Why stromatolites are important

Then, for 2 billion years, photosynthesising Stromatolites pumped oxygen into the oceans like underwater trees, before trees existed. Close up of Stromatolites at Hamelin Pool. Living Stromatolites are no longer widely distributed. There are only two well-developed marine Stromatolite areas in the world: in the Bahamas and at Hamelin Pool in the Shark Bay area of Western Australia.

A group gathers to discuss Stromatolites at Hamelin boardwalk. Photo Ben Parkhurst. But sea grass meadows are being damaged by the runoff caused by floods and extreme temperature events. Climate change is likely to lead to more frequent tropical storms and more frequent flooding events in the area, 4 threatening the Stromatolites of Hamelin Pool.

Human interference is another threat. To protect the delicate structures, visitors are restricted to the boardwalk. From here they may be underwhelmed: Stromatolites look a little like cow pats from that vista.

OTU abundances were normalized to relative abundance in percentage. The morphology of unconsolidated precipitates was analyzed via scanning electron microscopy SEM , while the chemical elemental composition was assessed with energy dispersive X-ray spectroscopy EDS. Samples from the crystalline layer Figure 1 were rinsed with ultrapure water and dried for 2 days at room temperature RT. To remove halite residues, samples for later SEM runs were soaked in ultrapure water overnight.

The final pellet was dried at RT. Both unconsolidated precipitates and lithified deposit were further studied for C and O stable isotopes. Isotopic values were established in four samples per layer. A sample of the microbial mat was horizontally cut along the crystalline layer. The upper half consisting of green and pink layers was aerobically incubated in room-tempered sterile hypersaline solution under natural lighting.

Additionally, the preparation procedure was adapted to avoid abiotic calcium carbonate precipitation Supplementary Table S1. After 10 weeks, each plate was individually wrapped in a translucent plastic bag to avoid agar desiccation. The plastic bags were punctured with a needle to allow gas exchange. A sterile microscope slide was added to provide a retrievable surface for cellular growth and carbonate nucleation. DNA extraction, 16S amplicon sequencing and taxonomic assignment were performed as described above.

Crystal nucleation and maturation were monitored with an optical microscope, while established carbonate crusts were analyzed via SEM-EDS. The pellet was then dried at RT. The material was put on mounts and coated with platinum as described above. All samples were treated ultra-sonically in a bath sonicator for 60 s.

EDS was conducted to assess the elemental composition of sample compounds and ED to assign a crystalline or amorphous character. In addition, carbonate crusts from solid cultures were subjected to stable isotope analysis. The beakers were covered in a plastic wrap punctured with a fine needle to allow gas exchange. A sterile microscope slide was added to the liquid cultures as retrievable surface for cellular growth.

The cultures were subjected to 12 h of both continuous UV-C and photosynthetically active radiation PAR combined 40 W LED, also at 40 cm distance , followed by 12 h of darkness, for a total period of 6 weeks. To avoid natural light exposure throughout the day, the chamber was completely covered in tinfoil. Four weeks after the start of the experiment, one of the positive agar controls was added to the UV-C exposed experiment to monitor UVR effect on intact cyanobacterial mats.

In response to the appearance of red precipitate in the liquid UVR-exposed experiments, negative controls of ASN-III-UL medium and sterile demineralized water covered in non-punctured plastic wrap were added to examine external contamination. Cyanobacterial mat cover was monitored over a period of 6 weeks in all cultures. While the number of archaeal reads increased strongly from the green top layer to the black bottom layer, the bacterial reads showed a reverse tendency with most reads obtained in the top layer and least in the bottom layer.

Archaeal diversity was overall low with an estimated Chao-1 richness ranging from 6 in the top layer to Figure 2. Taxonomic composition of microbial mat layers showing A archaeal read counts and B bacterial read counts. Diversity estimators for the bacterial community revealed a slight increase in Chao-1 richness from 96 to from the top to the bottom layer Supplementary Table S2. The evenness was overall low 0. This provides the taxonomic identifier of the best hits but does not give the percent identity to these hits.

These layers are dominated by methanogens of the genus Methanohalophilus and to a lesser extent of Halobacteria. The lower layer is characterized by a relative higher abundance of potential sulfate reducing bacteria Desulfosalsimonas , a novel Desulfobacteraceae genus and Desulfovibrio. Sequences derived from potential sulfur oxidizing bacteria were mainly found in the lowest black layer at 0. Among these abundant genera we can recognize members that increase or decrease in abundance with depth of the layers and those that are more abundant in the two middle layers Supplementary Table S4.

