## 3 Results and Discussion
### In Vitro Microbiological Study
#### 3.2.2 Experiments with Substrates of Soil and Biochar  
The results of growth assays for the two selected strains (11C and 15C) on different substrates with varying soil/biochar ratios will be discussed below. The proportions used are those indicated in Table 1 (Section 2.3.4).  
For strain 11C at 15 days, the count in SB\({}_{10}\) was 3.92 \(\times\) 10\({}^{4}\) CFU and in SB\({}_{20}\) was 2.42 \(\times\) 10\({}^{4}\) CFU (Figure 7). At the same time, substrates S and B yielded very similar counts, around 1.02-1.04 \(\times\) 10\({}^{4}\) CFU. After 30 days of incubation, bacterial growth was also observed, but with differences depending on the substrate: SB\({}_{10}\) showed the highest count at 1.4 \(\times\) 10\({}^{4}\) CFU, followed by B with 1.11 \(\times\) 10\({}^{4}\) CFU. After 60 days, a significant decline in microbial count was observed across all substrates, likely due to nutrient depletion. Although the behaviour of _Bacillus cabrialesii_ at 60 days has not been specifically described in the literature, some _Bacillus_ strains have been reported to exhibit population decreases in soil under low-nutrient conditions [78].  
For strain 15C, the highest counts were also recorded after 15 days of incubation across all substrates (Figure 7). The highest value (1.787 \(\times\) 10\({}^{4}\) CFU) corresponded to substrate B. Elevated counts (8.2 \(\times\) 10\({}^{3}\) CFU) were also observed in substrate SB\({}_{20}\). After 30 days, the counts were reduced by approximately by half in all substrates compared to those observed after 15 days. In this case, substrate B again yielded the best results, with 8.56 \(\times\) 10\({}^{3}\) CFU. Over the same period, SB substrates also supported bacterial growth, though to a lesser extent. In contrast, in soil without biochar (substrate S), no bacterial growth was detected  
Figure 7: Microbial counts in substrates soil/biochar (CFU) vs. incubation time (days). Legend: S (soil 100%), SB\({}_{10}\) (soil/biochar 90/10%), SB\({}_{20}\) (soil/biochar 80/20%) and B (biochar 100%). (**A**) Strain 11C (_Bacillus cabrialesii_) and (**B**) strain 15C (_Pseudomonas neuropathica_).  
after 30 days. After 60 days, as observed with strain 11C, bacterial growth was negligible. This decline might be attributed again to nutrient depletion.  
Bacterial counts for the two strains (_Bacillus cabrialesii_ and _Pseudomonas neuropathica_) demonstrated their ability to colonise substrates of soil and biochar, with biochar exerting a positive influence on their growth. However, a notable difference was observed: strain 15C growth was considerably higher in substrates with pure biochar (B) at 15 days (805 CFU vs. 17.875 CFU, Figure 7), despite the high toxicity commonly reported for fresh biochar [72; 79].  
In recent studies, the combined application of biochar inoculated with _Bacillus subtilis_, nanomaterials, and proline was proven to enhance wheat's tolerance to salinity [80]. This approach might be promising, as biochar improves soil structure and water retention capacity, while PGP bacteria facilitate nutrient uptake and modulate plant hormonal responses (e.g., ethylene). The use of this biochar/PGP bacteria amendment can modulate the hormonal signalling pathways, thereby improving plants' ability to withstand water and salt stress related to drought and salinity [81].  
However, our growth trial was conducted under in vitro conditions, with specific moisture conditions and in the absence of other biotic and abiotic factors typically encountered in agricultural soils (in vivo). Consequently, further research focused on field and plant experiments is required.