Inoculation methods that enhance the survival of Bacillus strains in rice husk biochar for use as bacterial carriers

Storing biochar in soil is an effective method for improving the soil environment and reducing atmospheric greenhouse gases. Some strains of Bacillus species have been utilized as plant growth-promoting bacteria or biological control agents against plant diseases and pests. By colonizing biochar with beneficial bacteria, highly functional biochar that contributes to increased crop yields can be developed. In this study, we investigated the survival of type strains of eight Bacillus species (Bacillus amyloliquefaciens, B. licheniformis, B. nakamurai, B. pumilus, B. siamensis, B. subtilis, B. thuringiensis, and B. velezensis) in rice husk biochar, based on the sporulation state of vegetative cells or endospores. The bacterial density in the biochar significantly reduced when inoculated with a high concentration of vegetative cells, whereas it remained high when inoculated with a high concentration of spores. When water-washed biochar was inoculated with a high concentration of vegetative cells, survival significantly improved compared to that when unwashed biochar was inoculated. When a high concentration of vegetative cells was inoculated into the biochar water extract (biochar: water = 1:10), the bacterial densities of most species, except B. nakamurai and B. licheniformis, were significantly lower than those in alkali-sterilized water at pH 10.2 (same pH as biochar) and normal sterilized water (pH 6.7). These results suggest that the survival of Bacillus vegetative cells in biochar is inhibited by water-soluble inhibitors contained in the biochar rather than by alkaline conditions. When endospores were inoculated onto unwashed biochar, all strains maintained high viability for at least 12 weeks.