The genus Marinobacter is especially abundant in the two middle layers. Finally, we identified genera which nearly exclusively reside in one of the four layers with an on average 50 times higher abundance than the sum of the other three layers or were absent in the other layers.

SEM-EDS analysis of the crystalline, third layer from the top identified the unconsolidated precipitates as magnesium calcite. Aggregates were closely associated with each other, forming textures appearing poorly crystallized Figures 3C,D. The delta values of stable 16 O and 18 O were relatively constant throughout the layers, fluctuating between 0.

Figure 3. Table 1. Mean isotope delta values throughout the layer of crystalline precipitates in the mat Top 0 and the primary Top 1 , porous Top 2 , compact Top 3 , and bottom Top 4 layer of the carbonate deposit. The cyanobacteria formed dense, filamentous mats on agar Supplementary Figure S2. In liquid medium, cellular growth was monitored on available surfaces culture vessel, microscope slide first, while more mature filaments conglomerated in floating mats.

After 8 weeks of incubation, the bacterial cover started to die off on the increasingly desiccated agar base, while liquid cultures were maintained.

Both light- and dark-green mats showed similar morphological characteristics. The single trichomes consisted of aligning rectangular cells about 2. The majority of cultured cells showed strong red fluorescence when excited at nm and not when excited at — nm, confirming the presence of phycoerythrin pigmentation characteristic for cyanobacteria Figure 4. Molecular analysis identified the cyanobacterium as Geitlerinema sp. Since molecular analysis identified no other cyanobacteria in the enrichment cultures, the cultures are mono-phototrophic.

Figure 4. Morphology of isolates from mixed agar culture under an optical microscope in standard adjustment smaller pictures and equipped with a green filter big picture.

The EPS appears as white to yellowish sheaths around the cyanobacterial cells. Incubation of the Geitlerinema enrichment revealed nucleation of micro-globules after 17 days of incubation on seven agar plates. In close proximity to many of the nucleation sites with denser filament coverage, the agar adopted cloudy spots of darker discoloration Figures 5A,B. Over the course of the following weeks, the spheroids amalgamated in granulated textures and continued to grow in diameter Figures 5C,D.

Sixty days post-inoculation, white crusts visible to the naked eye and morphologically different from halite appeared on top of bacterial mats Figure 5E. Imaging disclosed plate-shaped microstructures Figure 6A conglomerated in needle and cauliflower aggregates, which were in turn associated in granulated textures Figures 6B,C.

ED patterns were composed of few distinct rings and scattered spots of varying intensities, which were indexed corresponding to the d-spacing values of calcite after Downs et al. Furthermore, completely calcified cells were disclosed via imaging and chemical elemental analysis Figure 6C. Figure 5. Crystal nucleation and growth observed under an optical microscope. A Nucleation of globules in association with bacterial filaments and development of darker spots indicated with arrows in the medium, B increased number of nucleating crystals around filaments attracted to a CO 2 or O 2 bubble in the medium, C amalgamation of globules into granulated textures growing into D larger crystals on the surface of filament clusters and eventually E into carbonate crusts visible to the naked eye.

Figure 6. SEM imaging and EDS analysis of carbonate crusts from agar cultures illustrating A needle morphology, B aggregate texture including nucleating spheroids light blue arrows in EPS sheaths dark blue arrows and C sheaths of EPS with nucleating spheroids as well as a calcified cell white arrow associated with spheroids on the far left, and D indexed ED patterns of calcite sample compounds.

Neither extracellular crystals nor crusts could be observed in liquid cultures. TEM imaging revealed approximately round, intracellular inclusions of a range of diameters below 0. The inclusions were often associated with each other, stayed in place upon tilting of the sample and featured similar atomic weights in HAADF-mode.

Figure 7. Intracellular inclusions from liquid culture experiments. B Map and exemplary ED of intracellular granules. Liquid cyanobacterial cultures exposed to UV-C radiation revealed the formation of red flakes within a week after the start of the experiment Figures 8B , 9A.

After 6 weeks, cyanobacterial growth could be observed in two liquid UV-C exposed cultures, albeit exclusively beneath the added microscope slides. In liquid controls without UV-C, cyanobacterial growth was observed in one culture only, and exclusively on the top side of the microscope slide Figures 8A,B.

When subjected to UV-C, the surface bacterial cover of a positive agar control significantly decreased within days and completely diminished after a week. Similar intracellular inclusions as in the culture experiments could be observed in both UV-C exposed and positive control filaments Figure 9E. Correspondingly, the analysis of elemental composition and structure rendered approximately round structures peaking in the elemental P, Ca, Mg, S, and sporadically K spectra Figures 9A—D.

