Hector Castro

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.

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.

Structure and Function of Methanogens along a Short-Term Restoration Chronosequence in the Florida Everglades

ABSTRACT The removal of plants and soil to bedrock to eradicate exotic invasive plants within the Hole-in-the-Donut (HID) region, part of the Everglades National Park (Florida), presented a unique opportunity to study the redevelopment of soil and the associated microbial communities in the context of short-term primary succession and ecosystem restoration. The goal of this study was to identify relationships between soil redevelopment and activity and composition of methanogenic assemblages in HID soils. Methane production potentials indicated a general decline in methanogenic activity with restoration age. Microcosm incubations strongly suggested hydrogenotrophic methanogenesis as the most favorable pathway for methane formation in HID soils from all sites. Culture-independent techniques targeting methyl coenzyme M reductase genes (mcrA) were used to assess the dynamics of methanogenic assemblages. Clone libraries were dominated by sequences related to hydrogenotrophic methanogens of the orders Methanobacteriales and Methanococcales and suggested a general decline in the relative abundance of Methanobacteriales mcrA with time since restoration. Terminal restriction fragment length polymorphism analysis indicated methanogenic assemblages remain relatively stable between wet and dry seasons. Interestingly, analysis of soils across the restoration chronosequence indicated a shift in Methanobacteriales populations with restoration age, suggesting genotypic shifts due to site-specific factors.