Syntrophic-methanogenic associations along a nutrient gradient in the Florida Everglades

Abstract

Nutrient runoff from the Everglades Agricultural Area resulted in a well-documented gradient of phosphorus concentrations in soil and water, with concomitant ecosystem-level changes, in the northern Florida Everglades. It was recently reported that sulfate-reducing prokaryote assemblage composition, numbers, and activities are dependent on position along the gradient (H. Castro, K. R. Reddy, and A. Ogram, Appl. Environ. Microbiol. 68:6129-6137, 2002). The present study utilized a combination of culture- and non-culture-based approaches to study differences in composition of assemblages of syntrophic and methanogenic microbial communities in eutrophic, transition, and oligotrophic areas along the phosphorus gradient. Methanogenesis rates were much higher in eutrophic and transition regions, and sequence analysis of 16S rRNA gene clone libraries constructed from samples taken from these regions revealed differences in composition and activities of syntroph-methanogen consortia. Methanogens from eutrophic and transition regions were almost exclusively composed of hydrogenotrophic methanogens, with approximately 10,000-fold-greater most probable numbers of hydrogenotrophs than of acetotrophs. Most cultivable strains from eutrophic and transition regions clustered within novel lineages. In non-culture-based studies to enrich syntrophs, most bacterial and archaeal clones were either members of novel lineages or closely related to uncultivated environmental clones. Novel cultivable Methanosaeta sp. and fatty acid-oxidizing bacteria related to the genera Syntrophomonas and Syntrophobacter were observed in microcosms containing soil from eutrophic regions, and different lines of evidence indicated the existence of novel syntrophic association in eutrophic regions.

FIG. 1.

Quantification of weekly depletion of 20 mM spiked fatty acids (filled symbols) in WCA-2A soils leading to formation of methane (open symbols). (A) Propionate; (B) butyrate. Symbols: triangles, F1 (eutrophic); circles, F4 (transition); squares, U3 (oligotrophic). Analyses were conducted in triplicate, and mean values are presented; error bars represent ±1 standard deviation.

FIG. 2.

Quantification of weekly depletion of 20 mM formate or acetate (filled symbols) in WCA-2A soils leading to formation of methane (open symbols). (A) Formate; (B) acetate. Symbols: triangles, F1 (eutrophic); circles, F4 (transition); squares, U3 (oligotrophic). Analysis was conducted in triplicate, and mean values are presented; error bars represent ±1 standard deviation.

FIG. 3.

Phylogenetic tree of partial 16S rRNA gene sequences from domain Archaea, constructed with PAUP version 4.0b8 using maximum parsimony. Clones were obtained from F1 (eutrophic), F4 (transition), and U3 (oligotrophic) microcosms, spiked with P (propionate) or B (butyrate). Numbers at nodes represent bootstrap values (100 times resampling); only values of >50 are presented. Halobacterium salinarum was used as the outgroup.

FIG. 4.

Phylogenetic tree of partial 16S rRNA gene sequences from domain Bacteria, constructed with PAUP version 4.0b8 using maximum parsimony. Clones were obtained from F1 (eutrophic), F4 (transition), and U3 (oligotrophic) microcosms, spiked with P (propionate) or B (butyrate). Numbers at nodes represent bootstrap values (100 times resampling analysis); only values of >50 are presented. Arthrobacter globiformis was used as the outgroup.

FIG. 5.

DIC microscopy of consortia obtained from F1 (eutrophic), F4 (transition), and U3 (oligotrophic) microcosms. (A) Consortium from F1 (eutrophic) microcosm, showing the Methanosaeta characteristic of long filaments with blunt ends, juxtaposed with butyrate-oxidizing syntrophic bacteria; (B) consortium from F4 (transition) microcosm; (C) consortium from U3 (oligotrophic) microcosm, showing Methanosarcina in clumps with very few presumed fatty acid-oxidizing bacteria.

FIG. 5.

DIC microscopy of consortia obtained from F1 (eutrophic), F4 (transition), and U3 (oligotrophic) microcosms. (A) Consortium from F1 (eutrophic) microcosm, showing the Methanosaeta characteristic of long filaments with blunt ends, juxtaposed with butyrate-oxidizing syntrophic bacteria; (B) consortium from F4 (transition) microcosm; (C) consortium from U3 (oligotrophic) microcosm, showing Methanosarcina in clumps with very few presumed fatty acid-oxidizing bacteria.

FIG. 5.

DIC microscopy of consortia obtained from F1 (eutrophic), F4 (transition), and U3 (oligotrophic) microcosms. (A) Consortium from F1 (eutrophic) microcosm, showing the Methanosaeta characteristic of long filaments with blunt ends, juxtaposed with butyrate-oxidizing syntrophic bacteria; (B) consortium from F4 (transition) microcosm; (C) consortium from U3 (oligotrophic) microcosm, showing Methanosarcina in clumps with very few presumed fatty acid-oxidizing bacteria.