# Changes in Soil Chemical Properties Due to Long-Term
## 4 Discussion
In addition to nutrient limitations, the activity of methane-oxidizing bacteria is mainly regulated by the availability of oxygen and methane and the presence of mineral nitrogen, where there is a strong positive correlation between the application of nitrogen fertilizer and methane oxidation [50; 51]. [PERSON] et al., [51] also observed that methanotrophic populations and methane oxidation rates were significantly stimulated by urea as a result of the relief of nitrogen constraint. PCA plot and k-means clustering performed in this study supported a grouping according to compost fertilization treatments. There were distinct and separate clusters of the different treatments under long-term compost fertilization, when soil chemical properties and gene abundance were used to assess variabilities. The PCA analysis showed distinct clustering mainly by the effects of PC1, PC2 and PC3, explaining up to 88.57% cumulative loading. The implications of these mean that all three treatments showed variabilities in soil chemical properties and the abundance of _mcrA_ and _pmoA_ genes. Similar changes in the paddy fields occur across studies where different types of fertilizers change multiple aspects of the soil in terms of the physical, chemical, or biological components [12; 38]. These changes occur over short-term fertilizer perturbations [10; 11; 44] or long-term fertilization [12; 15; 36; 40] and resulted in different paddy fields with particular soil chemical characteristics [38; 48]. The unfertilized paddy field in the current study was characterized by low nutrient soils with low abundances of _mcrA_ and _pmoA_ genes. The NPKCom and the Com treatments were generally characterized by soils with high nutrient contents and higher abundances of _mcrA_ and _pmoA_ genes with differences in pH and DOC content. Our results were consistent with previous studies where long-term compost fertilization resulted in paddy fields with high nutrient content with more diverse and abundant microbial communities [12; 38], which ultimately drive different soil biochemical processes, including methanogenesis and methane oxidation [8; 10; 44; 44]. To give an overview of microbial communities attributed to the _mcrA_ and _pmoA_ genes, clones of the genes were sequenced and reference sequences were obtained. Previous studies have observed that Methanobacteriaceae in anoxic rice paddics are associated with the rice roots and rhizosphere or on the bulk soil [52; 53; 54]. The results of the current study on the detection of Methanobacteriaceae could be attributed to straw amendments [55] and manure [56], whereby their input increased the diversity of methanogens in rice paddics with a predominance of the Methanobacteriaceae. Type I and type II methylotrophs occur in paddy fields with unsaturated water content and oxidize methane when conditions were suitable [57]. The predominance of type I MOB in the current study was consistent with other studies showing that long-term organic fertilization increased the abundance of type I MOB [36], while the abundance of type II decreased in manure and NPK+manure treatments [44]. Consequently, type I was found in paddy fields supplemented with fertilizers, while type II methanotrophs are generally found in native soils with low or no fertilization [58; 59], which is in line with the results of the current study, where more sequences of type I MOB were detected. The \(mcrA\) and \(pmoA\) gene sequences observed in the current study were mainly attributed to reference sequences known to respond well with compost or NPK+Compost application.