Magnesium and bio-slurry enhance the properties of bio-tiles produced using an automated microbial calcium carbonate precipitation process

This study presents a novel, modular 3D printing system for the scalable production of microbially induced calcium carbonate (CaCO 3) precipitation (MICP) produced bio-materials. Through modular design principles and operation automation, the technique offers a biochemical route to cementitious materials with the potential for lower energy and carbon footprints compared to traditional kiln-fired ceramics. The bio-tiles produced using this technique exceed international standards of breaking strength and modulus of rupture. By leveraging freeze-dried urease-active bio-slurry powder, the process ensures uniform bacterial distribution within the sand matrix, enhancing reproducibility and scalability. To establish a technical baseline for this automated platform, the effect of CaCO 3 seed supplementation, magnesium-enriched cementation solution, and manual daily nutrient broth treatments was evaluated as a function of ureolytic activity and mineralization performance. Results revealed that magnesium addition paired with nutrient broth supplementation was a notable factor associated with the observed strength enhancement, achieving an average of 622 ± 84 N. While individual seed loadings showed variability consistent with gravity-driven infiltration, the aggregate performance of magnesium-supplemented tiles established a clear general technical trend, achieving strengths 3.3 times higher than bio-tiles treated without magnesium and only nutrient broth. However, nutrient broth treatments were found to be redundant as ureolytic requirements were likely satisfied by the bio-slurry itself. Additional CaCO 3 seeds minimally influenced strength but improved edge solidification. Despite challenges in achieving uniform solidification without specific additives, the automated system successfully integrated submersion and pumping methodologies. The bio-tiles exceeded strength standards for tiles with a water absorption of greater than 10%, underscoring their potential for sustainable construction applications. This proof-of-concept highlights the mechanical automation as a key enabler for scalable MICP-based manufacturing, reducing labour intensity and optimising bio-cementation for sustainable, high-performance materials. Future research should focus on enhancing solidification consistency and advancing MICP-based bio-materials for structural applications. The integration of automation, modularity, and optimised MICP conditions marks a significant advancement toward the commercial-scale production of materials with improved environmental profiles.

Energy consumption accounts for up to 30% of production costs in ceramic manufacturing (European Commission, 2019), with ceramic tile production being a notable contributor to carbon emissions regardless of the energy source. As the global population grows and infrastructure ages, the demand for sustainable construction materials with lower embodied carbon becomes increasingly critical. Bio-cementation, particularly microbially induced calcium carbonate (CaCO 3) precipitation (MICP), is emerging as a promising alternative. Among the various pathways for MICP, microbial urea hydrolysis is the most widely adopted due to its high efficiency in calcium carbonate precipitation (Achal and Pan, 2011). This process binds particles such as sand through the formation of CaCO 3 crystals, finding applications in soil erosion prevention (Whiffin et al., 2007), creation of subsurface barriers against pollutant spread (Cunningham et al., 2011), dust control (Hamdan and Kavazanjian, 2016), concrete crack repair (Jonkers et al., 2010; Seifan and Berenjian, 2018), and the production of bio-materials (Bu et al., 2018; Dikshit et al., 2022). From a chemical engineering perspective, the development of automated bioprocesses for construction materials represents a convergence of biochemical reaction engineering, process control, and sustainable manufacturing principles.

Several techniques have been explored to produce bio-materials using MICP, the more notable including injection (Cheng et al., 2013), spraying (Dosier, 2016), pumping (Horn et al., 2024) and submersion (Horn et al., 2023). While each has shown promise at the laboratory scale, large-scale implementation remains constrained by practical limitations(Gomez et al., 2017; Nassar et al., 2018). Injection methods, while effective for small-scale studies, often suffer from clogging and uneven reagent distribution, limiting their scalability(Cheng and Cord-Ruwisch, 2014; Wang et al., 2022; Whiffin et al., 2007). Spraying techniques—used commercially by BioMASON (Dosier, 2016)—present challenges with fluid penetration and cementation consistency when explored for soil stabilisation and road hardening (Kim et al., 2022) and literature is scarce for its application to bio-materials. Submersion approaches simplify reagent management and have demonstrated success in producing bio-tiles that meet ISO strength standards (Horn et al., 2023). However, diffusion limitations during CaCO 3 precipitation lead to inhomogeneous cementation in thicker materials (Bu et al., 2018; Cheng et al., 2020), and the manual packing and unpacking of moulds hinders throughput and reproducibility (Horn et al., 2023). Pumping methods offer more controlled reagent delivery through sealed moulds (Lambert and Randall, 2019), improving cementation uniformity (Horn et al., 2024). Innovations such as timed mould inversion and CaCO 3 seeding have enabled the production of bio-tiles compliant to two ISO classes (Horn et al., 2024). Nevertheless, the technique still relies on labour-intensive processes, including manual mould assembly and handling, which remain significant barriers to scale-up. These limitations across techniques highlight the need for automation to enable consistent, high-throughput production of bio-tiles.

