Cyanobacterial exopolymer properties differentiate microbial carbonate fabrics

Although environmental changes and evolution of life are potentially recorded via microbial carbonates, including laminated stromatolites and clotted thrombolites, factors controlling their fabric are still a matter of controversy. Herein, we report that the exopolymer properties of different cyanobacterial taxa primarily control the microbial carbonates fabrics in modern examples. This study shows that the calcite encrustation of filamentous Phormidium sp. secreting acidic exopolymers forms the laminated fabric of stromatolites, whereas the encrustation of coccoid Coelosphaeriopsis sp. secreting acidic exopolymers and poor calcification of filamentous Leptolyngbya sp. secreting non-acidic exopolymers form peloids and fenestral structures, respectively, i.e. the clotted fabric of thrombolites. Based on these findings, we suggest that the rise and decline of cyanobacteria possessing different exopolymer properties caused the expansion of thrombolites around the Proterozoic/Cambrian boundary.

Bacterial composition. The composition of the bacterial community estimated by 16 S rRNA gene clone library analysis shows that the phylum Cyanobacteria is dominant in both the stromatolites and thrombolites ( Supplementary Fig. S2). However, the bacterial diversity in the stromatolites is lower than that in the thrombolites possibly due to the higher flow velocity at the stromatolite surface: it is experimentally demonstrated that the bacterial diversity in freshwater biofilms inversely correlates with the flow velocity 11 . This hydrodynamic effect would also differentiate the dominant cyanobacterial genera: genus Phormidium is dominant in the stromatolites, whereas genus Leptolyngbya is dominant in the thrombolites. This trend of cyanobacterial phylotype composition is consistent with that of morphotypes identified by the microscopic observations described below.
Metabolic influence on CaCO 3 precipitation. Microelectrode measurements show similar trends in both the stromatolites and thrombolites (Supplementary Fig. S3). Under light conditions, increases of O 2 , CO 3 2− and Ω and decreases of CO 2 and Ca 2+ are observed at the microbialite surface. Under dark conditions, a decrease of O 2 and an increase of CO 2 are observed, whereas Ca 2+ , CO 3 2− and Ω exhibit no detectable shift. These results indicate that CaCO 3 precipitation at both microbialites is primarily induced by cyanobacterial photosynthesis 9 . Depositional and mineralogical characteristics of stromatolite. The surface of stromatolites is represented by patches of light green and purple colour, and several millimetres think laminations are recognised in the cross section (Fig. 1a, b). Confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM) observations indicate that the dominant cyanobacterium in stromatolites is a filamentous Phormidium sp. that secretes a thin (ca. 0.2 μm) exopolymer sheath ( Fig. 1c; Supplementary Fig. S4a). Lectin binding analysis (LBA) suggests that these sheaths contain abundant acidic sugars with carboxyl groups and the sheath exterior is surrounded by a significant number of fine minerals (Fig. 1c). TEM and scanning transmission X-ray microscopy (STXM) observations reveal that the minerals larger than ca. 1 μm in diameter are mostly calcite ( Fig. 1d-j). Magnesium is undetectable in these calcite crystals ( Supplementary Fig. S5), which is consistent with the feature of carbonates precipitated around the exopolymers 12 . In the vicinity of the exopolymer sheath, minerals exhibit the characteristics of an amorphous CaCO 3 (ACC) precursor reported by ref. 13 : they show aragonite-like NEXAFS (near edge X-ray absorption fine structure) spectra (Fig. 1f,g), and are unstable under an electron beam, and decompose into polycrystalline calcium oxide during TEM observations (Fig. 1i). These features suggest that calcite nucleation occurred on the surface of a Phormidium sheath. Conversely, minerals that are ca. 200 nm in diameter are mostly clay minerals (Fig. 1f,h; Supplementary Fig. S4a) representing the absorption of suspended clay from turbulent water onto the acidic exopolymer via a divalent cation bridge 14 . Heavy calcification of Phormidium filaments results in sheath encrustation 15 , and abundant, upward oriented filaments at the stromatolite surface (Fig. 1k,l) cause horizontally uniform precipitation of calcite at the mesoscopic scale. The spaces between calcified filaments are left as growth cavities, and the alternation of porous and dense layers forms laminations (Fig. 1m).
