Andrew Ogram

Composition and Function of Sulfate-Reducing Prokaryotes in Eutrophic and Pristine Areas of the Florida Everglades

ABSTRACT As a result of agricultural activities in regions adjacent to the northern boundary of the Florida Everglades, a nutrient gradient developed that resulted in physicochemical and ecological changes from the original system. Sulfate input from agricultural runoff and groundwater is present in soils of the Northern Everglades, and sulfate-reducing prokaryotes (SRP) may play an important role in biogeochemical processes such as carbon cycling. The goal of this project was to utilize culture-based and non-culture-based approaches to study differences between the composition of assemblages of SRP in eutrophic and pristine areas of the Everglades. Sulfate reduction rates and most-probable-number enumerations revealed SRP populations and activities to be greater in eutrophic zones than in more pristine soils. In eutrophic regions, methanogenesis rates were higher, the addition of acetate stimulated methanogenesis, and SRP able to utilize acetate competed to a limited degree with acetoclastic methanogens. A surprising amount of diversity within clone libraries of PCR-amplified dissimilatory sulfite reductase (DSR) genes was observed, and the majority of DSR sequences were associated with gram-positive spore-forming Desulfotomaculum and uncultured microorganisms. Sequences associated with Desulfotomaculum fall into two categories: in the eutrophic regions, 94.7% of the sequences related to Desulfotomaculum were associated with those able to completely oxidize substrates, and in samples from pristine regions, all Desulfotomaculum-like sequences were related to incomplete oxidizers. This metabolic selection may be linked to the types of substrates that Desulfotomaculum spp. utilize; it may be that complete oxidizers are more versatile and likelier to proliferate in nutrient-rich zones of the Everglades. Desulfotomaculum incomplete oxidizers may outcompete complete oxidizers for substrates such as hydrogen in pristine zones where diverse carbon sources are less available.

Phylogenetic Characterization of Methanogenic Assemblages in Eutrophic and Oligotrophic Areas of the Florida Everglades

ABSTRACT Agricultural activities have produced well-documented changes in the Florida Everglades, including establishment of a gradient in phosphorus concentrations in Water Conservation Area 2A (WCA-2A) of the northern Everglades. An effect of increased phosphorus concentrations is increased methanogenesis in the eutrophic regions compared to the oligotrophic regions of WCA-2A. The goal of this study was to identify relationships between eutrophication and composition and activity of methanogenic assemblages in WCA-2A soils. Distributions of two genes associated with methanogens were characterized in soils taken from WCA-2A: the archaeal 16S rRNA gene and the methyl coenzyme M reductase gene. The richness of methanogen phylotypes was greater in eutrophic than in oligotrophic sites, and sequences related to previously cultivated and uncultivated methanogens were found. A preferential selection for the order Methanomicrobiales was observed in mcrA clone libraries, suggesting primer bias for this group. A greater diversity within the Methanomicrobiales was observed in mcrA clone libraries than in 16S rRNA gene libraries. 16S rRNA phylogenetic analyses revealed a dominance of clones related to Methanosaeta spp., an acetoclastic methanogen dominant in environments with low acetate concentrations. A significant number of clones were related to Methanomicrobiales, an order characterized by species utilizing hydrogen and formate as methanogenic substrates. No representatives of the orders Methanobacteriales and Methanococcales were found in any 16S rRNA clone library, although some Methanobacteriales were found in mcrA libraries. Hydrogenotrophs are the dominant methanogens in WCA-2A, and acetoclastic methanogen genotypes that proliferate in low acetate concentrations outnumber those that typically dominate in higher acetate concentrations.

Soil molecular microbial ecology at age 20: methodological challenges for the future.

The year 2000 marks the twentieth anniversary of the publication in Soil Biology & Biochemistry by Vigdis L. Torsvik (University of Bergen) of the first procedure for isolation of bacterial DNA from soil (Torsvik, 1980), arguably initiating the subdiscipline of soil molecular microbial ecology. Since 1980, great strides have been made in the development of methods and in the application of genetic tools to analysis of soil microbial communities, and many soil microbiology laboratories routinely incorporate these tools in their research. It is likely that the concept of soil molecular ecology will soon disappear as a subdiscipline of microbial ecology, and that these tools will become as routine and indispensable as are genetic tools in microbial physiology. However, even though increasing numbers of soil microbiologists use molecular biology in their research, some fundamental obstacles must be overcome before these tools become as routine as are, for example, many soil chemical methods. This anniversary provides an opportunity for retrospection on the applicability of genetic tools to soil microbial ecology, and of methodological needs for the immediate future. 7 2000 Elsevier Science Ltd. All rights reserved.

