surface area and proportion of the core used in replicate studies (Supplementary Data 1). The accretion rate (mm m−2) was estimated based on the total amount of 13C labeled HCO3- that was incorporated into the inorganic (87%) versus organic C fractions (Supplementary Data 1). Differentiation between inorganic and organic fractions was accomplished using the acid fumigation method45. The rate of bicarbonate uptake within the inorganic fraction (Supplementary Fig 3.) was used to confirm movement/ precipitation of captured C into the particulate inorganic pool. Composition and porosity from CR22 were used to obtain maximal annual growth estimates detailed below.

C accretion in mineralized parts of microbialites
Stromatolites exhibited varying abundances of pore space, detrital silicates, and biogenic precipitates of calcium phosphate and calcite. Their relative proportions were determined from back scattered electron (BSE) images, on which different phases and pore space show different signal intensities (e.g., calcite white, pore space black, Ca phosphate dark gray, silicate detritus variably light gray). BSE images were converted into 8-bit grayscale images. Using the gray-scale Threshold tool of the ImageJ 1.53k software (http://imagej.nih.gov/ij), domains of uniform BSE signal intensity can be selected and converted into area proportions. We determined phase and pore space abundances for seven selected domains from four CR samples (Fig. 7A). Detailed sample descriptions and phase analysis can be found in Büttner et al. 22.

Combining the phase proportions with the known phase compositions22 and phase densities, the CO2 proportion in the bulk sample can be calculated using the RockMaker software46 (Supplementary Data 2). The software converts the weight percentage of each oxide compound and the abundance of each phase into proportional totals of each oxide species, from which the bulk composition of the whole rock can be obtained by normalization. From the CO2 weight percentage of the whole rock, the weight percentage of carbon (Fig. 7B) was obtained by molar weight conversion. Our calculations assume water-filled pore space. The carbon content of the analyzed stromatolite precipitates increases from 7.3 wt% (average of all data) to 9.4 wt% if the pore space is filled with air. Pore-free calcite has a carbon content of 12 wt%. The relevant parts of the Rock Maker software showing all calculation steps in spreadsheet cell references and the obtained results are available as electronic Supplementary Data 2. Further detail on the elemental content calculations is provided in the associated publication46. Annual growth/ accretion estimates (Supplemental Spreadsheet 1, column AI) are presented in mm per year and were calculated as the annual C mineralization (Kg C m−2 yr−1) divided by the average C content per mm m−2 [161 Kg C m−3 (Fig. 7 and described in Supplemental Spreadsheet 2) divided by 1000 to convert into mm sections over m−2 areas], providing maximal annual growth/ accretion estimates in mm yr−1.

Nutrient analyses
Filtered (0.3 µm GF-75) nutrient samples were stored frozen until analysis. All samples were analyzed in triplicate and are reported as the average ± the standard deviation. Ammonium concentrations were measured using the phenol-hypochlorite method47. Nitrate, NO2- and PO43- concentrations were measured on a Lachat QuikChem 8500 autoanalyzer48. Total dissolved nitrogen (TDN) and dissolved organic C (DOC) concentrations were measured by high-temperature combustion on a Shimadzu TOC-VTNM autoanalyzer49 and instrument calibration was verified using the consensus reference material program at the University of Miami (http://yyy.rsmas.miami.edu/groups/biogeochem/CRM.html). Dissolved organic nitrogen (DON) was calculated by subtracting dissolved inorganic nitrogen (NH4+, NO3-, and NO2-) from TDN.

DNA extraction and 16S rRNA amplicon sequencing
Genomic DNA (gDNA) was extracted from microbialite samples using the Zymo quick-DNA™ fecal/soil microprep kit (Catalog No. D6012) as per the manufacturer’s instructions. DNA was extracted from ~500 mg of microbialite material, yielding between 2–4 µg of gDNA. PCR amplification of template DNA was performed as described in Waterworth et al. 27. Samples were sequenced on the South African Institute for Aquatic Biodiversity (SAIAB) Aquatic Genomics Research Platform (AGRP) Illumina Miseq platform, generating ~250 nt amplicon libraries (only forward reads were of a sufficient quality to use in analysis). Data curation was performed using the mothur software platform50 as in Waterworth et al. 27. The 50 most numerically abundant OTUs were used for analysis (Fig. 3B).

Shotgun metagenomic sequencing
gDNA was sequenced using the Ion-Torrent Proton P1 sequencing platform at the Central Analytical Facility of the University of Stellenbosch (Supplementary Taable 6). Metagenomic data was assembled into contiguous sequences with the SPAdes assembler (version 3.12.0)6 using the “-iontorrent” and “-only-assembler” options with default kmer values. Scaffolds were classified at kingdom level using Autometa51 and extracted using custom Python script pull_bacterial_contigs.py. Genes were identified in bacterial scaffolds with Prodigal and annotated against the KEGG database using KOFamScan. Reliable KEGG annotations (score > threshold) were extracted and counted per sample using custom Python script strict_kegg_parser.py. In all samples, total identified genes were counted and used to correct KEGG-annotated gene counts to relative gene abundance per sample. Finally, putative taxonomic origin of each annotated gene was assigned using Autometa classifications and counted to determine from which bacteria different metabolisms may stem from. This was achieved using the kegg_tax_count.py script. All scripts for analysis and visualization of this data can be found in the GitHub repo: (https://github.com/samche42/C_uptake_manuscript).

Statistical analysis
The open-source R Studio program (version 0.99.490) was used for all statistical tests. The Shapiro-Wilk test was used to check whether data were normally distributed; data that were not normally distributed were log-transformed prior to further statistical analyses. Statistical differences were determined using ANOVAs followed by post-hoc Tukey’s tests to determine if the means from different treatments were significantly different from one another. Treatments were considered significantly different if the p value was ≤ 0.05.

X-ray powder diffraction spectroscopy
X-ray powder diffraction patterns were recorded on a Bruker D8 Discover XRD system housed at the Tebello Nyokong Institute for Nanotechnology Innovation at Rhodes University. The instrument is equipped with a proportional counter, using Cu-Kα radiation (λ=1.5405 Å, nickel filter). Data were collected in the range from 2θ=10° to 60°, scanning at 1.5° min−1 with a filter time-constant of 0.38 s per step and a slit width of 6.0 mm. Samples were placed on a plastic powder specimen holder. The X-ray diffraction data were treated using the Eva (evaluation curve fitting) software. Baseline correction was performed on each diffraction pattern by subtracting a spline function fitted to the curved background.