Depositional and mineralogical characteristics of thrombolite. The surface of thrombolites is orange, and no laminations are observed in its cross section (Fig. 2a,b); instead, the submillimetre to 1-cm-sized fenestral structures are conspicuous (Fig. 2n). CLSM and TEM observations indicate that the dominant cyanobacteria are filamentous Leptolyngbya sp. and coccoid Coelosphaeriopsis sp., which secrete a thin (ca. 0.3−0.5 μm) exopolymer sheath and a relatively thick (ca. 1.5−3.0 μm) capsule, respectively (Fig. 2c,d; Supplementary  Fig. S4b,c). LBA suggests that Leptolyngbya sheaths lacking a detectable amount of acidic sugars are mostly free from mineralisation (Fig. 2c), whereas Coelosphaeriopsis capsules containing acidic sugars are enclosed by minerals (Fig. 2d,e). TEM and STXM observations reveal that calcite is the encrusting mineral on Coelosphaeriopsis capsules ( Fig. 2f-k). In a conventional thin section, calcified Coelosphaeriopsis colonies exhibit peloids, which are internally structureless microcrystalline carbonate sands 16 (Fig. 2l-n). The peloids are scattered around tangled Leptolyngbya filaments at the thrombolite surface (Fig. 2l,m). They become more condensed in the deeper parts of thrombolites, and the spaces occupied by non-mineralised Leptolyngbya filaments are left as irregular fenestral structures, with overall formation of clotted fabrics at the mesoscopic scale (Fig. 2n). In addition, a filamentous cyanobacterium Scytonema sp. that secretes a relatively thick (ca. 3-5 μm) and acidic exopolymer sheath is locally visible in the thrombolite, and the sheath interior is impregnated with calcite crystals (Fig. 2c) 15,17 .
The acidic sugar detected by LBA was confirmed by a lectin blocking assay ( Supplementary Fig. S6) and the fluorescence labelling of carboxyl groups ( Supplementary Fig. S7).

Discussion
Factors controlling the microbial carbonate fabrics. Among the observations at the Ueno tufa site, the spatial proximity of heavily and poorly calcified cyanobacteria that secret acidic and non-acidic exopolymers, respectively, in the thrombolites is critical toward understanding the microbial carbonate formation mechanism. The crystallisation process generally comprises crystal nucleation and growth, and both the nucleation and growth rates are proportional to mineral supersaturation 18,19 . Supersaturation at the microbialite surface, which is the major crystallisation site, is elevated by the combination of CO 2 degassing in the water column and cyanobacterial photosynthesis at the deposit surface ( Supplementary Fig. S3). However, a diffusive boundary layer (DBL) blankets the deposit surface 20 , and a significant difference in supersaturation cannot be expected at the mesoscopic scale. In contract, supersaturation of water retained in the exopolymer sheaths/capsules is likely much higher under light conditions due to a reduced diffusion rate 21 . Very high supersaturation in the exopolymer sheaths/capsules per se is common to most cyanobacterial taxa; however, the calcite nucleation rate on polysaccharides is proportional to their acidity under such conditions 22 . Therefore, the observed difference in the  degree of cyanobacterial calcification can be primarily attributed to the difference in their exopolymer acidity, i.e. the nucleation rate. This interpretation also applies to stromatolites, although cation absorption by clay minerals may affect the formation and stability of the ACC precursor. Higher flow velocity at the stromatolite-depositing site would reduce the DBL thickness 23 ; however, it reduces the hydrochemical difference between the stromatolite surface and the water column, which cannot create a significant difference of supersaturation at the mesoscopic scale. The calcification styles of acidic exopolymer sheaths/capsules, either as encrustation or impregnation, are not apparently related to their thickness, which indicates the influence of other factors such as their internal structure.
