# Evaluating Soil Bacteria for the Development of New Biopreparations with Agricultural Applications
## 3 Results  
### Isolation and Identification of Soil Isolates  
The bacteria were isolated from soil under a no-till farming system, where all crop residues were left in the field in the form of mulch after harvesting the main crop. The pH of the soil samples was slightly acidic or acidic (pH 3.9-5.7). The samples varied in elemental composition and contained 28.15-57.68% carbon, 0.09-0.30% nitrogen, 0.02-0.14% phosphorus, 4.56-8.55% hydrogen, and 0.02-0.69% sulphur. The average carbon to nitrogen (C:N) ratio was 259. The number of spore-forming bacteria ranged from 10\({}^{6}\) to 10\({}^{7}\) CFU/g (Table S1), there were not any statistically important connections between bacterial count and soil constitution. Based on morphological differences between the colonies on agar plates, 24 isolates were selected for identification and further study based on their macroscopic features: growth intensity, colony size, colony edges, color and pigment production, consistency, and texture. The spore-forming bacteria were isolated from plates with TSA medium (tryptic soy agar, Merck-Millipore, Poland) and spread on pure cultures.  
Different approaches were used to identify the bacteria isolated from soil. After basic diagnostic activities, API biochemical tests and the MALDI TOF-MS procedure were performed. However, these did not always give consistent or acceptable results. Therefore, 16S rDNA gene sequencing was performed on the strains with the greatest biotechnological potential.  
The vast majority of the isolated bacteria belonged to the genus _Bacillus_ (Table 1). Bacteria of the genera _Lysinibacillus_, _Paenibacillus_, and _Prestia_ were also identified. These bacteria constitute a large heterogeneous group within the phylum Firmicutes and order Bacillus.  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**No.** & **WK No.** & **Identification (Percentage Compliance, \%)** \\ \hline
1 & WK-1 & _Bacillus subtilis_ (99.8) \({}^{\text{A}}\) \\ \hline
2 & WK-2 & _Bacillus megaterium_ (99.5) \({}^{\text{A}}\)_/B. megaterium/cogulans_ (92.0) \({}^{\text{M}}\) \\ \hline
3 & WK-3 & _Bacillus_ sp. (82.0) \({}^{\text{M}}\) \\ \hline
4 & WK-4 & _Bacillus circulans_ (87.0) \({}^{\text{A}}\) \\ \hline
5 & WK-5 & _Bacillus megaterium_/cogulans_ (84.0) \({}^{\text{M}}\)_/Pristia megaterium_ (99.9) \({}^{\text{S}}\) \\ \hline
6 & WK-6 & _Solibacillus silvestris_ (48.6) \({}^{\text{M}}\) \\ \hline
7 & WK-7 & _Bacillus lentus_ (99.8) \({}^{\text{A}}\)_/B. megaterium/cogulans_ (88.0) \({}^{\text{M}}\) \\ \hline
8 & WK-8 & _Bacillus mycoides_ (83.9) \({}^{\text{A}}\)_/B. cereus group_ \({}^{\text{M}}\) (85.5) \\ \hline
9 & WK-9 & _Bacillus circulans_ (92.4) \({}^{\text{A}}\)/_Paenibacillus_ sp. (50.0) \({}^{\text{M}}\)/_Paenibacillus_ \\  & _amylolytica_ (99.9) \({}^{\text{S}}\) \\ \hline
10 & WK-10 & _Bacillus megaterium_ (99.8) \({}^{\text{A}}\)_/B. megaterium/cogulans_ (92.0) \({}^{\text{M}}\) \\ \hline
11 & WK-11 & _Bacillus_ sp. (82.5) \({}^{\text{M}}\) \\ \hline
12 & WK-12 & _Bacillus licheniformis_ (98.8) \({}^{\text{A}}\)/B. licheniformis_ (80.4) \({}^{\text{M}}\) \\ \hline \hline \end{tabular}
\end{table}
Table 1: Taxonomic affiliations of the tested soil bacteria.  
The strains WK-16 and WK-21, identified as strains of the food-borne pathogen _B. cereus_, were excluded from further studies. However, we also recognize that until the whole-genome sequencing of given _B. cereus_ isolates of interest and bioinformatical analysis for the toxin-encoding genes (_nheABC_, _hbICDA_, _entFM_, _cytK_) presence will be performed, one cannot be sure if the isolated _B. cereus_ representative is in fact a pathogenic strain. For that reason, we excluded the potentially dangerous strains from further study.