Fungal colonization and penetration of mortar as a suitable simulant for concrete: Implications for fungal biodeterioration in the built environment.

A range of fungal species showed variable abilities to colonize and penetrate a mortar substrate. Calcium biomineralization was a common feature with calcium-containing crystals deposited in the microenvironment or encrusting hyphae, regardless of the specific mortar composition. Several species caused significant damage to the mortar surface, exhibiting burrowing and penetration, surface etching, and biomineralization. In some cases, extensive biomineralization of hyphae, probably by carbonatization, resulted in the formation of crystalline tubes after hyphal degradation on mortar blocks, including those amended with Co or Sr carbonate. Ca was the only metal detected in the biomineralized formations with Co or Sr undetectable. Aspergillus niger, Stemphylium sp. and Paecilomyces sp. could penetrate mortar with differential responses depending on the porosity. Fluorescent staining of thin sections recorded penetration depths of ∼530 um for A. niger and ∼620 um for Stemphylium sp. Penetration depth varied inversely with porosity and greater penetration depths were achieved in mortar with a lower porosity (lower water/cement ratio). These results have provided further understanding of biodeteriorative fungal interactions with cementitious substrates that can clearly affect structural integrity. The potential significance of fungal colonization and such biodeteriorative phenomena should not be overlooked in built environment contexts, including radionuclide storage and surface decontamination.

Advances in microbial self-healing concrete: A critical review of mechanisms, developments, and future directions.

The self-healing bioconcrete, or bioconcrete as concrete containing microorganisms with self-healing capacities, presents a transformative strategy to extend the service life of concrete structures. This technology harnesses the biological capabilities of specific microorganisms, such as bacteria and fungi, which are integral to the material's capacity to autonomously mend cracks, thereby maintaining structural integrity. This review highlights the complex biochemical pathways these organisms utilize to produce healing compounds like calcium carbonate, and how environmental parameters, such as pH, temperature, oxygen, and moisture critically affect the repair efficacy. A comprehensive analysis of recently published peer-reviewed literature, and contemporary experimental research forms the backbone of this review with a focus on microbiological aspects of the self-healing process. The review assesses the challenges facing self-healing bioconcrete, including the longevity of microbial spores and the cost implications for large-scale implementation. Further, attention is given to potential research directions, such as investigating alternative biological agents and optimizing the concrete environment to support microbial activity. The culmination of this investigation is a call to action for integrating self-healing bioconcrete in construction on a broader scale, thereby realizing its potential to fortify infrastructure resilience and sustainability.

Evaluation of the microbial wet-vacuum system (M-Vac®) for DNA sampling from rough, porous substrates, and its compatibility with fully automated platforms.

DNA retrieval methods traditionally used during forensic evidence recovery including swabbing and tape lifting, can have limited effectiveness when used on porous, rough substrates such as bricks and carpet. This is possibly due to the DNA material being dispersed and unreachable for surface sampling techniques. In this evaluation we investigated the effectiveness of the Microbial Wet-Vacuum System (M-Vac®; M-Vac® Systems, Inc., Sandy, UT), as it has been reported to retrieve greater amounts of DNA material from challenging exhibits. A four-stage evaluation was conducted, starting with seeding carpet and brick substrates with a known donor's saliva in two dilutions and comparing the DNA recovery of tape lifting, swabbing, and the M-Vac®. A victim struggle scenario on carpet was then mimicked to compare trace DNA recovery by each method. Two mock scenarios were also conducted; a shirt was submerged in a creek bed for a period of five days to sample for the wearer's DNA, and a car boot was sampled to assess the possibility of recovering a victim's DNA amongst background DNA from the usual car occupants. Finally, the compatibility of the M-Vac® sampling process was optimised for the fully automated DNA lysis and extraction platforms used in the NSW (Australia) jurisdiction by comparing filter subsampling methods. The results from the study were mixed. For bricks, none of the collection methods were effective in retrieving DNA. On carpet, the M-Vac® retrieved the greatest quantities of DNA from the saliva-seeded samples, however, tape lifts outperformed all methods for 'touch' DNA recovery. The M-Vac® retrieved the greatest amount of DNA from the t-shirt recovered from a creek bed as it was able to retrieve the embedded DNA. The final mock case car boot scenario resulted in greater victim DNA recovery from tape lifts, with the M-Vac® more likely to recover mixtures too weak and/or complex to be interpreted. Finally, operational considerations regarding the compatibility of the M-Vac® system with fully automated DNA lysis and extraction are discussed. Considering the substantial time and cost to deploy the M-Vac®, it is recommended to be utilised in casework only after swabbing and tape lifting methods have failed to yield sufficient DNA material, where the substrate properties would likely benefit from the M-Vac's® niche capabilities for retrieving embedded DNA, and low levels of background DNA may be anticipated.

