Abundance and community composition of methanotrophs in a Chinese paddy soil under long-term fertilization practices

Background, aim, and scope As the second most important greenhouse gas, methane (CH4) is produced from many sources such as paddy fields. Methane-oxidizing bacteria (methanotrophs) consume CH4 in paddy soil and, therefore, reduce CH4 emission to the atmosphere. In order to estimate the contribution of paddy fields as a source of CH4, it is important to monitor the effects of fertilizer applications on the shifts of soil methanotrophs, which are targets in strategies to combat global climate change. In this study, real-time polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) based on 16S rRNA and pmoA genes, respectively, were used to analyze the soil methanotrophic abundance and community diversity under four fertilization treatments: urea (N), urea and potassium chloride (NK), urea, superphosphate, and potassium chloride (NPK), and urea, superphosphate, potassium chloride, and crop residues (NPK+C), compared to an untreated control (CON). The objective of this study was to examine whether soil methanotrophs responded to the long-term, different fertilizer regimes by using a combination of quantitative and qualitative molecular approaches. Materials and methods Soil samples were collected from the Taoyuan Experimental Station of Agro-ecosystem Observation at Changde (28 degrees 55' N, 111 degrees 26' E), central Hunan Province of China, in July 2006. Soil DNAs were extracted from the samples, then the 16S rRNA genes were quantified by real-time PCR and the pmoA genes were amplified via general PCR followed by DGGE, cloning, sequencing, and phylogenetic analysis. The community diversity indices were assessed through the DGGE profile. Results Except for NPK, other treatments of N, NK, and NPK+C showed significantly higher copy numbers of type I methanotrophs (7.0-9.6x10(7)) than CON (5.1x10(7)). The copy numbers of type II methanotrophs were significantly higher in NPK+C (2.8x10(8)) and NK (2.5x10(8)) treatments than in CON (1.4x10(8)). Moreover, the ratio of type II to type I methanotrophic copy numbers ranged from 1.88 to 3.32, indicating that the type II methanotrophs dominated in all treatments. Cluster analyses based on the DGGE profile showed that the methanotrophic community in NPK+C might respond more sensitively to the environmental variation. Phylogenetic analysis showed that 81% of the obtained pmoA sequences were classified as type I methanotrophs. Furthermore, the type I-affiliated sequences were related to Methylobacter, Methylomicrobium, Methylomonas, and some uncultured methanotrophic clones, and those type II-like sequences were affiliated with Methylocystis and Methylosinus genera. Discussion There was an inhibitory effect on the methanotrophic abundance in the N and a stimulating effect in the NK and NPK+C treatments, respectively. During the rice-growing season, the type II methanotrophs might be more profited from such a coexistence of low O-2 and high CH4 concentration environment than the type I methanotrophs. However, type I methanotrophs seemed to be more frequently detected. The relatively complex diversity pattern in the NPK+C treatment might result from the strong CH4 production. Conclusions Long-term fertilization regimes can both affect the abundance and the composition of the type I and type II methanotrophs. The inhibited effects on methanotrophic abundance were found in the N treatment, compared to the stimulated effects from the NK and NPK+C treatments. The fertilizers of nitrogen, potassium, and the crop residues could be important factors controlling the abundance and community composition of the methanotrophs in the paddy soil. Recommendations and perspectives Methanotrophs are a fascinating group of microorganisms playing an important role in the biogeochemical carbon cycle and in the control of global climate change. However, it is still a challenge for the cultivation of the methanotrophs, although three isolates were obtained in the extreme environments very recently. Therefore, future studies will be undoubtedly conducted via molecular techniques just like the applications in this study.