METHODS OF BIOTIC DIRECTED CALCITE PRECIPITATION BY DIAZOTROPHIC CYANOBACTERIA

CROSS-REFERENCE TO RELATED APPLICATIONS

This International PCT application claims the benefit of and priority to U.S. Provisional Application No. 63/420,190 filed October 28, 2022, the specification, claims and drawings of which are incorporated herein by reference in their entirety.

GOVERNMENT INTEREST

This invention was made with government support under grant number DE-SC0020361 awarded by the U.S. Department of Energy (DOE). The U.S. Government has certain rights in this invention.

TECHNICAL FIELD

The present invention relates to novel systems, methods and compositions for calcite precipitation, and in particular contact-mediated precipitation of calcium carbonate by nitrogen fixing heterocyst cells in filamentous cyanobacteria. In a preferred embodiment, the present invention relates to novel systems, methods and compositions for the production of biocement, and in particular biocement produced using filamentous cyanobacteria placed under conditions to induce calcium carbonate precipitation.

BACKGROUND

Microbiologically induced calcium carbonate precipitation (MICP) is the process through which microbiota cause the precipitation of calcium carbonate, forming crystals which can result in solidification of sand/sediment. MICP results from the metabolic activities of microorganisms. For cyanobacteria, MICP is thought to result due to cells sequestering bicarbonate to drive the photosynthetic process. This results in a lowering of the pH within the surrounding media, causing precipitation of calcium. However, the mechanisms by which photosynthetic cyanobacteria cause MICP is still poorly understood. Here Applicants show new evidence of MICP caused by filamentous Anabaena cyanobacteria using quantitative microscopy. Cells of this species differentiate into photosynthetic vegetative cells and nitrogen-fixing heterocysts.

In contrast to previous results, Applicants data suggests that MICP occurs due to two distinct mechanisms: Firstly, mechanical stress on vegetative cells can cause leakage and/or lysis, releasing sequestered bicarbonate into the environment, resulting in formation of new crystals. Secondly, contact between a heterocyst and a calcite crystal seed appears to cause rapid crystal growth. These results are of interest to optimizing the production of bio-cement, a living, green construction material, which could assist with carbon sequestration and reduce the impact of climate change. These results also suggest an evolutionary benefit of contact-mediated precipitation as an anchoring tool to secure cyanobacteria growing in beach sands or as bacterial mats.

As noted above, while many different types of organisms can perform calcium carbonate (CaCOs) precipitation (e.g., shell and coral), the underlying mechanisms that result in structures with specific properties (e.g., shape, size, chemical composition, arrangement) are not well understood. Multiple factors play a major role in determining the properties and rate of mineral formation. These include the chemical (e.g., pH, calcium levels, alkalinity) and physical (temperature, rigidity) environment, the biochemical pathways involved, and the availability and type of nucleation site. CaCCh has been shown to nucleate on protein scaffolds as well as on polysaccharides and other biopolymers. The chemical and physical environments are often linked with the biochemical pathways because different environments can promote the use of different biochemical pathways.

Under certain environmental conditions, the cyanobacterial CCb-concentrating mechanism (CCM) can also shift the pH of the extracellular environment, resulting in the precipitation of CaCCh minerals including calcite, aragonite, and vaterite in a process termed microbially-induced carbonate precipitation (MICP). In addition, many ureolytic organisms, including Sporocarcma pasteurii, are known to induce the precipitation of CaCCh as a byproduct of urea hydrolysis. Other microbial nutrient assimilation pathways that consume protons and elevate the extracellular pH also have the potential to induce MICP. While all known free-living cyanobacteria contain a CCM, many diverse cyanobacteria also encode urease in addition to myriads of other biochemical pathways that could in principle result in MICP, including nitrogen fixation. As such, there is a long-felt need to better characterize the chemical and biological foundations of MICP, and in particular efficient and cost-effective means to initiate and control MICP for commercial and environmental purposes.

The present inventors have further demonstrated that cyanobacterium Anabaena sp. PCC ATCC 33047 is able to precipitate calcium carbonate in the form of calcite under nitrogen-fixing conditions where external nitrogen sources (besides atmospheric N2) are not needed. In a preferred embodiment, this allows for low resource production of biocalcite for building materials such as biocement, carbon sequestration, and other industrial uses. For example, traditional Portland cement based concrete releases a large amount of CO2 into the environment by burning limestone (calcium carbonate). To the contrary, the biotic directed calcium carbonate crystallization processes described herein instead can sequester CO2 into biomass and minerals for renewable building and carbon storing materials

SUMMARY OF INVENTION

The present inventors demonstrate that heterocysts induce calcium carbonate crystal formation on contact, and that vegetative cells did not induce calcium carbonate crystal formation on contact, but did upon mechanical stress/lysis. The present inventors further demonstrated that no fixed nitrogen or fixed carbon source was required to induce calcite precipitation with a diazotrophic cyanobacteria. As a results, the present inventors demonstrated that diazotrophic cyanobacteria results in calcium carbonate precipitation either in cases of mechanical stress (i.e., washed against a rock) or when a heterocyst encounters a calcium carbonate crystal when in growth conditions.

In one aspect, the inventive technology describes systems, methods, and compositions for the biotic directed formation of calcite. In another aspect, the inventive technology describes systems, methods and compositions to facilitate nitrogen fixation by diazotrophic cyanobacteria. In one preferred embodiment, the present invention describes the use of contact-mediated precipitation of calcium carbonate by nitrogen fixing heterocyst cells in filamentous cyanobacteria. In another preferred embodiment, the present invention describes the process of mechanical cellular lysis as a method to induce calcium carbonate precipitation.

