Microbial phase analysis of soil created by Multiple Parallel Mineralization
The multiple parallel mineralization (MPM) method can promote both ammonification and nitrification because nitrifying bacteria and dystrophic microorganisms can be co-cultured in water or non-soil substrates even in the presence of organic matter. The purpose of this project was to clarify the difference in microbial phase between microbial inoculum and MPM-prepared cultures and soils. In this project, bark compost was used as a microbial inoculation source, and rock wool was used as a non-soil substrate. First, a tube filled with 100 mL of rock wool was prepared and rinsed with 100 mL of distilled water. Next, 1 g of bark compost was added as an inoculum, 1 mL of 10% w / v fish fertilizer diluted with distilled water was added as an organic matter, and the tube was incubated overnight at 25 Â° C in the dark. The next day, the tube was rinsed with 100 mL of distilled water and 1 mL of 10% w / v fish fertilizer was added again. Fish fertilizer addition, overnight incubation, and water rinsing were repeated daily for 3 weeks until nitrates were detected in the leachate. Rock wool granules carrying microbial communities were sent to the TechnoSuruga Laboratory (Shizuoka, Japan) for amplicon sequence analysis. Total DNA was extracted using the ISOIL for Beads Beating Kit (Nippon Gene Co., Ltd., Toyama, Japan). Total DNA was purified with the DNeasy PowerClean Pro Cleanup Kit (QIAGEN, GER). The V3-V4 region of bacterial and archaeal 16S rRNA was amplified using the Pro 341F / 805R primers and dual index method. Barcoded amplicons were paired-end sequenced on 2Ã?284-bp cycle using the MiSeq system with MiSeq Reagent Kit version 3 (600 Cycle) chemistry.Paired-end sequencing reads were merged using fastq-join program with default settings. Only joined-reads that had quality value score of 20 or greater for more than 99 % of the sequence were extracted using FASTX-Toolkit. The chimeric sequences were deleted with usearch6.1. Identification from sequences Analyses of sequence reads were performed manually using the Ribosomal Database Project (RDP) Multiclassifier tool, which is available from the RDP website (http://rdp.cme.msu.edu/classifier/). Bacterial species identification from sequences was performed using the Metagenome@KIN Ver 2.2.1 analysis software (World Fusion, Japan) and the TechnoSuruga Lab Microbial Identification database DB-BA 13.0 (TechnoSuruga Laboratory, Japan) with homology for 97 % or greater.We also analyzed and compared the microbial phase of bark compost and MPM liquid culture. To prepare the MPM liquid culture medium, 100 mL of sterile distilled water inoculated with 1 g of bark compost was placed in a flask, and fish fertilizer was placed in a flask to a final concentration of 1 g / L and cultured in an orbital shaker (120 rpm) 2 weeks at 25 Â° C in the dark. The culture was then centrifuged at 10000 xg for 5 minutes. Cell pellets were analyzed by TechnoSuruga Laboratory using the same method described above in this section.