Data availability
The unassembled metagenomic data generated in this study have been deposited in the NCBI database under bioproject PRJNA706574 (https://www.ncbi.nlm.nih.gov/bioproject/) and assembled metagenomic data can be accessed on MG-RAST under the accession numbers mgm4790047.3, mgm4790043.3, mgm4784267.3, mgm4784118, mgm4876328.3, mgm4876691.3 and mgm4876904.3. 3. 16S rRNA amplicon library data can be accessed on NCBI under the bioproject PRJNA611746. C uptake and CO2 data and calculations are provided as Excel Spreadsheets in the Supplementary Data 1 and 2.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This paper has many major flaws, unfortunately. Although it is an interesting paper in which 13C-incorporation and microbial diversity was measured, in my opinion, major flaws include:\n1) These are not stromatolites; maybe thrombolitic microbialtes. No thin sections are shown (or other evidence) that the surface mats shown in Fig 1 actually contribute to the structure underneath;\n2) the shallow water overhead appears to be flowing, so there is degassing, which impacts the alkalinity - in addition to the metabolism considered, this must play a role in precipitation. In fact, Fig 1D is a prime example of a travertine deposit\n3) the claim that the rates measured here are uniquely high is not true. I used Pace et al. 2018 (Geobiology) and found that assuming 10-12 hr daylight, the C-fixation rates reported there are 7.2-14.4 g/m2.d; there are numerous studies that report net photosynthetic rates in microbialites and microbial mats; Jorgensen and coworkers, De Beer, Kuhl, Visscher, Wieland and others have used micro electrodes (which prevents the issue of depletion of the C-pool; see below)\n4) a very selective, poor number of papers is referenced when it comes to comparisons with existing literature (of the last 5 years); also, there is more involved in carbonate precipitation than an alkalinity change (see Kaczmeirczak)\n5) predicted values cannot be compared with measured ones (e.g., for \"stromatolite\" growth\")\n6) as the authors know, the cycling of element is very tight in microbial mats like the ones they report on; this makes isotopic studies a bit difficult, especially those using carbon isotopes, because there is no isotopic discrimination as the C pool is virtually depleted;\n7) Van Gemerden 1993 (Marine Geology) exactly describes his model of photosynthesis and chemolithotrophy as the authors of this paper do; what the authors do not mention is that several metabolisms dissolve (by lowering the alkalinity) carbonates;\n8) slurries (homogenates) have of course much higher metabolic areas;in fact, in real mats (like the superficial mats of this study), the DIC replenishment is limited (the exact reason for an increase in alkalinity - see papers by Kempe & Kazmeirczak);\n9) is a four-hour incubation in the middle of the day (peak sunlight) representative for the daytime period? DIC measurement from a similar investigation cannot be used - there may be fluctuations, or other factors; this can cause a problem in the calculations; it is unclear what \"SP:\" stands for.\n10) for accretion rates, perhaps thin sections should be viewed; most likely, the precipitate is micrite with a different density (not a major issue, though).\nThese structures are not stromatolites like the ones in the Bahamas and Shark Bay and certainly do not resemble any stromatolite found in the fossil record. There is no consideration for other metabolic processes that play a role (either stimulating precipitation or dissolution of carbonates), not that simply an increase in alkalinity would suffice (there are other critical factors involved, e.g., nucleation sites for minerals to form).", "reviewer_2": "Review of \"Integration of multiple metabolic pathways supports unprecedented rates of carbon sequestration in living stromatolites\"\nThank you for the opportunity to review this manuscript. In this study of South African stromatolites, the authors state that they have measured rates of carbon sequestration using 13C labeled bicarbonate tracer experiments and linked these rates to the metabolic function and genetic diversity of the microbial communities assessed by 16S rRNA and shotgun metagenomic analysis. In their results, the authors highlight an exceptionally high rate of carbon sequestration (higher than tropical evergreen and deciduous forests) that is facilitated by non-photosynthetic mechanisms bolstering known carbon uptake by the light-dependent photosynthetic pathway. They attribute the high rates to a combination of mineralization through alkalinization, and chemoautotrophic metabolism. An interesting potential link was made to NH4 concentrations in some settings.\nOverall, the manuscript text is clearly written. The approach used in this study relies on published methods, and the authors use conservative estimates in their calculations based on previous published measurements of water chemistry in the study sites. In these calculations, I would suggest defining all variables in the rate calculations and including scale bars in the field photos.\nHowever, I have concerns about the authors definition of sequestration and their accretion rate estimates, which are fundamental components of their analysis. If the conclusions are better supported and confirmed by measurement rather than estimate, this will be a high impact contribution that highlights the previously unrecognized importance of stromatolite environments in the suite of known global carbon sinks. I agree with the authors, this would be a highly significant observation if supported.\nI am not a microbiologist, so my comments will be made on the aspects of sequestration and carbonate mineralization. I summarize those concerns in the following:\n1. It seems the authors are using “uptake” and “sequestration” interchangeably. They interpret high uptake rates in the measurements of the 13C labeled experiments to reflect chemoautotrophy and carbonate precipitation, but how is this differentiated? How much precipitation occurred? How long is the carbon sequestered, or does it get quickly recycled through other microbial metabolisms?\n2. To produce calcium carbonate, HCO3 needs to be broken into CO3 + H. The release of this proton can reduce pH and influence net carbonate production. How is this H+ shuttled away from precipitation to produce such high rates of proposed precipitation? If this is completed through the alternative metabolic processes, please clarify. Balanced reactions of each showing how this is expected to occur can be helpful to the reader.\n3. The assumption that the density of stromatolite carbonate is equal in density to a non-porous pure calcite need further consideration. When viewed in thin section or SEM, it is apparent that carbonate in stromatolite is highly heterogeneous, with many voids that produce high porosity, in some instances high permeability. Depending on the environment and the stromatolite formation mechanism, multiple minerals can also be present in the stromatolite. I would encourage the authors to consider this in the context of their estimates of accretion rate before comparing to other settings.\n4. The Bahamian stromatolites accrete via trapping and binding, while the accretion mechanism of these stromatolites in South Africa is not mentioned. Should it be precipitation as it might appear from the photos, Shark Bay stromatolites are a better analogue. It would be expected that trapped and bound carbonate accretes faster than microbial precipitation, but would not have the same magnitude of impact on bicarbonate uptake. This is an important area of comparison to discuss, and I encourage the authors to explain why the South African stromatolites behave so anomalously when it comes to carbon sequestration potential. This has implications for the applicability of the findings to the geological record.\n5. The stromatolites in the field photos appear to be at least partially subaerially exposed. How do periods of subaerial exposure alter the continuity of the accretion rates proposed here? How does this impact estimates of carbon uptake and subsequent sequestration?\nIn its current form, I cannot recommend this manuscript for publication. Differentiation between uptake and sequestration is needed, and a mechanism to deal with the proton released during the transformation of bicarbonate to carbonate should be presented to warrant stromatolites as a carbon sink. Finally, I suggest the authors either measure precipitation rate (difficult in such short time spans) or produce a measured accretion rate curve for the samples. These results can be compared estimated accretion rate results calculated in the present study once the porosity and heterogeneity of stromatolites is accounted for. If this accretion rate curve agrees with the proposed revisions to the accretion rate estimates based on uptake, the conclusions of the paper would be strengthened.", "reviewer_3": "The work entitled \"Integration of multiple metabolic pathways supports unprecedented rates of carbon sequestration in living stromatolites\" offers new insights into these ecosystems. With a combination of fieldwork, bioinformatic analyses, and wet-lab experiments, the study proposes an unrecognized role for stromatolites. The study is well designed and the data support the conclusions. I only have minor remarks on the manuscript, but I have not received supplementary information: the zip file with these data had only another copy of the manuscript. I would not recommend publication before seeing the full data.\nSome comments from the manuscript\nLine 256: define NPP\nLine 257-261: You compare predicted growth only based on your carbon sequestration measurements. Is this valid? Additional factors might be involved. For example, the Highbourne Cay and Shark Bay systems you cite have similar growth rates, but one order of magnitude differences in carbon sequestration rates.\nLine 329: I understand ambient temperature can be variable and was not controlled, however, please provide at least a range of approximate temperature.