Bio-tiles represent a promising MICP application, addressing both technical challenges of cementation uniformity and solution penetration, and commercial viability (Horn et al., 2023). Their potential for commercialisation is bolstered by the possibility of automating production processes where labour costs are a concern, which could significantly enhance the scalability of MICP by reducing human intervention and combining the strengths of existing methods while mitigating their limitations. A fully automated process would add the required amount of sand, smooth and compact it and then solidify it into a bio-material through the addition of cementation solution. This technique would be useful to add to the body of MICP knowledge and finally address some of the scalability concerns of MICP (Rahman et al., 2020; Zhu and Dittrich, 2016).

Combining the automation and localised and homogeneous cementation of the pumping technique with the submerging of the bio-material and simple operation of the submersion method, a form of 3D printing technique could offer a unique opportunity for making an optimised production process that draws on the benefits of each technique. Additive manufacturing technologies also have the potential to be modular and therefore highly scalable. They can also offer a layered approach that could overcome fluid penetration challenges for larger bio-materials, expanding on the options for bio-materials that can be bio-cemented and therefore makes it a good option for an MICP technique to grow bio-materials. Commercialisation and performance of a manufacturing process revolves around the ability to scale which extends to the ease of operation, to what extent a production process can be automated and ultimately, making the process more economical by increasing throughput (Kromann and Sørensen, 2019). There has been little success with scaling up MICP processes and even fewer instances reported in literature. The advantages and disadvantages of each of these bio-material production techniques are given in Table 1.

Binder jet 3D printers are a type of additive manufacturing machine where a liquid binder (cementation solution in combination with bacteria) is added to loose particles (inoculated/pure sand) to form a solid material (bio-material). It is possible to produce spatial bio-cemented structures by selectively depositing sand inoculated with a urease-active bio-slurry in a lattice-like pattern amongst plain sand, which improves fluid penetration and increases the homogeneity of cementation (Nething et al., 2020). The same authors used an automated version of the injection method to produce small-scale bio-columns in small syringe tubes using which resulted in compressive strengths of up to 23 MPa (Nething et al., 2020). Erdmann et al. (2022) instead used a pressurised dispensing unit for both bacteria and cementation solutions to prove that a layer of CaCO 3 could be printed. The sand was packed and the surface smoothed by hand, and therefore the process was not fully automated. Apart from these instances, the literature on 3D printing using MICP is scarce and none to the authors knowledge on 3D printing construction bio-materials. Although the small bio-columns of Nething et al. (2020) are a start, the method does not account for fluid penetration challenges and is likely not very scalable.

The urease active bio-slurry used by Nething et al. (2020) was effective for 3D printing the small bio-columns and spatial structures. The bio-slurry was formed from the addition of calcium and urea directly to the bacteria solution, causing the encapsulation of bacteria cells with CaCO 3 shells. The wet bio-slurry was then freeze-dried to make a dry powder. There is potential for the bio-slurry to also act as CaCO 3 seeds to initiate CaCO 3 precipitation. The presence of seeds, in combination with other surfaces, offers supplementary nucleation sites for crystal formation and thereby, lowers the activation energy required for nucleation of further CaCO 3 crystals (Mullin, 2001). In addition, CaCO 3 crystals themselves lower the supersaturation concentration required for nucleation, further increasing the rate of nucleation (Lioliou et al., 2007). The use of bio-slurry for MICP applications is not common, however, Cheng and Shahin (2016) who developed the technique found its application to soil bio-cementation to be controllable, reproducible and homogenous. Previous work (Horn et al., 2024) identified the beneficial effect of seeding with CaCO 3 crystals for bio-materials with a pumping technique. Higher seed loadings improved the breaking strength of bio-tiles by 21%–82%, with no sign of a threshold being reached (Horn et al., 2024). In addition, the encapsulated bacteria have been shown to be more resistant to wash out as the CaCO 3 seeds keep them bound between the sand particles (Nething et al., 2020). Freeze-dried bio-slurry has been proven to maintain its ureolytic activity for over 6 months in cold storage at 20 °C (Nething et al., 2020), making it advantageous for scaling up production and transporting the bacteria where needed, increasing accessibility to the technology.

Considering the challenges with achieving satisfactory strength properties with bio-cemented materials, there are further supplements that can be added to the system which warrant investigation for new production methods. Some researchers have added nutrient broth to the cementation solution to support the metabolism and rapid growth of the bacteria during MICP (Feng et al., 2021; Rittmann et al., 2011; Wen et al., 2020) which can increase urease production (Chuo et al., 2020). Lambert and Randall (2019) found that nutrient broth was the most expensive input when manufacturing bio-bricks. It has also been found that the addition of nutrient broth can severely affect the compressive strength of bio-materials produced with a submersion technique (De Oliveira et al., 2021). Therefore, while potentially beneficial, the applicability of nutrient broth treatments needs to be assessed for different production techniques.

Another supplement which was found to enhance strength properties of bio-materials, was the addition of magnesium to the cementation solution (Horn et al., 2023; Xu et al., 2020). It is hypothesized, consistent with prior literature (Putra et al., 2016; Xu et al., 2020; Zhang et al., 2015), that the mechanism may involve enhancing the rate of calcium ion removal such that smaller crystals are formed while also promoting the formation of aragonite polymorph of CaCO 3 over calcite. Magnesium has improved the compressive strength of bio-columns by 40% (Xu et al., 2020) and the breaking strength of bio-tiles from 546 N to 620 N, a 13.6% increase (Horn et al., 2023).