Comparison of calcium carbonate production by bacterial isolates from recycled aggregates.

The new technology of microbially induced calcium carbonate precipitation (MICP) has been applied in construction materials as a strategy to enhance their properties. In pursuit of solutions that are more localized and tailored to the study's target, this work focused on isolating and selecting bacteria capable of producing CaCO3 for posterior application in concrete aggregates. First, eleven bacterial isolates were obtained from aggregates and identified as genera Bacillus, Lysinibacillus, Exiguobacterium, and Micrococcus. Then, the strains were compared based on the quantity and nature of calcium carbonate they produced using thermogravimetric analysis, X-ray diffraction, and scanning electron microscopy with energy dispersive spectroscopy. Bacillus sp. dominated the cultured isolates and, along with Lysinibacillus sp., exhibited the highest CaCO3 conversion (up to 80%). On the other hand, Exiguobacterium and Micrococcus genera showed the poor ability to MICP (21.3 and 20.3%, respectively). Calcite and vaterite were the dominant carbonate polymorphs, with varying proportions. Concrete aggregates have proven to be a source of microorganisms capable of producing stable calcium carbonates with a high conversion rate. This indicates the feasibility of using microorganisms derived from local sources for application in construction materials as a sustainable way to enhance their characteristics.

Dominance by cyanobacteria in the newly formed biofilms on stone monuments under a protective shade at the Beishiku Temple in China.

Following the installation of a protective shade, rapid propagation of microorganisms showing in black and grey colors occurred at Beishiku Temple in Gansu Province of China. This study employed a combination of high-throughput sequencing technology, morphological examinations, and an assessment of the surrounding environmental condition to analyze newly formed microbial disease spots. The investigation unveiled the responsible microorganisms and the instigating factors of the microbial outbreak that subsequently to the erection of the shade. Through comparison of bioinformatics, the ASV method surpasses the OTU method in characterizing community compositional changes by the dominant microbial groups, the phylum Cyanobacteria emerged as the most dominant ones in the microbial community accountable for the post-shade microbial deterioration. The black spot and grey spot are predominantly composed of Mastigocladopsis and Scytonema, respectively. Validation analysis, based on the active RNA-level community results, supported and validated these conclusions. Comparative scrutiny of the microbial community before shade installation and the background environmental data disclosed that the erection of the shade prompted a decrease in temperatures and an increase in humidity within the protected area. Consequently, this spurred the exponential proliferation of indigenous cyanobacteria in the spots observed. The outcomes of this study carry considerable significance in devising preventive conservation strategies for cultural heritage and in managing the process of biodeterioration.

Impact of bacterial admixtures on the compressive and tensile strengths, permeability, and pore structure of ternary mortars: Comparative study of ureolytic and phototrophic bacteria.

The addition of bacterial biomass to cementitious materials can improve strength and permeability properties by altering the pore structure. Photoautotrophic bacteria are understudied mortar bio-additives that do not produce unwanted by-products compared to commonly studied ureolytic species. This study directly compares the impact of the addition of heterotrophic Bacillus subtilis to photoautotrophic Synechocystis sp. PCC6803 on mortar properties and microstructure. Cellulose fibers were used as a bacteria carrier. A commercial concrete healing agent composed of dormant bacterial spores was also tested. Strength, water absorption tests, mercury intrusion porosimetry, differential scanning calorimetry, thermogravimetric analysis, and scanning electron microscopy were applied to experimental mortar properties. The photoautotrophic modifications had a stronger positive impact on mortar strength and permeability properties than sporulated heterotrophic modifications due to differences in surface properties and production of exopolysaccharides. The findings provide support for photoautotrophic species as additives for mortars to move away from ammonia-generating species.

Exploring a high-urease activity Bacillus cereus for self-healing concrete via induced CaCO3 precipitation.