\nLines 344-348: please define all abbreviations used in your formulas.\nLine 370: \"concentrations of between 2 – 4 μg of gDNA\". Concentrations should be in μg/ml, I think that word should be changed. I understand 2 - 4 μg is the total amount of DNA you obtained.\nIn lines 384-385, please clarify which genes are used for each category."}, "author_response": "Nature Communications Manuscript NCOMMS-21-27695\nRESPONSE TO THE REVIEWER COMMENTS\nWe thank the reviewers for their insightful comments and suggestions. We have\ndone a careful review of their comments and have edited the manuscript accordingly\nor have provided a detailed response as to why changes were not made. Our\nresponses are in bold below each comment.\nReviewer #1 (Remarks to the Author):\nThis paper has many major flaws, unfortunately. Although it is an interesting paper in\nwhich 13C-incorporation and microbial diversity was measured, in my opinion, major\nflaws include:\n1) These are not stromatolites; maybe thrombolitic microbialtes. No thin sections are\nshown (or other evidence) that the surface mats shown in Fig 1 actually contribute to\nthe structure underneath;\nResponse: Two of the sites in this study (CR and SK) have been identified in the\nliterature as stromatolites (Buttner et al. 202121, Smith et al. 201119; Smith et al.\n201820, Rishworth et al. 202022). However, while the OV745 and TP formations\ndisplay the characteristic layered structure, they still need to be confirmed as true\nstromatolites. Therefore, we have changed the term “stromatolites” in the title to\n“microbialites” and edited the text where appropriate (Introduction: additional\nreferences cited lines 87-89, Results lines 111-119).\n2) the shallow water overhead appears to be flowing, so there is degassing, which\nimpacts the alkalinity - in addition to the metabolism considered, this must play a role\nin precipitation. In fact, Fig 1D is a prime example of a travertine deposit\nResponse: Yes, the water is flowing over the sampling stations at all four\nformations. To determine if the carbon uptake observed was via abiotic reactions, we\nperformed mercuric chloride (HgCl2)-killed controls and found that carbon uptake in\nthese treatments accounts for a relatively small proportion ~15% of the rates\nobserved in both day and night time treatments. If formation is solely, abiotically\nfacilitated by degassing then one would expect the carbon uptake rates within the\nHgCl2 killed controls to account for a larger proportion of the total carbon uptake\nfound in the live incubations. This was not the case, please see Figure 4.\n3) the claim that the rates measured here are uniquely high is not true. I used Pace\net al. 2018 (Geobiology) and found that assuming 10-12 hr daylight, the C-fixation\nrates reported there are 7.2-14.4 g/m2.d; there are numerous studies that report\nnet photosynthetic rates in microbialites and microbial mats; Jorgensen and coworkers,\nDe Beer, Kuhl, Visscher, Wieland and others have used micro electrodes (which\nprevents the issue of depletion of the C-pool; see below)\nResponse: We agree with the reviewer that some microbial mats have higher\nphotosynthetic rates and have therefore made the appropriate edits to address this\n1\nrecommendation. However, our study is unique in that the shotgun metagenomic\ndata provides insights into the potential mechanisms that enable high rates of light\nindependent (night time) uptake. The literature recommended by the reviewer has\nbeen helpful and we have cited some of the relevant papers.\nLine 45-48: Abstract\n“Through the integration of these mechanisms, stromatolites can sequester\ninorganic carbon over a full 24-hour cycle at a rate (6.87-12.26 gC m-2 24h-1)\namong the most productive microbial mats and exceeding the most productive\nterrestrial systems.”\nLines 82-86: Introduction\n“Unlike microbial mats, microbialites precipitate carbonate through biological\nalkalinization, potentially aided by the trapping and release of cations by\nexopolymers to form C-containing minerals, such as calcite, aragonite and\ngypsum2,16,17.This process can sequester carbon for extended periods on the order\nof 106 years18.”\nLine 235-248: Discussion\n“The presence of gene indicators of chemoautotrophic metabolism, such as the WL\npathway, raise the possibility that carbon is being actively taken up at night for\nchemoautotrophic carbon fixation. In addition, various metabolic guilds of\nmicroorganisms such as sulphur-cycling bacteria, methanogens, fermenters and\nheterotrophs, can promote either precipitation or dissolution of carbonate by\nchanging alkalinity16,17. In stromatolites, these metabolisms can also be linked in a\nvariety of syntrophic relationships34. Thus, it is probable that night-time carbon\nuptake is driven by both active HCO -uptake to fill biological carbon demand, and\n3\nbiologically driven mineralization, as a by-product of a system where net metabolic\nactivity is balanced in favour of alkalinisation. It is also possible that various genes\nrelated to H+ transport (Na+:H+ antiporters), H production (bidirectional\n2\nhydrogenases), and electron bifurcation which can be linked to H+ import, may aid\ncarbonate precipitation by removing H+ 35.”\nLine 256-260: Discussion\n“These rates are consistent with photosynthetic rate estimates in previous studies\nusing intact stromatolite material28. These rates are significantly higher than a\nnumber of extremely productive microbial mats such as those of Tengchong and\nLittle Hot Creek (4.6 mgC gTOC-1 and 1.82 mgC gC-1h-1 for non-lithifying and\nlithifying mats, respectively)26,2.”\n4) a very selective, poor number of papers is referenced when it comes to\ncomparisons with existing literature (of the last 5 years); also, there is more involved\nin carbonate precipitation than an alkalinity change (see Kaczmeirczak)\nResponse: We concur that there is more to carbonate precipitation than alkalinity\nalone. Therefore we explored the roles of various metabolisms and carbon\nconcentrating pathways that may facilitate/ promote precipitation. We also conducted\nHgCl2 “kill” control incubations to assess abiotic precipitation. We assessed light\ndependent (day) and light independent (night) metabolisms and analysed shotgun\nmetagenomic data sampled from each site. We also described the potential role of\nEPS in concentrating cations and increase precipitation with references to the work\nof Kaczmierczak. We have also added additional references.\n“Unlike microbial mats, microbialites precipitate carbonate through biological\nalkalinization, potentially aided by the trapping and release of cations by\nexopolymers to form C-containing minerals, such as calcite, aragonite and\ngypsum2,16,17.This process can sequester carbon for extended periods on the order\nof 106 years88.”\nLine 238-248\n“In addition, various metabolic guilds of microorganisms such as sulphur-cycling\nbacteria, methanogens, fermenters and heterotrophs, can promote either\nprecipitation or dissolution of carbonate by changing alkalinity16,17. In microbialites,\nthese metabolisms can also be linked in a variety of syntrophic relationships34. Thus,\nit is probable that night-time carbon uptake is driven by both active HCO -uptake to\n3\nfill biological carbon demand, and biologically driven mineralization, as a by-product\nof a system where net metabolic activity is balanced in favour of alkalinisation. It is\nalso possible that various genes related to H+ transport (Na+:H+ antiporters), H\n2\nproduction (bidirectional hydrogenases), and electron bifurcation which can be linked\nto H+ import, may aid carbonate precipitation by removing H+ 35.”\n5) predicted values cannot be compared with measured ones (e.g., for \"stromatolite\"\ngrowth\")\nResponse: We agree with the reviewer and have clarified the text to show that our\ncalculation is at the lower range of possible accretion rates based on porosity and\ncomposition of each individual formation.\nLine 269-274\n“BBased upon daytime rates of CaCO precipitation reported by du Plooy et al\n3\n(2020)32, the SK stromatolites may be sequestering more than 90% of their carbon\nuptake. Therefore, the uptake rates measured in this study over 24 hours could be\nequivalent to approximately 10 mm m-2 of pure calcite growth per year, although the\ntrue accretion rate is likely higher given the known heterogeneity and porosity of\nmicrobial carbonates.”\n6) as the authors know, the cycling of element is very tight in microbial mats like the\nones they report on; this makes isotopic studies a bit difficult, especially those using\ncarbon isotopes, because there is no isotopic discrimination as the C pool is virtually\ndepleted;\nResponse: The volume (water) to mass (microbial community) ratio was\nintentionally high in our incubations to account for rapid removal and replenishment\nof bicarbonate. Using the known bicarbonate additions, calculated rates and total\nmass of particulate carbon in each incubation, we can confirm that our samples did\nnot run out of available bicarbonate and thus complete removal of 13C during the 4\nhour bioassay was not an issue.\nLine 316-319\n“To ensure that the microbial community did not run out of bicarbonate during the\nincubation, 1 mL of the slurry (100-500 µg microbialite carbon) was added to 500 mL\nof filtered station water in acid washed (10% HCl) 500 mL PETG incubation bottles.”\n3\n7) Van Gemerden 1993 (Marine Geology) exactly describes his model of\nphotosynthesis and chemolithotrophy as the authors of this paper do; what the\nauthors do not mention is that several metabolisms dissolve (by lowering the\nalkalinity) carbonates;\nResponse: We appreciate the reviewer’s comment and drawing our attention to van\nGemerden 1993. This is an interesting perspective, however if dissolving\nmetabolisms are dominant without a tightly coupled counter metabolism or reaction,\nthat should lead to a net release of bicarbonate and dilution of the total bicarbonate\npool, which in turn would cause an underestimation of actual bicarbonate uptake\nrates. This supports the contention that our work represents a conservative estimate\nof carbon uptake.