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.

Genetic and Functional Variation in Denitrifier Populations along a Short-Term Restoration Chronosequence

ABSTRACT Complete removal of plants and soil to exposed bedrock, in order to eradicate the Hole-in-the-Donut (HID) region of the Everglades National Park, FL, of exotic invasive plants, presented the opportunity to monitor the redevelopment of soil and the associated microbial communities along a short-term restoration chronosequence. Sampling plots were established for sites restored in 1989, 1997, 2000, 2001, and 2003. The goal of this study was to characterize the activity and diversity of denitrifying bacterial populations in developing HID soils in an effort to understand changes in nitrogen (N) cycling during short-term primary succession. Denitrifying enzyme activity (DEA) was detected in soils from all sites, indicating a potential for N loss via denitrification. However, no correlation between DEA and time since disturbance was observed. Diversity of bacterial denitrifiers in soils was characterized by sequence analysis of nitrite reductase genes (nirK and nirS) in DNA extracts from soils ranging in nitrate concentrations from 1.8 to 7.8 mg kg−1. High levels of diversity were observed in both nirK and nirS clone libraries. Statistical analyses of clone libraries suggest a different response of nirS- and nirK-type denitrifiers to factors associated with soil redevelopment. nirS populations demonstrated a linear pattern of succession, with individual lineages represented at each site, while multiple levels of analysis suggest nirK populations respond in a grouped pattern. These findings suggest that nirK communities are more sensitive than nirS communities to environmental gradients in these soils.

Characterization of methanogenic and methanotrophic assemblages in landfill samples

A greater understanding of the tightly linked trophic groups of anaerobic and aerobic bacteria residing in municipal solid waste landfills will increase our ability to control methane emissions and pollutant fate in these environments. To this end, we characterized the composition of methanogenic and methanotrophic bacteria in samples taken from two regions of a municipal solid waste landfill that varied in age. A method combining polymerase chain reaction amplification, restriction fragment length polymorphism analysis and phylogenetic analysis was used for this purpose. 16S rDNA sequence analysis revealed a rich assemblage of methanogens in both samples, including acetoclasts, H2/CO2-users and formate-users in the newer samples and H2/CO2-users and formateusers in the older samples, with closely related genera including Methanoculleus, Methanofollis, Methanosaeta and Methanosarcina. Fewer phylotypes of type 1 methanotrophs were observed relative to type 2 methanotrophs. Most type 1 sequences clustered within a clade related to Methylobacter, whereas type 2 sequences were broadly distributed among clades associated with Methylocystis and Methylosinus species. This genetic characterization tool promises rapid screening of landfill samples for genotypes and, therefore, degradation potentials.

Fatty Acid-Oxidizing Consortia along a Nutrient Gradient in the Florida Everglades†

ABSTRACT The Florida Everglades is one of the largest freshwater marshes in North America and has been subject to eutrophication for decades. A gradient in P concentrations extends for several kilometers into the interior of the northern regions of the marsh, and the structure and function of soil microbial communities vary along the gradient. In this study, stable isotope probing was employed to investigate the fate of carbon from the fermentation products propionate and butyrate in soils from three sites along the nutrient gradient. For propionate microcosms, 16S rRNA gene clone libraries from eutrophic and transition sites were dominated by sequences related to previously described propionate oxidizers, such as Pelotomaculum spp. and Syntrophobacter spp. Significant representation was also observed for sequences related to Smithella propionica, which dismutates propionate to butyrate. Sequences of dominant phylotypes from oligotrophic samples did not cluster with known syntrophs but with sulfate-reducing prokaryotes (SRP) and Pelobacter spp. In butyrate microcosms, sequences clustering with Syntrophospora spp. and Syntrophomonas spp. dominated eutrophic microcosms, and sequences related to Pelospora dominated the transition microcosm. Sequences related to Pelospora spp. and SRP dominated clone libraries from oligotrophic microcosms. Sequences from diverse bacterial phyla and primary fermenters were also present in most libraries. Archaeal sequences from eutrophic microcosms included sequences characteristic of Methanomicrobiaceae, Methanospirillaceae, and Methanosaetaceae. Oligotrophic microcosms were dominated by acetotrophs, including sequences related to Methanosarcina, suggesting accumulation of acetate.

