## 4 Discussion
These studies underscore the versatility of biochar as a valuable tool in bioremediation and agricultural applications. By providing a stable surface for microbial attachment and improving microbial survival rates, biochar can significantly increase the effectiveness of microbial interventions in contaminated soils, wastewater treatment, and plant growth. Evaluating the adsorption properties of biochar is important when considering biochar as a functional ingredient. However, the maintenance of functional properties following the digestive process also needs to be assessed. This study focuses on the antimicrobial and prebiotic properties since the main goal was to evaluate biochar as a functional ingredient that could promote the beneficial microorganisms in the gut microbiota of animals and reduce the occurrence of diseases. Our results revealed that the in vitro digestion process could release functional compounds with modulating activity toward the microorganisms tested. Pathogens such as _E. coli_ F4+ and F18+ were inhibited without a negative influence on probiotics, such as _Lactobacillus_ sp. Using biochar as animal feed requires an in vitro study of simulated digestion in order to assess the potential interactions with the gut environment and ensure safety and efficacy [39]. In vitro studies are lacking on the effects of digested biochar on microorganisms. However, some in vitro studies have highlighted the functional properties of biochar, highlighting its antimicrobial and probiotic potential [21]. However, in vivo studies are also limited. [PERSON] et al., (2013) [PERSON] et al., (2013) [40] found a lower number of coliform bacteria in feces of pigs fed 0.3% of bamboo biochar, thus suggesting the possibility that biochar may have an effect on the proliferation of _E. coli_, which is the dominant microorganism responsible for inducing diarrhea in young pigs. Our results highlight the dual role of biochar in reducing pathogens and supporting beneficial microbes, thus making biochar a potential functional ingredient in animal feed. _Lactobacillus_ sp. or, in general, lactic acid bacteria (LAB) are used to prevent or treat intestinal infection or diarrhea caused by pathogenic bacteria through different mechanisms (e.g., stimulation of immunity, competition for nutrients, inhibition of epithelial and mucosal adherence, inhibition of epithelial invasion, and production of antimicrobial substances) [41]. The impact of probiotics can be augmented with the inclusion of biochar. This synergy exploits the adsorptive properties of biochar, which create a more favorable gut environment, in conjunction with the direct benefits of probiotics in strengthening the gut microbiota and improving gastrointestinal health. On the other hand, _Escherichia coli_ is a common pathogen in livestock farming, particularly in pig production, where it causes gastrointestinal diseases that impair animal growth and disrupt production cycles, leading to significant economic losses [22]. The results obtained in this study, although limited to the laboratory setting, highlight the potential of biochar as a functional ingredient, which could become a valuable addition to animal feedstuffs by helping to reduce bacterial infections. This, in turn, could reduce the need for antibiotics and alleviate the economic pressures caused by infectious diseases on livestock farms. However, the safety, efficacy, and practical operation of biochar need to be thoroughly evaluated in in vivo studies in order to verify its applicability in real-world settings.