\nLine 241-248: Discussion\n“In microbialites, these metabolisms can also be linked in a variety of syntrophic\nrelationships34. Thus, it is probable that night-time carbon uptake is driven by both\nactive HCO -uptake to fill biological carbon demand, and biologically driven\n3\nmineralization, as a by-product of a system where net metabolic activity is balanced\nin favour of alkalinisation. It is also possible that various genes related to H+ transport\n(Na+:H+ antiporters), H production (bidirectional hydrogenases), and electron\n2\nbifurcation which can be linked to H+ import, may aid carbonate precipitation by\nremoving H+ 35.”\n8) slurries (homogenates) have of course much higher metabolic areas;in fact, in real\nmats (like the superficial mats of this study), the DIC replenishment is limited (the\nexact reason for an increase in alkalinity - see papers by Kempe & Kazmeirczak);\nResponse: We agree with the reviewer that slurries can display enhanced metabolic\nrates therefore we were conservative with all calculations. That said, our observed\nrates are consistent with daytime primary production estimates conducted on intact\nmaterial from site SK (Du Plooy et al, 202032).\nLine 256-260\n“These rates are consistent with photosynthetic rate estimates in a previous study\nusing intact stromatolite material from SK32 and are significantly higher than a\nnumber of extremely productive microbial mats such as those of Tengchong and\nLittle Hot Creek (4.6 mgC gTOC-1 and 1.82 mgC gC-1h-1 for non-lithifying and\nlithifying mats, respectively)2,28”\n9) is a four-hour incubation in the middle of the day (peak sunlight) representative for\nthe daytime period? DIC measurement from a similar investigation cannot be used -\nthere may be fluctuations, or other factors; this can cause a problem in the\ncalculations; it is unclear what \"SP:\" stands for.\nResponse: We purposely targeted the highest light during the day and the darkest\ntime at night to isolate the carbon uptake mechanisms of the diel cycle. By targeting\nthe peak daylight and middle of the night, we were better able to isolate the\nprocesses and rates of light dependent and light independent processes. We used\nan estimate of 12hrs of daylight and 12 hours of night in our estimates to account for\n4\ntwilight periods. Running longer incubations (24 hours) may have resulted in a\ndepletion of the bicarbonate pool and our inability to determine an accurate rate, as\nthe reviewer indicated in point 6. Combining peak daylight and night time rates is\nfrequently done for diel studies that are not able to run the full 24 hours.\nIt is truly unfortunate that the bicarbonate concentration samples we collected were\ncompromised during processing at a national laboratory, but we used the lowest\nobserved bicarbonate concentration from published representative samples from this\nregion (Dodd et al, 2020), although all sites had relatively similar concentrations.\nTherefore, while not ideal, using the lowest observed bicarbonate concentration\nwould result in potential underestimations not over estimations of actual rates. We\nhave been as conservative as possible with our findings and again while not ideal,\nusing the lowest reported value of these relatively consistent DIC numbers enables\nus to show the minimal potential impact of these systems.\nLine 367\nSP is specific carbon uptake rate changed to for clarity.\n𝑣\n𝑷𝑪 𝒂𝒕% 𝒙𝒔\n𝝆 = × [𝑷𝑪]\n𝒗 𝒂𝒕%𝒙𝒔 × 𝑻𝒊𝒎𝒆\n10) for accretion rates, perhaps thin sections should be viewed; most likely, the\nprecipitate is micrite with a different density (not a major issue, though). These\nstructures are not stromatolites like the ones in the Bahamas and Shark Bay and\ncertainly do not resemble any stromatolite found in the fossil record. There is no\nconsideration for other metabolic processes that play a role (either stimulating\nprecipitation or dissolution of carbonates), not that simply an increase in alkalinity\nwould suffice (there are other critical factors involved, e.g., nucleation sites for\nminerals to form).\nResponse: We respect the reviewer’s point of view, but it seems that they are\nunfamiliar with the well documented South African Tufa stromatolite formations that\nare, as the review points out, very different from those found in marine and\nhypersaline systems and also, not travertine deposits (Rishworth et al. 202022).\nWhile outside the scope of this study, which focuses on biologically driven\nbicarbonate uptake, we have cited our recent paper in Geobiology (Buttner et al.\n202121) showing that the CR formations are in fact phosphatic stromatolites that are\ndirectly comparable to those in the fossil record. Further validation can be found in\nRishworth et al. 202022, Smith et al. 201119 Smith et al. 201820. Smith et al. 2018\nspecifically identifies features native to these formations which are shared with\nstromatolites in the fossil record.\nReviewer #2 (Remarks to the Author):\nReview of \"Integration of multiple metabolic pathways supports unprecedented rates\nof carbon sequestration in living stromatolites\"\nThank you for the opportunity to review this manuscript. In this study of South African\nstromatolites, the authors state that they have measured rates of carbon\n5\nsequestration using 13C labeled bicarbonate tracer experiments and linked these\nrates to the metabolic function and genetic diversity of the microbial communities\nassessed by 16S rRNA and shotgun metagenomic analysis. In their results, the\nauthors highlight an exceptionally high rate of carbon sequestration (higher than\ntropical evergreen and deciduous forests) that is facilitated by non-photosynthetic\nmechanisms bolstering known carbon uptake by the light-dependent photosynthetic\npathway. They attribute the high rates to a combination of mineralization through\nalkalinization, and chemoautotrophic metabolism. An interesting potential link was\nmade to NH4 concentrations in some settings.\nOverall, the manuscript text is clearly written. The approach used in this study relies\non published methods, and the authors use conservative estimates in their\ncalculations based on previous published measurements of water chemistry in the\nstudy sites. In these calculations, I would suggest defining all variables in the rate\ncalculations and including scale bars in the field photos.\nHowever, I have concerns about the authors definition of sequestration and their\naccretion rate estimates, which are fundamental components of their analysis. If the\nconclusions are better supported and confirmed by measurement rather than\nestimate, this will be a high impact contribution that highlights the previously\nunrecognized importance of stromatolite environments in the suite of known global\ncarbon sinks. I agree with the authors, this would be a highly significant observation\nif supported.\nI am not a microbiologist, so my comments will be made on the aspects of\nsequestration and carbonate mineralization. I summarize those concerns in the\nfollowing:\n1. It seems the authors are using “uptake” and “sequestration” interchangeably. They\ninterpret high uptake rates in the measurements of the 13C labeled experiments to\nreflect chemoautotrophy and carbonate precipitation, but how is this differentiated?\nHow much precipitation occurred? How long is the carbon sequestered, or does it\nget quickly recycled through other microbial metabolisms?\nResponse: We thank the reviewer for pointing this out and have edited the text\naccordingly, using “uptake” when referring to our incubations and the term\n“sequestration” in the literature review and in the discussion where appropriate.\n2. To produce calcium carbonate, HCO3 needs to be broken into CO3 + H. The\nrelease of this proton can reduce pH and influence net carbonate production. How is\nthis H+ shuttled away from precipitation to produce such high rates of proposed\nprecipitation? If this is completed through the alternative metabolic processes,\nplease clarify. Balanced reactions of each showing how this is expected to occur can\nbe helpful to the reader.\nResponse: We appreciate the reviewer’s suggestion to expand on this connection.\nThe comment has encouraged us to look deeper into the mechanisms by which this\ncould be accomplished. Based on the presence of genes encoding bidirectional\nhydrogenases, it is possible that cyanobacteria or other bacterial species could be\nproducing H during dark periods. Based on the presence of H+ Na+ antiporters and\n2\n6\nheterodisulfide reductases it is possible that H+ can be shuttled into cells to maintain\npH homeostasis in an alkaline environment or as a means of energy conservation in\nbacteria utilizing the WL pathway. There may also be a pairing of contrasting\nmetabolisms to take advantage of various metabolic by-products. This combination\nsupports our observations of diverse metabolisms across formations.\nLine 245-248\n“It is also possible that various genes related to H+ transport (Na+:H+ antiporters), H\n2\nproduction (bidirectional hydrogenases), and electron bifurcation which can be linked\nto H+ import, may aid carbonate precipitation by removing H+ 35.”\n3. The assumption that the density of stromatolite carbonate is equal in density to a\nnon-porous pure calcite need further consideration. When viewed in thin section or\nSEM, it is apparent that carbonate in stromatolite is highly heterogeneous, with many\nvoids that produce high porosity, in some instances high permeability. Depending on\nthe environment and the stromatolite formation mechanism, multiple minerals can\nalso be present in the stromatolite. I would encourage the authors to consider this in\nthe context of their estimates of accretion rate before comparing to other settings.\nResponse: This point was also made by Reviewer 1. We have clarified the text to\nshow that our accretion calculation is at the lower range of possible accretion rates\nbased on porosity and composition of each individual formation. We have also\nincluded CaCO precipitation data reported by du Plooy et al (2020)32 for the SK\n3\nstromatolites that supports our estimation of accretion rates.