Our project design process started off aiming to solve local problems through a lens of sustainability. One pressing Australian problem is the demand for housing far exceeding the supply, with there being only 400 dwellings per 1000 people according to a Gratton report. The Australian Government has set a target of building 1.2 million homes in the next five years and for that and future needs, there is a need to significantly ramp up the production capacity. But it is pivotal to balance out the environmental concerns with the goal of human health and prosperity which at times contradict each other. The more environmentally conscious options are more expensive in the short term, but people often fail to consider the long-term costs.

Wood is touted to be a sustainable building material but is met with problems of scaling ability and fire safety. One way to tackle this problem without sacrificing growth is to make the current standard materials more sustainable. Multiple sources like Greenhouse Gas Control Technologies - 6th International Conference , Renewable and Sustainable Energy Reviews and Sixth Assessment Report of the Intergovernmental Panel on Climate Change identify that the most damaging aspect of the traditional construction industry is the Green House Gas (GHGs) emitted through the production of cement, which amounts for 5-8% to nearly 10% of global GHG emissions and this has reinforced our belief of needing to tackle this problem. The major element of GHG contribution within cement production is the need to heat up limestone, through the process of calcination, to produce clinker and the CO₂ released during this process. To counteract this environmental cost, we take inspiration from nature.

Humans have been using limestone for thousands of years in the process of building culminating into a scalable industrial process through the invention of Portland cement in 1824. But we have only more recently understood the biological mechanisms behind calcium carbonate precipitation through the understanding gained from Microbially Induced Calcium Carbonate Precipitation (MICPs) which is utilized by corals and stromatolites. These processes happen at ambient temperatures, solving for the problem of the need for artificially high temperatures and the carbon released to get to those temperatures. We also avoid the CO₂ liberated from the limestone which amounts to 55% of the emissions. Going along the path of MICPs, one drawback of the urealytic hydrolysis pathway is the toxic by-product ammonia, which causes a rise in environmental eutrophication levels, risking the proliferation of algal blooms. For this reason, we incorporated a nitrification/denitrification pathway to break down the ammonia to nitrogen gas.

Our project design was developed with certain environmental, social, moral, and scientific values at mind. The global housing crisis is a social concern that requires maintaining the basic human need for shelter that is in odds with our moral obligation to avert the negative effects of anthropogenic climate change. There is a need to strike a balance between these two values as the current cement industry contributes to nearly 10% of the global GHG emissions. To reach this optimum environmental balance, we employed the scientific values of curiosity, diligent observation, creativity, and collaboration in problem solving to iterate through the design-build-test-learn cycle to arrive at a synthetic biology solution which achieves cement production at much lower energy cost and GHG emissions.

Our project identifies that the reliance on ordinary Portland cement (OPC), the ubiquitous constituent of concrete used by the global construction industry, amounting for around 4.2 billion tonnes on average in the last five years. This is significantly contributing to worldwide GHG emissions, as for every unit weight of OPC produced, an equivalent amount of GHGs is emitted.

In light of this, substituting OPC with MICP solves for the GHG emissions during the production process. In the traditional cement industry, limestone needs to be broken down/calcinated to Calcium oxide (CaO) to go ahead with reaching calcium carbonate. This top-down process of breakdown and incorporation with other materials like gravel to make up concrete results in GHGs in two ways; the cumulative CO₂ liberated along the production process and CO₂ emitted upstream to generate energy for heating up of limestone during calcination, which combine to contribute to about 90% of the GHG emissions.

Our solution is more of a bottom-up process wherein through the enzymatic action of urease breaking down urea to ammonia and CO₂ and carbonic anhydrase using CO₂ and H2O alongside calcium ions sourced from calcium chloride to precipitate out calcium carbonate (CaCO₃). This precipitation accumulates over the bacterial nucleation sites, forming mesocrystals as they latch onto the desired substrate and the mesocrystals combine to follow through with cementation. This eliminates GHG emissions during and upstream of the traditional cement production as MICP happens at ambient temperatures in contrast.

But in the process of solving for GHG emissions from OPC, we realised the GHG burden, albeit a relatively smaller burden, is now being shifted to the upstream carbon costs levied through the production of urea and more importantly calcium chloride of lab-grade purity, as well as other environmental damage.

That environmental damage comes in the form of eutrophication caused by the ammonia released from the breakdown of urea. Eutrophication negatively affects water bodies through the excess accumulation of in this case ammonia serving as food for algae and macrophytes and their subsequent abnormal growth blocking sunlight and reducing oxygen levels to the extent of posing danger to aquatic life as well as possibly posing risk to humans if that water body is put to human use as it is toxic. Alongside external concerns, there is also a concern that if ammonia under charged conditions can accumulate and prove to be cytotoxic within Enzocrete.

For this reason, we have decided to incorporate the nitrifying enzymes of ammonium monooxygenase and hydroxylamine oxidoreductase and denitrifying enzymes of nitrite, nitric and nitrous oxide reductases to break down the ammonia to less damaging nitrogen gas. In this way, we hope to make our project more responsible by not introducing a new problem to solve for a pre-existing problem and this step is also crucial in the future attempts to scale this process up from its lab origins for viability reasons.