The structural integrity and esthetic appeal of concrete can be compromised by concrete cracks. Promise has been shown by microbe-induced calcium carbonate precipitation (MICP) as a solution for concrete cracking, with a focus on urease-producing microorganisms in research. Bacillus cereus was isolated from soil and employed for this purpose in this study due to its high urease activity. The strain exhibited strong tolerance for alkaline media and high salt levels, which grew at a pH of 13 and 4% salt concentration. The repair of concrete cracks with this strain was evaluated by assessing the effects of four different thickeners at varying concentrations. The most effective results were achieved with 10 g/L of sodium carboxymethyl cellulose (CMC-Na). The data showed that over 90% repair of cracks was achieved by this system with an initial water penetration time of 30 s. The study also assessed the quantity and sizes of crystals generated during the bacterial mineralization process over time to improve our understanding of the process. KEY POINTS: • MICP using Bacillus cereus shows potential for repairing concrete cracks. • Strain tolerates alkaline media and high salt levels, growing at pH 13 and 4% salt concentration. • Sodium carboxymethyl cellulose (CMC-Na) at 10 g/L achieved over 90% repair of cracks.

Effect of Bacillus subtilis on mechanical and self-healing properties in mortar with different crack widths and curing conditions.

This research aimed to investigate the effectiveness of Bacillus subtilis (B. subtilis) in self-healing cracks in concrete and enhancing concrete strength through microbial induced calcium carbonate precipitation (MICP). The study evaluated the ability of the mortar to cover cracks within 28 days, taking into account the width of the crack, and observed the recovery of strength after self-healing. The use of microencapsulated endospores of B. subtilis was also examined for its impact on the strength of concrete. The compressive, splitting tensile, and flexural strengths of normal mortar were compared to those of biological mortar, and it was found that biological mortar had a higher strength capacity. Microstructure analysis using SEM and EDS showed that bacterial growth increased calcium production, contributing to the improved mechanical properties of the bio-mortar.

Use of bacterial binder in repair mortar for micro-crack remediation.

Micro-cracks are one of the types of stone deterioration which can propagate and lead to surface detachments and larger cracks in the long run. The present study developed a sustainable and environmentally friendly infill material-biological mortar (BM), as an alternative to conventional approaches. Using a biomineralization approach, this BM was explicitly designed for healing micro-cracks (less than 2 mm) in historic travertines. To this end, the mortar was prepared using a calcifying Bacillus sp. isolated from thermal spring water resources in Pamukkale Travertines (Denizli), stone powder gathered from travertine quarries in the vicinity, and a triggering solution specifically designed to set off calcium carbonate precipitation reaction. After setup, BM was applied to micro-cracks of artificially aged test stones for testing. Scanning electron microscopy revealed calcium carbonate-coated Bacillus sp. bodies in the BM matrix, optical microscopy showed secondary calcite minerals throughout the BM applied micro-cracks, and stereomicroscopy and nanoindentation analyses demonstrated bonding of BM with stone due to microbial calcification activities. Furthermore, BM and original material contact showed a continuous and coherent structure in all samples. Within this context, BM could be considered a promising and alternative approach for the remediation of micro-cracks of historic stones. KEY POINTS: A binder was produced by the MICP of Bacillus sp. Pamukkale. Physical, mineralogical, and nanomechanical characterization demonstrated microbial calcite precipitates in BM. A significant bond was determined between the grains and matrix of BM due to Bacillus sp. calcite production activities.

Impact of the immobilized Bacillus cereus MG708176 on the characteristics of the bio-based self-healing concrete.

Novel carrier units were evaluated for their bio-healing benefits in our study to increase the efficacy of concrete healing. Bacillus cereus MG708176, an alkali-tolerant, calcite precipitating, endospore-forming strain was added as a bio-healing agent after its immobilization on wood ash units. A spore concentration of [1.3 × 107 spore/cm3] combined with 2.5% w/w urea was added to cement. Beams of 40 × 40 × 160 mm were used and tested for completely damaged mortar specimens after 7, 14, and 28 days of water treatment. Using wood ash bacterial mortars, totally destructed specimens were fully healed in all time intervals. Positive changes in concrete mechanical properties in bacterial wood ash treatment that were 24.7, 18.9, and 28.6% force for compressive, flexural, and tensile strengths more than control. The micro-images of the Scanning Electron Microscope (SEM) showed the dense concrete structure via calcite, Bacillafilla, and ettringite formation. Our results have shown improvements in the concrete healing efficiency and the mechanical concrete properties by filling the concrete cracks using a calcite-producing bacterium that is immobilized on wood ash units.

Performance of biological methods on self-healing and mechanical properties of concrete using S. pasteurii.