\nLine 260-276\n“Based upon daytime rates of CaCO precipitation reported by du Plooy et al (2020)32,\n3\nthe SK stromatolites may be sequestering more than 90 % of their carbon uptake.\nTherefore, the uptake rates measured in this study over 24 hours could be equivalent\nto approximately 10 mm m-2 of pure calcite growth per year, although the true accretion\nrate is likely higher given the known heterogeneity and porosity of microbial\ncarbonates. This is double that estimated for the microbialites of Clinton Creek40 and\norders of magnitude higher than the Highbourne Cay and Shark Bay systems (0.33\nmm and 0.4 mm per year, respectively)41-43.\n4. The Bahamian stromatolites accrete via trapping and binding, while the accretion\nmechanism of these stromatolites in South Africa is not mentioned. Should it be\nprecipitation as it might appear from the photos, Shark Bay stromatolites are a better\nanalogue. It would be expected that trapped and bound carbonate accretes faster\nthan microbial precipitation, but would not have the same magnitude of impact on\nbicarbonate uptake. This is an important area of comparison to discuss, and I\nencourage the authors to explain why the South African stromatolites behave so\nanomalously when it comes to carbon sequestration potential. This has implications\nfor the applicability of the findings to the geological record.\nResponse: Identifying the type of accretion was outside of the scope of this study\nbut the reviewer makes an important point that should be the focus of future work for\nuse in interpreting the geological record. We don’t believe that the South African\nstromatolites are anomalous but likely represent dynamics in peritidal stromatolites\nelsewhere (Rishworth et al. 2020)22. The difference between pertidal (supratidal) and\n7\nmarine/hypersaline systems may be the community composition (Waterworth et al.\n2021) and source water that result in differences in carbon uptake metabolisms and\nrates. We are hoping to broaden this study to compare our findings to microbialites in\nother systems in the near future. We have included mention of Buttner et al. 2021\nwho found few detrital grains in material from CR indicating that precipitation is most\nlikely the primary form of accretion in that system.\nLine 260-268\n“The rates measured for OV745 were also higher than stromatolites, such as the\nHamelin pool, predominantly formed through carbonate precipitation and Bahamian\nformations that are formed through trapping and binding (113 mgC m-2 h-1 and ~10\nmgC m-2 h-1 respectively)12,29. When night-time rates are taken into account, and the\nprocess is extrapolated to a 24-hour day, the absolute rates of carbon uptake by the\nmicrobialites examined in this study (6.87-12.26 gC m-2 day-1) are significantly higher\nthan the average net primary production of tropical evergreen and tropical deciduous\nforests (3.83-3.89 gC m-2 day-1)37-39”\n5. The stromatolites in the field photos appear to be at least partially subaerially\nexposed. How do periods of subaerial exposure alter the continuity of the accretion\nrates proposed here? How does this impact estimates of carbon uptake and\nsubsequent sequestration?\nResponse: This is a very insightful observation and one that we are currently\ndevising systems to test experimentally. For this study, we only collected samples\nthat were under constant flow and not subaerially exposed.\nIn its current form, I cannot recommend this manuscript for publication.\nDifferentiation between uptake and sequestration is needed, and a mechanism to\ndeal with the proton released during the transformation of bicarbonate to carbonate\nshould be presented to warrant stromatolites as a carbon sink. Finally, I suggest the\nauthors either measure precipitation rate (difficult in such short time spans) or\nproduce a measured accretion rate curve for the samples. These results can be\ncompared estimated accretion rate results calculated in the present study once the\nporosity and heterogeneity of stromatolites is accounted for. If this accretion rate\ncurve agrees with the proposed revisions to the accretion rate estimates based on\nuptake, the conclusions of the paper would be strengthened.\nResponse: We have addressed the uptake vs sequestration issue and have\nproposed mechanisms for proton release. While an interesting point, specifically\nidentifying the accretion rate is outside of the scope of this manuscript, which\nfocuses on linking carbon uptake to metabolic potential (genomics). We used a very\nnarrow view of accretion rate to establish a baseline minimal increase in depth per\nformation to determine if our estimated rates could be close to realistic and to set the\nstage for future studies of accretion. We are currently devising experimental\napproaches to test true accretion rates in the field, which as the reviewer points out\ncould take some time.\nThe work entitled \"Integration of multiple metabolic pathways supports\nunprecedented rates of carbon sequestration in living stromatolites\" offers new\ninsights into these ecosystems. With a combination of fieldwork, bioinformatic\nanalyses, and wet-lab experiments, the study proposes an unrecognized role for\nstromatolites. The study is well designed and the data support the conclusions. I only\nhave minor remarks on the manuscript, but I have not received supplementary\ninformation: the zip file with these data had only another copy of the manuscript. I\nwould not recommend publication before seeing the full data.\nWe appreciate the reviewer’s kind words and will make sure that the\nsupplementary data is uploaded correctly.\nSome comments from the manuscript\nLine 25: define NPP\nLine 266-268\n“…are significantly higher than the average net primary production of tropical\nevergreen and tropical deciduous forests (3.83-3.89 gC m-2 day-1)37-39”\nLine 257-261: You compare predicted growth only based on your carbon\nsequestration measurements. Is this valid? Additional factors might be involved. For\nexample, the Highbourne Cay and Shark Bay systems you cite have similar growth\nrates, but one order of magnitude differences in carbon sequestration rates.\nResponse: Thank you for raising this point, which has been addressed in the text.\nPlease see below.\nLine 255-263\n“The daytime uptake rates measured in OV745 averaged 11.5 mgC gC h-1 or 408\nmgC m2 h-1. These rates are consistent with photosynthetic rate estimates in a\nprevious study using intact stromatolite material from SK32 and are significantly\nhigher than extremely productive microbial mats such as those of Tengchong and\nLittle Hot Creek (4.6 mgC gTOC-1 and 1.82 mgC gC-1h-1 for non-lithifying and\nlithifying mats, respectively)28,2. The rates measured for OV745 were also higher\nthan stromatolites, such as the Hamelin pool, predominantly formed through\ncarbonate precipitation and Bahamian formations that are formed through trapping\nand binding (113 mgC m-2 h-1 and ~10 mgC m-2 h-1 respectively)12,29\nLine 269-274\n“Based upon daytime rates of CaCO precipitation reported by du Plooy et al\n3\n(2020)32,the SK stromatolites may be sequestering more than 90 % of their carbon\nuptake. Therefore, the uptake rates measured in this study over 24 hours could be\nequivalent to approximately 10 mm m-2 of pure calcite growth per year, although the\ntrue accretion rate is likely higher given the known heterogeneity and porosity of\nmicrobial carbonates.”\nhowever, please provide at least a range of approximate temperature.\nResponse: We thank the reviewer for their suggestion. We have added this\ninformation to the text as outlined below.\nLines 291-293\n“Daytime temperature in freshwater microbialite pools typically average between\n23˚C and 24˚C during these months19.”\nLines 351-352\n“Temperatures fluctuated in the barrage pool by approximately 5 oC over the full diel\ncycle”\nLines 34-348: please define all abbreviations used in your formulas.\nResponse: We thank the reviewer for catching this error. SP is specific carbon\nuptake rate changed to for clarity.\nLine 367 𝑣\n𝑷𝑪 𝒂𝒕% 𝒙𝒔\n𝝆 = × [𝑷𝑪]\n𝒗 𝒂𝒕%𝒙𝒔 × 𝑻𝒊𝒎𝒆\nLine 370: \"concentrations of between 2 – 4 μg of gDNA\". Concentrations should be\nin μg/ml, I think that word should be changed. I understand 2 - 4 μg is the total\namount of DNA you obtained.\nResponse: This has been changed.\nLine 394-397\n“DNA was extracted from approximately 500 mg of stromatolite material, yielding\nbetween 2 – 4 μg of gDNA. PCR amplification of template DNA was performed as\ndescribed in Waterworth et al26.”\nIn lines 384-385, please clarify which genes are used for each category.\nResponse: Thank you for your comment, the Kegg orthology numbers are contained\nin\nsupplementary data.\n10"}, {"round": 2, "reviewer_comments": {"reviewer_2": "The revisions to the manuscript \"Integration of multiple pathways supports unprecedented rates of carbon sequestration in living microbialites\" generally addressed many of the comments on the original manuscript.\nThe measurements document bicarbonate uptake rate, and the revised text reflects that in most of the text. The addition of microbial metabolisms to remove and recycle H was a useful addition.\nHowever, carbon sequestration remains to be shown and is a central point of the manuscript's relevance and importance (see lines 102-105, 218-222, and 277-286). In addition to the release of the proton, the precipitation of carbonate minerals is known to reduce alkalinity, and thus may even release CO2 back to the atmosphere (see papers by Elderfield, Archer, Broecker and Peng, etc). This is fundamental to address in order to contextualize these results and support the author's proposal that the carbonate produced by microbialites are the most efficient carbon sink that sequesters carbon on geologically relevant time periods. The revised manuscript cites a review of carbon sequestration mechanisms (Fuss et al., 2018), and not measurements of sequestration via mineralization.