In another preferred aspect, the present inventors have demonstrated that contact between nitrogen fixing cells (termed heterocysts) are able to induce calcium carbonate precipitation. Other microbes including strains of cyanobacteria can produce calcite. These non-nitrogen fixing cyanobacteria require nitrate or other nitrogen sources to grow, while Anabaena sp. ATCC 33047 does not. Other bacteria that use ureolytic calcium carbonate precipitation also require reduced carbon sources, nitrogen, and urea. Nitrogen fixing cyanobacteria are therefore able to produce this valuable mineral with minimal resources. The patterning of the heterocysts also enables patterning of crystal growth in 3D space based on the position of the heterocyst cell.

Additional aspects of the present invention include systems, methods, compositions, and kits for the production of biocement. In a preferred embodiment, the biocement of the invention can include a quantity of diazotrophic cyanobacteria in a growth media under conditions that induce MICP and a quantity of aggregate and water. The biocement of the invention can further be formed in a frame to produce one or more construction materials and the link.

Additional aspects of the invention will be evidenced from the specification, figures and claims set forth herein.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1A-I: Single-cell analysis of Anabaena sp. ATCC 33047 growth and differentiation, a-c: Representative frames showing Anabaena growing on the microscope in BG- 11 media without supplemental nitrogen (BG11-N). Images were collected in the brightfield and chlorophyll fluorescence channels. A merged image is also shown (chlorophyll fluorescence shown in red). The arrows indicate heterocysts, which are identified both by their increased size and the loss of chlorophyll fluorescence. The magenta yellow indicates a cell in the early stages of differentiation. Scale bars are 10 pm. d-g: Representative frames showing cells growing in BG-11 media supplemented with calcium chloride and bicarbonate (BGl l-N+Ca). Initial seed crystals were observed (indicated by arrows). We observed initial crystal growth (seen in frame e), likely due to temperature change as the sample was placed in the growth chamber. Some crystals (yellow arrow) showed increased growth due to interaction with the cells. Scale bars are 20 pm. h and i: Crystal growth was tracked over time, h shows the merged frame after 50.5 hours and i shows the mask labeling the crystals, j shows the crystal area over time. Scale bars are 50 pm. The zoomed-in region shown in d-g is indicated in magenta.

Fig. 2A-G: Physical contact with Anabaena cells causes crystals to grow, a: Time-lapse images showing crystal growth as a result of Anabaena 33047 cell lysis within a crystal, b: The average chlorophyll intensity of the two cells tracked and the corresponding growth of crystal area. Dotted lines indicate the time when the individual cells lysed, c: Time-lapse images showing nucleation and growth of a crystal at a sharp bend of a fdament. The crystal growth appeared to be caused by cellular leakage due to mechanical stress at the bend, d and e: Representative frames and plot showing that crystal growth does not occur due to proximity or contact with a vegetative cell, f: Representative frames showing growth of a crystal after contact with a heterocyst (indicated by an arrow), g: Plot showing crystal area over time. Dotted line indicates time of heterocyst contact at 37 hours. All plots show the brightfield image in grayscale and chlorophyll fluorescence in red. All scale bars are 20 pm. Fig. 3A-G: Correlated live-cell and Raman imaging of calcite precipitation by Anabaena sp. ATCC 33047 at single-crystal and single-cell resolution, a-c: Area Raman scan with calcite peak (1085 cm' ¹ ) intensity in gray and chlorophyl fluorescence from final frame of live growth experiment in red and an overlay of the two channel. Scale bar is 20 pm. e: Raman scan of this crystal showing calcite and carotenoid presence. f,g: Two Raman scans of crystals distant from cellular growth showing calcite and vaterite with no carotenoid presence, and d: corresponding image of crystals in final frame of time lapse experiment. All scale bars are 20 pm.

Fig. 4A-C: Calcite precipitation acts as an anchoring mechanism against oxygen bubble- induced cell washout, a: Time-lapse images showing cells being washed away by an oxygen bubble in non-calcium carbonate precipitating conditions. b,c: Time lapse images showing a bubble passing over cells in calcium carbonate precipitating conditions All scale bars are 20 pm.

Fig 5A-F: a,b Raman scans of aragonite and calcite control, c-d: Raman scans of targeted crystals from live growth microscopy experiment, f: Raman scans from distal region of same growth pad used in live growth microscopy experiment shown in c-d, but with no cells within view.

Fig 6A-I: a: Final time point of crystal tracking mask used to determine crystal area over time, d-i: Plots showing the mean chlorophyll fluorescence surrounding crystals over time for the x micron area surrounding the crystal, used as a proxy for cell density/growth.

DETAILED DESCRIPTION OF INVENTION

Organisms across all kingdoms of life have been shown to influence minerals, including calcium carbonate, as part of their growth. In eukaryotes, organisms such as shellfish utilize calcium carbonate present in the aquatic environment to form vital structures. Microbiota, such as fungi, archaea and bacteria, have also been shown to cause the precipitation of calcium carbonate through different mechanisms, collectively known as microbiologically induced calcium carbonate precipitation (MICP). Bacteria in particular have been shown to cause MICP through a range of metabolic processes, including urea hydrolysis, denitrification, methane oxidation, sulfate reduction, iron reduction, degradation of calcium oxalate, degradation of amino acids, and photosynthesis. Photosynthetic cyanobacteria are key players which affect the precipitation of calcium carbonate in the ocean and other aquatic environments. Fossilized cyanobacteria have been found in microbial mats preserved in siliciclastic rocks and in silicified stromatolites. This record points to the role of cyanobacteria influencing calcite precipitation. However, the exact mechanism of MICP caused by cyanobacteria is still unknown.