The cyanobacterial exopolymer properties described so far are further responsible for the differentiation of the fabrics of the investigated microbialites. The dominance of filamentous cyanobacteria that secrete acidic exopolymers provides mesoscopically uniform nucleation sites, owing to which the laminated fabric of stromatolites is produced. In contrast, a combination of coccoid and filamentous cyanobacteria that secrete acidic and non-acidic exopolymers, respectively, provides mesoscopically heterogeneous nucleation sites, owing to which the clotted fabric of thrombolites is formed. These observations indicate that three-dimensional structures of biofilms strongly affect microbialite fabrics. The cyanobacterial cellular morphology, either coccoid or filamentous, has a subordinate influence by affecting the distribution pattern of nucleation sites. In addition, small coccoid cyanobacteria observed inside peloids (Fig. 2e) are hardly recognisable in a conventional thin section (ca. 50-μm thick; Fig. 2l-n), which potentially resolves the controversy regarding the relation between dominant cellular morphology and microbialite fabrics 2,4,10 .
These results have improved our knowledge of the fundamental mechanisms involved in the formation of microbial carbonates, as follows: (1) Confirmation of the long-held view that cyanobacterial acidic exopolymers provide CaCO 3 mineral nucleation sites 15 . (2) Recognition that non-acidic exopolymers are relatively unsuitable for nucleation, which overwhelms the inhibitory effect of acidic exopolymers 21,24 . (3) Supersaturation at crystallisation sites primarily contributes to the precipitation quantity rather than localizes the nucleation sites, which suggests that it is a prerequisite for microbial carbonate formation, as previously assumed 5,21,24 .

Implications for the fossil record. These observations from modern examples provide significant insights
into the interpretation of the fossil records of microbial carbonate, particularly from the perspective of their fabric and quantity. For example, a substantial change of microbial carbonate fabric occurred around the Proterozoic/ Cambrian boundary when both thrombolites and calcified cyanobacteria first expanded 4-6,15,25 . A number of factors have been proposed to explain these changes: those for thrombolites include dominant microbial cellular morphology 4 , a framework construction mechanism 2 and a microbial growth/calcification ratio 10 , whereas those for calcified cyanobacteria include the ambient water Mg 2+ /Ca 2+ ratio 26 , temperature 27 , CaCO 3 supersaturation 28 , dissolved inorganic carbon (DIC) concentration 21 and equilibrium CO 2 partial pressure (pCO 2 ) 29 . However, our observations from modern processes underscore the potential importance of cyanobacterial exopolymer properties to the expansions of both of thrombolites and calcified cyanobacteria around the Proterozoic/Cambrian boundary. If this is the case, an evolutionary/extinction event of cyanobacteria that drastically changed their exopolymer properties is expected at that time.
Conversely, long-term quantitative changes recognised in the fossil record 3,21 would have been largely, if not entirely, controlled by supersaturation at the crystallisation sites. Indeed, this view has been experimentally corroborated 9 . By evaluating factors other than supersaturation (such as precipitation inhibitors 26 and metazoan competition 3 ), the quantitative records of microbial carbonate would provide a proxy for oceanic pH and DIC 9 .
These interpretations echo the perceptive view presented in ref. 6 that the history of microbial carbonates reflects the superimposition of prokaryote evolutionary/extinction events onto environmental fluctuations. In any case, future studies must evaluate the applicability of knowledge from freshwater microbialites to their seawater counterparts.
Methods XRD analysis. The surface part (ca. 5 mm) of microbialite samples were air-dried, powdered using a mortar and pestle, and analyzed using a powder X-ray diffractometer with Cu Kα radiation (40 kV, 40 mA) and a graphite monochromator (MultiFlex, Rigaku).

Water chemistry analysis.
For characterization of the creek water chemistry, pH and temperature were measured in the field using a portable pH meter (D-51, Horiba). Alkalinity was determined by acid-base titration using a hand-held titrator and a 1. were adjusted to 2% HNO 3 , and cation concentrations (Ca 2+ , Mg 2+ , Na + , and K + ) were estimated using inductively coupled plasma optical emission spectroscopy (ICP-OES; iCAP7200, Thermo Fisher Scientific). The measured values were processed with the PHREEQC 30 computer program to calculate the DIC concentration, pCO 2 , and Ω.