Since our principal goal is to limit GHG emissions from the cement industry to combat climate change, it would be amiss to not consider the upstream costs involved in the production of the reagents that make MICP possible. Current studies on MICP have been using lab-grade calcium chloride and urea as large-scale case studies of MICP implementation are rarer.

But usage of lab-grade reagents come with their own significant carbon footprint and there is a need to replace these reagents with less polluting alternative production processes. For urea, an alternative is to use environmental wastewater as the source for nitrates. Single step electrocatalysis can be utilized to arrive at urea with ambient conditions and room temperature, ideally meeting its relatively lower energy needs through renewable energy. As for calcium chloride, an alternative is to shift to commercial-grade purity by substituting the lab-grade Solvay process with hyperchlorination of alkyl chloride, which has shown to have a carbon footprint reduction of 26.7 to 82%, relative to the lab-grade reagent.

As we put together a synthetic biology solution that solves for eutrophication and carbon-heavy reagent production cycles roadblocks impeding the wider adoption and the resulting increased viability and incentives that economies of scale confer on the MICP technology. We hope that we adequately reached a balance between addressing the immediate need for housing while addressing the negative externalities of the traditional cement industry and MICP through Enzocrete.

The results of this project must by design be suitable for commercial application. As such economics must be considered when determining the potential end users for the bacteria. The most obvious one being the existing concrete industry, especially those who already have their own investigations into greener alternatives. Examples in Australia are Wager, Fineform Concrete, and Holcim who all invest into sustainable production. These companies would have the resources to cultivate, purify and transport our MICP limestone at large scales, fitting into their existing supply lines. Having an inhouse source of calcium carbonate is also useful for cutting costs and reducing supply inconsistencies that affect customers. The ease of replication and ability for smaller production levels also opens up rural regions and farms as an area of applicability. The lower needs of these communities combined with their often-isolated status means the decrease in efficiency is not a major detriment. Indeed, many of the reagents required like Urea could also be sourced internally at livestock farms, allowing a high degree of self-sufficiency and recycling. Especially when combined with our automated machinery. Cheap, pre-set and flexible, this system has the broadest possible users. From being integrated into small- or large-scale production or helping future iGEM teams' assay MICP enzymes, there is a multitude of applications. Outside of this, laboratories could gain a precise and accurate way of running long and broad tests, eliminating both human error and labour-intensive work.

We envision others using our project as an integrated part of the cement process, with as little modification as possible. This applies to both the Enzocrete bacteria and the traditional cement making process. As changes must be rapid to prevent ongoing CO₂ emissions, the simpler our project is to use and the more conveniently it can be slotted into the existing structures the greater positive effect we can have.

The bacterial production and pathway to use in cement is self-contained. The MICP bacteria will be replicated on mass but can also be done at scale. Then the preliminary reagents of urea and calcium chloride will be added to allow the production of the calcium carbonate crystals around the bacteria. The E. coli cells will not have to be treated or lysed, the crystalline structures are what allows the bypassing of limestone burning. So, after the limestone has been precipitated out of the broth, it can then be filtered and mixed with regular cement components, such as silica and iron oxide.

Identification of the ideal enzymes for MICP, including enzymes that are active in the pH, temperature, and mineral conditions of cement production are important for efficient and economical MICP cement production. This could be done at a multitude of scales, thanks to the flexibility and low cost of our Bio Foundry concept. Using only three machines; the Xarm6 robotic arm, OT2 liquid handler, and thermocycler, enables fast and high-throughput synthetic biology, including screening enzyme activity, assembling DNA that encodes enzymes, and propagating engineered bacterial strains. As our system uses off the shelf components, it is vastly cheaper than existing Bio Foundry setups, allowing for a much lower entry for MICP development but can also be adapted to other synthetic biology applications. The modularity allows for the expansion of unit to include many of these machines working in parallel, greatly increasing testing and cultivation capacity. This system has other potential uses as well, the code allows labs to increase precision over large scale pipetting and analysis.

Overall, we imagine that our MICP limestone can replace the existing cement procedures, while preserving and even expanding the ease, cost and sufficiency of the industry as a whole. Going from cell to construction with one bacterium.

To implement our project into the real world would take a series of experiments and steps. The completion of the full Enzocrete bacterium with all three pathways; Urease, Carbonic Anhydrase and Nitrification/Denitrification, would allow further testing on the speed, quality and quantity of the limestone produced. Once this has been achieved materials and tensile testing would be the main priority to ensure long term safety and efficacy with traditionally produced cement. This could be conducted internally as UNSW has extensive Architectural and Engineering faculties. Once comparable performance could be shown, we could then start the process of consulting with cement and concrete companies. As the bacteria is inert by the time it is integrated into the cement mixture, the traditionally considerable legal restrictions around the usage of genetically modified organisms will be reduced and only the storage and replication would need to be shown to be environmentally safe. The BioFoundry process would also continue to be refined, a standardised interface allowing customisation without intensive coding knowledge would allow use by a much greater section of the population.

The detailed elaboration of our integrated human practises is displayed on an independant integrated HP page.