## 4 Discussion
Yet, spores from pathogenic species like _Clostridium botulinum_ and _Clostridium tetani_ are already present in the soil, suggesting that fertilization with digestate is unlikely to increase the risk of diseases caused by these organisms [33; 61]. Favorable growth conditions can emerge in aerobic soil through the formation of transient anaerobic micronuclehes, which offer a suitable environment for these obligate anaerobic bacteria. For instance, increased respiratory activity by one or more aerobic bacteria can create anaerobic conditions allowing obligate anaerobic bacteria to survive and operate effectively [62]. Nevertheless, an ongoing debate surrounds the potential risks associated with the repeated use of digestate as fertilizer. Interestingly, our observations indicate that the addition of biochar led to a substantial reduction in the presence of Clostrtiaceae, particularly those belonging to Cluster I and II. Similarly, decreases in the relative abundances of Clostrtiaceae due to biochar application were also observed in studies by [PERSON] et al. [63] and [PERSON] et al. [17]. This suggests that biochar may be effective in mitigating the impact of Clostrtiaceae resulting from the spread of digestate. While the mechanism by which biochar achieves this reduction is not fully clear, it could be related to an increase in soil aeration [64] that interferes with _Clostritium_ survival in the soil, or to the ability of biochar to facilitate electron transfer in the surrounding environment, acting as an electron mediator of exoelectrogenic microorganisms as proposed by several authors [9; 65; 66]. Further research is needed to explore the potential of biochar as a valuable tool in minimizing the impact of Clostrtiaceae in soil ecosystems influenced by digestate application. In terms of GHG emissions, the dosage of biochar played a crucial role in CO\({}_{2}\) emissions, resulting in a divergent trend with a significant increase at lower concentrations (2%) and a substantial reduction at higher concentrations (10%). In lower concentrations, biochar may stimulate microbial activity by serving as a substrate for microbial growth, influencing nutrient availability, and promoting microbial metabolism [67]. Conversely, the strong reduction in CO\({}_{2}\) emissions at higher biochar concentrations suggests the suppression of microbial metabolic activities or carbon turnover rates [68]. However, this observed divergent pattern underscores the importance of considering biochar dosage as a crucial factor in its impact on soil processes. In the soil, a consistent trend of CH\({}_{4}\) uptake is evident throughout the incubation period, indicating a prevailing microbial activity responsible for CH\({}_{4}\) consumption. This pattern is suggestive of the presence of methanotrophic microorganisms known for utilizing CH\({}_{4}\) as both C and energy source [48]. The biochar amendment, however, resulted in a general reduction in CH\({}_{4}\) uptake. Surprisingly, contrasting differences were noted in _pmaO_ gene abundance, a marker associated with methane oxidation processes. These results suggest that biochar application may have influenced the activity of methanotrophic microbial community rather than its abundance and dynamic. The observed pattern of CH\({}_{4}\) consumption appears to contradict the significant increase in _mcrA_ gene copy number in soil amended with digestate. The _mcrA_ gene is commonly used as an indicator of methane production potential [47]. However, the simultaneous prevalence of methanotrophs, aerobic microorganisms that oxidize CH\({}_{4}\), may have led to a net reduction in CH\({}_{4}\) emissions. This suggests that although digestate may increase CH\({}_{4}\)-producing microorganisms, the application of biochar might aid in aerating the soil and promoting the activity of CH\({}_{4}\)-consume microorganisms [14]. Furthermore, it is crucial to assess the persistence, encompassing both resistance and resilience, of the introduced microorganisms in the soil following digestate application.