Biological methods (adding bacteria to the concrete mixtures) among the most recently investigated procedures increase the durability of concrete and repair concrete cracks. In the present study, different biological methods were used to heal the cracks of concrete and the most suitable method was subsequently introduced as the main aim of the research. For this purpose, the culture medium, various sources of calcium salts as bacterial nutrients, and the effect of air-entrained agent on the healing process were studied. The results showed that the use of bacterial nutrient inside the concrete mixes has an affirmative impact on the mechanical properties and self-healing characteristics of concretes. Simultaneous use of Sporosarcina pasteurii bacteria and calcium nitrate-urea or calcium chloride-urea as a bacterial nutrient in the concrete mixture increased the 28 days compressive strength of concretes by 23.4% and 7.5%, respectively. The utilization of bacterial cells, nutrients, and culture in the concrete mixture provided the ability to heal wide cracks where the healing time was significantly reduced (about 8 days). On the other hand, separation of the bacterial culture medium slightly reduced the self-healing performance of the concretes.

Performance Evaluation of Bio Concrete by Cluster and Regression Analysis for Environment Protection.

The focus of this research is to isolating and identifying bacteria that produce calcite precipitate, as well as determining whether or not these bacteria are suitable for incorporation into concrete in order to enhance the material's strength and make the environment protection better. In order to survive the high "potential of hydrogen" of concrete, microbes that are going to be added to concrete need to be able to withstand alkali, and they also need to be able to develop endospores so that they can survive the mechanical forces that are going to be put on the concrete while it is being mixed. In order to precipitate CaCO3 in the form of calcite, they need to have a strong urease activity. Both Bacillus sphaericus and the Streptococcus aureus bacterial strains were evaluated for their ability to precipitate calcium carbonate (CaCO3). These strains were obtained from the Department of Biotechnology at GLA University in Mathura. This research aims to solve the issue of augmenting the tension and compression strengths of concrete by investigating possible solutions for environmentally friendly concrete. The sterile cultures of the microorganisms were mixed with water, which was one of the components of the concrete mixture, along with the nutrients in the appropriate proportions. After that, the blocks were molded, and then pond-cured for 7, 28, 56, 90, 120, 180, 270, and 365 days, respectively, before being evaluated for compressibility and tensile strength. An investigation into the effect that bacteria have on compression strength was carried out, and the outcomes of the tests showed that bacterial concrete specimens exhibited an increase in mechanical strength. When compared to regular concrete, the results showed a maximum increase of 16 percent in compressive strength and a maximum increase of 12 percent in split tensile strength. This study also found that both bacterial concrete containing 106, 107, and 108 cfu/ml concentrations made from Bacillus sphaericus and Streptococcus aureus bacteria gave better results than normal concrete. Both cluster analysis (CA) and regression analysis (RA) were utilized in this research project in order to measure and analyze mechanical strength.

Strain Screening and Particle Formation: a Lysinibacillus boronitolerans for Self-Healing Concrete.

Microbial-induced calcite precipitation is a promising technology to solve the problem of cracks in soil concrete. The most intensively investigated microorganisms are urease-producing bacteria. Lysinibacillus that is used as urease-producing bacteria in concrete repair has rarely been reported. In this study, Lysinibacillus boronitolerans with a high urease activity was isolated from soil samples. This strain is salt- and alkali-tolerance, and at pH 13, can grow to ~OD600 2.0 after 24 h. At a salt concentration of 6%, the strain can still grow to ~OD600 1.0 after 24 h. The feasibility of using this strain in self-healing concrete was explored. The data showed that cracks within ~0.6 mm could be repaired naturally with hydration when spores and substrates were added to the concrete in an appropriate proportion. Moreover, the number and morphology of CaCO3 crystals that were produced by bacteria can be influenced by the concrete environment. An efficiency method to elucidate the process of microbial-induced calcium carbonate crystal formation was established with Particle Track G400. This study provides a template for future studies on the theory of mineralization based on microorganisms. IMPORTANCE The formation of calcium carbonate crystals in concrete by urease-producing bacteria is not understood fully. In this study, a Lysinibacillus boronitolerans strain with a high urease activity was isolated and used to analyze the counts and sizes of the crystals and the relationship with time. The data showed that the number of crystal particles increases exponentially in a short period with sufficient substrate, after which the crystals grow, precipitate or break. In concrete, the rate-limiting steps of calcium carbonate crystal accumulation are spore germination and urease production. These results provided data support for the rational design of urease-producing bacteria in concrete repair.