This work therefore aimed to manufacture bio-tiles that meet ISO strength standards using an automated MICP process. A powderised bio-slurry was assessed for its efficacy in producing bio-tiles that meet strength standards and the ureolytic activity compared for different media concentrations to liquid bacteria. The automated MICP system represents a novel approach to bioprocess automation in bio-manufacturing, addressing key chemical engineering challenges including labour-intensive material handling and process scalability through modular design. The key objectives of the study were to investigate supplementing the process with different masses of additional CaCO 3 seeds, magnesium addition in the cementation solution, and a daily cementation solution treatment enriched with nutrient broth. Finally, the effect of each supplement on the bio-tile breaking strength was quantified. In addition, water absorption and CaCO 3 content was assessed.

Microbiological cultivation

The ureolytic soil bacteria, S. pasteurii (ATCC 11859), was cultured as described by Henze and Randall (2018). In summary, a colony was cultivated under sterile aerobic batch conditions in 10 mL of ammonium-yeast medium in a shaking incubator (120 rpm) at 30 °C for 24 h. This culture was then added to 90 mL of yeast growth medium in a 1 L conical flask and returned to the incubator for 16 h until the late exponential growth phase.

The required number of flasks were combined and then ureolytic activity of the batch was determined as described by Horn et al. (2023). In short, the concentration of ammonium ions produced over half an hour was measured in duplicate through the addition of 15 mL of bacteria solution to 15 mL of 1 M synthetic urea (≥99.5% purity, Sigma-Aldrich, Darmstadt, Germany) solution. The initial ammonium ions present and final concentration, after 30 min shaking at 120 rpm, were measured.

Urease active bio-slurry powder preparation protocol

Following ureolytic activity tests, the remaining bacteria solution (1.17–1.47 L) was used to make bio-slurry. The urease-active bio-slurry powder was prepared from freeze-drying the conventional bacteria solution after mixing in calcium and urea to form protective CaCO 3 capsules around the bacteria. Once powderised, the bio-slurry essentially delivers the bacteria as well as CaCO 3 seeds.

To prepare the bio-slurry, the pH of the bacteria culture was corrected to 7.9 with 85% H 3 PO 4 (Nething et al., 2020). The solution was added to an autoclaved 5 L Schott bottle to allow a headroom with sufficient oxygen for the aerobic bacteria. Synthetic urea and calcium chloride dihydrate (CaCl 2 ·2H 2 O; ≥99.1% purity, Kimix, Cape Town, South Africa) resulting in 0.4 M concentrations (Cheng et al., 2020; Nething et al., 2020) were added to the bacteria in portions while mixing. The lid of the bottle was lightly screwed on. The solution was stirred at 60% speed for 6 h on a magnetic stirrer (Labcon M5250, Laboratory Equipment, Chamdor, South Africa). The precipitated material was then allowed to settle for 14 h (Nething et al., 2020). At this point, as much of the supernatant as possible was extracted by syringe and the remainder drained using a Buchner funnel. The bio-slurry was then freeze dried (Varian 801, Vacuum Technologies, Cape Town, South Africa) for 72 h at 450 mTorr and −33 °C. The lyophilised bio-slurry was gently powderised in a mortar and pestle, and then vacuum sealed and stored at −20 °C (Nething et al., 2020). About 46 g of bio-slurry was made from 1 L of bacteria solution.

For quantifying the ureolytic activity of the freeze-dried bio-slurry, a similar procedure as for liquid bacteria was followed where 1 g of bio-slurry powder was resuspended in 15 mL of deionised water in an autoclaved flask and mixed with 15 mL of 1 M urea solution, the flask was sealed and shaken at 120 rpm for 30 min. The change in ammonium ion concentration was once again compared.

To assess ways of improving bio-slurry performance, the bacteria was prepared with growth medium consisting of a double and triple concentration of yeast extract and ammonium sulphate (Nething et al., 2020) and the ureolytic activity was compared to bacteria grown with single concentrated medium. Bio-slurry was produced from each bacteria solution, lyophilised and ureolytic activity compared as previously stated.

Bioprocess system inputs

Dune sand from the Cape Flats in Cape Town, considered relatively well-graded with a D 50 of 469 nm, was used as the aggregate for this study due to its ideal particle size distribution (Horn et al., 2023). Cementation solution consisted of an equimolar concentration of 0.3 M urea and calcium in the form of calcium chloride dihydrate and when added, 0.3 M anhydrous magnesium chloride (MgCl 2, ≥98% purity, Sigma-Aldrich). The nutrient-enriched cementation solution contained 0.3 M urea and calcium, as well as 3 g/L No. 1 Nutrient Broth (Sigma-Aldrich) (Stocks-Fischer et al., 1999). The nutrient broth contained 15 g/L peptone, 3 g/L yeast extract, 6 g/L sodium chloride, 1 g/L D-glucose and was mixed into a small amount of deionised water and autoclaved before use. The seeded geotextiles used to provide additional CaCO 3 seeds to the bio-tiles were non-woven needle-punched geotextile recovered from previous work (Horn et al., 2024).

