## 4 Discussion  
The addition of biochar generally enhances soil properties, including CEC, water retention, TC, or TN in soils. However, studies evaluating the first years of biochar applications and their relationship to the microbial community have received less attention than its influences on soil physicochemical properties ([PERSON], 2018; [PERSON], 2018). Our previous study showed that the first two years of pinewood biochar application to the sandy soil investigated experienced an 18% increase in TC, and showed signs of improvements in C sequestration and SOM dynamics ([PERSON], 2018). The TC increase in biochar incorporated (10%-Inc) in the 0-15 cm soils support claims reported in other studies that suggest a contributing role of biochar in C sequestration ([PERSON], 2018). The beneficial effect produced by the biochar amendments could be due to the stimulation of soil microorganisms' metabolism, consequently leading to increased cycling of nutrients trapped in biomass residue. This study provides an insight into the compositional and functional responses of the soil microbial community to biochar amended sandy soils. Our results indicated that the total FAMEs, an index of microbial community size, increased in the three biochar amended soil treatments by the second year (2019). Increased soil microbial community size in the pinewood biochar treatments after two years may be explained by the changes in SOM dynamics and soil aggregation (significant and positive correlation). After the first year, the soil health indicator of microbial transformations, C/N ratios, were higher in the biochar-treated soil than the control. This trend continued, being more pronounced in the second year, potentially due to biochar's microbial use as a substrate. However, elevated C/N ratios suggest slow transformation and recycling of organic matter ([PERSON] and [PERSON], 2018). Biochar contains a small amount of labile organic C, caused by  
Figure 4: Redundancy analyses (RDA) of the correlations between (**a**)-soil enzyme activities to soil properties, (**b**)- the correlations between soil enzyme activities and microbial community composition indicated by FAME indicators (mol%), and (**c**)-the correlations between soil properties and microbial community composition indicated by FAME indicators (mol%). AMF-Arbuscular mycorrhiza fungi; SaproFung-Saprophytic fungi; Actico-actinomycetes; GramNeg-Gram-negative; GramPos-Gram-positive; C/N-Total organic carbon to total nitrogen; TN-total nitrogen, TC-total carbon; POXC-permanganate oxidable carbon; moisture-gravimetric soil moisture; MWD-mean weight diameter; AS-arylsulfatase; NAG-\(\beta\)-glucosidaseaminidase; PME; acid phosphatase, and BG-\(\beta\)-glucosidase. the breakdown of the pyrolyzed material's original chemical structure [49]. However, the biochar's woody parent material has a C-rich structure (67.6%) with a high proportion of aromatic compound structures, explaining why it took until the second year for the effects to reach this detection level. The additional, high surface area of this type of biochar may adsorb water-nutrients such as soluble C that facilitate microbial colonization [50] and enhance soil aggregation [11]. The greater microbial abundance detected can contribute to increases in microbial extracellular polymeric substances (EPS), classified as polysaccharides that can heighten soil particles' aggregation and benefit plants by maintaining moisture and trapping nutrients [29; 51]. Slight changes in microbial composition occurred after the first year of the biochar additions. However, a significant change appeared after the second year. These results suggest that time is a critical factor controlling soil microbial biomass and composition [52]. In our research, both total bacteria and fungi increased similarly, but only gram-positive bacteria increased significantly with the addition of pine-biochar. Gram-positive bacteria play an essential role in the degradation of aromatic soil C and can utilize biochar C for growth [14; 53]. [PERSON] et al.