Previous studies have proposed different theories for MICP caused by cyanobacteria. In aquatic environments, the components of calcium carbonate exist in equilibrium in between salt and ionic forms (i.e., Ca ²⁺ + 2HCO ₂ CaCO ₂ + CO ₂ + H ₂ O). Cyanobacteria sequester carbon dioxide, needed to drive the photosynthetic process, within their cells through the activity of the rubisco enzyme. This activity increases the pH of the surrounding medium through the release of OH’ ions which then drive the equilibrium towards the precipitation of calcium carbonate. However, other authors have shown that calcium carbonate precipitation occurs even in the absence or inhibition of photosynthesis and theorize instead that cell-surface proteins are responsible for crystal nucleation. Thus, there is still considerable debate about how cyanobacteria cause MICP.

Better understanding of MICP processes in cyanobacteria is important to produce green concrete alternatives. Concrete is formed by mixing cement with aggregates, such as sand and gravel. Cement typically consists of calciferous silicates, which form crystals when dried, giving the concrete its hardness and chemical stability to withstand environmental conditions. Portland cement is the most common type of cement used in construction around the world. However, the creation of Portland cement requires baking limestone and other minerals in a kiln and is a major source of global CO2 emission. Some estimates suggest that cement production contributes -5-8% of the total CO2 emissions and is a major contributor of global warming. As an alternative, MICP is currently being developed to generate biocement.

In contrast to typical cement, biocement manufacturing uses MICP-causing bacteria to precipitate calcium carbonate. Doing so eliminates the need for baking limestone, thereby reducing carbon emissions. Current biocement manufacturing typically utilizes bacteria such as Sporosarcina pasteurii, which cause MICP through urea hydrolysis. In this process, fixed nitrogen in the form of urea is supplied to the bacteria. The urea is hydrolyzed by the cells to form ammonia and carbonic acid, which results in a pH increase and bicarbonate concentration increase, eventually resulting in calcium carbonate precipitation. However, use of these bacteria have two significant drawbacks: (1) the fixation of nitrogen, typically through the Haber-Bosch process, is also a major contributor to global warming, with estimates suggesting that the industry consumes up to 1% of the global energy production and accounts for -1% of the global annual CO2 emissions as of 2010, and (2) the by-products of urea hydrolysis releases ammonia and ammonium pollutants into the environment which can adversely affect human and environmental health.

In contrast, Anabaena, a species of filamentous cyanobacteria, is capable of MICP without the need for added urea, reducing both energy requirements and carbon emissions. Additionally, some proportion of cells within Anabaena filaments differentiate from photosynthetic (so-called vegetative) cells into specialized cells called heterocysts. Heterocysts are responsible for fixing nitrogen, which is then transported to the vegetative cells for growth. Thus, using this species to generate biocement would result in net carbon and nitrogen fixation. It is therefore essential to gain a better understanding of how MICP is caused by this species in order to optimize production yields.

Here, Applicants utilized time-lapse and multispectral microscopy to film Anabaena sp. ATCC 33047 (hereafter Anabaena) growing under environmental conditions favoring MICP. This species is a non-model organism and difficult to perform genetic experiments with, rendering typical biochemical assays challenging. Applicants results show growth behaviors involved with heterocyst differentiation and allow for observation of calcium carbonate precipitation mechanisms with a resolution as to the impact of cell types. From this, Applicants observed two distinct phenomena which suggest individual mechanisms of calcium carbonate precipitation caused by vegetative cells and heterocysts. These results are the first to identify these separate mechanisms as the cellular heterogeneity in Anabaena makes it difficult to study in traditional biochemical assays, where both cell types are averaged into a single population.

The present inventors have demonstrated that cyanobacterium is able to precipitate calcium carbonate in the form of calcite under nitrogen-fixing conditions where external nitrogen sources (besides atmospheric N2) are not needed. In a preferred embodiment, this allows for low resource production of biocalcite for building materials, carbon sequestration, and other industrial uses. For example, traditional Portland cement based concrete releases a large amount of CO2 into the environment by burning limestone (calcium carbonate). To the contrary, the biotic directed calcium carbonate crystallization processes described herein instead can sequester CO2 into biomass and minerals for renewable building and carbon storing materials

As described herein, using quantitative long-term time lapse microscopy, the present inventors provide visual evidence that nitrogen fixation by diazotrophic cyanobacteria could play a major and yet underappreciated role in MICP. In one aspect, the present inventors sought to confirm that nitrogen fixation through nitrogenase will result in calcium carbonate crystallization, and that this crystallization will be specific to nitrogen fixing conditions. In another embodiment, the present inventors demonstrated that biotic directed formation of calcium carbonate is in the form of calcite, whereas abiotic-directed calcium carbonate formation contains a mixture of calcite and vaterite, for example. In another embodiment, the present inventors sought to confirm that precipitation calcium carbonate will occur without the presence of urea/urease activity, and that the crystals resulting from nitrogenase activity are discernable from crystals precipitated through abiotic means.

As generally shown in FIG. 13, calcite precipitation occurred through multiple distinct mechanisms, specifically through: 1) heterocyst contact with calcium carbonate resulting in crystal growth; and 2) vegetative cell lysis/leakage resulting in calcite nucleation and growth. In one embodiment, the invention include the use of endogenous or heterologously expressed nitrogenase in bacteria, diazotrophic cyanobacteria such as a calcite precipitator, and may further be used to reduce nitrogen feeds in certain industrial applications. In another embodiment, biologically- directed calcite crystallization by diazotrophic cyanobacteria can be applied to sequester carbon, and in particular atmospheric CO2.

As noted above, diazotrophic cyanobacteria has the ability to induce a cement material that can fuse loose aggregate, such as grains of sand. Other aggregates such as glass beads, recycled glass foam, fly ash composite, soil, small stones, basalt, fibers, and mixtures of the above may also be used. Ideally, local aggregate would be used from, distilled directly from the location where the units are to be manufactured. If the pieces of aggregate, such as sand, are fused in a formwork or deposited in layers and treated in accordance with the teachings of this invention, construction materials, which are preferably masonry units such as brick, blocks, or any size and shape of a structural component may be manufactured and, as desired, easily mass produced. The teachings of this invention could further be used to produce pre-cast elements such as panels, columns, tiles, counter-tops, and/or any other construction unit commonly produced using sand, gravel, asphalt, clay, brick, concrete, and/or stone, any of which may be recycled material. A hardened material is formed in a process known as MICP as generally described herein.