Reactor design of the automated MICP system

The automated MICP prototype was designed with the principle of a binder jet 3D printer. Sand that was inoculated with bioslurry (the activator) was used as the solid base. Cementation solution was deposited on the sand in automated sequential treatments, causing a reaction to occur that resulted in the production of bio-cement (the binder).

The prototype was designed by Amnova Tech, Johannesburg, South Africa. The crucial parts of the prototype for understanding its operation are given in Figure 1. Detailed drawings are available in the Supplementary Material; Supplementary Figures S1A–D. In the material selection process for the prototype, as far as possible, metal parts that can rust were avoided and instead, acrylic, silicone, stainless steel and aluminium were used. The 3D printed parts used in the prototype were primarily made from PLA filament, as well as PET-G and ABS.

The reaction chamber consisted of a square platform (100 × 100 mm) with drainage holes and a silicone edge, such that the platform could raise and lower vertically within an acrylic chute. The silicone edging allowed a seal to be maintained around the sides where it meets the chute, keeping sand and solution from escaping at the edges.

When the platform was in a raised position, the holes within the platform allowed spent solution to drain through the bio-tile and the platform. In the lowered position, the platform rested against a registering silicone seal, blocking the holes and preventing solution from draining. The acrylic chute was secured to the main assembly frame through a custom 3D printed part. The main silicone seal was secured in place by the same 3D printed part.

The linear actuator (200 mm stroke, Robotzone, Suzhou, Taiwan) that powers the vertical movement of the platform was paired with a second matching linear actuator that powers a second platform that compacted the bio-tile from above as desired. These linear actuators could each apply 80 kg of force.

The cementation solution deposition was carried out using a diaphragm pump (600–800 L/h, AHEADPUMP AW500S, Qingdao Ahead Electric Co., Shandong, China), and a custom 3D printed showerhead (with a dosing area of 90 × 90 mm), and airlocks were maintained at the feed container and at the outlet. A pair of solenoids (0.02–0.8 MPa, Meishuo, Yueqing City, China) in series at the showerhead, were used to ensure accurate dosing of cementation solution and no leaks. The solution was showered over the bio-tile using a 3D printed shower head. Stepper motors (NEMA17) were used to roll the showerhead into place over the tile for each deposition and rolled out of the way following the deposition.

The automated control system integrates feedback mechanisms through G-code programming, enabling precise control of process variables including solution deposition rates, retention times, and compaction forces. This systematic approach to process control represents a significant advancement in bioprocess automation.

Additive manufacturing process design

For each bio-tile of 100 × 100 × 10 mm, freeze-dried bio-slurry (3 wt%, 3.28 g) was carefully mixed into the required amount of sand (Nething et al., 2020), resulting in a visually even distribution of the white bioslurry throughout the dry sand mixture. The walls of the chute were coated with petroleum jelly and lined with clear acetate for structural support of the bio-tile edges and its easy removal from the chute. A non-woven needle-punched polypropylene geotextile lined the interface between the platform and the inoculated sand, preventing sand from passing through the platform holes. The inoculated sand was carefully packed on to the platform, flattened and compressed into the chute. A geotextile was also placed on top of the inoculated sand, this was critical in preventing the cementation solution depositions from disturbing the cementation of the surface of the bio-tile.

The working instructions for the automated system were written in a G-code file, which was processed by a 3D printing motion software (Duet Web Control, United Kingdom), sent to the microprocessor (Duet 3 Main Board 6XD, Duet3D, Peterborough, United Kingdom). The microprocessor then sent control commands to the various system outputs including microcontrollers (Arduino Mega and Uno, Arduino, Ivrea, Italy) that control the actuators. Each deposition of cementation solution was run for 3.5 s which equated to 84 ± 2.5 mL, such that there was about 15 mm of pooling above the surface of the bio-tile. Over a run-time of 8 days, each treatment cycle was held for a retention time of 1 hour and 45 min, followed with 15 min for draining of the spent solution. The bio-tile was compacted after every second treatment for the first 2 days of treatment cycles. Given the 10 mm thickness of the tile, each treatment effectively acted as a single-layer saturation event, rather than a high-pressure spraying process, providing a consistent dosing resolution across the 90 × 90 mm showerhead area.

Initially, the bio-slurry-sand mixture was completely dry and therefore, a double deposition was carried out at the start of the program. However, when the process was supplemented with additional nutrients, the bio-tile was first treated manually with a single deposition (84 mL) of nutrient-enriched cementation solution, and then a single deposition of regular cementation solution to start. Thereafter, manual nutrient-enriched treatments were carried out daily in place of the corresponding treatment of the regular cementation solution.

Analytical methods

A Thermoscientific Gallery (TSG) (Automated Chemistry Analyser: 861, ThermoFisher Scientific, United States) was used to measure the concentration of calcium, ammonia and magnesium. Before inserting into the TSG for analysis, all samples were diluted to 1 in 100.

