Revealing nutritional requirements of MICP-relevant Sporosarcina pasteurii DSM33 for growth improvement in chemically defined and complex media

Microbial induced calcite precipitation (MICP), particularly ureolysis-based MICP, offers promising applications in various fields, including soil reinforcement, construction material restoration (limestone, concrete), metal and radionuclide remediation, and CO2 sequestration. Ureolysis is particularly attractive because it directly produces carbonate and ammonium ions upon urea hydrolysis, increasing pH and facilitating calcium carbonate (calcite) precipitation in the presence of calcium ions. Sporosarcina pasteurii (formerly Bacillus pasteurii) is widely used for MICP due to its high urease activity, endospore formation, and alkaliphilic/halophilic properties, making it suitable for applications on construction materials like concrete. Despite its importance, S. pasteurii cultivation is almost exclusively performed in complex media (yeast extract or peptone), yielding only moderate biomass concentrations (OD600 below 5). This limitation hinders the economic feasibility of MICP for industrial applications. Complex media complicate the identification of growth-limiting substrates or inhibitory components due to their undefined composition, which varies greatly among different batches and suppliers. Chemically defined media, in contrast, allow for straightforward identification of auxotrophic deficiencies and nutritional requirements, facilitating optimization of cultivation performance. While some information on the auxotrophic deficiencies and nutritional requirements of S. pasteurii (amino acids, thiamine, biotin, nicotinic acid, ammonia, glutamine) exists, variability among strains is reported. This study focused on the detailed investigation of the nutritional requirements and auxotrophic deficiencies of S. pasteurii DSM33, utilizing a high-throughput online-monitoring microbioreactor system (BioLector) for efficient screening of nutrient demands. This involved systematically omitting or substituting medium components, identifying useful carbon sources, and ultimately applying the findings to improve both chemically defined and inexpensive complex media for enhanced biomass production. The improved media were then assessed for their impact on oxygen transfer rate (OTR) and bacterial growth using shake flask cultivation with online monitoring.

Numerous studies have explored MICP and its applications. Rahman et al. (2020) provide a state-of-the-art review of MICP and its sustainability. de Muynck et al. (2010) review microbial carbonate precipitation in construction materials. Other research highlights the use of MICP for heavy metal removal (Jalilvand et al., 2020), radionuclide remediation (Brookshaw et al., 2012), and CO2 sequestration (Okyay et al., 2016). The metabolic pathways involved in carbonate precipitation are diverse, but ureolysis is particularly promising due to its direct carbonate production (Mondal & Ghosh, 2019). S. pasteurii stands out for its high urease activity (Zhu & Dittrich, 2016), making it a popular choice for MICP studies focused on increasing urease activity or growth (Ma et al., 2020; Omoregie et al., 2017; Williams et al., 2016; Achal et al., 2009). Most cultivation protocols use complex media (van Paassen, 2009; Cuzman et al., 2015; Kahani et al., 2020), with some employing wastewater as a low-cost alternative (Cuzman et al., 2015). However, published media typically result in low bacterial growth, highlighting the need for efficient cultivation protocols to make MICP economically viable (Silva et al., 2015; Omoregie et al., 2019; Mujah et al., 2017). The use of complex media hinders systematic process optimization due to their undefined and variable composition (Aller et al., 2014; Diederichs et al., 2014). Chemically defined media offer advantages for identifying growth limitations and auxotrophies (Kim et al., 2012; Chervaux et al., 2000; Müller et al., 2018). Previous studies have described some amino acid, thiamine, biotin, nicotinic acid, ammonia, and glutamine requirements for S. pasteurii, but strain-to-strain variation exists (Knight & Proom, 1950).