As such, the present invention is further directed to systems, methods, and compositions for the production of biocement. In one preferred embodiment, the biocement of the invention can include a quantity of diazotrophic cyanobacteria in a growth media under conditions that induce MICP, and a quantity of aggregate and water. The conditions that induce MICP can include one or more of the following: 1) conditions that apply mechanical stress on the diazotrophic cyanobacteria resulting in calcite crystal growth; conditions that contact diazotrophic cyanobacteria with calcium carbonate resulting in calcite crystal growth; 3) comprises conditions that contact a heterocyst of the diazotrophic cyanobacteria with calcium carbonate resulting in calcite crystal growth; 4 ) nitrogen fixing conditions where nitrogen, apart from atmospheric nitrogen is necessary for bacterial growth. In one preferred embodiment, the diazotrophic cyanobacteria of the invention can include lysed diazotrophic cyanobacteria, and preferably mechanically lysed diazotrophic cyanobacteria, that can be contacted with calcium carbonate resulting in calcite nucleation and crystallization.

As used herein, “cyanobacteria” and “blue-green algae” refer to a group of photosynthetic bacteria widely distributed throughout aquatic environments, including freshwater and marine (saltwater) environments, and in some soils. Cyanobacteria may occur as single cells, thread-like filaments, or as colonies of various sizes and shapes composed of groups of many filaments or cells. In one embodiment, the diazotrophic cyanobacteria of the invention includes Anabaena sp. ATCC 33047. As used herein, “Anabaena,” describes a genus of nitrogen-fixing blue-green algae with beadlike or barrel -like cells and interspersed enlarged spores (heterocysts), found as plankton in shallow water and on moist soil.

Compositions of the invention may contain nutrient media to maintain and/or allow the cells to flourish and proliferate and further allow the cells to be subject to conditions that induce MICP or allow MICP to be induced. The various types of nutrient media for cells, and in particular, cyanobacteria cells of the invention are known and commercially available and include at least minimal media (or transport media) typically used for transport to maintain viability without propagation, and yeast extract, molasses, and com steep liquor, typically used for growth and propagation.

The biocement of the invention can include a quantity of aggregate combined with the diazotrophic cyanobacteria in a growth media under conditions that induce MICP. In a preferred embodiment, the aggregate of the invention can be selected from sand, gravel, glass beads, recycled glass foam, fly ash composite, soil, small stones, basalt, fibers and mixtures thereof. Moreover, the biocement of the invention can be formed in a frame, also generally referred to as a form. In this embodiment, a composition containing diazotrophic cyanobacteria can be combined with aggregate and water in a frame forming a construction material. Embodiments of invention utilizes MICP, and methods are defined to fabricate full-scale construction materials, including load bearing masonry which may be pre-cast. The benefits of a construction material that can be “grown” go beyond issues of economy and sustainability. As this is a material made by aggregation, additional materials can be added to the composite for additional performance traits, such as fibers for additional strength, Titanium Dioxide [TiCh] for pollution absorption, glass beads for the transmission of light, and/or air-entrained aggregates for insulation. MICP materials mimic the properties of natural sandstone and are composed of similar crystalline formations.

As used herein, the term “frame” includes frames, forms, molds, and other apparatus which may be used to hold loose pieces of aggregate together before the pieces are bonded in accordance with the teachings of this invention, and which can more preferably generate a construction material. In a preferred embodiment, material remains in the mold and cures for seconds to minutes to hours or days, depending upon the amount and type of material, the temperature, pressure, humidity, environmental conditions, and the amount of diazotrophic cyanobacteria, and the conditions that induce MICP. Preferably curing times are on the order of seconds, minutes, hours or days for larger building materials. Alternatively, partially cured material may be transferred to an atmospheric chamber for further curing under defined conditions. As further used herein, a construction material of the invention can be selected from: concrete masonry units, cinder blocks, bricks, foundation blocks, breeze blocks, hollow blocks, solid blocks, besser blocks, clinker blocks, high or low density blocks, aerated blocks, tile or pre-cast veneers. Also as used herein, the term “construction material” includes construction material, which is porous and non-porous, and can further be formed from being pressed into molds, or from being extruded among other methods generally known in the construction arts.

As used herein the term “construction material” means a composite material formed from cement and a particulate starting material as defined below (e.g., an aggregate). The cement is formed of cyanobacteria generated precipitated CaCCh (calcite) crystals, which upon precipitation from solution bind the particles of the particulate starting material together to form a hardened material. The construction material is thus the consolidated product of the method. The construction material may be, or may be like, mortar or concrete. The construction material may thus be used, for instance, in masonry, to bind together stones or bricks or suchlike. Alternatively, the construction material may be used in (or may be produced as) concrete-like masonry units, blocks, bricks, slabs etc. Tn certain embodiments, the construction material may be used in ground stabilization or in fundaments. For instance, the construction material may be used in the foundations of a building or suchlike. The construction material may be used for any purpose for which a cement-containing composite material is commonly used. It will be seen, therefore, that the construction material may be a solid product, or component, and may be provided in unit form (in the sense of a unit being an individual item of a masonry or construction component), e.g., a brick, block, panel, tile, column, pillar, counter-top, pre-cast veneer or any pre-cast element used in building or manufacture etc. It can thus be any shaped solid construction component. Methods of shaping or forming construction material into such a solid or shaped form are well known in the art, and any of these could be used, for example forming the construction material in a mold, compression, drying etc.