Standards for tiles

The International Organisation of Standards (ISO) sets comprehensive requirements for tiles, encompassing criteria such as dimensional and surface quality, and more, depending on factors like water absorption, manufacturing method, and finish (ISO 2018 ISO 13006, 2018). The standards referenced in this study align with those detailed by Horn et al. (2023), given the similarities between the bio-tiles produced here and those in their research. Specifically, these bio-tiles fall into the extruded tiles category with water absorption greater than 10%. For this proof-of-concept study, the primary focus was on breaking strength and modulus of rupture, which were considered essential benchmarks. Briefly, breaking force (F) was measured using a three-point bending test on a ProLine Z100 (ZwickRoell 1,474, Ulm, Germany), as per ISO guidelines (ISO 2019 ISO 10545-4, 2019). A cylindrical bearing plate was lowered at a rate of 2 mm/min towards the bio-tile on the bending device which was mounted on a broad bearing plate. Once the bio-tile fractured, the breaking strength (N) was calculated using the dimensions of the bending device, following the approach and dimensions (D = 12; L = 100; l 1 = 19; l 2 = 62; t = 3 mm) outlined by Horn et al. (2023). The minimum thickness of the bio-tile at the broken edge was measured with callipers, and the modulus of rupture (flexural strength, MPa) was then calculated. Therefore, for bio-tiles with a water absorption greater than 10%, the breaking strength to be met or exceeded is 600 N and the modulus of rupture must be a minimum of 8 N/mm 2 (ISO 2019 ISO 10545-4, 2019).

Water absorption is an index that indicates a tile’s frost and weathering resistance (Zhang et al., 2015). Water absorption plays a critical role in determining a tile’s classification regarding the required minimum breaking strength and modulus of rupture. The method used to analyse water absorption was adapted from the SANS1449 standard (SABS, 2012) as described by Horn et al. (2023). Briefly, tile halves were inserted into a water bath vertically and heated to boiling from room temperature, boiled for 2 hours and then left to cool overnight. After patting dry, the wet mass could be recorded.

Performance of the urease-active bio-slurry powder compared to conventional liquid bacteria solution

The ureolytic activity of regular bacteria solution was compared to freeze-dried bio-slurry made from the same batch (Figure 2). Bio-slurry was found to have almost double the ammonium production of regular liquid bacteria solution. The consistent performance of the bio-slurry across varied growth medium concentrations provides a clear indication of its efficacy as a standardized, stabilized biocatalyst for automated processes.

Nething et al. (2020) utilised ammonium-yeast growth medium constituted from double the concentration of yeast extract and ammonium sulphate for growing the bacteria solution used in the bio-slurry. When compared to original growth medium, the ammonium produced was similar for the bacteria solution prepared with double and triple concentrated growth medium, however, the bio-slurry activity decreased by 29% and halved, respectively (Figure 2). This suggests that bio-slurry activity does not benefit from double or triple concentration of growth medium but rather reduces with increasing concentration of medium. Hence, the original growth medium recipe was followed for bio-slurry preparation.

For the ureolytic activity tests, the final concentration of bio-slurry solids was 33.3 mg/mL, which was similar to the concentration used in the activity tests ran by Cheng and Shahin (2016) who mixed 5 g bio-slurry (about 15% solid content) with 15 mL 3 M urea solution and 10 mL deionised water, to give a final concentration of 25.6 mg bio-slurry solids/mL. When Nething et al. (2020) adapted the method, 540 µL resuspended freeze-dried bio-slurry was added to 60 µL 0.1 M urea solution, to give a final bio-slurry solids concentration of 1.17 mg/mL. Therefore, these bio-slurry ureolytic activity tests were more similar to those conducted by Cheng and Shahin (2016).

Bioprocess supplementation with calcium carbonate seeds, magnesium and nutrient broth

The bio-tiles produced with the automated technique were initially relatively poor in breaking strength and needed additives for the corners to be intact and to form more solidly, such that breaking strength tests could be conducted. The inability to calculate the modulus of rupture for specific samples serves as a critical diagnostic result: it quantitatively demonstrates that without specific additives, gravity-driven deposition fails to achieve the structural integrity required for ISO standards. Even with some additives, solidification was so poor in some cases that a minimum thickness could not be measured, and the modulus of rupture was incalculable. The water absorption of bio-tiles supplemented with magnesium was 10.3% ± 0.9% (n = 5, Supplementary Material; Supplementary Figure S2), and therefore, the breaking strength and modulus of rupture targets were 600 N and 8 N/mm 2, respectively.

The effect of additional CaCO 3 seed loading (over and above that provided by the bio-slurry), a daily manual nutrient broth-enriched treatment and cementation solution supplemented with magnesium are shown in Figure 3. Across the varying seed loadings tested, the data demonstrates a clear and significant performance enhancement when magnesium is present, regardless of individual sample dispersion. Most of the bio-tile breaking strengths were widely dispersed over the seed loadings tested, with few exceptions. This was likely influenced by uneven seeding of the geotextiles, the fluid dynamics of the process and reliability concerns of the software, although there was a clear enhancement of breaking strength when supplementing with magnesium. Previous studies (Horn et al., 2024; Li et al., 2020) have also reported variability in the strength of MICP-produced bio-materials ranging 2%–27%, especially those with low strengths.

