# Changes in Soil Chemical Properties Due to Long-Term
## 4 Discussion  
The long-term fertilization with compost and NPK+Compost containing rice straw and cattle manure altered the soil chemical properties of the paddy fields. These significant changes, particularly soil OM and nutrient contents under long-term fertilization, have also been observed in previous studies [11; 35; 36; 37; 38; 39]. The increase in OM was mainly attributed to the significant increase in carbon input [40]. In the first year of organic treatment in the study by [PERSON] et al. [41] OM and N were seen to increase by 0.67% and 0.64%, respectively. However, under long-term simulation, there is a net accumulation of OM and residual N concentrations, generally increasing the overall OM and N contents in paddy fields even after the end of the growing season. Labile and dissolved organic carbon pools also increased in the current study. This is in agreement with other studies, where amending paddy fields with organic fertilizers provided a potent source of labile C content rich in nutrients; thereby building higher labile organic C [12; 42; 43], including organic acids [10; 12]. For instance, a continuous manure application increased OM, DOC and various nutrient contents by 1.4-3.9 times higher than the unfertilized field [11]. The results of the current study showed that _mcrA_ and _pmoA_ gene abundance significantly increased in both Com and NPKCom treatments relative to the unfertilized paddy fields. In most cases, methane emission is positively correlated to the abundance of methanogens or the _mcrA_ gene [11; 15; 44; 45]. At the tillering stage, where methane emission was previously observed to be high in the study of [PERSON] et al., [11], _mcrA_ gene abundances were all significantly higher in paddy fields with continuous manure application by 17-55 times than the unfertilized field and resulted in higher methane pro  
Figure 3: Phylogenetic tree showing the relationship between the partial _pmoA_ DNA sequences of methane oxidizing bacteria determined in this study with their closest identified sequences retrieved from GenBank. Bootstrap values are shown at the branch points. The tree is drawn to scale, with the branch lengths in the same unit as those of the evolutionary distances used to infer the phylogenetic tree. duction potential. In addition, the abundance of methanotrophs and methane oxidation related genes have been observed to correlate with methane emission or an abundance of methanogenesis-related genes [10; 45; 46; 47]. The significant increase in both _mcrA_ and _pmoA_ gene abundance in compost-fertil fallow paddy fields in the current study indicated their potential for enhanced methanogenesis and methane oxidation. Multiple factors affect methanogenesis, the abundance of the _mcrA_ gene and methane oxidation, or the abundance of the _pmoA_ gene [4; 8]. In the current study, the significant differences in soil chemical properties between the unfertilized and compost-fertilized paddy fields and the stronger PCA main loading factors attributed to TN, K, OM, Ca, pH and DOC implied that these variables cumulatively affected the abundance of the _mcrA_ and _pmoA_ genes. Under long-term fertilization, these variables were the most important factors affecting methanogenic, methanotrophic and microbial communities [10; 11; 38; 44], and methane emission [48]. The increase in the nutrient content of paddy soils could relieve the nutrient limitation leading to an overall increase in microbial population; or the increased OM and labile DOC drives the enhanced activity of methanogenic archaeal communities, consequently increasing the _mcrA_ gene abundance. These situations were similarly observed in other studies, where the increase in OM, TN, labile C pools, and other soil nutrients led to increased microbial diversity and abundance [38; 40; 49]. These scenarios could also be true for methane-oxidizing bacterial communities.