Our study demonstrates a strong inhibition of dry/wet alternation on CH4
production and emission, similar to findings observed in previous studies (2
). To obtain a mechanistic understanding of the drainage effect, we analyzed soil biogeochemistry, as well as the abundance and composition of methanogenic community, in the samples from four soil compartments. To the best of our knowledge, this is the first comprehensive investigation of methanogenic functional genes and their transcription in association with CH4
emission and production. Furthermore, we analyzed the dynamics of these genes in the context of the turnover of ferric ion and sulfate and with other soil environmental factors in rice field soil.
In the permanent flooding treatment, the abundance of mcrA
gene copies was relatively stable over the growing period of rice plants (). This stability of methanogen populations has also been observed previously (19
). In the drainage treatment, however, the growth of methanogenic populations, as revealed by mcrA
gene abundance, was suppressed in four soil compartments during the second dry/wet cycle. The first dry/wet cycle did not significantly affect the mcrA
gene copies, probably due to insufficient drainage (27
). Apparently, methanogenic populations can survive the moderate drainage or aeration of paddy field soil.
However, the mcrA
transcripts in the DR treatment substantially decreased during both the first and the second dry/wet cycles. On the other hand, it stayed relatively stable in the FL treatment. In the first drainage, the mcrA
transcripts in the bulk and rhizosphere soils decreased by 2 orders of magnitude. The transcript/gene ratio decreased to close to zero on day 62 (b and c), indicating that the expression of the mcrA
gene was completely repressed by the drainage treatment. This inhibition coincided with the suppression of methane production (b). A recent study of peat soil showed that the transcript/gene ratio of the mcrA
gene correlated linearly with the CH4
). Hence, it may be a general characteristic that transcription of a functional gene is a more sensitive descriptor of activity in environmental samples than the abundance of the gene.
Ratios of mcrA transcript to gene in surface soil (a), bulk soil (b), rhizosphere soil (c), and washed root material (d) (n = 3).
Correlation analysis revealed that both mcrA
gene and transcript abundance were significantly correlated positively with methane production potentials, the concentration of extractable Fe(II), and the soil water content and negatively with the concentration of extractable sulfate in the bulk and rhizosphere soils. The correlation coefficients were more significant for the transcripts than the gene copies. This result indicated that the transcripts explained the inhibitory effect of dry/wet alternation on methanogenic dynamics better than the gene abundance. The negative correlations of mcrA
transcript and gene abundance with the concentrations of extractable Fe(III) and sulfate suggested that the iron or sulfate reducers activated by Fe(III) and sulfate regenerated and probably outcompeted the methanogens for substrates. In addition, O2
stress could also repress the mcrA
transcription, although it recovered to some extent after reflooding (52
Alternate dry/wet cycles did not influence the structure of the methanogenic community, a finding consistent with several earlier studies demonstrating the stable structure of methanogenic archaea in rice field soil under different conditions (17
). A recent study using the traditional culture method also showed no effects of water management on the methanogenic community (14
). However, DNA analysis and cultivation might only show the existence of methanogens and not their activity in rice field soil. In contrast, the T-RFLP profiles of the mcrA
transcripts showed that alternate dry/wet cycles altered the composition of the metabolically active methanogens.
The transcription of some methanogens such as Methanobacteriaceae
(T-RF of 504 bp), which were low in abundance under continuous flooding, was stimulated by drainage and reflooding. Of specific interest, the transcripts of Methanocellaceae
increased in abundance in the bulk soil at the end after the alternate dry/wet cycles. A recent study targeting mcrA
transcripts in Japanese rice field soil also showed that Methanocellaceae
survived better during the dry season (47
). Furthermore, the mcrA
transcripts of these organisms in the slurry incubation were more resistant to oxygen exposure than other methanogens (52
). It is currently unclear why Methanocellales
can cope with the soil aeration. However, according to the genome sequence available for one strain (9
), these organisms possess a unique set of genes encoding antioxidant enzymes and oxygen-insensitive fermentation enzymes, which probably facilitate them to be better adapted under alternate dry/wet conditions.
The rates of CH4
emission gradually increased with time and were highly correlated with the rates of CH4
= 0.978, P
< 0.001). These parameters were also highly correlated with the methane production potentials in soil compartments (i.e., bulk soil, rhizosphere soil, and roots) except for surface soil. Interestingly, the CH4
production potential in the FL treatment was greater in the rhizosphere and the washed root material than in the bulk soil, indicating that methanogenesis was active in these environments, albeit the potentially oxic conditions. This was in line with previous findings that root-derived substrates served as major carbon and energy sources for CH4
production despite the possibility of O2
release into the rhizosphere soil (24
). The temporal patterns of methane production potentials were very similar among the bulk soil, rhizosphere soil, and washed root material and were strongly inhibited by intermittent drainage. The dry/wet alternation also markedly reduced the concentration of Fe(II) [hence the increase in Fe(III)] and increased the concentration of sulfate in the soil. These results were in agreement with previous laboratory incubation experiments showing that short-term aeration or O2
exposure significantly increased the concentrations of Fe(III) and sulfate (34
). Correlation analyses indicated that methane production potential in all soil compartments was positively correlated with the concentration of Fe(II) [hence negatively with Fe(III)] and negatively correlated with the concentration of sulfate. These results indicated that alternate dry/wet cycling inhibited CH4
production because of the regeneration of Fe(III) and sulfate, which in turn allowed iron- and sulfate-reducing bacteria to outcompete methanogens for the substrates H2
In conclusion, our study showed that intermittent drainage significantly reduced the rates of CH4 production and emission in rice field soil by primarily suppressing transcription in some members of the methanogenic archaeal community, whereas the abundance of methanogens was comparatively little affected. The inhibitory effects were probably related to the regeneration of Fe(III) and sulfate, which activated iron and sulfate reducers and hence outcompeted methanogens for available substrates. Our study indicated that the analysis of functional genes at the mRNA level is more powerful than at the DNA level if the biogeochemical process is to be linked to microbial identity in the environments.