The breaking strength is mostly independent of additional CaCO 3 seed loading (Figure 3). In bio-tiles supplemented with nutrient broth, the breaking strength was 188 ± 60 N for all tiles (n = 8), across the various seed loadings. This standard deviation is 32% of the average breaking strength, which shows the inhomogeneity of the bio-cementation that occurred, albeit across the seed loadings. The high standard deviation (32%) in lower-strength tiles reflects the sensitivity of gravity-fed infiltration to local sand-packing variations. However, the consistent performance enhancement in the magnesium-supplemented group, regardless of additional seed loading, suggests that magnesium supplementation acts as a significant chemical variable associated with strength in this automated system, effectively overriding the stochastic variability associated with manual seeding. In contrast, when no additional seeds where added, the bio-tile had a breaking strength of 198 N. While this is a singular result, it is likely that the bio-cementation was limited and so variable that seeds had little effect on the low breaking strength of bio-tiles supplemented with nutrient broth. When no additional seeds were used in the presence of 0.3 M magnesium, a bio-tile with the lowest and highest strength of all magnesium-supplemented bio-tiles occurred (498 and 715 N), indicating that additional seeds had little effect on the strength of bio-tiles produced with this technique.

There was already 3.28 g of bio-slurry (∼3 wt% of bio-tile), or urease active CaCO 3 seeds, added internally to each bio-tile. It follows that the additional external seed loading of geotextiles would have had less of an effect on the strength of bio-tiles because there is a threshold beyond which nucleation can be bypassed entirely (Kim et al., 2020; Lioliou et al., 2007) and crystals grow bigger instead. At this point, the advantage of seeding to increase the rate of nucleation no longer applies. However, previously (Horn et al., 2024) bio-tiles were seeded with up to a total of 14.5 g of additional seeds (0.07 g/cm 2 geotextile) and consistently saw increasing breaking strength, with no threshold reached. In this study, a maximum of 36.5 g of additional seeds (0.18 g/cm 2) were supplemented to the bio-tile on the geotextiles, with external seed loading having little effect on breaking strength. It is possible that the threshold for the effect seeding has on breaking strength was lower when seeds are added internally to the bio-material. This was because the seeds were mixed amongst the bio-tile particles, in comparison to applied on the surface of the bio-tiles in both geotextiles, as in the pumping technique (Horn et al., 2024). Therefore, surface application of CaCO 3 seeds is less effective than internal seed loading. Additionally, the urease-active nature of bio-slurry seeds, preserved at −20 °C until use, could be compounding the seeding benefit. Other factors, such as the flow dynamics and mixing, could have also played a role in this finding, and the improved efficacy of internally mixed seeds over surface loading would need to be verified for other production techniques.

It was hypothesised that the nutrient broth would improve the bacterial metabolism and enhance the breaking strength of the bio-tiles. While some authors found that supplementing all cementation solution with nutrient broth was deleterious to the compressive strength measured (De Oliveira et al., 2021), others also noted nutrient broth would be one of the highest contributors to overall cost of production should the process commercialise (Lambert and Randall, 2019). Consequently, a daily treatment using 3 g/L nutrient broth-enriched cementation solution was utilised in this study to lower costs and still offer a boost to bacterial metabolism.

The observed performance of nutrient broth suggests that additional enrichment may be redundant or even deleterious in an automated system where the bio-slurry provides a concentrated nutrient matrix. Preliminary tests showed that the breaking strength of bio-tiles benefit from the addition of daily nutrients (Supplementary Material; Supplementary Figure S3). When the final tests were run after the bulk of the seed loading experiments, nutrient broth was found not to improve breaking strength. Instead, bio-tiles without daily nutrient broth treatments and without magnesium supplementation (n = 2) had breaking strengths that were 100% greater than those with only nutrient broth supplementation (n = 8) and for bio-tiles supplemented with magnesium (n = 2), an improvement of 12% was found (Figure 3). These results underscore that the nutrient requirements for ureolysis were likely satisfied by the bio-slurry itself, and the manual nature of these treatments served primarily as an exploratory probe for process optimization.

The effect of supplementing the cementation solution with 0.3 M magnesium ions was the most remarkable of the three supplements. On average, supplementary magnesium and daily nutrient broth treatments resulted in breaking strengths of 622 ± 84 N across seed loadings (n = 7). This standard deviation is 13% of the average breaking strength for magnesium-supplemented bio-tiles, supporting that bio-tiles with higher strengths have less strength variability. The standard deviation reported here reflects the variation across different seed loadings, indicating that higher-strength bio-tiles achieve a more stable cementation profile. The presence of magnesium improved the breaking strength of nutrient broth-enriched bio-tiles 3.3 times. This increase was greater than a previous bio-tile study (Horn et al., 2023) using the submersion technique (13%) as well as the two-fold increase in compressive strength of bio-columns produced with an injection technique (Xu et al., 2020). It is hypothesized, consistent with prior literature, that magnesium may favourably interact with CaCO 3 precipitation by potentially reducing crystal size (Putra et al., 2016) and promoting the selection of the aragonite polymorph over others (Nayanthara et al., 2019; Xu et al., 2020). Literature suggests that the prevalence of aragonite increases the density of the precipitated CaCO 3 and the Moh’s hardness from 3 or less to 3.5 (Seifan and Berenjian, 2018), which could provide a theoretical basis for the higher breaking strengths observed in the magnesium-supplemented bio-tiles, although further microstructural characterization is required to confirm this. The CaCO 3 content of bio-tiles supplemented with both magnesium and nutrient broth (Supplementary Material; Supplementary Figure S2) was marginally higher (39.7% ± 1.8%) than those supplemented with nutrient broth alone (35.7% ± 1.3%). Both are within the range of CaCO 3 contents of bio-tiles grown with the pumping technique (Horn et al., 2024). Supplementing with magnesium alone could be a more cost-effective strategy than adding nutrient broth.