Figure 8. Effects of UVR on cyanobacterial growth. Cultures featuring bacterial growth are marked with a green dot, continuously sterile cultures with a red dot. B Cyanobacterial growth occurred exclusively beneath the microscope slide in liquid culture subjected to UV-C, and on only top of the slide in the control. C Development of an intact cyanobacterial mat in agar culture after exposure to UVR. Figure 9. Intracellular inclusions from the UVR experiment.

B Map of a filament from an agar control culture. C Representative EDS of the intracellular inclusions. D Representative ED of the intracellular inclusions.

The Lagoa Vermelha microbial mat has been maintained for several years under controlled laboratory conditions to serve as a model ecosystem far from its place of origin Vasconcelos et al. Since most natural communities change and adapt to laboratory conditions, it is difficult to extrapolate laboratory results to the field. However, here we show that several of the key functions photosynthesis, presence of sulfur cycling taxa and calcification remain intact with several of the natural key species performing these tasks Supplementary Tables S3 , S4.

The number of reads recovered by 16S rRNA amplicon sequencing using archaeal and bacterial primer sets revealed an inverse relationship in abundance between Archaea and Bacteria that, respectively, increase and decrease with depth Figure 2. Although not quantitative, a potential higher archaeal abundance in the interior is consistent with studies on microbial mats in the natural stromatolite-forming Shark Bay area Papineau et al.

Also the overall microbial composition and the characteristic layering including a green photosynthetic layer, a white crystalline layer and a black sulfide rich layer are common for calcifying microbial mats such as from Shark Bay and Lagoa Vermelha Vasconcelos et al. Hence, we consider the laboratory-incubated mats a suitable reference for natural stromatolite forming systems.

The considerable incidence of Archaea in the bottom layer Figure 2 is mostly attributed to the abundance of a novel group of Archaea that could only be assigned at the class level to Lokiarchaeia of the Asgard phylum using the SILVA version as reference database. At this moment, we cannot give a conclusive identification, but members of the Asgard group, including Lokiarchaeia, have been observed before in stromatolites Wong et al.

Little is known about this group of Archaea, but they may form a symbiotic association with Methanomicrobia with whom they often co-occur Xiang et al. The genus Methanohalophilus within the order Methanosarcinales, is the most abundant archaeal genus in the top three layers and hints to an important contribution of potentially hydrogenotrophic methanogenesis in the mats.

Hydrogenotrophic methanogenesis is considered an ancestral form of methane production Bapteste et al. The Thermoplasmata, represented by the Marine Benthic Group D and DHVEG-1 are found especially in the lower layers and may contribute to the sedimentary cycling of carbon, which might assign a key role of these organisms in lithifying mats Zhou et al.

The overall composition is typical for microbial mats with a dominance of Proteobacteria, Bacteroidetes and Cyanobacteria, dependent on the sampling depth Bolhuis and Stal, Primary production in the stromatolite type microbial mats is performed by oxygenic photosynthetic Cyanobacteria of the genus Geitlerinema , the dominant, filamentous cyanobacterium, Dactylococcopsis , a unicellular species and the filamentous genus of Halomicronema Supplementary Table S4.

Each of these salt tolerant cyanobacterial genera are frequently found in hypersaline environments Oren, and stromatolites Samylina and Zaytseva, Sulfur cycling is performed in the deepest layer by sulfur oxidizing bacteria Thiohalospira and sulfate reducing bacteria order Desulfobacterales and Desulfovibrionales Supplementary Table S4.

Sulfate reduction has been suggested to play a key role in the precipitation of carbonates in modern calcifying microbial mats, and recently a novel member of the Desulfovibrionaceae family has been linked to potential calcium carbonate deposition in a hypersaline environment Spring et al. Those genera may be involved in lamination formation requiring anoxic conditions and EPS nucleation sites Vasconcelos et al.

Anaerobic, halophilic Halanaerobium taxa forming hydrogen and metabolizing C6 sugars, as well as Spirochaeta are potential fermenting bacteria. Fermentation can potentially counteract the calcification process, but may be prone to diel fluctuations Dupraz et al. The presence of anaerobic phyla in the oxic top layers may be explained with sampling cross-overs or scattered anaerobic micro-zones Wong et al.

However, sulfate reducers, often considered as anaerobes, have also been found in micro- oxic regions of a mat suggesting active sulfur cycling within the upper layers Minz et al. The hypersaline nature of the microbial mat is reflected in the occurrence of halophilic species amongst the Archaea Halomarina and Halomicroarcula and halotolerant bacterial members e.

