Effect of temperature on structure and function of the methanogenic archaeal community in an anoxic rice field soil

Soil temperatures in Italian rice fields typically range between about 15 a nd 30 degrees C. A change in the incubation temperature of anoxic methanoge nic soil slurry from 30 degrees C to 15 degrees C typically resulted in a d ecrease in the CH4 production rate, a decrease in the steady-state H-2 part ial pressure, and a transient accumulation of acetate, Previous experiments have shown that these changes were due to an alteration of the carbon and electron flow in the methanogenic degradation pathway of organic matter cau sed by the temperature shift (K, J, Chin and R. Conrad, FEMS Microbiol. Eco l, 18:85-102, 1995). To investigate how temperature affects the structure o f the methanogenic archaeal community, total DNA was extracted from soil sl urries incubated at 30 and 15 degrees C. The archaeal small-subunit (SSU) r RNA-encoding genes (rDNA) of these environmental DNA samples were amplified by PCR with an archaeal-specific primer system and used for the generation of clone libraries. Representative rDNA clones (n = 90) were characterized by terminal restriction fragment length polymorphism (T-RFLP) and sequence analysis. T-RFLP analysis produced for the clones terminally labeled fragm ents with a characteristic length of mostly 185, 284, or 392 bp. Sequence a nalysis allowed determination of the phylogenetic affiliation of the indivi dual clones,vith their characteristic T-RFLP fragment lengths and showed th at the archaeal community of the anoxic rice soil slurry was dominated by m embers of the families Methanosarcinaceae (185 bp) and Methanosaetaceae (28 4 bp), the kingdom Crenarchaeota (185 or 284 bp), and a novel, deeply branc hing lineage of the (probably methanogenic) kingdom Euryarchaeota (392 bp) that has recently been detected on rice roots (R Gro ss kopf, S, Stubner, a nd W. Liesack, Appl. Environ. Microbiol, 64:4983-4989, 1998). The structure of the archaeal community changed when the temperature was shifted from 30 degrees C to 15 degrees C, Before the temperature shift, the clones (n = 3 0) retrieved from the community were dominated by Crenarchaeota (70%), "nov el Euryarchaeota" (23%), and Methanosarcinacaeae (7%). Further incubation a t 30 degrees C (n = 30 clones) resulted in a relative increase in members o f the Methanosarcinaceae (77%), whereas further incubation at 15 degrees C (n = 30 clones) resulted in a much more diverse community consisting of 33% Methanosarcinaceae, 23% Crenarchaeota, 20% Methanosaetaceae, and 17% novel Euryarchaeota. The appearance of Methanosaetaceae at 15 degrees C was cons picuous, These results demonstrate that the structure of the archaeal commu nity in anoxic rice field soil changed with time and incubation temperature .