Performance of the automated MICP system

The automated system which was designed for the integration of biological and mechanical systems and tested for an approach to combining the beneficial aspects of the submersion and pumping bio-processes. While the full experimental workflow included manual nutrient broth interventions, the underlying mechanical platform, governed by G-code and automated solenoid control, represents a valid transition to automated bio-manufacturing. However, the pumping technique has one important aspect which was not incorporated into the design: that of forcing solution through the pore space during pumping (Horn et al., 2024). This action of solution being driven through interparticle channels allows them to be more thoroughly filled by precipitate than by gravity alone, as is the case with the automated technique. Due to this gravity-driven mechanism of the automated technique, supplemental magnesium was required to produce bio-tiles with a breaking strength higher than the standard. In comparison, the bio-tiles produced by the pumping technique (Horn et al., 2024) met the standard with just 4.2 g (0.021 g/cm 2 geotextile) of additional CaCO 3 seeds and no supplementary magnesium. Compared to the pumping technique, which requires manual daily inversion and high labour intensity per unit, the automated platform executes 96 automated treatments and 12 compaction cycles without manual intervention. This is calculated over the 8-day (192-h) run-time with a 2-h treatment cycle (1.45 h retention and 0.15 h drainage) and compaction occurring after every second treatment for the first 48 h. While the pumping method achieved higher strengths without additives, the automated system’s value lies in its modular scalability and reduced human error, establishing a roadmap for high-throughput production. Therefore, the amount of inputs for the automated system was greater than for the pumping system. While the previous pumping method achieved higher absolute strengths (940 ± 92 N) without magnesium (Horn et al., 2024), it relied on manual, labour-intensive processes, including mould packing, daily mould inversion and monitoring and dealing with blockages. The current study prioritizes the automation of these processes to address scalability barriers. Because gravity-driven infiltration in the automated prototype is inherently less efficient at filling pores than forced-flow pumping, magnesium was utilized as a necessary chemical supplement to ensure the bio-tiles still met ISO strength benchmarks. Consequently, this approach represents a trade-off where a slight reduction in peak strength, compared to manual methods, is accepted in exchange for a significant increase in process automation and modularity. Magnesium was included in this system with the intent of investigating its potential to enhance the localized density of the precipitate, addressing the inherent inhomogeneity of gravity-driven cementation. While the specific inhibitory effects of magnesium on calcite nucleation are well-documented in literature, the priority in this study was the empirical achievement of structural integrity. By utilizing magnesium as a chemical intensifier, the bio-tiles successfully surpassed the 600 N ISO breaking strength threshold despite the lack of forced-flow infiltration. To further improve material performance in future applications, automation strategies should focus on optimizing the synchronization between the 12 compaction cycles and reagent dosing through the G-code programmable control loops. This would allow for a multi-layered approach to incrementally increase localized density and fluid penetration, potentially reducing the reliance on chemical supplements while maintaining high-performance standards in a fully automated framework.

The water absorption tests yielded an average value of 11.2% ± 0.8% across the tested bio-tiles (as shown in Supplementary Figure S2 of the Supplementary Material). According to the ISO 13006:2018 classification for ceramic tiles, these results place the automated bio-tiles in the category of tiles with a water absorption >10%, which is typically suitable for indoor wall applications. The relatively high porosity is a direct consequence of the gravity-driven infiltration method used in the automated platform, which lacks the high-pressure compaction found in industrial ceramic manufacturing. However, this level of absorption is consistent with other MICP-based materials and confirms the structural viability of the tiles for non-load-bearing architectural elements. The relatively high-water absorption (>10%) observed in these bio-tiles is a result of the gravity-driven deposition, which lacks the pore-filling efficiency of forced-flow methods. To address this and expand the application scope to lower-absorption ISO classes, future iterations of the automated process should utilize the programmable control loops to optimize compaction forces and introduce multi-layered dosing strategies. By refined scheduling of the 12 compaction cycles (calculated as one compaction every second treatment for the first 48 h) and adjusting deposition rates, internal porosity could be systematically reduced. Such a reduction in water absorption is targeted to potentially improve the material’s resistance to weathering and frost should the bio-tiles be used outdoors.

