Metagenomic analysis of pro- and eukaryotic components of microbiota of biologically active preparation bags

The vast majority of microbial preparations are based on pure cultures of microorganisms. The use of such preparations is quite reasonable and effective for such functions as nitrogen fixation in symbiosis of rhizobia with legumes or biocontrol properties of bacilli. However, when it comes to soil processes that determine the cycle of biogenic elements, in particular the transformation of plant residues, the microbial preparations based on pure cultures are not stable and sufficiently effective, even when consortia containing a mixture of several microbial species are used. This is due to the fact that microbiological processes in soil are carried out not by individual species, but by microbial communities with complex network organization. Therefore, a more effective alternative is the mobilization of native cellulose-degrading communities in the form of microbial preparations. This is exactly the case with the microbial preparation BAGS (biologically active substrate on straw), the development of the precursor of which began at the All-Russian Research Institute of Agricultural Microbiology in the 1930s. In the present work the metagenomic data on the taxonomic composition of prokaryotic and eukaryotic communities of two consecutive batches of BAGS preparation, one of which acted as an inoculum, are presented for the first time. In addition, comparison of the microbial composition of mature BAGS, the major components used in its preparation (peat, straw) and soil as a source of humate-degrading bacteria made it possible to suggest the origin of major prokaryotic components of the microbiota of the preparation. The aim of the work was to use a metagenomic approach to study the taxonomic composition of the microbial consortium in two batches of BAGS and components for its production. We used ready mature BAGS microbial preparation obtained by composting peat-silage-mineral mixture with inoculum for 11 months which was the last of a series of perennial periodic renewals for at least 20 years, and mature BAGS from the previous 15-month composting that served as inoculum. Lme-softened sphagnum peat (village of Fornosovo, Leningrad Province) was 15 % decomposed, dry oat straw was shredded; soil was sod-podzolic light loamy (Belogorka settlement, Leningrad Province). For BAGS preparation, the peat was added with dolomite meal to pH 6.4, oat straw (10 % of peat weight), Azophoska (10 g of nitrogen/kg of straw) and inoculum (20 % of peat weight). The mixture was moistened to 60 % of full moisture capacity, mixed, packed into plastic 90 l containers and incubated for 11 months at 28 °С, periodically moistening and mixing. Microbiological analysis, including bacterial and fungal components, was performed by deep sequencing of the 16S rRNA gene (prokaryotes) and ITS (fungi) amplicon libraries. Data were analyzed using the dada2 package, and basic analyses (alpha and beta diversity, bar graphs and heatmaps) were performed using the phyloseq package and tidyverse in the R software environment. PERMANOVA analysis was performed using the vegan package. According to the analysis of the taxonomic composition of BAGS, the main components of bacterial communities of both batches of BAGS preparation, one of which acted as an inoculum, belonged to representatives of phyla Pseudomonadota (27-32 %), Planctomycetota (11-14 %) and Bacteroidota (10-11 %), and the main components of fungal communities were representatives of classes Sordariomycetes, Mucoromycetes and Leotiomycetes . By the end of the composting period, the bacterial part of the BAGS community appeared to be more stable than the fungal one and very close to the composition of the inoculum, while the eukaryotic communities were influenced by the peat microbiome. Meanwhile, years of BAGS reproduction have shaped its own unique microbiome. Approximately 12 % of all taxa in BAGS were unique and specific to it, and the microbial mass of unique taxa in the preparation community accounted for 93 % of their total number.