The particulate starting material used will determine many of the properties of the construction material, and thus may be chosen based on the desired properties of the construction material. For instance, the use of an aggregate consisting of a fine material, such as sand, may yield a construction material with properties similar to mortar or low-strength concrete; use of a coarser aggregate, such as gravel, may yield a construction material with properties similar to a stronger concrete. If necessary, the construction material may be reinforced, for instance using rebar or other reinforcements such as natural (e.g., cellulose) or synthetic fibers (e.g., glass fibers, rock wool, synthetic polymer fibers, etc.).

Thus, depending on the desired characteristics of the construction material, any suitable particulate material may be used as the particulate starting material, and in particular any aggregate material (namely an aggregate material used in the construction industry, for instance soil, sand, sawdust, asphalt, a rock aggregate such as crushed stone, gravel or slag, or sintered clay or shale). The material may be natural or synthetic. Recycled materials may be used, e.g., recycled building materials, including e.g., recycled concrete.

As further used herein “cement” is a preparation which may be used to bind materials together, including notably particulate starting materials to prepare construction materials. Cement may be used to prepare a concrete-like material. However, it may also be used to bind together already-prepared construction materials (e.g., in unit form, such as blocks or bricks). In one embodiment cement may be used to prepare a mortar to bind construction materials together. As used herein, “biocemenf ’ means cement that is produced by the biotic-directed calcite precipitation methods and compositions as described generally herein, which can further be used to create a construction material.

Another embodiment of the invention is directed to compositions of the invention that are useful in 3D printing. Compositions can be combined in a controlled fashion to be continuously layered forming a structure as determined by the 3D replication software. 3D printers are commercially available and can be modified by one skilled in the art to utilize compositions of the invention. For example, a quantity biocement containing a quantity of diazotrophic cyanobacteria in a growth media under conditions that induce microbially induced calcite precipitation (MICP) with a quantity of aggregate and water can be applied to a 3D printer to produce various construction materials, and in particular shaped construction materials.

As used herein, the singular terms “a,” “an,” and “the” include the plural reference unless the context clearly indicates otherwise. The term “or” as used herein, refers to a non-exclusive or, unless otherwise indicated.

The invention now being generally described will be more readily understood by reference to the following examples, which are included merely for the purposes of illustration of certain embodiments of the embodiments of the present invention. The examples are not intended to limit the invention, as one of skill in the art would recognize from the above teachings and the following examples that other techniques and methods can satisfy the claims and can be employed without departing from the scope of the claimed invention. Indeed, while this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

EXAMPLES

Example 1 : Overview and Experimental Summary,

Single cell analysis of live growth microscopy has illuminated behaviors not before seen in filamentous, diazotrophic cyanobacteria. This approach to studying the organism Anabaena 33047 (Anabaena ) has resulted in the characterization of two calcium carbonate mechanisms in growth conditions requiring no fixed nitrogen. These findings were made possible by the novel single cell analysis and live growth correlated microscopy of Anabaena. Notably, these methods allowed for the observation of single-cell and single-crystal growth kinetics and unveiling the spatial interactions between cells and crystals for the first time, allowing for the relationship between cell growth and crystal formation to be understood.

Calcite precipitation has been shown to result from the growth of Anabaena. Specifically, crystal nucleation and growth occur because of vegetative cell lysis or leakage, and crystal growth occurs due to heterocyst contact with crystal seeds. Both are likely resulting from metabolic processes occurring within the relevant cells.

Vegetative cells concentrate bicarbonate as a part of carbon fixation. Additionally, cells tend to maintain a consistent internal calcium concentration compared to their environment. The rapid leakage of these components into the environment surrounding the cell could result in an increase in partial pressure sufficient to cause precipitation, resulting in crystal nucleation and growth.

Multiple potential evolutionary benefits of calcite precipitation have been proposed, including acting as a water buffering system that can be controlled by precipitation and dissolution. Links between calcite and diazotrophs are numerous, including calcite dissolving organisms which are vital for maintaining rice paddies homeostasis. Based on the evidence provided, Applicants believe the most promising evolutionary benefit of calcium carbonate precipitation by Anabaena is as an anchoring mechanism. This mechanism relies on the presence of calcium carbonate in locations that the marine cyanobacteria can encounter, and which would provide an optimal environment for growth. Calcium carbonate has been found to be an important contributor in coastal waters and resulting in sand or gravel deposits on beaches. These environments would provide a suitable environment with sufficient light and nutrient exchange to sustain the growth of an organism like Anabaena, so anchoring could provide an alternative means of maintaining optimal conditions, as compared to an approach such as motility in Geitlerinema.

Further supporting this hypothesis is the identification of Anabaena in intertidal zone sediments. Specifically, it has been identified with oncolites, which form from biogenic precipitation of calcium carbonate by cyanobacterial films. These could arise as a result of the continued precipitation inside of a cyanobacterial mat, potentially arising from the mechanisms described in this manuscript. Calcium carbonate precipitation has been previously observed within cyanobacterial mats to support this idea.

These observations support the idea of a contact mediated calcium carbonate precipitation method as serving as a mechanism of anchoring an organism either to costal calcium carbonate sands or cyanobacterial films and could offer significant evolutionary benefits to an organism that can biocement themselves to an existing crystal when metabolically active. The filamentous nature of Anabaena would mean that while a crystallization event most likely results in the death of the cell responsible, and likely some of those around it, this could still be overall beneficial for anchoring the rest of the filament and offering an advantage to the organism as a whole.

The evolutionary potential of calcium carbonate is varied and still under investigation. The present invention offers new insight into potential benefits to ana33047 and filamentous calcium carbonate precipitators in general. In the field of calcium carbonate precipitation, current focuses trend towards understanding the potential of calcium carbonate mechanisms as a method for cementation due to its massive potential environmental impact.

