Evaluation of different strategies for efficient sporulation and germination of the MICP bacterium Lysinibacillus sphaericus strain MB284 (ATCC 13805)

Abstract

AbstractEnvironmental harsh conditions are one of the main challenges to the survivability of bacteria during microbially induced calcium carbonate precipitation (MICP) process. Due to the high resistivity of endospores against inhospitable conditions in comparison with vegetative cells, different sporulation methods were applied to Lysinibacillus sphaericus strain MB284 by changing the environmental conditions to investigate the growth of germinated cells. It was found that the sporulation yield was more when both carbon source starvation and the thermal shock process were applied to this bacterium. In addition, extending the sporulation time of cells into the minimal medium at 2 °C for a couple of weeks had a great impact on improving the sporulation yield. Comparing the growth rate of germinated endospores in natural conditions (pH 7 and 25 °C) and harsh conditions (pH 12, temperature of -10 to 60 °C, salinity up to 100 g/l) showed that endospores generated by thermal shock are able to germinate in almost every inhospitable condition except at low pH (∼3). Finally, exposing generated endospores before germination to harsh conditions (carbon source starving, high and low pH and temperature, and desiccation) for a nearly long period (to 100 days) showed that only low pH(∼3) had a negative effect on the germination process and bacterial growth curve that indicated endospore of strain MB284 can be an appropriate solution for the problem of the survivability of bioagents in MICP. These results will provide helpful information about preparing and applying endospores of L. sphaericus for crack healing in concrete.ImportanceIn the bio-self-healing process, bacterial cells are responsible for the production of calcium carbonate to fill cracks in the concrete. Since cracks can happen at any time, cells must survive under harsh conditions in concrete for a long period. This study for the first time investigates different endosporulation methods to find the best well-formed endospores for microbial-inducing calcium carbonate precipitation (MICP). This study shows that the endospores of strain MB284 formed by the thermal shock can survive under inhospitable conditions including different ranges of temperatures (−4 to 60 °C), pH (3 to 14), salinity (up to 100 g/l), and starvation for about 100 days. Furthermore, the bacterial growth rate and the kinetics of calcium carbonate production by germinated endospores and vegetative cells were similar to each other that indicate endospores of strain MB284 formed by the thermal shock method developed in this study are good candidates for the MICP process.Abstract Figure