## 4 Discussion
[54] examined bacterial assimilation of 13C labeled biochar substrates and reported greater radiolabel incorporation within gram-positive bacteria. Additionally, gram-positive bacteria and saprotrophic fungi are generally responsible for SOM and litter breakdown, while gram-negative bacteria often fill a more specialized niche as nitrifiers or methane oxidizers [55]. The gram-negative bacteria and actinomycete groups in our peach-tree soils increased with biochar, but the differences were not significant. G+/G\(-\) ratios revealed the bacterial community shift to a more gram-positive bacteria-dominated community. Gram-negative bacteria use C sources that are relatively labile, while Gram-positive bacteria use C sources derived from soil organic matter that is more recalcitrant [56]. The initial addition of biochar may result in more significant soil recalcitrant C in the first year. After its breakdown and utilization by gram-positive bacteria, the remaining labile C source may have contributed to the increased detection of gram-negative bacteria during the second year [56]. With the slight increase in microbial biomass due to adding biochar to the study soils, the microbial diversity increased proportionality, particularly when 5%-Sur and 10%-Inc biochar were added. The effect of biochar on soil microbial communities is very complex. [PERSON] et al. [11] showed that biochar had a more significant impact on microbial community composition than microbial biomass. Our results showed that biochar has a differential impact on different bacterial types, e.g., the increase of gram-positive bacteria during the first year might to lead an increase in the Shannon diversity index. Additionally, one of the limitations of EL-FAME is the difficulty of differentiating dead microorganisms or plant origin residues [57]. The cyclopropyl fatty acids reportedly increases during the stationary phase of growth that follows substrate depletion in many species and is used as starvation stress indicators [58]. The cy/pre ratios in the biochar added samples were significantly lower than the control treatment for both years in the 0-15 cm soils (Table 2), indicating an actively growing microbial community, as evidenced by the increase in the total FAME indicators (Table 1). The PCA separated the biochar amended plots from the control treatments along the first axis, particularly in 2019. The separation appeared to be influenced by higher FAME indicator concentrations. RDA analysis of the microbial community showed a strong association with enzyme activities (Figure 4b), suggesting that the microbial community's functional activities drive the metabolic cycling status of nutrients in soils. The RDA analysis did not show a correlation between microbial communities and the soil physicochemical parameters (Figure 4c); however, the Pearson correlation did show a significantly positive correlation of TC, TN, and POXC (Figure 3). Several authors report that organic amendments influenced soil microbial communities' biomass and structure, and these changes were related to the soil C and N content [59]. Activities of microbial-derived soil enzymes mediate many rate-limiting nutrient cycling processes in soils and play essential roles in soil organic matter dynamics [60; 61]. Adding biochar has a significant effect on SOM decomposition depending on soil type, biochar origin, and pyrolysis temperature [6; 7]. Our results suggest that biochar inhibited EAs involved in carbon mineralization, particularly \(\beta\)-glucosidase. This enzyme strongly reflects C mineralization in soils and is considered a general index for evaluating soil microbial activity [62], and is often used to measure labile C's incorporation rate. The biochar amended soils had the lowest TN and POXC, and both soil attributes correlated directly with \(\beta\)-glucosidase activities (Figure 3). The decrease in labile C may also imply noticeable consumption by microorganisms. Similar decreases in \(\beta\)-glucosidase activity were reported [31; 32; 63].