Comparison of Bacterial Community in Three Soils for Making Saltpeter, Kodo-hou, Baiyo-hou and Shousekikyu-hou

Objective: From the 16th to 19th centuries in Japan, saltpeter (potassium nitrate: KNO3), a major ingredient of gunpowder, was produced using three different methods: Kodo-hou, Baiyo-hou, and Shousekikyu-hou. We previously reported that Baiyo-hou had the advantage of affording high amounts and quality of saltpeter due to the available abundance of ammonium-nitrogen in the soil. In this study, we compared these three soils using metagenomic approach. Methods: We exhaustively analyzed the bacterial community in three soils conserved underground for 20 years: Kodo-hou, relic soil (Baiyo-hou of the Edo period) from underneath a Gassho residence, and cow manure substituted for Shousekikyu-hou, using 16S rRNA genes from soil bacteria. The nitrate ion (NO3-) concentration in the tested soils was also measured. Results and Discussion: A high concentration of NO3- was detected in the relic soil, cow manure and the soil from beneath the Gassho residence, while the soil from a general field was not. The metagenomic analyses of soils using 16S rRNA revealed that a similar abundance ratio of phyla was represented in both the Kodo-hou soil and cow manure when compared to the field soil. The profile of phyla in the Kodo-hou soil was extensively different from those of the other soils. The phylum Actinobacteria shared in the bacterial community of the Kodo-hou soil was 97%. The phylum Protobacteria shared in the bacterial communities was 30% from the field soil, 13% from relic soil and 46% from cow manure, respectively, while it was only 2% in the Kodo-hou soil. We further analyzed the genus level of soil bacteria that contributed to the nitrification and denitrification in the bacterial database. The genera Nitrospira, JG37-AG-70, Nitrobacter and Nitrosovibrio were identified as the bacteria for nitrification. These bacteria were found in the relic soil, cow manure and general field soil. No bacteria contributing to nitrification was detected in the Kodo-hou soil. The sequence read count of operational taxonomic units (OTUs) belonging to Nitrospira was much higher in Baiyo-hou soil than the other soils. The bacteria having the function of denitrification found in the soils were assigned to the general soils Rhodobacter, Pseudomonas, Paracoccus, and Bacillus. Taken together, the manufacturing of saltpeter might be a high biotechnological method for making saltpeter utilizing soil bacteria.