Calcite precipitation offers a carbon sequestration opportunity through the replacement of cement. Cement is a globally expensive product, both energetically and environmentally. Finding a mechanism to utilize this process without the requirements of fixed nitrogen would further push the reaction in an energy neutral direction, eliminating the need for fixed nitrogen for growth and thereby reducing the reliance on the Haber Bosch process. Both cement production and the Haber Bosch processes are leading impacts on global warming. In certain embodiments, the mechanisms described in the present invention can form the basis of an environmentally friendly alternative to Portland cement, one that can be sustained with light as its exclusive energy source.

Example 2: Time-lapse fluorescence microscopy enables individual cells to be tracked.

To demonstrate the feasibility of the present invention, Anabaena was grown in BG11 media without supplemental nitrogen (denoted BG11-N) and filmed under typical laboratory culturing conditions. Cells were grown under a soft agar pad, which constrains cell growth in a 2- dimensional plane. Figure la-c shows representative frames of an Anabaena filament. Brightfield and chlorophyll fluorescence images were recorded (see Methods). As the filament grows, some cells differentiate into heterocysts, which can be identified both by size and the loss of chlorophyll fluorescence over time (indicated by arrows).

To observe calcium carbonate crystal growth, 25 mM of calcium chloride and sodium bicarbonate was added to the BG11-N media (denoted BG11-N+Ca). Frames from a representative movie are shown in Figure Id-g. Applicants utilized computational software to track individual crystal growth, as shown in Figure Ih-j. As shown in Figure Ij, Applicants data showed that all initial seed crystals grew when the sample was placed on the microscope. This was likely due to the temperature change when the room temperature pad was placed in the 37 °C growth chamber. Similar growth was observed in experiment replicates. In these experiments, Applicants did not observe global crystal growth beyond the first two hours. Since the cells continued to grow during this time, Applicants data suggests that the hypothesis that photosynthesis causes crystal growth was incorrect, at least under these conditions. After this initial growth, the crystal area stayed constant for approximately 20 hours. After this time, some crystals showed growth upon interaction with cyanobacterial cells. Applicants identified two specific interactions which caused crystal growth. These interactions are discussed in the following sections.

Example 3: Cyanobacterial cell lysis or leakage results in crystal growth.

From these data, Applicants were able to identify two mechanisms that cause crystal growth due to cell interactions. In this section, Applicants show evidence that calcium carbonate crystal precipitation results from the release of cellular contents which occurs due to mechanical stress. Applicants observed two distinct conditions in which this release occurs.

First, cyanobacteria cells can lyse if mechanically trapped against a hard object. In Figure 2a, Applicants observed an instance in which a vegetative cell grows adjacent to a seed crystal. As the cell grows, one of its progenies becomes confined by its siblings and eventually lyses, likely due to contact against the rough surface of the crystal. This causes crystal precipitation approximately 60 minutes later. In a second instance, shown in Figure 2c, a filament is stressed at a bend. A new crystal nucleates at the bend and grows at the cellular junction.

Applicants proposed model for these precipitation events is that mechanical confinement or stress causes cells to leak or release their contents into the surrounding media. Since cyanobacteria cells sequester carbon primarily as carbonate ions, the release of these causes the crystal to grow. Shortly before cell lysis and crystal growth, an increase in chlorophyll fluorescence is observed, which is consistent with Applicants previous observations of mechanical stress on cyanobacteria. Similarly, as Anabaena cells are joined internally within a filament, mechanical stress at the bend likely allowed cell material to leak out.

Interestingly, Applicants found that precipitation was induced by these mechanical interactions and is not caused simply by proximity to growing vegetative cells. Figure 2d shows examples of vegetative cells growing close to or against a seed crystal. The crystal does not increase in size despite ~26 hours of cellular growth. This result contrasts with previously reported observations which suggested that cyanobacteria cause MICP due to photosynthetic processes alone.

Example 4: Heterocyst contact results in calcium carbonate crystal growth.

Applicants also observed that crystal growth occurred upon contact with a heterocyst cell. A representative example is shown in Figure 2f. Here, a vegetative cell differentiates into a heterocyst, which then physically contacts a seed crystal. After this contact, the crystal grows rapidly. As shown previously in Figure 2d, this crystal growth does not occur through contact with a vegetative cell alone. This observation of heterocyst contact MICP has not been previously observed. Applicants theorize that MICP occurs due to nitrogen-fixing reactions by the heterocysts. These cells rapidly catalyze the fixation of nitrogen gas into ammonia. This reaction results in major consumption of protons, (A ₂ + 16ATP + 8e“ + 8H ⁺ 2NH ₃ + H ₂ + 16ADP + 16Pj) followed by (2NH ₃ + 2H ⁺ 2NH ), or 10 protons are consumed per molecule of nitrogen gas fixed into two molecules of ammonium. This results in an alkalinity change in the environment inside a heterocyst, and likely a local area gradient of proton consumption at its surface. It is important to note here that these observations show that MICP only appears to occur when a heterocyst is in physical contact or in extremely close proximity with an existing crystal.

Example 5: Chemical composition of biotic and abiotic crystals.

To identify the polymorph form of the crystals, the same sample was imaged using a Raman-capable microscope. The sample were manually registered to identify the crystals tracked in the movie. An area Raman scan was then performed, as shown in Figure 3b, along with chlorophyll fluorescence from the final frame of the live growth movie shown in Figure 3a. The images are shown overlayed in Figure 3c to show overlap of crystal location from Raman microscope data with the end of live growth experiment.

Based on the Raman spectra, heterocyst is in physical contact or in extremely close proximity with an existing crystal found that the crystals consisted primarily of calcite. Interestingly, the crystals also showed traces of carotenoids (3e), indicating the presence of cell debris within. In comparison, abiotic crystals from a different region in the same sample were much smaller, and appeared to be composed of either calcite or vaterite (2d,f,g).