\nThe authors also propose that microbialite mineralization could be an effective tool for mitigating carbon emissions. If sequestration as carbonate minerals can be demonstrated as described above, this idea should be supported further. Based on the distribution of modern peritidal stromatolites, what is the magnitude of the potential flux and sequestration period? For example, absolute rates of carbon uptake by microbialites were compared to NPP of tropical evergreen and deciduous forests, the areas of which are significantly different, and thus the flux of carbon is significantly different.\nIn its current form, the carbon sequestration potential has not been documented, and thus the data do not support the central relevance proposed for these observations.", "reviewer_3": "The revised manuscript of Professor Dorrington and colleagues addresses my concerns. Based mostly on carbon sequestration measurements and metagenomics data, the study proposes an unrecognized role for stromatolites/microbialites. The study is well designed and the data support the conclusions. Checking on the supplementary data now provided I have only a few inquiries/suggestions. I would recommend the publication of a revised version.\n* Figure 3 requires the supplementary data to understand what specific genes are shown. I'd provide at least an abbreviation of the genes represented on each row.\n* Following your discussion, on lines 232-235 you hypothesize on the role of CAs, which seem to be abundant in your dataset. Are there extracellular CAs in your data? How abundant are they?\n* You mention the WL pathway for chemoautotrophic carbon fixation at night. And it's present in the OV745 samples as shown in Fig 3. However, which organisms would bear it? I can not tell only from the OTUs in Fig 2. Are these WL-bearing organisms abundant? I doubt how much is this contributing to the observed sequestration, but defining the relative contributions is beyond this work."}, "author_response": "Nature Communications Manuscript NCOMMS-21-27695C\nResponse to Reviewer’s Comments\nNote: Line numbers refer to the track changes version of the manuscript\nReviewer #4 (Remarks to the Author):\nThe revisions to the manuscript “Integration of multiple metabolic pathways supports\nunprecedented rates of carbon sequestration in living microbialites” by Dorrington et\nal. generally addressed many of the comments on the last version of the manuscript.\nThe authors provided the requested details on the methods and on the calculations.\nHowever, considering the equation of carbonate precipitation 2HCO3- + Ca2+ -->\nCaCO3 + CO2 + H2O, no robust carbon sequestration is demonstrated if the\nreleased CO2 is not tracked and quantified during the precipitation of CaCO3. The\nauthors demonstrate the transfer of carbon between the dissolved (HCO3-) and\nparticulate fraction (CaCO3) and do not account for CO2 release. This crucial aspect\nmust be clearly stated in i) the abstract ii) the results section iii) the discussion. This\ntransfer of carbon between HCO3- and CaCO3 should not be referred as carbon\nsequestration since CO2 is released during the process. In this frame, the title must\nbe replaced by “Integration of multiple metabolic pathways supports unprecedented\nrates of carbon transfer in living microbialites”.\nResponse:\nWe have carefully considered the points raised by the reviewer and respond in full\nbelow. However, based on the context provided below, we do not agree that our\nabstract should be amended, since our findings have not changed.\nWe have toned down the final assertion in the abstract to read: “Hence,\ncontemporary microbialites provide a highly effective biological mechanism to\nprecipitate dissolved CO as geologically stable carbonate mineral deposits.”. (Lines\n2\n46-48).\nWe have also added text to the Results (lines 177- 179) and Discussion (lines 303 –\n311) sections as articulated below.\nThis aspect on the CO2 release during the precipitation must be discussed regarding\n1) the dissolved C speciation, 2) the bacterial communities populating the biofilm and\n3) the type of carbonate precipitating. On this last point, Raman analyses is an\nimportant addition. However, the quality of the 3 spectra provided now in\nsupplementary is particularly low and it is quite difficult to distinguish the peaks of\ncalcite from the aragonite peaks for instance. In addition, whether the Raman\nanalyses were done on all the studied samples or only on one sample is unclear. I\nsuggest to do additional Raman analyses on the studied samples from the different\nstudied sites, to decipher about the mineralogical composition of the microbialites,\n1\nwhich is a crucial aspect to quantify the carbon transfer during carbonate\nprecipitation. I also suggest combining in situ Raman analyses and SEM with bulk\nXRD and/or FTIR, to correlate microscopy (SEM and Raman) with the bulk\nmineralogy of the studied samples and highlight the presence of potential additional\ncarbonate minerals. Last, the accretion rate determination is not clearly explained. I\nrecommend more details on this result which is one of the important results of the\nstudy.\nResponse:\nIn aqueous solutions, dissolved inorganic carbon (DIC) exists primarily as HCO -,\n3 2 2 3\nintermediate. Bicarbonate is the dominant species in most natural systems (typically\n> 90% of DIC) and accounts for ~98% of DIC in our microbialite system. This\nbalance is maintained based on the following well-established carbonate equilibrium\nreactions:\nCO (aq)↔H CO ↔HCO −↔CO 2−\n2 2 3 3 3\nAs the reviewer describes, when CaCO precipitates it consumes HCO - and\n3 3\nproduces CO . However, we point out that in buffered aqueous systems such as the\n2\none in this study, CaCO₃ precipitation does not necessarily result in net CO₂ release\ninto the atmosphere. The removal of HCO - and the subsequent release of CO\n3 2\ndrives equilibrium reactions that strongly favour the production of HCO -, meaning\n3\nthat most of the produced CO quickly re-equilibrates with water into H CO which\n2 2 3\ndissociates to produce HCO -.\n2 2 2 3 3\nThe reviewer is justified in highlighting the release of CO during the CaCO\n2 3\nprecipitation reaction. However, this process needs to also be considered in relation\nto equilibrium reactions and the buffering capacity of the system. In our system the\npH remains relatively stable within a pH range of 8.5 – 8.7 along the freshwater to\nmarine gradient (Dodd et al. 2018), indicating a highly buffered system that does not\ndramatically change with exposure to actively growing microbialites. Thus, even\nthough CaCO formation liberates CO into the aqueous phase, the equilibrium\n3 2\nchemistry ensures that most, if not all of that CO is quickly converted back into\n2\nmicrobial communities actively fixing carbon (Beer et al. 2020), like those shown in\nour study, further reducing the likelihood of substantial CO release from the system.\n2\nand recycled back into the HCO - pool or taken up in its CO form, that does not\n3 2\nnegate our finding in that we are measuring and reporting the amount of 13C taken\nup and precipitated as calcite from a HCO - source. Our data therefore documents\n3\nnot just transfer, but biologically mediated stabilization of carbon into solid mineral\nform. While direct CO₂ fluxes were not measured in situ, the strong coupling\nbetween bicarbonate uptake (this study), O₂ production (du Plooy et al. 2020), and\naccretion rates (this study) demonstrates that these microbialites likely act as\nefficient carbon sinks.\ntrack carbon in a manner that is not possible using less precise, bulk or flux\nmethods, (Cullen et al. 2001). We used this well-established approach (> 41,400\nstudies for primary production alone based on a perfunctory Google Scholar search)\nto measure the proportion of the C supplied as H13CO - that was directly captured\n3\n(Figure 6). We were then able to show that 87% of the C captured was precipitated\nas inorganic carbon (lines 235-240 in the text). We also show that the microbialite\nsystems used in this study primarily precipitate this carbon as calcite instead of other\nforms (revised manuscript lines 246-260, Figure 7; supplementary Figure 4), the\ngrowth/C accretion of nascent colonies over time (mass and volume of microbialites\nincreased over time, Figure 8) and we explore the microorganisms (Figure 3) and\nmetabolisms (Figure 4, 5) that likely support these rates. This work represents\nsubstantial evidence, multiple analytical approaches and reproducibility of these\nefforts that support our assertions that carbon is in fact being biologically captured\n(see comparison to abiotic assimilation in Figure 5) and then precipitated in a stable\nmineral form as calcite (see below).\nWe agree with the reviewer that our Raman spectra may be insufficiently robust to\nconclusively identify the CaCO polymorph (calcite vs. aragonite, vaterite) may be\n3\ninsufficiently robust. We therefore replaced the Raman spectra with powder XRD\nspectra, which provide unequivocal evidence (Supplementary Figure 4; lines 246-\n250). Following the reviewer’s suggestion, we analysed multiple samples from each\nof three sampling sites (Cape Recife, Schoenmakerskop and Cape St Francis\nOV745), all of which yielded identical results - the precipitating phase is calcite. In\norder to avoid redundancy, we removed the Raman data from the manuscript.\nFinally, we are not in agreement with the reviewer about the use of sequestration in\nthe title, but are willing to accommodate their view by using the term “precipitation”,\nwhich we think is appropriate, instead. We have revised the title to read “Integration\nof multiple metabolic pathways supports unprecedented rates of carbon\nprecipitation in living microbialites”."