Sporosarcina pasteurii DSM33 was obtained from the DSMZ. Shake flask cultivation was performed in 250 mL flasks at 30°C, 200 rpm, with online monitoring of bacterial growth via backscatter measurements. Cryopreservation involved harvesting cells at the exponential growth phase, adding 15% glycerol, and freezing at -80°C. Precultivation involved inoculating chemically defined medium with cells from the glycerol stock to achieve an OD600 of 0.05-0.1. The BioLector I system, combined with 48-well microplates and DO optodes, was used for microplate cultivation at 30°C (1200 rpm, 3 mm shaking diameter, 800 µL volume). The system monitored bacterial growth via backscatter (620 nm) and dissolved oxygen. Experiments involved omitting individual or groups of medium components to determine nutritional requirements and auxotrophies. The chemically defined medium's composition (Table 1) included glucose, sodium acetate, K2HPO4, urea, NaCl, (NH4)2SO4, trace elements, various amino acids (grouped according to Müller et al., 2018), and vitamins. Experiments to identify useful carbon sources involved replacing glucose with other substrates (10 g/L). Shake flask cultivations (250 mL flasks, 10 mL volume, 30°C, 300 rpm) using the KuhnerTOM system monitored OTR and backscatter to analyze cultivation performance in various media. Optical density at 600 nm (OD600) was measured using a microplate reader, and cell dry weight (CDW) was calculated using a predetermined correlation with OD600. Urease activity was determined using a conductivity assay. All chemicals were of analytical grade.

Systematic omission of medium components in chemically defined medium revealed that S. pasteurii DSM33 requires phosphate and trace elements for growth. While ammonia is often reported as essential, urea could substitute it due to the organism's ureolytic activity. Omitting amino acids resulted in growth deceleration, indicating the importance of amino acid availability. Further analysis pinpointed L-cysteine and L-methionine as auxotrophic requirements. Genome analysis of a related strain (BNCC337394) corroborated the absence of genes responsible for sulphate assimilation and reduction for L-cysteine and L-methionine biosynthesis. The strain also exhibited auxotrophy for thiamine and nicotinic acid but not for biotin, consistent with previous observations on strain variability. A linear growth phase observed in the chemically defined medium was found to be due to a limitation of glutamate group amino acids. Supplementing the chemically defined medium with these amino acids (tripled concentration) significantly improved growth (51% increase in OD600, reaching 17.5 ± 1.2). Further experiments using different carbon sources indicated that glucose, maltose, lactose, fructose, sucrose, acetate, L-proline, and L-alanine were all usable, though glucose remained the most efficient. Supplementing two commonly used complex media (CaSo and YE) with the identified essential components resulted in a fivefold increase in optical density at the end of cultivation. The best results were achieved with the improved YE medium, yielding a maximum OD600 of 23.2 ± 0.6. Shake flask cultivation with online OTR monitoring showed a higher maximum OTR in the supplemented media. The improved YE medium reached a maximum OD600 of 26.6 ± 0.7, the highest reported for a S. pasteurii batch culture to date. While OD600 increased significantly, urease activity did not increase proportionally, leading to reduced specific urease activity in the improved chemically defined medium. However, supplementing the complex medium resulted in significantly higher specific urease activity.

This study successfully established optimal nutritional conditions for S. pasteurii DSM33 cultivation, leading to a significant improvement in biomass production. The identification of auxotrophic requirements and growth-limiting substrates in chemically defined media enabled the development of a highly effective chemically defined medium for high cell density cultivation. The findings were then successfully translated to commonly used complex media, resulting in substantial yield improvements. The increased biomass production is crucial for making MICP economically feasible for industrial applications. The observation of a linear growth phase, indicative of substrate limitation, emphasizes the importance of precisely controlling nutrient supply. The superior performance of the supplemented complex medium highlights the practical relevance of these findings. Further investigation is warranted to understand the interplay between nutrient supply and urease activity regulation, as well as to optimize urease production independently of biomass. This optimized cultivation approach has significant implications for the development and implementation of MICP-based technologies across various fields.

This research significantly advanced S. pasteurii cultivation by identifying critical nutritional requirements and auxotrophic deficiencies. The developed chemically defined and optimized complex media achieved record-high biomass yields. These findings hold promise for enhancing the economic viability and scalability of MICP applications. Future research should focus on further optimizing nutrient supply strategies to further improve biomass and urease activity, exploring different complex media and waste materials, and conducting biocementation experiments to evaluate the improved media in practical applications. Investigating the regulatory mechanisms controlling urease expression in the context of the defined medium could also lead to more effective strategies for enhanced MICP efficiency.

This study focused on a single strain of S. pasteurii (DSM33). The findings might not be directly generalizable to other strains, which could have different nutritional requirements. While analytical-grade chemicals were used in this research, the cost-effectiveness of using lower-grade chemicals should be investigated for large-scale applications. The correlation between OD600 and CDW was determined using a limited set of data. This correlation might need to be refined with more extensive data from various cultivation conditions. Only batch cultivations were performed. Future studies may benefit from exploring different cultivation strategies, such as continuous cultivation, to further enhance biomass and urease production.