The design chart in Figure 4 shows that multiple bio-tiles that were supplemented with nutrient broth, additional CaCO 3 seeds as well as magnesium, meet the breaking strength and modulus of rupture targets for extruded tiles with a water absorption of greater than 10%.

Water absorption of tiles is classified into tiles exceeding a water absorption of 10%, those between 6% and 10%, between 3% and 6% and those with a water absorption of less than 3%. Each class has use in different applications. For example, bathrooms require tiles with almost no water absorption, such as vitrified tiles. All but one of the bio-tiles were produced with a water absorption exceeding 9.6%, as with bio-tiles produced with the submersion technique (Horn et al., 2023), and therefore do not have as broad applicability as bio-tiles produced with the pumping technique (Horn et al., 2024). These bio-tiles could therefore be used for wall and floor tiles in more general areas of buildings, especially where bespoke products with interesting aesthetics would be appreciated.

Bio-slurry was found to be beneficial to the formation of bio-tiles and improved solidification, when liquid bacteria solution was used instead, bio-tiles were not fully formed and had two distinct layers that were not bio-cemented together (Figure 5A). Bio-slurry and liquid bacteria both deliver the bacteria throughout the bio-tile, bio-slurry offers an additional benefit of providing CaCO 3 seeds within the sand matrix which lowers the need for nucleation of CaCO 3 crystals, allowing them to form more easily exactly where they are needed. Finally, the CaCO 3 -encapsulated bacteria also prevents wash out, ensuring continued reaction within the bio-tile (Nething et al., 2020).

While additional seeds had minimal effect on the breaking strength of bio-tiles produced with the prototype, aesthetically, they did aid in overall solidification of bio-tile and formation of CaCO 3 at the corners and edges. Figures 5B–D were all grown with geotextiles without seeds. However, even the strongest bio-tiles produced with magnesium require improvement to dimensional and surface quality (Figure 5E), compared to those produced with the pumping technique (Horn et al., 2024). Therefore, the addition of extra seeds is still recommended for bio-tiles produced with this automated technique. It is speculated that the additional seeds at the edges and corners allowed precipitation to form there better than without through improved support of the edges with the seeded geotextile being more structurally supportive with the CaCO 3 deposits.

Conceptualising scalability

The prototype was designed to be automated and modular. While this prototype had a single reaction chamber, it was postulated that it could be configured to have an array of reaction chambers along the rails that are serviced by a row of showerheads corresponding to the number of reaction chambers in a row (Figure 6). This conceptualized modular array represents the technical roadmap required to achieve the high-throughput replication necessary for robust statistical validation of this bioprocess in industrial-scale applications. The showerheads would deposit row by row and return ‘home’. At the specified timing, the platforms would be raised for draining, and so on. This would allow scalability and time efficiency. This system has only been tested for thin materials, such as tiles, however, there is potential for larger bio-materials to be grown. Future research should investigate ways of layering such that larger bio-materials can be bio-cemented.

Based on the 8-day (192-h) treatment cycle, a modular configuration consisting of a 12-chamber array (as shown in Figure 6) would yield a batch of 12 bio-tiles every 8 days. This results in a steady-state production rate of approximately 45 tiles per month. From a resource perspective, a full 12-tile batch would automatically process approximately 97 L of cementation solution (calculated as 84 mL per treatment × 96 treatments × 12 chambers). This modular array represents the technical roadmap required to achieve the high-throughput replication necessary for robust statistical validation of this bioprocess in industrial-scale applications. Future research will focus on the capture and treatment of the resulting ammonium-rich effluent, potentially utilizing the recovered nitrogen for secondary fertilizer applications (Lambert and Randall, 2019), which aligns with the environmental objectives of MICP-based manufacturing.

A mechanically automated and modular MICP prototype was developed and represents an innovative approach to the integration of biochemical reaction engineering and mechanical systems, focusing on automation and modularity for bio-manufacturing. While providing a preliminary technical baseline, its design and operations were iterated such that bio-tiles that meet international breaking strength and modulus of rupture standards were produced using freeze-dried urease active bio-slurry. The gravity-driven process introduces stochastic variability across different parameters; however, the results demonstrate a distinct general trend where 0.3 M magnesium was found to have the greatest impact on the breaking strength of bio-tiles produced with the mechanically automated 3D printing technique. Magnesium supplementation served as a dominant factor observed in strength enhancement, increasing the breaking strengths of bio-tiles 3.3 times to 622 ± 84 N. Seeding with additional CaCO 3 crystals was found to have little effect on breaking strength, however, was important for improving the solidification on the bio-tiles at the corners and edges. Nutrient broth treatments were determined to be redundant for this specific system, as bio-tiles grown without daily nutrient broth-enriched cementation solution treatments were 12%–100% stronger. Multiple bio-tiles that were produced through the automated process supplemented with all three additives had breaking strengths that exceeded the ISO standard, for example, 681, 671 and 702 N and modulus of ruptures of 13.1, 12.6 and 11.9 N/mm 2 for and water absorptions of greater than 10%. The modularity and operational flexibility of this system has the potential to significantly contribute to the advancement of sustainable manufacturing systems, provided future iterations utilize the conceptualized modular array to achieve the high-throughput replication necessary for robust statistical validation.