Analysis of mold and mycotoxins in naturally infested indoor building materials.

Health issues of residents of mold-infested housing are reported on a regular basis, and reasons for the arising impairments can be manifold. One possible cause are the toxic secondary metabolite produced by indoor microfungi (mycotoxins). To enable a more thorough characterization of the exposure to mycotoxins in indoor environments, data on occurrence and quantities of mycotoxins is essential. In the presented study, 51 naturally mold-infested building material samples were analyzed applying a previously developed method based on ultra-high performance liquid chromatography (UHPLC) separation in combination with triple-quadrupole mass spectrometry (TQMS) detection. A total of 38 secondary metabolites derived from different indoor mold genera like Aspergillus, Fusarium, Penicillium, and Stachybotrys were analyzed, of which 16 were detectable in 28 samples. As both the spectrum of target analytes and the investigated sample matrices showed high chemical varieties, an alternative calibration approach was applied complementary to identify potentially emerging matrix effects during ionization and mass spectrometric detection. Overall, strong alterations of analyte signals were rare, and compensation of considerable matrix suppression/enhancement only had to be performed for certain samples. Besides mycotoxin determination and quantification, the presence of 18 different mold species was confirmed applying microbiological approaches in combination with macro- and microscopic identification according to DIN ISO 16000-17:2010-06. These results additionally highlight the diversity of mycotoxins potentially arising in indoor environments and leads to the assumption that indoor mycotoxin exposure stays an emerging topic of research, which has only just commenced.

Physiological suitability of sulfate-reducing granules for the development of bioconcrete.

Regular monitoring and timely repair of concrete cracks are required to minimize further deterioration. Self-healing of cracks has been proposed as an alternative to the crack maintenance procedures. One of the proposed techniques is to use axenic cultures to exploit microbial-induced calcite precipitation (MICP). However, such healing agents are not cost-effective for in situ use. As the market for bio-based self-healing concrete necessitates a low-cost bio-agent, nonaxenic sulfate reducing bacterial (SRB) granules were investigated in this study through cultivation in an upflow anaerobic sludge blanket reactor. The compact granules can protect the bacteria from adverse conditions without encapsulation. This study investigated the microbial activities of SRB granules at different temperatures, pH, and chemical oxygen demand concentrations which the microbes would experience during the concrete casting and curing process. The attenuation and recovery of microbial activities were measured before and after the exposure. Moreover, the MICP yield was also tested for a possible use in self-healing bioconcrete. The results consistently showed that SRB granules were able to survive starvation, high temperature (50-60°C), and high pH (12), together with scanning electron microscope/energy dispersive spectrometry/X-ray diffraction analysis evidence. Microbial staining analysis demonstrated the formation of spores in the granules during their exposure to harsh conditions. SRB granule was thus demonstrated to be a viable self-healing nonaxenic agent for low-cost bioconcrete.

Attachment on mortar surfaces by cyanobacterium Gloeocapsa PCC 73106 and sequestration of CO2 by microbially induced calcium carbonate.

Cyanobacterial carbonate precipitation induced by cells and extracellular polymeric substances (EPS) enhances mortar durability. The percentage of cell/EPS attachment regulates the effectiveness of the mortar restoration. This study investigates the cell coverage on mortar and microbially induced carbonate precipitation. Statistical analysis of results from scanning electron and fluorescence microscopy shows that the cell coverage was higher in the presence of UV-killed cells than living cells. Cells are preferably attached to cement paste than sand grains, with a difference of one order of magnitude. The energy-dispersive X-ray spectroscopy analyses and Raman mapping suggest cyanobacteria used atmospheric CO2 to precipitate carbonates.

A critical review on microbial carbonate precipitation via denitrification process in building materials.

The naturally occurring biomineralization or microbially induced calcium carbonate (MICP) precipitation is gaining huge attention due to its widespread application in various fields of engineering. Microbial denitrification is one of the feasible metabolic pathways, in which the denitrifying microbes lead to precipitation of carbonate biomineral by their basic enzymatic and metabolic activities. This review article explains all the metabolic pathways and their mechanism involved in the MICP process in detail along with the benefits of using denitrification over other pathways during MICP implementation. The potential application of denitrification in building materials pertaining to soil reinforcement, bioconcrete, restoration of heritage structures and mitigating the soil pollution has been reviewed by addressing the finding and limitation of MICP treatment. This manuscript further sheds light on the challenges faced during upscaling, real field implementation and the need for future research in this path. The review concludes that although MICP via denitrification is an promising technique to employ it in building materials, a vast interdisciplinary research is still needed for the successful commercialization of this technique.