In combination with this, it was observed that in locations without cell growth (even on a different location of the same pad as other experiments with cell and crystal growth), there is no coordinated crystal growth observed, having only the early abiotic crystal growth previously observed and no further growth following. These observations strongly point towards local cell growth being necessary for crystal growth in the given calcium carbonate precipitating conditions. Example 6: Calcium carbonate crystals anchor cells during live microscopy experiments.

Here, Applicants show examples which suggest that MICP could play a role in anchoring cells. In microscopy datasets, Applicants sometimes observe that a gas bubble will grow across the field of view, particularly in conditions where cells have been growing for 12 or more hours. These gas bubbles grow because of oxygen release during photosynthesis. The bubbles push cells around in a movie and often wash them out of the field of view, shown in Figure 4a.

In Figure 4b and c, Applicants observe that the presence of calcium carbonate crystal seeds results in cells anchoring themselves against movement. When given the proper conditions for calcium carbonate crystal growth, even when no crystal seed is initially visible, the movement of the bubble is resisted by cells through nucleation and growth of calcium carbonate crystals. Example 7: Materials and Methods.

Strain and culturing: Anabaena sp. ATCC 33047 obtained from Dr. Himadri Pakrasi’s lab, grown at 37°C in BG11 media. All preculturing occurring in 25 mL liquid cultures with 100 rpm orbital shaking in 125 ml baffled flasks with foam stoppers. Liquid and agar cultures were grown in an AL-41 L4 Environmental Chamber (Percival Scientific, Perry, IA) at 37°C under continuous (24-hour) illumination at 150 pmol photons m ^-2 s ^-1 from cool white fluorescent lamps in air (0.04% CO2).

Time-lapse microscopy: All long-term observation images and videos were obtained using a Nikon TiE inverted wide-field microscope with Perfect Focus System, controlled using NIS Elements AR software (version 5.11.00; 64-bits) with Jobs package. Images were acquired using a digital sCMOS camera (Hamamatsu ORCA-Flash4.0 V2+) with a 20x air objective (Nikon CFI Plan Apochromat Lambda D). Temperature during cell growth in all images was maintained at 37 °C using an Okolab cage Incubator (Okolab). Growth light and trans-illuminating imaging light were supplied from a light-emitting diode (LED) light source (LIDA Light Engine, Lumencor, Beaverton, OR). Epifluorescence imaging light was supplied from a custom-filtered LED light source (Spectra X Light Engine, Lumencor, Beaverton, OR) and delivery was controlled using a synchronized hardware-triggered shutter. Cells for imaging were grown in liquid culture to -1.00 OD 730 nm. 25 mM CaCh (25 pL IM) was added to the sample side of imaging pad (1 mL) and allowed to fully dry/ dissolve. 3-5 x 2 pL drops of cells were added to the imaging side of the pad and allowed to dry. The pad was flipped into the imaging dish, and an additional 25 mM (25 pL IM) sodium bicarbonate was added to the top of the pad. The imaging dish (Ibidi p-dish 35mm glass) was immediately sealed with parafdm, and placed into the microscope environmental chamber at 37 °C. Cells were grown under 37 °C and 150 pmol photons m ^-2 s ^-1 red light at 640 nm; this light was turned on at all times except during fluorescence imaging.

All imaging performed on Nikon Plan Apo (wavelength) 20x. Brightfield illuminated by Lida atExW: 450, power 2.0, ExW: 550, power 2.0, ExW: 640, power 2.0. Cy5 excited at 640 nm, power 5.0. When present, CFP excited at 440nm, power 50.0. RFP excited at 555 nm, power 10.0. Time points either every 20 or 30 minutes.

Correlated live-cell and Raman microscopy: Live growth microscopy was performed as described in time-lapse microscopy. A stitched image was taken including the regions imaged during the live growth microscopy. The sample was then transferred to the Raman microscope. Raman images taken with lOx and lOOx objectives, and scanning performed on lOOx objective. Raman scans were performed using a 532 nm laser and scanning from 84 to 1786 wavenumbers shift. All Raman scans were performed the same day as live growth microscopy and samples were stored in the dark during transfer. Crystals on Raman were identified back to live growth microscopy using the stitched image and comparing the crystal fingerprint to provide crosslocalization.

Image analysis: Image segmentation was performed using a modified version of CyAN that has been further customized for filamentous cyanobacteria. Filament analysis and measurements performed using MATLAB scripts based on functions present within CyAN.

REFERENCES

1. Schrder, L., Boon, N., De Kock, T. & Cnudde, V. The capabilities of bacteria and archaea to alter natural building stones - A review. International Biodeterioration & Biodegradation 165, 105329 (2021).

2. Ismail, R. et al. Synthesis and Characterization of Calcium Carbonate Obtained from Green Mussel and Crab Shells as a Biomaterials Candidate. Materials 15, 5712 (2022).

3. Hammes, F., Boon, N., de Villiers, J., Verstraete, W. & Siciliano, S. D. Strain- Specific Ureolytic Microbial Calcium Carbonate Precipitation. Appl Environ Microbiol 69, 4901- 4909 (2003).

4. Er§an, Y. ., Belie, N. de & Boon, N. Microbially induced CaCO3 precipitation through denitrification: An optimization study in minimal nutrient environment. Biochemical Engineering Journal 101, 108-118 (2015).

5. Luff, R., Wallmann, K. & Aloisi, G. Numerical modeling of carbonate crust formation at cold vent sites: significance for fluid and methane budgets and chemosynthetic biological communities. Earth and Planetary Science Letters 221, 337-353 (2004).