Carbon and oxygen stable isotope analysis. Water samples were filtered through a 0.2 μm membrane and collected in gas-tight glass bottles. Tufa samples were collected by scraping the surface part (ca. 0.5 mm) with a knife, and air-drying. Carbon and oxygen isotopes were measured with a mass spectrometer, as described previously 31 . 16 S rRNA gene analysis. Almost full-length 16 S rRNA genes of bacteria were obtained from stromatolite and thrombolite, for which the methods described previously 32 were applied for sampling, DNA extraction, and polymerase chain reaction (PCR) amplification. The PCR products were purified, cloned into vector pTAC-1 (BioDynamics Laboratory Inc.), and then transformed into chemically competent Escherichia coli (Competent Quick DH5α, Toyobo). Inserts of randomly selected colonies were used for bidirectional sequencing with flanking vector primer M13BDFw (5′ CAG GGT TTT CCC AGT CAC GAC 3′) and M13BDRev (5′ CGG ATA ACA ATT TCA CAC AGG 3′). DNA sequencing was performed on a DNA analyzer (ABI3730, Applied Biosystems) with the BigDye terminator version 3.1 cycle sequencing kit. Closest relatives were determined using SINA online 16 S rRNA sequence classifier 33 based on the Greengens database 34 . The obtained sequences (76 clones from the stromatolite, and 99 clones from the thrombolite) were checked for their chimeras using the Bellerophon server 35 . Representative gene sequences in this study were deposited in the DNA Data Bank of Japan (DDBJ) database under accession numbers AB862884-AB862938, and LC215056-LC215137.
Microelectrode measurements. Microbial metabolism and CaCO 3 precipitation at the microbialite surface was evaluated using O 2 , CO 2 , Ca 2+ , and CO 3 2− microelectrodes, as described previously 9 . Construction and handling of the CO 3 2− microelectrode were performed according to ref. 36 . Creek water collected at Site 1 was used for the measurement.
Thin section observations. Vertical sections of the microbialite surfaces were observed using thin sections.
Microbialite samples were first fixed using phosphate-buffered saline (PBS) containing 3.7% formaldehyde for 2 days, after which the solution was replaced with 50% ethanol in PBS and the sample was stored at 4 °C until further processing. Thin sections were then prepared from resin-embedded samples, as described previously 37 . Transmitted, cross-polarized, and fluorescent light images were acquired using CLSM (LSM700, Zeiss) equipped with a CCD camera (AxioCam MRc, Zeiss) and ZEN2010 software (Zeiss). Fluorescent light images consisted of two channels, one acquired by excitation at 488 nm with a BP505-600 nm emission filter, and another by excitation at 555 nm with an LP615 nm emission filter. Composites of cross-polarized and fluorescent light images were generated by the Lighten mode of Adobe Photoshop CS6. Transmitted light images of lower magnification were acquired using a conventional microscope (Eclipse LV100 POL, Nikon).

LBA.
The distribution pattern of acidic sugars was investigated using a lectin from Limulus polyphemus (LPA, Cosmo Bio), which is known to have binding specificity to glucuronic acid and N-acetylneuraminic acid 38 . Either small blocks of the surface part (ca. 5 mm 3 ) or a slurry were prepared from fresh microbialite samples (within 48 h after collection), and soaked in 50 ng μL −1 of FITC-conjugated LPA lectin for 20 min at room temperature. Unbound lectin was thoroughly removed by washing with a buffer [88 mM NaCl, 20 mM Tris (pH 8.0), 0.01% (w/v) SDS], and block samples were submerged in distilled water while slurry samples were enclosed by a cover slip with mounting media (AF2, Citifluor). Fluorescent and reflected light (488 nm excitation with an SP490 nm emission filter) images were then acquired using CLSM. For block samples, image stacks of optical slices were acquired, and plane views of the rough microbialite surface were generated by maximum intensity projection mode of Imaris software (Bitplane). Negative control of LBA was conducted by applying FITC-conjugated lectin from Phaseolus vulgaris (PHA, Cosmo Bio), which has binding specificity to none of the tested sugars 38 . In addition, untreated samples were observed for comparison.