Extremophilic taxa predominate in a microbial community of photovoltaic panels in a tropical region.

Photovoltaic panels can be colonized by a highly diverse microbial diversity, despite life-threatening conditions. Although they are distributed worldwide, the microorganisms living on their surfaces have never been profiled in tropical regions using 16S rRNA high-throughput sequencing and PICRUst metagenome prediction of functional content. In this work, we investigated photovoltaic panels from two cities in southeast Brazil, Sorocaba and Itatiba, using these bioinformatics approach. Results showed that, despite significant differences in microbial diversity (p < 0.001), the taxonomic profile was very similar for both photovoltaic panels, dominated mainly by Proteobacteria, Bacteroidota and lower amounts of Cyanobacteria phyla. A predominance of Hymenobacter and Methylobacterium-Methylorubrum was observed at the genus level. We identified a microbial common core composed of Hymenobacter, Deinococcus, Sphingomonas, Methylobacterium-Methylorubrum, Craurococcus-Caldovatus, Massilia, Noviherbaspirillum and 1174-901-12 sharing genera. Predicted metabolisms focused on specific genes associated to radiation and desiccation resistance and pigments, were detected in members of the common core and among the most abundant genera. Our results suggested that taxonomic and functional profiles investigated were consistent with the harsh environment that photovoltaic panels represent. Moreover, the presence of stress genes in the predicted functional content was a preliminary evidence that microbes living there are a possibly source of metabolites with biotechnological interest.

Improvement of Biomineralization of Sporosarcina pasteurii as Biocementing Material for Concrete Repair by Atmospheric and Room Temperature Plasma Mutagenesis and Response Surface Methodology.

Microbially induced calcium carbonate precipitation (MICP) has recently become an intelligent and environmentally friendly method for repairing cracks in concrete. To improve on this ability of microbial materials concrete repair, we applied random mutagenesis and optimization of mineralization conditions to improve the quantity and crystal form of microbially precipitated calcium carbonate. Sporosarcina pasteurii ATCC 11859 was used as the starting strain to obtain the mutant with high urease activity by atmospheric and room temperature plasma (ARTP) mutagenesis. Next, we investigated the optimal biomineralization conditions and precipitation crystal form using Plackett-Burman experimental design and response surface methodology (RSM). Biomineralization with 0.73 mol/l calcium chloride, 45 g/l urea, reaction temperature of 45°C, and reaction time of 22 h, significantly increased the amount of precipitated calcium carbonate, which was deposited in the form of calcite crystals. Finally, the repair of concrete using the optimized biomineralization process was evaluated. A comparison of water absorption and adhesion of concrete specimens before and after repairs showed that concrete cracks and surface defects could be efficiently repaired. This study provides a new method to engineer biocementing material for concrete repair.

Characterization of a Novel CaCO3-Forming Alkali-Tolerant Rhodococcus erythreus S26 as a Filling Agent for Repairing Concrete Cracks.

Biomineralization, a well-known natural phenomenon associated with various microbial species, is being studied to protect and strengthen building materials such as concrete. We characterized Rhodococcus erythreus S26, a novel urease-producing bacterium exhibiting CaCO3-forming activity, and investigated its ability in repairing concrete cracks for the development of environment-friendly sealants. Strain S26 grown in solid medium formed spherical and polygonal CaCO3 crystals. The S26 cells grown in a urea-containing liquid medium caused culture fluid alkalinization and increased CaCO3 levels, indicating that ureolysis was responsible for CaCO3 formation. Urease activity and CaCO3 formation increased with incubation time, reaching a maximum of 2054 U/min/mL and 3.83 g/L, respectively, at day four. The maximum CaCO3 formation was achieved when calcium lactate was used as the calcium source, followed by calcium gluconate. Although cell growth was observed after the induction period at pH 10.5, strain S26 could grow at a wide range of pH 4-10.5, showing its high alkali tolerance. FESEM showed rhombohedral crystals of 20-60 µm in size. EDX analysis indicated the presence of calcium, carbon, and oxygen in the crystals. XRD confirmed these crystals as CaCO3 containing calcite and vaterite. Furthermore, R. erythreus S26 successfully repaired the artificially induced large cracks of 0.4-0.6 mm width.