ORIGINAL ARTICLE High level of calcium carbonate precipitation achieved by mixed culture containing ureolytic and nonureolytic bacterial strains P. Harnpicharnchai 1 , S. Mayteeworakoon 1 , S. Kitikhun 1 , S. Chunhametha 1 , S. Likhitrattanapisal 1 , L. Eurwilaichitr 2 and S. Ingsriswang 1 1 National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pahonyothin Road, Pathum Thani, Thailand 2 National Science and Technology Development Agency, Phahonyothin Road, Pathum Thani, Thailand Significance and Impact of the Study: This study characterized specific Bacillus and related species that are capable of microbial-induced calcium carbonate precipitation (MICP), especially at high pH and tem- perature. Furthermore, some of the selected microorganisms can generate moderately high levels of MICP even in the absence of urea. Thus, this may help lower the cost of the MICP process. Most impor- tantly, an especially high level of MICP is achieved with a mixed culture containing TBRC 1396 (Priestia megaterium), TBRC 8147 (Neobacillus drentensis) and ATCC 1159 (Sporosarcina pasteurii). The findings promote the utilizing microorganisms in MICP process as environment-friendly and sustainable technol- ogy for applications such as bioconcrete. Keywords Bacillus spp., microbially induced calcium carbonate precipitation, mixed-culture bacteria, nonureolytic bacteria, ureolytic bacteria. Correspondence Supawadee Ingsriswang, National Center for Genetic Engineering and Biotechnology, National Science and Technology Develop- ment Agency, 113 Thailand Science Park, Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120 Thailand. E-mail: supawadee@biotec.or.th 2021/LAMICRO-2021-0278.R2: received 8 October 2021, revised 13 May 2022 and accepted 17 May 2022 doi:10.1111/lam.13748 Abstract This study demonstrates a remarkably high level of microbial-induced calcium carbonate precipitation (MICP) using a mixed culture containing TBRC 1396 (Priestia megaterium), TBRC 8147 (Neobacillus drentensis) and ATCC 11859 (Sporosarcina pasteurii) bacterial strains. The mixed culture produced CaCO 3 weights 14 times higher than those obtained from S. pasteurii, the gold standard for efficient MICP processes. The three strains were selected after characterization of various Bacillus spp. and related species for their ability to induce the MICP process, especially in an alkaline and high-temperature environment. Results showed that the TBRC 1396 and TBRC 8147 strains, as well as TBRC 5949 (Bacillus subtilis) and TBRC 8986 (Priestia aryabhattai) strains, could generate calcium carbonate at pH 9–12 and temperature 30– 40°C, which is suitable for construction and consolidation purposes. The TBRC 8147 strain also exhibited CaCO 3 precipitation at 45°C. The TBRC 8986 and TBRC 8147 strains are nonureolytic bacteria capable of MICP in the absence of urea, which can be used to avoid the generation of undesirable ammonia associated with the ureolytic MICP process. These findings facilitate the successful use of MICP as a sustainable and environmentally friendly technology for the development of various materials, including self-healing concrete and soil consolidation. Introduction Microbially induced calcium carbonate precipitation (MICP) is a metabolic interaction between certain microorganisms and organic or inorganic compounds, resulting in the deposition of carbonate compounds in the environment (Castro-Alonso et al. 2019; Rahman et al. 2020). MICP has aided in the development of bio- concrete (self-healing concrete) and the improvement of concrete properties (Rajasekar et al. 2017). Bioconcrete is a promising sustainable technology that reduces the nega- tive environmental impact caused by CO 2 emissions and Letters in Applied Microbiology 75, 888--898 © 2022 The Society for Applied Microbiology. 888 Letters in Applied Microbiology ISSN 0266-8254 Downloaded from https://academic.oup.com/lambio/article/75/4/888/6989401 by guest on 20 January 2023