6. Baumgartner, L. K. et al. Sulfate reducing bacteria in microbial mats: Changing paradigms, new discoveries. Sedimentary Geology 185, 131-145 (2006).

7. DeJong, J. T., Mortensen, B. M., Martinez, B. C. & Nelson, D. C. Bio-mediated soil improvement. Ecological Engineering 36, 197-210 (2010).

8. Braissant, O., Verrecchia, E. & Aragno, M. Is the contribution of bacteria to terrestrial carbon budget greatly underestimated? Naturwissenschaften 89, 366-370 (2002).

9. Rodriguez-Navarro, C., Rodriguez-Gallego, M., Ben Chekroun, K. & Gonzalez- Munoz, M. T. Conservation of Ornamental Stone by Myxococcus xanthus- Induced Carbonate Biomineralization. Appl Environ Microbiol 69, 2182-2193 (2003).

10. Dupraz, C. et al. Processes of carbonate precipitation in modem microbial mats. Earth-Science Reviews 96, 141-162 (2009).

11. Demoulin, C. F. et al. Cyanobacteria evolution: Insight from the fossil record. Free Radic Biol Med 140, 206-223 (2019).

12. Zhang, J.-Z. Cyanobacteria blooms induced precipitation of calcium carbonate and dissolution of silica in a subtropical lagoon, Florida Bay, USA. Sci Rep 13, 4071 (2023). 13. Riding, R. Cyanobacterial calcification, carbon dioxide concentrating mechanisms, and Proterozoic-Cambrian changes in atmospheric composition. Geobiology 4, 299-316 (2006).

14. Zhu, T. & Dittrich, M. Carbonate Precipitation through Microbial Activities in Natural Environment, and Their Potential in Biotechnology: A Review. Front Bioeng Biotechnol 4, 4 (2016).

15. Jiang, H.-B., Cheng, H.-M., Gao, K.-S. & Qiu, B.-S. Inactivation of Ca2+/H+ Exchanger in Synechocystis sp. Strain PCC 6803 Promotes Cyanobacterial Calcification by Upregulating CO2-Concentrating Mechanisms. Applied and Environmental Microbiology 79, 4048-4055 (2013).

16. Dittrich, M. & Sibler, S. Calcium carbonate precipitation by cyanobacterial polysaccharides. Geological Society, London, Special Publications 336, 51-63 (2010).

17. Liang, A., Paulo, C., Zhu, Y. & Dittrich, M. CaCO3 biomineralization on cyanobacterial surfaces: Insights from experiments with three Synechococcus strains. Colloids and Surfaces B: Biointerfaces 111, 600-608 (2013).

18. Options for the future of cement | Request PDF. https://www.researchgate.net/publication/286378556_Options_f or_the_future_of_cement.

19. Boden, T. A., Andres, R. J. & Marland, G. Global, Regional, and National Fossil- Fuel CO2 Emissions. (2017).

20. Omoregie, A. I. et al. Soil bio-cementation treatment strategies: state-of-the-art review. Cieotechnical Research 1-25 (2023) doi: 10.1680/jgere.22.00051.

21. Taking the CO2 out of NH3. C&EN Global Enterp 97, 18-21 (2019).

22. An Ecological Risk Assessment of Ammonia in the Aquatic Environment. https://www.tandfonline.com/doi/epdf/! 0.1080/71360992 l?needAccess=true doi: 10.1080/713609921.

23. Moore, K. A. et al. Mechanical regulation of photosynthesis in cyanobacteria. Nat Microbiol 5, 757-767 (2020).

24. Jehlicka, J. et al. Potential and limits of Raman spectroscopy for carotenoid detection in microorganisms: implications for astrobiology. Philos Trans A Math Phys Eng Sci 372, 20140199 (2014). 25. Gabrielli, C., Jaouhari, R , Joiret, S. & Maurin, G. In situ Raman spectroscopy applied to electrochemical scaling. Determination of the structure of vaterite. Journal of Raman Spectroscopy 31, 497-501 (2000).

26. Buzgar, N. & Apopei, A. I. The Raman Study of Certain Carbonates. Analele Stiintifice de Universitatii A.I. Cuza din Iasi. Sect. 2, Geologic 55, 97-112 (2009).

27. Torrecilla, I., Leganes, F., Bonilla, I. & Fernandez -Pinas, F. Use of Recombinant Aequorin to Study Calcium Homeostasis and Monitor Calcium Transients in Response to Heat and Cold Shock in Cy anobacteria 1. Plant Physiology 123, 161-176 (2000).

28. Goel S, Gautam & M, Kaushik, (second). Nitrogen fixation and protein profiles of halotol erant Nostoc muscorum-R strain isolated from rice fields and ARM-221 strain. Indian J Exp Biol 746-750 (1997).

29. Woodroffe, C., Farrell, J., Hall, F. & Harris, P. Calcium Carbonate Production and Contribution to Coastal Sediments. Faculty of Science, Medicine and Health - Papers: part A 149- 158 (2017) doi: 10.1017/9781108186148.010.

30. Anagnostidis, K. Geitlerinema, a new genus of oscillatorialean cyanophytes. Plant SystEvol 164, 33-46 (1989).

31. Alshuaibi, A., Duane, M. J. & Mahmoud, H. Microbial-Activated Sediment Traps Associated with Oncolite Formation Along a Peritidal Beach, Northern Arabian (Persian) Gulf, Kuwait. Geomicrobiology Journal 29, 679-696 (2012).

32. Kremer, B., Kazmierczak, J. & Stal, L. J. Calcium carbonate precipitation in cyanobacterial mats from sandy tidal flats of the North Sea. Geobiology 6, 46-56 (2008).

33. Tay, J. W. & Cameron, J. C. Computational and biochemical methods to measure the activity of carboxysomes and protein organelles in vivo. Methods Enzymol 683 , 81-100 (2023).