Lectin blocking assay. Binding specificity of LPA lectin was checked by lectin blocking assay 38 . 12 competing sugars were tested: 11 were selected from 12 different monosaccharides identified from cyanobacterial exopolymers to date (glucuronic acid, galacturonic acid, arabinose, fructose, fucose, galactose, glucose, mannose, rhamnose, ribose, and xylose) 39 , and 1 was N-acetylneuraminic acid. First, 50 ng μL −1 of FITC-conjugated LPA lectin was pre-incubated for 15 min with competing sugars at three different concentrations (0.1, 1, and 10 mg mL −1 ), followed by LBA using stromatolite samples as described above. The same microscopic settings were applied for all samples.
Fluorescence labeling of carboxyl groups. To cross-check the results of the LBA, carboxyl groups were fluorescently labeled by modification of the procedure described previously in ref. 40 . Either block or slurry samples fixed with 3.7% formaldehyde/PBS were soaked in 2 mL of 0.1 M 2-morpholinoethanesulfonic acid (MES) buffer, pH 5.5, and 100 μL of 50 mM EZ-Link Pentylamine-Biotin (Thermo Fisher Scientific) was added, followed by 25 μL of 100 mg mL −1 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride in 0.1 M MES buffer. Samples were incubated at room temperature for 2 h using a rotary shaker, and washed three times with PBS. Samples were then soaked in 500 μL of 1/10 fluorescein-conjugated streptavidin (GeneTex) diluted with PBS, incubated at room temperature for 1 h in the dark, and washed three times with PBS. Fluorescent and reflected light images were acquired using CLSM, as described above.
SciEntific REPORTS | 7: 11805 | DOI:10.1038/s41598-017-12303-9 TEM analysis. Microbialite samples fixed with 2.5% glutaraldehyde/creek water were post-fixed with 1.5% OsO 4 in 100 mM cacodylate buffer, pH 7.4, for 90 min, and embedded in an epoxy resin (EPOK 812, Oken). 800 nm thin sections were first obtained using an ultramicrotome (Ultracut E, Reichert-Jung), and transmitted and cross-polarized light images were acquired after toluidine blue staining. 70−80 nm thin sections were obtained, stained with 2% uranyl acetate and lead citrate, and montage images were acquired using TEM (JEM-1400, Jeol) operated at an accelerating voltage of 80 kV. To analyze the relationship between the exopolymers and minerals, thin-foil sections were prepared from resin embedded samples using a focused-ion beam (FIB) apparatus (SMI4050, Hitachi), and observed with TEM (JEM-ARM200F, Jeol) operated at an accelerating voltage of 200 kV, as described previously 41 . The elemental composition was examined using energy-dispersive X-ray spectroscopy (EDS) installed with the TEM, and combined elemental maps were generated by scanning TEM (STEM) with Analysis Station 3.8 software.
STXM analysis. STXM-based NEXAFS analysis of carbon (1s) and calcium (2p) were performed using the BL13A beamline at KEK-PF (Tsukuba, Japan), of which the general experimental setup has been described previously 42 . Model compounds for Ca NEXAFS measurements were obtained from Nichika Inc. (calcite and aragonite) and Wako Pure Chemical Ltd. (calcium oxide), and the ground powders were deposited onto a carbon-coated copper grid (Cu 200 mesh, Jeol). For sample analysis, the surface of fresh stromatolite was scraped with a sterile knife, suspended in distilled water, dropped onto a carbon-coated copper grid, and air dried at room temperature. Sample preparation and STXM analysis were conducted within 24 h and 56 h after sampling, respectively. Compositional images were generated with the RGB composite mode of aXis 2000 software 43 using images of specific absorption edges for protein (288.2 eV), acidic polysaccharide (288.6 eV) [44][45][46][47][48][49] , and calcium (352.6 eV) 13 . In addition, the same thin-foil sections used for TEM analysis were also analyzed using STXM.