Biocementing Bacteria

This page outlines the development of engineered microorganisms capable of microbially induced calcite precipitation (MICP) through surface-displayed carbonic anhydrase. By combining enzyme optimization, chassis selection, and surface localization strategies, we aim to create bacteria that can convert atmospheric CO₂ into stable calcium carbonate for sustainable biocementation on Earth and Mars.

Microbially induced calcite precipitation

Metabolic pathways to MICP

Microbially induced calcite precipitation (MICP) is a process in which microorganisms facilitate formation of calcium carbonate [1]. This occurs when microbial activity alters the local chemical environment in a way that drives the precipitation of insoluble carbonate salts, particularly calcium carbonate, or calcite. This pathway is accelerated by microbial enzymes that catalyze otherwise slow chemical equilibria. The two most widely studied pathways are urea hydrolysis and the carbonic anhydrase pathway

Urea hydrolysis by urease

Some bacteria express urease, which hydrolyzes urea into ammonia and carbon dioxide. This process increases the pH of the environment, while carbonate is simultaneously converted into bicarbonate and carbonate. This creates the condition for calcium carbonate crystals to form once calcium is present. However as mentioned, this pathway releases ammonia as a byproduct. While effective in driving calcite precipitation, excess ammonia can lead to environmental concerns such as nutrient imbalance or potential toxicity Yu et al., 2021.

Carbon dioxide hydration by carbonic anhydrase

The carbonic anhydrase pathway utilizes a different microbial strategy. Here, the microbes express the enzyme carbonic anhydrase (CA) catalyzes the reaction of carbon dioxide into bicarbonate. This happens under normal conditions within water, but CA accelerates the reaction. The bicarbonate produced can then deprotonate to form carbonate, which combines with calcium ions to precipitate calcium carbonate. Unlike urea hydrolysis, this pathway avoids the release of ammonia, directly utilizing carbon dioxide as a carbon source. This makes this approach more attractive for carbon capture and sequestration, where excess carbon dioxide can be mineralized into a stable, solid form. Although less established than urea hydrolysis, CA precipitation is emerging as a more sustainable and environmentally friendly approach.

S-layer Display

How does surface display enable enhanced MICP activity?

Surface display is a mechanism employed by many prokaryotic organisms to localize proteins to the cell surface. By fusing carbonic anhydrase to an organism’s native surface layer anchoring protein, our enzyme of interest can be exported and presented on the cell’s surface. This enhances MICP activity by increasing substrate accessibility.

Surface display systems

Surface layer (S-layer) proteins are typically anchored to the outer membrane through their N-terminal domains while their C-terminal domains serve as signal sequences for secretion. In S. elongatus UTEX 2973, the VCBS S-layer protein facilitates export using a type I secretion system [2]. C. crescentus CB2A expresses the rsaA S-layer protein, which also utilizes type I secretion for export [herdmanCellCycleDependent2024a?]. Although E. coli does not have an S-layer, surface display can be achieved by introducing the ice nucleation protein (INP), which anchors to the outer membrane [3].

Proof of Concept

Core CA candidates

⁠We began identifying the key characteristics of an ideal carbonic anhydrase (CA) for biocementation through extensive literature review. Studies showed that surface-expressed CA outperforms intracellularly expressed CA in promoting CaCO₃ formation [4], as the enzyme can directly interact with environmental CO₂ and Ca²⁺ ions. Ideal CAs should also be thermostable, active across a broad pH range, and resistant to denaturation, which are traits crucial for performance in variable Martian or terrestrial conditions.

From previous works, we noted that SazCA (from Sulfurihydrogenibium azorense) is regarded as the benchmark CA due to its exceptional catalytic efficiency (kcat/Km ≈ 4.4 × 10⁶ M⁻¹s⁻¹) and thermostability (active up to 100 °C) ([5] ). Other bacterial CAs from Brucella and Helicobacter species were also reported to maintain robust activity under physiological or mildly alkaline conditions suitable for microbially induced calcium carbonate precipitation (MICP).

After shortlisting candidate enzymes based on literature data (temperature stability, pH tolerance, catalytic efficiency), we fed these sequences into our Dry Lab’s bioinformatics pipeline. This computational workflow performed:

• Phylogenetic analysis across diverse CA families to identify evolutionary robustness and potential functional similarities.
• Structural prediction (AlphaFold-based) to visualize and model the enzymes when fused to surface display scaffolds (e.g., VCBS).
• RMSD calculations to evaluate conformational changes in fusion proteins, lower RMSD indicates less distortion and potentially higher retained activity.
• Molecular dynamics simulations to predict enzyme stability and pH-dependent activity across varying environmental conditions. This allowed us not to just test the enzyme in isolation, but as it would realistically appear on the cell surface as part of a larger display complex fusion. This approach helped us prioritize CAs that would remain active when surface-displayed and accessible for carbonate precipitation.

