# Evaluating Soil Bacteria for the Development of New Biopreparations with Agricultural Applications
## 4 Discussion  
It is important to note that in Bacillus, phenotypic characters may vary within and between species, often leading to inconsistent species identification [46]. Standard MALDI-TOF MS is faster, but reliable differentiation within the \(B\). _cereus_ group is difficult using commercial databases [47], especially for closely related environmental isolates with metabolic profile variations in comparison to collection strains. This technique can be significantly improved with development and application of novel, genus, or group specific MALDI-TOF MS identification protocols or dedicated databases, containing reference mass spectra of well-characterized representatives of targeted microorganisms isolated from various environments. Species distinction by 16S ribosomal DNA gene sequencing also does not guarantee correct taxonomic assignment [48]. Alternative diagnostic approaches include specific PCR targeting toxins, virulence plasmids, and chromosomal markers. However, these also do not always provide completely satisfactory results [49,50]. Identification of Bacillus bacteria is therefore difficult and should be performed using different approaches to obtain the most reliable results. Bacteria from the \(B\). _cereus_ group are commonly found in soil and food and are often an etiological factor causing opportunistic food poisoning [50]. Therefore, the isolates from this group (WK-16 and WK-21) should not be used in biopreparations for industrial applications. Most of the tested bacterial strains preferred TSA medium and a growth temperature of 30 \({}^{\circ}\)C. However, some of the isolated soil bacteria (characterized by an OGT of 20 \({}^{\circ}\)C and the ability to grow at 10 \({}^{\circ}\)C) may possess adaptations to survive at lower temperatures. Many bacteria isolated from soil samples exhibit resistance to temperature fluctuation, which is regarded as one of the key properties for survival in seasonally changing and highly competitive environments. Such microorganisms are known to play an important role in reducing cold stress in plants, including crops, by improving the bioavailability of nutrients and minerals, reducing osmotic stress, and regulating phytohormones [51,52]. During the screening of enzymatic activity, most of the tested strains exhibited proteolytic, cellulolytic, and amyloid activity. However, no xylanolytic or ligninolytic activity was observed. The strains WK-14 and WK-15 exhibited relatively high cellulolytic and  
Figure 7: Cytotoxicity of soil bacteria metabolites after 24 h exposure to insect Sf-9 cells. Each data point represents the mean absorbance value from four replicates. Results are presented as mean \(\pm\) SD. IC50 (mg/mL), unless no IC50 was obtained. amyloidtic activities in all types of submerged cultures. Strain WK-15 also exhibited high xylanolytic and pectinolytic activity, but interestingly did not exhibit high amyloidity activity when grown on solid screening medium with starch or pectinolytic potential when grown with pectin. Similarly, no clear zones were observed around WK-14 colonies cultured on CMC- and starch-supplemented solid media during on-plate assays. This suggests that submerged cultivation with complex crop residues may support excretion of hydrolytic enzymes that were not detected by on-plate screening conducted with high-purity grade reagents. On the other hand, the broad hydrolytic potential exhibited during on-plate tests by strain WK-13 was not confirmed by the results of enzymatic activity assays in post-culture liquids. This may be linked to various factors, such as the media composition (e.g., carbon and nitrogen sources), different oxygenation levels in each cultivation method, and the presence of other biotic or abiotic inhibitors. During the saccharification of lignocellulosic substrates (such as wheat, tritticale, and winter barley residues) by microorganisms and their various enzymes, complex carbohydrates are broken down to oligo- and monosaccharides [53].