}, {"round": 3, "reviewer_comments": {"reviewer_4": "Review of manuscript titled:\n“Integration of multiple metabolic pathways supports unprecedented rates of carbon sequestration in living microbialites”\nby Eric W. Isemonger, Rosemary A. Dorrington and collaborators\nThis manuscript addresses an important question about the carbonatogenesis potential of microbialites and the\ncorresponding metabolic pathways. This study aims to link the relationships between the taxonomic diversity and functional\ncapacity with C uptake and precipitation rates of microbialites. To this end, the authors performed DNA extraction from\nmicrobialites, 16S rRNA amplicon and metagenomic sequencing, together with laboratory-based experiments. The\ncharacterization of the microbial communities, and the metabolic pathways associated with microbialites is very valuable\nand the aim to combine both metabolic pathways and microbialite accretion rate and C uptake is novel. I recommend\nclarifying and modifying several points, in particular on clarifying the methods and on the discussion of the limits of the\nmethods, before considering its publication to Nature Communications.\n1. The core of the paper, i.e., the estimations of C uptake and the precipitation rates are both not robust enough. This is\nprobably because the successive calculations are not provided in the manuscript nor in supplementary information. All the\ncalculations must be provided in supplementary information as an excel file with all the calculations visible. In addition,\nplease consider providing a workflow with the type of experiment/the samples used, the estimation from natural samples and\nthe techniques/methods/calculations as well as the results obtained to better follow the study.\n2. This study describes the accretion of existing carbonate grains, incubated as a slurry (crushed microbialite with water) in\nnatural water collected from a natural site. The experiments do not demonstrate the in-situ formation of carbonate and\nlithification of these grains. However, this is now accepted by the community that microbialites form via different\nmineralization pathways, with the most prominent pathways being in situ precipitation of mineral (mainly carbonate) via (1)\nthe metabolic activity of microorganisms (induced biomineralization) and (2) the presence of extracellular organic molecules\n(influenced biomineralization). An experiment never reproduces the complexity of Nature. The question is whether a solution\nwith a microbialite slurry mimics the natural processes of growth of microbialite. I believe a point of discussion on the limits of the\nexperiment conducted here regarding the “forced” accretion experiment applied here is needed.\n3. The experimental approach conducted here leads to a very high accretion rate (i.e., 1 mm per week, figure 7 and lines\n235-238) which I believe overestimates the reality. Accretion rates have been measured in situ by different groups (i.e.,\nincubating substrates within lakes or by datation) for microbialites from both continental and marine settings and the studies\nshow a range of accretion rate from 0.33 mm/year to up to actually 5 mm/yr (after Power et al., 2011). Again, the high\naccretion rate provided here from the experiments should be criticized in the manuscript.\n4. Line 59: A point in the introduction on the role of physico-chemical conditions allowing the formation of microbialites is\nmissing.\n5. Lines 56, 81, 97: Different terms are used to refer to modern microbialites: extant, living, contemporary: please uniformize\nthe nomenclature along the manuscript\n6. For the uptake measurement: What was the 13C content of the collected microbialite used in the experiment? It is\nexpected to have a consequent mass of 13C already coming from the slurry (the microbialites grains incubated within the\nbottle). This data is not mentioned in the method section nor in the result and is crucial to consider in the rate of 13C uptake.\n7. There is very likely an overestimation of 13C on the filters after the experiments linked to the saturation of the filter with\n13C-rich water. The filters from 2018 and 2019 experiments collected before the mass spectrometer analyses need to be\nrinsed with DI water to be reflective of the real 13C uptake by biomass and carbonate. This step is not mentioned in the\nmethod section.\n8. Figure 7: under which type of laboratory culture?\n9. Lines 45, 219, 233, 471, 721: Ca-carbonate is not necessary calcite. Ca-carbonate has different polymorphs. Ca-\ncarbonate can be aragonite, calcite, vaterite, monohydrocalcite. This manuscript and the past study (Buttner et al., 2021)\ndo not decipher about the type of Ca-carbonate. Please remove the name calcite and replace by “Ca-carbonate”.\n10. Line 144: write as “microbialite”.\n11. Line 232: How the accretion of 9.20 to 16. 41 kg CO2/m2/yr was determined?\n12. Lines 392-402: DIC is not only HCO3- this is also CO32- and CO2. Moreover, it is necessary to mention that the released\nCO2 during the formation of CaCO3 from 2(HCO3-) has not been measured and considered in the calculation of the C rate\ncalculation.\n13. Line 474: What are precisely the 7 selective domains?\n14. Line 474: What are the proportions of Ca-carbonate in the 4 samples?\n15. Lines 476-477: Provide the calculations (equations) made by the software RockMaker to calculate C contents of\nmicrobialites. Using which data? There is a lack of clarity on how the Ca-carbonate was quantified within the CR\nmicrobialite.\n16. Line 475: After checking the link, different threshold plugins exist on ImageJ. What is exactly the name of the used\nthreshold plugin?\n17. A method paragraph is missing for the electron microscopy observations: how the samples were prepared? Under which\nanalytical conditions?"}, "author_response": "Manuscript ID: NCOMMS-21-27695B-Z\n\"Integration of multiple metabolic pathways supports unprecedented rates of carbon\nsequestration in living microbialites” by Sipler, Isemonger, Waterworth et al.\nRESPONSE TO REVIEWER 4\nReviewer #4 (Remarks to the Author):\nThis manuscript addresses an important question about the carbonatogenesis potential of\nmicrobialites and the corresponding metabolic pathways. This study aims to link the\nrelationships between the taxonomic diversity and functional capacity with C uptake and\nprecipitation rates of microbialites. To this end, the authors performed DNA extraction from\nmicrobialites, 16S rRNA amplicon and metagenomic sequencing, together with laboratory-\nbased experiments. The characterization of the microbial communities, and the metabolic\npathways associated with microbialites is very valuable and the aim to combine both\nmetabolic pathways and microbialite accretion rate and C uptake is novel. I recommend\nclarifying and modifying several points, in particular on clarifying the methods and on the\ndiscussion of the limits of the methods, before considering its publication to Nature\nCommunications.\n1. The core of the paper, i.e., the estimations of C uptake and the precipitation rates are both\nnot robust enough. This is probably because the successive calculations are not provided in\nthe manuscript nor in supplementary information. All the calculations must be provided in\nsupplementary information as an excel file with all the calculations visible. In addition, please\nconsider providing a workflow with the type of experiment/the samples used, the estimation\nfrom natural samples and the techniques/methods/calculations as well as the results\nobtained to better follow the study.\nResponse: This has been done. We have expanded the methods section to better represent\nthe workflow and as requested, provide an excel file with all the data and calculations used\nin this study. In light of the additional information, we believe a workflow is unnecessary.\n2. This study describes the accretion of existing carbonate grains, incubated as a slurry\n(crushed microbialite with water) in natural water collected from a natural site. The\nexperiments do not demonstrate the in-situ formation of carbonate and lithification of these\ngrains.\nResponse: We have addressed the question of whether experiments conducted with\ncrushed microbialites can be directly related to in situ (intact) microbialite by providing\nadditional data referred to in the methods (lines 412-416) as follows:\n“To confirm that the use of a slurry did not artificially inflate or deflate rate evaluations, we\ncompared the daytime rates of whole (intact) versus slurry microbialite preparations. This\neffort revealed no significant difference in specific uptake rates between crushed-slurry and\nwhole microbialite source material (p=0.4; Supplementary Figure 5)”\nHowever, this is now accepted by the community that microbialites form via different\nmineralization pathways, with the most prominent pathways being in situ precipitation of\nmineral (mainly carbonate) via (1) the metabolic activity of microorganisms (induced\nbiomineralization) and (2) the presence of extracellular organic molecules\n(influenced biomineralization). An experiment never reproduces the complexity of Nature. The question\nis whether a solution with a microbialite slurry mimics the natural processes of growth of\nmicrobialite. I believe a point of discussion on the limits of the experiment conducted here\nregarding the “forced” accretion experiment applied here is needed.\nbiomineralization versus influenced biomineralization is shown Figure 5A and B, where the\nmercury chloride “killed” controls represented approximately 15% of C uptake during the day\nand at night. These controls account for both influenced biomineralization and abiotic\nmineralisation, therefore in our experimental system approximately between 85 92% of C\nuptake can be attributed to induced biomineralization.\nTo emphasise this, we have revised the text in the results section as follows:\nLines 193-196: “Mercuric chloride (HgCl2) was used in parallel control treatments to halt\nbiological (induced) incorporation to determine the fraction of observed uptake that was due\nto the presence of extracellular organic molecules (influenced) and abiotic precipitation.”\nLines 207-211: “Furthermore, HgCl2-treated control experiments placed abiotic\nincorporation/precipitation at only 8 to 12% of the daytime rates and 11 to 15% of the night-\ntime rates (Fig 5A; Fig. 5B), significantly lower (p<0.00506) than all 13C uptake\nmeasurements, indicating that the rates observed are biologically driven.”