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

Abstract

Soil temperatures in Italian rice fields typically range between about 15 and 30 degrees C. A change in the incubation temperature of anoxic methanogenic soil slurry from 30 degrees C to 15 degrees C typically resulted in a decrease in the CH4 production rate, a decrease in the steady-state H2 partial 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 caused by the temperature shift (K. J. Chin and R. Conrad, FEMS Microbiol. Ecol. 18:85-102, 1995). To investigate how temperature affects the structure of the methanogenic archaeal community, total DNA was extracted from soil slurries incubated at 30 and 15 degrees C. The archaeal small-subunit (SSU) rRNA-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 fragments with a characteristic length of mostly 185, 284, or 392 bp. Sequence analysis allowed determination of the phylogenetic affiliation of the individual clones with their characteristic T-RFLP fragment lengths and showed that the archaeal community of the anoxic rice soil slurry was dominated by members of the families Methanosarcinaceae (185 bp) and Methanosaetaceae (284 bp), the kingdom Crenarchaeota (185 or 284 bp), and a novel, deeply branching lineage of the (probably methanogenic) kingdom Euryarchaeota (392 bp) that has recently been detected on rice roots (R. Grosskopf, S. Stubner, and 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 = 30) retrieved from the community were dominated by Crenarchaeota (70%), "novel Euryarchaeota" (23%), and Methanosarcinacaeae (7%). Further incubation at 30 degrees C (n = 30 clones) resulted in a relative increase in members of 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 conspicuous. These results demonstrate that the structure of the archaeal community in anoxic rice field soil changed with time and incubation temperature.

FIG. 1

Effect of temperature change on the production of CH₄, H₂ and acetate in slurries of anoxic rice field soil. Values represent the mean ± standard deviation of three experiments. Dotted arrows indicate temperature shift; solid arrows indicate sampling for molecular analysis.

FIG. 2

Evolutionary distance dendrogram showing SSU rDNA sequences of representative soil clones (complete list in Table 1) in relation to known sequences of Crenarchaeota, including environmental sequences retrieved from a hot spring in the Yellowstone Park (pJP27, pJP78, pJP33, pJP41, and pJP89 [4]), from coastal marine environments (SBAR5, ANTARCTIC 12, and WHAR Q [15]), from forest soil (FFSB2 [21]), from agricultural soil (SCA1145, SCA1150, and SCA1180 [5]), from rice roots (ARR11 and ARR29 [18]), and bulk rice soil (ABS13 and ABS16 [17]). The scale bar indicates the estimated number of base changes per nucleotide sequence position.

FIG. 3

Evolutionary distance dendrogram showing SSU rDNA sequences of all the soil clones exhibiting a relation to known sequences of Euryarchaeota, including the environmental sequences to novel Euryarchaeota retrieved from rice roots (termed ARR) and bulk rice soil (termed ABS) (17, 18). The scale bar indicates the estimated number of base changes per nucleotide sequence position.

FIG. 4

Evolutionary distance dendrogram showing SSU rDNA sequences of representative soil clones (complete list in Table 1) in relation to known sequences of methanogenic Euryarchaeota and to environmental sequences retrieved from coastal marine environments (SBAR1A and WHAR N [15]), from rice roots (ARR19 and ARR16), and from bulk rice soil (ABS3, ABS9, ABS12, and ABS23 [17, 18]). The scale bar indicates the estimated number of base changes per nucleotide sequence position.

FIG. 5

T-RFLP patterns of archaeal SSU rDNA amplified from DNA extracts of anoxic rice soil slurries (A) before the temperature shift (ST1), (B) after further incubation at 30°C (S30), and (C) after further incubation at 15°C (S15). The x axis shows the length (base pairs) of the terminal restriction fragment, and the y axis shows the intensity of the bands in arbitrary units.

FIG. 6

Diversity of the major phylogenetic archaeal lineages in the soil samples ST1, S30, and S15 as represented by the numbers of SSU rDNA clones with characteristic sequence information.

FIG. 7

Cumulative operational taxonomic units represented by the SSU rDNA clones obtained from the soil samples ST1, S30, and S15. The plots were fitted to a hyperbolic function [y = ax/(b + x)] with a = 25.9 ± 1.6, 26.4 ± 2.8, and 49.6 ± 2.3, and b = 25.7 ± 2.7, 40.0 ± 6.5, and 40.9 ± 2.8, respectively.