Chassis selection and cloning strategies

Space Application

For use on Mars, we require an anaerobe that can utilize the abundant CO2 in the Martian atmosphere as a carbon source to propagate and precipitate calcite. Cyanobacteria fit the bill perfectly. When evaluating different strains for our project, our advisors Nannaphat (Patrik) Sukkasam and Kalen Dofher recommended model organisms for ease of engineering over extremophiles due to lack of developed tools and methodology around them.

With this in mind, we chose Synechococcus elongatus UTEX 2973, hereafter UTEX, as our cyanobacteria chassis. It is attractive because it is one of the fastest-growing cyanobacteria to have been discovered [6]. In an environment with very little resources, having an organism that can readily accumulate biomass and turn CO2 into useful substances is critical. Particularly relevant is that UTEX posseses an S-layer, and a recent publication was able to achieve S-layer display in this strain[2]. There are sufficiently developed toolkits, part collections, and experience around synthetic biology of UTEX and its close relative, S. elongatus PCC 7942, which made engineering this strain more approachable.

We sought to engineer synthetic constructs into the genome of UTEX via homologous recombination. This technique is well-established and preferred for engineering cyanobacteria [7]. Integrating into the genome also has the benefit of far greater expression stability compared to using shuttle vectors, especially without selection pressure, which is highly favourable for extraterrestrial use. We designed our S-layer display construct around this, and designed a suicide vector to integrate this and a kanamycin resistance cassette to the genome of UTEX.

Earth Application

Caulobacter crescentus CB2A, with a naturally occurring S-layer, is an ideal candidate for surface display. The S-layer of Caulobacter is well-characterized and has an established platform for displaying heterologous proteins [8], [9], [10]. In addition, due to the nature that it is a non-pathogenic and environmentally robust organism, CB2A is safe and practical for earth applications.

To achieve successful surface display application, a shuttle vector that replicates in both E. coli (for cloning/propagation) and C. crescentus (for expression) is used. The shuttle vector carries a gene of interest fused to the S-layer protein gene to ensure display of the engineered protein at the cell surface. This system allows easy manipulation in a standard host (E. coli) before transferring the construct into CB2A.

Benchmark

For a point of reference for our biocementing bacteria, we sought to display CAs in E. coli BL21(DE3) using the ice nucleation protein fragment. It was shown in E. coli that surface displaying CA is advantageous for activity over intracellular expression.

For all chassis, we employed multi-level Golden Gate Assembly and Gibson Assembly to generate synthetic constructs, propagated them in E. coli, and validated them via PCR and restriction digest. E. coli may be chemically transformed, but CB2A and UTEX both need to be transformed via electroporation. After successful antibiotic selection post transformation, we perform colony PCR to verify insertion as a final check before passing it onto functional validation.

See , , and for more information.

Expression of surface-displayed CA

A crucial factor driving CA catalytic activity is its access to the carbon dioxide. Anchoring the enzyme to the cell surface will expose it directly to atmospheric carbon, thereby reducing substance transfer limitation [11] and improving reaction speed. While a secreted, soluble form of the enzyme could achieve the same effect, display systems enhance enzyme stability and don’t require enzyme purification, thus simplifying the downstream process. To demonstrate that displayed CAs enables a faster rate of calcite precipitation, we will compare our display strains to an equivalent secretion strain where the secreted enzyme is not immobilized to the cell membrane. Additionally, we’ll engineer intracellular expression strains that retain the enzyme inside the cell. This third expression system acts as another control to compare against surface display.

Validation of CA and MICP activity

We developed a number of assays to verify surface display, quantify whole-cell CA activity, and test calcite precipitation. These assays build on one another to progressively link genotype to functional phenotype.

We can employ cell fractionation to extract the S-layer and other outer membrane proteins, and perform a Western Blot to confirm expression of our engineered S-layer proteins. All our fusion proteins feature a Myc tag for this purpose. Next, we employ a trypsin accessibility assay to ascertain whether the expressed fusion proteins are in fact immobilized to the cell membrane. After treating cells with or without trypsin, the surface protein fraction is once again recovered, and treated cells should be missing the expected Slp-CA fusion protein band in an SDS-PAGE gel.

The functional activity of biocementing bacteria can be ascertained using colorimetric assays - one to measure enzymatic activity using the esterase activity of carbonic anhydrases, the other to measure calcium depletion to assess precipitation. The concentration of soluble calcium from the supernatant will be measured using the o-Cresolphthalein complexone (o-CPC) assay, with a drop in calcium and a rise in pH indicating mineral precipitation. To confirm MICP and rule out CaCO₃ formation from free enzyme or calcium, a negative control will be included. To also determine whether surface displaying enzymes is beneficial for overall biocementing ability, we use cells intracellularly expressing CA as positive controls.

Verifying crystal morphology with an SEM analysis should show that whole-cell cultures with surface-displayed CAs produced smaller, well-formed crystals, whereas cell-free extracts with free CAs generate irregular crystals.

See for more information.