Distribution, activities, and interactions of methanogens and sulfate-reducing prokaryotes in the Florida Everglades

Abstract

To gain insight into the mechanisms controlling methanogenic pathways in the Florida Everglades, the distribution and functional activities of methanogens and sulfate-reducing prokaryotes (SRPs) were investigated in soils (0 to 2 or 0 to 4 cm depth) across the well-documented nutrient gradient in the water conservation areas (WCAs) caused by runoff from the adjacent Everglades Agricultural Area. The methyl coenzyme M reductase gene (mcrA) sequences that were retrieved from WCA-2A, an area with relatively high concentrations of SO4 (2-) (≥39 μM), indicated that methanogens inhabiting this area were broadly distributed within the orders Methanomicrobiales, Methanosarcinales, Methanocellales, Methanobacteriales, and Methanomassiliicoccales. In more than 3 years of monitoring, quantitative PCR (qPCR) using newly designed group-specific primers revealed that the hydrogenotrophic Methanomicrobiales were more numerous than the Methanosaetaceae obligatory acetotrophs in SO4 (2-)-rich areas of WCA-2A, while the Methanosaetaceae were dominant over the Methanomicrobiales in WCA-3A (with relatively low SO4 (2-) concentrations; ≤4 μM). qPCR of dsrB sequences also indicated that SRPs are present at greater numbers than methanogens in the WCAs. In an incubation study with WCA-2A soils, addition of MoO4 (2-) (a specific inhibitor of SRP activity) resulted in increased methane production rates, lower apparent fractionation factors [αapp; defined as (amount of δ(13)CO2 + 1,000)/(amount of δ(13)CH4 + 1,000)], and higher Methanosaetaceae mcrA transcript levels compared to those for the controls without MoO4 (2-). These results indicate that SRPs play crucial roles in controlling methanogenic pathways and in shaping the structures of methanogen assemblages as a function of position along the nutrient gradient.

FIG 1

(Left) Maximum likelihood tree representing the phylogeny of amino acid sequences (seqs) deduced from the sequences of mcrA genes retrieved from soils taken from sites F1, F4, and U3 in October 2009. Bootstrap values of ≥50% from 1,000 reassemblages are placed at the branch points. Gray, clades targeted by the group-specific primers designed in the present study; white, clades containing only sequences from this study; black, clades with reference sequences and not containing sequences from this study. (Right) Relative percentages of clades generated from the maximum likelihood tree and the clades targeted by the currently designed primers. MMC, Methanomasiliicoccales; MB, Methanobacteriales; MCEL, Methanocellales; MSR, Methanosarcinaceae; MM, Methanomicrobiales; MST, Methanosaetaceae.

FIG 2

Box-and-whisker plot (top) and temporal profile (bottom) of the copy numbers of mcrA from Methanomicrobiales, Methanosaeta (Methanosaetaceae), and Methanomicrobacteriaceae (Methanobacteriales) in WCA soils. Data in the temporal profile are presented in order of sampling: from the left, October 2009, April 2010, August 2011, and January, August, and December 2012 for WCA-2A sites F1, F4, and U3 and February 2012 and March and April 2013 for site W3. Error bars in the bar graph represent ±1 SE (n = 3). Box-and-whisker plots were generated from the pooled data from the temporal profile. Boxes depict the medians (horizontal lines in the boxes) and the lower and upper quartiles (bottoms and tops of the boxes, respectively). The vertical bars (whiskers) show the highest and the lowest values, excluding outliers. The different letters indicate a significant difference among the groups Methanomicrobiales, Methanosaetaceae, and Methanobacteriales (P < 0.05 by the Tukey-Kramer HSD test).

FIG 3

Box-and-whisker plot (top) and temporal profile (bottom) of the dsrB copy numbers compared with the T-M mcrA copy numbers estimated in the same sample. Data in the temporal profile are presented in order of sampling: from the left, April 2010, August 2011, and January, August, and December 2012 for WCA-2A sites F1, F4, and U3 and February 2012 and March and April 2013 for site W3. Error bars in the bar graph represent ±1 SE (n = 3). The box-and-whisker plot was constructed from the pooled data from the temporal profile. Boxes depict the medians (horizontal lines in the boxes) and the lower and upper quartiles (bottoms and tops of the boxes, respectively), while the whiskers show the highest and the lowest values, excluding outliers. The different letters indicate a significant difference between dsrB and T-M mcrA copy numbers (P < 0.05 by Student's t test).

FIG 4

Results of RDA presenting the correlation between mcrA and dsrB copy numbers and geochemical parameters obtained for the samples from sites F1, F4, and U3 collected in April 2010 and August and December 2012 and the samples from site W3 collected in February 2012 and April 2013. Arrows pointing in the same direction indicate positive correlations, and arrows pointing in opposite directions indicate negative correlations. The arrow length corresponds to the variance explained by the environmental variable. The first two axes explain 89.9% of the total canonical eigenvalues with a significant Monte-Carlo test value (P < 0.05).

FIG 5

Numbers of copies of the mcrA and dsrB genes and their transcripts measured from surface soils (0 to 4 cm depth) sampled from WCA-2A sites F1, F4, and U3 in August 2012. Error bars represent ±1 SE (n = 3).

FIG 6

CH₄ production rate (A) and composition of the δ¹³CH₄ and δ¹³CO₂ produced (B) during incubation of soils sampled in August 2012. Error bars represent ±1 SD (n = 3; note that the control incubation of site U3 soils did not produce a detectable amount of δ¹³CH₄). CT, control without MoO₄²⁻; Mo, addition of MoO₄²⁻ (20 mM).

FIG 7

mcrA transcript copy numbers, estimated using RT-qPCR, from the incubation of soils from sites F1 and F4 sampled in August 2012. For this RT-qPCR analysis, the RNA was isolated from the soils sampled on the same date that the gas samples were collected for the δ¹³CH₄ and δ¹³O₂ analysis. CT, control without MoO₄²⁻; Mo, addition of MoO₄²⁻ (20 mM). Error bars represent ±1 SE (n = 3).