Phylogeny of Acetate-Utilizing Microorganisms in Soils along a Nutrient Gradient in the Florida Everglades

ABSTRACT The consumption of acetate in soils taken from a nutrient gradient in the northern Florida Everglades was studied by stable isotope probing. Bacterial and archaeal 16S rRNA gene clone libraries from eutrophic and oligotrophic soil microcosms strongly suggest that a significant amount of acetate is consumed by syntrophic acetate oxidation in nutrient-enriched soil.

Syntrophs Dominate Sequences Associated with the Mercury Methylation-Related Gene hgcA in the Water Conservation Areas of the Florida Everglades

ABSTRACT The mechanisms and rates of mercury methylation in the Florida Everglades are of great concern because of potential adverse impacts on human and wildlife health through mercury accumulation in aquatic food webs. We developed a new PCR primer set targeting hgcA, a gene encoding a corrinoid protein essential for Hg methylation across broad phylogenetic boundaries, and used this primer set to study the distribution of hgcA sequences in soils collected from three sites along a gradient in sulfate and nutrient concentrations in the northern Everglades. The sequences obtained were distributed in diverse phyla, including Proteobacteria, Chloroflexi, Firmicutes, and Methanomicrobia; however, hgcA clone libraries from all sites were dominated by sequences clustering within the order Syntrophobacterales of the Deltaproteobacteria (49 to 65% of total sequences). dsrB mRNA sequences, representing active sulfate-reducing prokaryotes at the time of sampling, obtained from these sites were also dominated by Syntrophobacterales (75 to 89%). Laboratory incubations with soils taken from the site low in sulfate concentrations also suggested that Hg methylation activities were primarily mediated by members of the order Syntrophobacterales, with some contribution by methanogens, Chloroflexi, iron-reducing Geobacter, and non-sulfate-reducing Firmicutes inhabiting the sites. This suggests that prokaryotes distributed within clades defined by syntrophs are the predominant group controlling methylation of Hg in low-sulfate areas of the Everglades. Any strategy for managing mercury methylation in the Everglades should consider that net mercury methylation is not limited to the action of sulfate reduction.

Distribution and Stability of Sulfate-Reducing Prokaryotic and Hydrogenotrophic Methanogenic Assemblages in Nutrient-Impacted Regions of the Florida Everglades

ABSTRACT Although the influence of phosphorus loading on the Everglades ecosystem has received a great deal of attention, most research has targeted macro indicators, such as those based on vegetation or fauna, or chemical and physical parameters involved in biogeochemical cycles. Fewer studies have addressed the role of microorganisms, and these have mainly targeted gross informative parameters such as microbial biomass, enzymatic activities, and microbial enumerations. The objectives of this study were to characterize the dynamics of sulfate-reducing and methanogenic assemblages using terminal restriction fragment length polymorphism (T-RFLP) targeting the dissimilatory sulfite reductase (dsrA) and methyl coenzyme M reductase (mcrA) genes, respectively, and assess the impact of nutrient enrichment on microbial assemblages in the northern Everglades. T-RFLP combined with principal component analysis was a powerful technique to discriminate between soils from sites with eutrophic, transitional, and oligotrophic nutrient concentrations. dsrA T-RFLP provided a higher level of discrimination between the three sites. mcrA was a relatively weaker system to distinguish between sites, since it could not categorically discriminate between eutrophic and transition soil samples, but may be useful as an early indicator of phosphorus loading which is altering hydrogenotrophic methanogenic community in the transition zones, making them more similar to eutrophic zones. Clearly, targeting a combination of different microbial communities provides greater insight into the functioning of this ecosystem and provides useful information for understanding the relationship between eutrophication effects and microbial assemblages.