\n3. The experimental approach conducted here leads to a very high accretion rate (i.e., 1 mm\nper week, figure 7 and lines 235-238) which I believe overestimates the reality. Accretion\nrates have been measured in situ by different groups (i.e., incubating substrates within lakes\nor by datation) for microbialites from both continental and marine settings and the studies\nshow a range of accretion rate from 0.33 mm/year to up to actually 5 mm/yr (after Power et\nal., 2011). Again, the high accretion rate provided here from the experiments should be\ncriticized in the manuscript.\nResponse: Our experimental assessment of C uptake and accretion rates of 86% concur\nwith in situ measurements of daytime primary productivity and Ca accretion by du Plooy et al\n(2020). In the laboratory, the growth of laboratory cultures of SK microbialites at a rate of 1\nmm per week, at an accretion rate of 86%, would equate to growth of approximately 44 mm\nper year under ideal, laboratory conditions. The growth rate in natural, field formations is\nlower influenced by environmental factors such as seasonal fluctuations in freshwater inflow\nand nutrient concentration, desiccation, marine overtopping, weathering, invertebrate\ngrazing etc. This has been stated in the text (see lines 312-315).\nThe average daytime C uptake rate at OV745 is not that much higher that what has been\nmeasured at Hamelin Pool - 396 mg C m-2 h-1 vs.113 mg C m-2 h-1 (Bauld et al., 1976),\nrespectively and the growth rate of the Hamelin pool microbialites in turn, is an order of\nmagnitude higher than the Bahaman formations (Steppe et al., 2001), indicating significant\nvariation in the growth rate of different microbialite formations. What sets this study apart is\nour finding that C uptake in the South African microbialites continues at almost the same rate\nat night and precipitate at high precipitation rates (Supplementary Fig. 3). Ours is the first\ndiel study of microbialite primary production. It is entirely possible that other microbialites\nalso continue C uptake at night.\nFinally, the taxonomic and functional diversity of supratidal microbialite bacterial\ncommunities assessed in this study are distinct from other formations (e.g. Shark Bay,\nHighborne Cay, Pavillion lake and Little Creek). The composition of the bacterial\ncommunities together with environmental factors specific to their location in the supratidal\nzone, likely combine to support the exceptionally high rates of C uptake and C accretion\nobserved for the microbialites in this study.\n4. Line 59: A point in the introduction on the role of physico-chemical conditions allowing the\nformation of microbialites is missing.\nsummarising the known physico-chemical conditions of microbialite formations (temperature\nand pH) including those in this study. Ambient DIC in the South African microbialite\nformations as reported by Dodd et al 2018, are referred to in the methods section (Lines\n423-428).\n5. Lines 56, 81, 97: Different terms are used to refer to modern microbialites: extant, living,\ncontemporary: please uniformize the nomenclature along the manuscript\nResponse: This has been done.\n6. For the uptake measurement: What was the 13C content of the collected microbialite used\nin the experiment? It is expected to have a consequent mass of 13C already coming from\nthe slurry (the microbialites grains incubated within the bottle). This data is not mentioned in\nthe method section nor in the result and is crucial to consider in the rate of 13C uptake.\nResponse: Filtered site water and microbialite samples were used to determine the natural\nproportion of 13C in the microbialites at each site. The ambient 13C atom % for each site has\nbeen provided in the methods and the supplementary Data Excel Spreadsheet where they\nare used to obtain the atom % excess used in rate calculations. We also conducted parallel\ncontrol incubations that had no 13C added and incubated in bottles under the same\nconditions for the same incubation time. These control samples showed little difference from\nthe ambient (field) atom % and negligible impact on calculated rates (-0.0001 hr-1) in\ncomparison to enriched treatments that averaged 0.0112 h-1 and 0.0090 h-1 for day and\nnight incubations, respectively.\nWe have addressed this issue in the methods section as follows:\nLines 398-408: “The atom percent (atom %) enrichment, the proportion of the total carbon\nmass that is 13C, of the microbialite mass used in the incubations is 1.0975 for CR, 1.0975\nfor SK, 1.0970 for OV 745 and 1.0981 for TP. For reference, 13C exists in nature at 1.11%,\ntherefore all samples showed clear and consistent concentrations similar to the expected\nnatural abundance. Any bicarbonate released from the microbialite slurry would have diluted\nour additions, deflating the actual rates and thus making our estimates even more\nconservative. This is shown through the use of no-addition controls, prepared from the same\nslurry, incubated for the same amount of time in the same water but did not receive any\nH13CO . The average specific uptake rate for controls treatments was a negligible -0.0001 hr-\n3\n-1.”\n7. There is very likely an overestimation of 13C on the filters after the experiments linked to\nthe saturation of the filter with 13C-rich water. The filters from 2018 and 2019 experiments\ncollected before the mass spectrometer analyses need to be rinsed with DI water to be\nreflective of the real 13C uptake by biomass and carbonate. This step is not mentioned in\nthe method section.\nResponse: We agree that the methods need to be more detailed and have included this\ninformation in the methods.\nWith specific reference to this point, we have included the following text in the methods:\nLines 391-395: “Once on the filter, samples were rinsed with unamended, filtered site water\n(under vacuum) to wash away unincorporated isotope. Separate dedicated funnels and\nfiltration systems were used to produce filtered site water and to collect samples to\ndetermine the natural proportion of 13C in the microbialites at each site.”\n3\n8. Figure 7: under which type of laboratory culture?\nResponse: The title of Fig.7 (now Fig.8) was misleading and has been changed to read:\n“Nascent microbialites cultured in the laboratory.” We have also introduced new text into the\nmethods (Lines 359-365).\n9. Lines 45, 219, 233, 471, 721: Ca-carbonate is not necessary calcite. Ca-carbonate has\ndifferent polymorphs. Ca-carbonate can be aragonite, calcite, vaterite, monohydrocalcite. This\nmanuscript and the past study (Buttner et al., 2021) do not decipher about the type of\nCa-carbonate. Please remove the name calcite and replace by “Ca-carbonate”.\nResponse: We have provided MS. Ramen Spectroscopy data that indicates that indicates\nthat the C is accreted as calcite (New Supplementary Fig. 4)\n10. Line 144: write as “microbialite”.\nResponse: This has been done\n11. Line 232: How the accretion of 9.20 to 16. 41 kg CO2/m2/yr was determined?\nResponse: The methods section has been expanded to explain how the CO and C\n2\nconcentrations were determined.\n12. Lines 392-402: DIC is not only HCO3- this is also CO32- and CO2. Moreover, it is\nnecessary to mention that the released CO2 during the formation of CaCO3 from 2(HCO3-)\nhas not been measured and considered in the calculation of the C rate calculation.\nResponse: The reviewer is correct that HCO - is not the only form of DIC in aquatic\n3\nsystems, however, it is the most abundant form of DIC in most natural waters (85-95%)\nincluding our sample sites (Fig. 1). This high proportion in natural waters is why HCO -\nis\n3\noften used in aquatic carbon stable isotope tracer studies. The reviewer is also correct that\nCO release and transformations via chemical and biological reactions is expected.\nmicrobialite sites and light cycles. Movement of carbon between forms during the incubation\nperiod does not negate the carbon uptake and precipitation (movement from dissolved to\nparticulate) by the termination of the incubation. We added 13C tracer based on the most\ndominant form of DIC and assessed how much of that carbon was in the particulate form at\nthe end of the incubation. We propose several pathways that may contribute to this\nprecipitation but the key finding is that the precipitation occurred through predominantly\nbiological processes (Fig. 5) and into particulate inorganic forms (Supplementary Fig. 3) at\nrates that are similar to accretion rates observed in situ. We take the reviewers comment as\nan important next step on the path to exploring the factors underpinning the proposed\nmechanisms further, but that was outside the scope of this initial study as it was designed\nand executed.\n4\n13. Line 474: What are precisely the 7 selective domains?\nResponse: The ones listed in Fig 7B (and shown in 7A). We have added the reference to\nthe revised text in this section.\n14. Line 474: What are the proportions of Ca-carbonate in the 4 samples?\nResponse: We have this information only for SK and CR and the calcite proportions for the\nrelevant samples are listed in Fig. 7B. We do not have data for TP or OV745, Since the CR\nmicrobialites had the higher C uptake rates compared to OV745. We are likely\nunderestimating the accretion rates at CR.\n15. Lines 476-477: Provide the calculations (equations) made by the software RockMaker to\ncalculate C contents of microbialites. Using which data? There is a lack of clarity on how the\nCa-carbonate was quantified within the CR microbialite.\nResponse: More detailed experiments have been added to the methods section and as a\nnew Supplementary Data 5 and the old Supplementary Data 5 becomes Supplementary\ndata 6.\n16. Line 475: After checking the link, different threshold plugins exist on ImageJ. What is\nexactly the name of the used threshold plugin?\nResponse: There is only one “Threshold” tool. The other one is called “Colour Threshold\",\nbut since we are working with grey scale images there was no point in using this tool.\nExplanations on the use of “ImageJ” have been added into the Methods section.\n5"}]

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