Portland cement concrete is a vital construction material, but its production contributes significantly to global CO2 emissions (around 7-8%). While renewable energy and clinker substitution can reduce emissions, they don't eliminate process-related CO2 from limestone calcination. Bio-concrete offers a different approach, utilizing microbially induced calcium carbonate precipitation (MICP) to consolidate aggregates. This process is CO2-negative, storing CO2 in carbonate compounds. Ureolytic bacteria, specifically *Sporosarcina pasteurii*, catalyze the precipitation of calcium carbonate from urea and calcium salts, creating a calcite-cemented material similar to sandstone. This approach has potential for creating larger building components, and using renewable energy and circular economy principles could make it CO2-neutral. However, a significant challenge has been achieving compressive strengths comparable to conventional concrete while maintaining sufficient component depth. This research aims to overcome this limitation by optimizing aggregate packing density and cementation parameters using UACP (prepared by freeze-drying a bacterial-calcium carbonate slurry) and an automated pressure injection system to ensure homogeneous cementation.

Existing research on MICP has shown its application in self-healing concrete, soil consolidation, and bio-brick production. Studies have demonstrated a positive correlation between compressive strength and the amount of precipitated calcium carbonate. However, challenges remain in achieving high strength and depth due to limitations of reactant solubility, microbiological factors, and inhomogeneous cementation resulting from bacterial transport, chemical depletion, and clogging. Previous methods, like using "bioslurry" to embed bacteria in calcium carbonate crystals, have shown promise in improving bacterial retention, but compressive strengths and depths have remained below those of traditional concrete. This study builds on these advances to achieve superior results.

The study began by optimizing aggregate packing density using six different quartz sand fractions and the Elkem Material Mix Analyser (EMMA) software. The optimized mix was then combined with varying amounts of UACP (1.5% w/w relative to dry mix). Initial percolation experiments using gravity-based methods yielded insufficient cementation depth and reproducibility. To address this, an automated pressure-based injection system was developed to deliver cementation solutions (0.75 M equimolar urea and calcium chloride, unless otherwise noted) from the bottom at regular 4-hour intervals. Different pressures (0.1-0.75 bar) and UACP concentrations (0.5-7.5% w/w) were tested. Reproducibility was assessed using optimal parameter combinations, and a step-wise pressure increase was tested to manage clogging. Compaction pressure was also optimized (5 to 10 N/mm²). The characterization involved measuring specimen weight increase during biomineralization, flow rates, ammonium, calcium, and urea concentrations in effluent solutions, ultrasonic wave velocity, unconfined compressive strength (UCS), and calcium carbonate content. Environmental scanning electron microscopy (ESEM) was used to analyze the microstructure of selected specimens.

The optimized aggregate mix significantly improved packing density (φaggr = 0.35 compared to 0.45-0.47 for homogeneous sand). Initial percolation experiments resulted in cementation depths of less than 40 mm. The pressure injection system produced specimens with much greater cementation depths (up to 140 mm). A maximum unconfined compressive strength (UCS) of 57.4 MPa was observed (specimen 5.6), far exceeding previously reported values (2 MPa, 1.3 MPa, and 9.7 MPa in cited studies). However, inhomogeneities in UCS were observed in most specimens, with only specimens 1.2 and 5.3 showing consistent strength across their height. The cementation depth decreased with increasing UACP content up to 2% w/w, while excessive UACP (7.5% w/w) negatively impacted cementation. Ultrasonic wave velocity measurements correlated well with UCS values, indicating high homogeneity in specimens 1.2 and 5.3. Reproducibility studies (test series 6 and 7) showed that specimens with similar initial packing density and cementation parameters exhibited similar properties. Higher flow rates, observed in specimens with lower aggregate packing density, negatively affected strength. ESEM images showed uniform calcium carbonate coating thickness (around 20 µm) around sand grains, suggesting a growth limit in the crystallization process. The study also noted a plastic region in the stress-strain curve at ~4 MPa, possibly due to calcium carbonate bridge breakage and aggregate recompaction. The Young's modulus of specimens from test series 7 was around 11-12 GPa.

The findings demonstrate that maximizing aggregate content, optimizing cementation parameters, and using UACP greatly enhance bio-concrete strength and homogeneity. The exponential relationship between density and UCS highlights the importance of maximizing aggregate packing density. The plastic region in the stress-strain curve suggests potential for improvement through even higher aggregate packing density. The achieved strengths (51.2-52.5 MPa) approach the C20/25 strength class required for prefabricated reinforced structural elements, opening the door for partial substitution of traditional concrete. The use of UACP provides a significant advantage over pure bacterial cultures. Limitations regarding the flow rate and clogging suggest the necessity of a precise control system for large-scale production. The environmental impact of raw material sourcing and effluent recycling needs careful consideration, such as utilizing urine as a urea source and exploring effluent recycling strategies for a truly sustainable approach.

This study successfully produced high-strength, homogeneously cemented bio-concrete specimens with compressive strengths exceeding 50 MPa and cementation depths of 140 mm. This was achieved through optimized aggregate packing density, controlled pressure-based cementation, and the use of UACP. These findings open possibilities for using bio-concrete in load-bearing building components, partially replacing traditional concrete. Future research should focus on incorporating coarse aggregates and optimizing the use of fines to further improve strength and explore alternative calcium sources and effluent recycling methods to enhance sustainability.

The study's use of a relatively small-scale, laboratory-based system might limit the direct scalability to large-scale production. The choice of quartz sand might limit the generalizability of findings to other aggregate types. Further research is needed to fully optimize the mix design for different aggregate types and sizes and to address the potential corrosion issues associated with using calcium chloride as a calcium source.