The laboratory-controlled stromatolite continues to accrete carbonate mainly via in situ precipitation, which is similar to Precambrian systems and their modern analogs such as alkaline lake and Lagoa Vermelha stromatolites Kazmierczak and Kempe, ; Vasconcelos et al. Spadafora et al. In general, Lagoa Vermelha stromatolites are laminated on a sub-mm scale and have been described as good textural analogs to Precambrian forms Vasconcelos et al.

The mineralogical character of the studied precipitates Figure 3 is consistent with the results of earlier analyses of lithifying microbial mats in Lagoa Vermelha van Lith et al. A pronounced deviation from the natural system is the exclusive precipitation of magnesium calcite instead of additional dolomite. The periodic fluctuation of water levels is not mimicked in the laboratory-incubated microbial mat. The hypersalinity may thus not be pronounced enough for dolomite precipitation on the stable laboratory-incubated stromatolite.

The sediment presumably supplies a sufficient amount of Si to be sporadically trapped in the cyanobacterial EPS associated with magnesium calcite spheroids Figure 3B. Autotrophic carbon fixation features a strong preference for the lighter 12 C, which manifests in the OM itself after isotopic fractionation, e.

It is likely that diagenetic processing by aerobic heterotrophic bacteria consuming cyanobacterial necromass and EPS, as well as sulfate reduction in the anoxic zone of the microbial mat produces an increased 12 C ratio in primary precipitates. Degradation of OM has been suggested to lead to formation of microbialites over time, while the upper mat layers presumably produce carbonates of globular morphology Spring et al.

Especially the significance of sulfate reducers in Lagoa Vermelha carbonate diagenesis has been shown before, since in addition to the consumption of organic compounds, this metabolic pathway produces alkalinity Vasconcelos et al. Next to the consumption of autotrophic necromass, photosynthesis itself may mediate 13 C enriched DIC in a CO 2 limited system.

The laboratory growth conditions simulate the increased salinity of Lagoa Vermelha and thus decrease solubility of CO 2 Weiss, The cyanobacterial enrichment identified Geitlerinema sp. This species is also the most abundant cyanobacterium present in the top layer of the laboratory stromatolite Supplementary Table S4 and has been observed in natural stromatolites Samylina and Zaytseva, , while Vasconcelos et al. Geitlerinema sp.

While bioturbation of the sediment is mostly non-prevalent and higher eukaryotic organisms such as Vertebrates and Crustacea have merely been found in Lagoa Vermelha Vasconcelos et al. Culture growth on agar indicates a general adaption to the periodically semi-arid conditions with direct air exposure common in Lagoa Vermelha, with the gelatinous agar itself simulating a mat-like substrate. The mono-phototrophic community featured precipitation of magnesium calcite on agar plates Figures 5 , 6 , but not in liquid medium.

This suggests that a gel matrix favors mineral precipitation in the presence of Geitlerinema and that such media are good candidates to reproduce the substrate of natural microbial mats in general.

Carvalho et al. This indicates that the mat precipitates plus a section of the upper deposit layer of the stromatolite studied here have been formed in the laboratory and are especially valid to compare to the carbonates derived from cultures. Biominerals precipitated in those mono-phototrophic cultures are both chemically and morphologically similar to the ones precipitated in the natural microbial mat Figures 3 , 6.

While it is possible that other relevant cyanobacterial species were outcompeted in the experiment, this suggests a significant contribution of Geitlerinema to primary carbonate mineral precipitation on the studied stromatolite at least. A prominent difference in chemical composition, however, is the overall lower magnesium content in culture precipitates.

This phenomenon is most likely accounted for by a shortage of magnesium in the growth medium Supplementary Table S1. The solid, nutrient-rich agar surface promotes biomineralization in particular, since it is not subjected to fluid dynamics and allows a locally permanent change of the chemical milieu in an area beyond the bacterial EPS layers.

An abiotic factor further assisting ion concentration and thus calcite formation is evaporation. The overall higher culture volume renders this parameter less significant in liquid medium, but cannot be neglected on agar plates subjected to high temperatures for prolonged periods of time.

In the rock record, stromatolites can be recognized by characteristic laminar structures, such as those seen in the picture above. This is basically the cross section of a columnar stromatolite as seen in the Shark Bay picture.

Although simple, cyanobacteria were ultimately responsible for one of the most important "global changes" that the Earth has undergone. Being photosynthetic, cyanobacteria produce oxygen as a by-product.

Photosynthesis is the only major source of free oxygen gas in the atmosphere. As stromatolites became more common 2.