Structure and Functional Properties of Bacterial Communities in Surface Sediments of the Recently Declared Nutrient-Saturated Lake Villarrica in Southern Chile.

Lake Villarrica, one of Chile's main freshwater water bodies, was recently declared a nutrient-saturated lake due to increased phosphorus (P) and nitrogen (N) levels. Although a decontamination plan based on environmental parameters is being established, it does not consider microbial parameters. Here, we conducted high-throughput DNA sequencing and quantitative polymerase chain reaction (qPCR) analyses to reveal the structure and functional properties of bacterial communities in surface sediments collected from sites with contrasting anthropogenic pressures in Lake Villarrica. Alpha diversity revealed an elevated bacterial richness and diversity in the more anthropogenized sediments. The phylum Proteobacteria, Bacteroidetes, Acidobacteria, and Actinobacteria dominated the community. The principal coordinate analysis (PCoA) and redundancy analysis (RDA) showed significant differences in bacterial communities of sampling sites. Predicted functional analysis showed that N cycling functions (e.g., nitrification and denitrification) were significant. The microbial co-occurrence networks analysis suggested Chitinophagaceae, Caldilineaceae, Planctomycetaceae, and Phycisphaerae families as keystone taxa. Bacterial functional genes related to P (phoC, phoD, and phoX) and N (nifH and nosZ) cycling were detected in all samples by qPCR. In addition, an RDA related to N and P cycling revealed that physicochemical properties and functional genes were positively correlated with several nitrite-oxidizing, ammonia-oxidizing, and N-fixing bacterial genera. Finally, denitrifying gene (nosZ) was the most significant factor influencing the topological characteristics of co-occurrence networks and bacterial interactions. Our results represent one of a few approaches to elucidate the structure and role of bacterial communities in Chilean lake sediments, which might be helpful in conservation and decontamination plans.

Spatiotemporal distributions and relationships of phosphorus content, phosphomonoesterase activity, and bacterial phosphomonoesterase genes in sediments from a eutrophic brackish water lake in Chile.

Phosphorus (P) cycling by microbial activity is highly relevant in the eutrophication of lakes. In this context, the contents of organic (Po) and inorganic (Pi) phosphorus, the activity of acid (ACP) and alkaline (ALP) phosphomonoesterase (Pase), and the abundances of bacterial Pase genes (phoD, phoC, and phoX) were studied in sediments from Budi Lake, a eutrophic coastal brackish water lake in Chile. Our results showed spatiotemporal variations in P fractions, Pase activities, and Pase gene abundances. In general, our results showed higher contents of Pi (110-144 mg kg-1), Po (512-576 mg kg-1), and total P (647-721 mg kg-1) in sediments from the more anthropogenized sampling sites in summer compared with those values of Pi (86-127 mg kg-1), Po (363-491 mg kg-1) and total P (449-618 mg kg-1) in less anthropogenized sampling sites in winter. In concordance, sediments showed higher Pase activities (µg nitrophenyl phosphate g-1 h-1) in sediments from the more anthropogenized sampling sites (9.7-22.7 for ACP and 5.9 to 9.6 for ALP) compared with those observed in less anthropogenized sampling sites in winter (4.2-12.9 for ACP and 0.3 to 6.7 for ALP). Higher abundances (gene copy g-1 sediment) of phoC (8.5-19 × 108), phoD (9.2-47 × 106), and phoX (8.5-26 × 106) genes were also found in sediments from the more anthropogenized sampling sites in summer compared with those values of phoC (0.1-1.1 × 108), phoD (1.4-2.4 × 106) and phoX (0.7-1.2 × 106) genes in the less anthropogenized sites in winter. Our results also showed a positive correlation between P contents, Pase activities, and abundances of bacterial Pase genes, independent of seasonality. The present study provided information on the microbial activity involved in P cycling in sediments of Budi Lake, which may be used in further research as indicators for the monitoring of eutrophication of lakes.

Campylobacterota dominate the microbial communities in a tropical karst subterranean estuary, with implications for cycling and export of nitrogen to coastal waters.

Subterranean estuaries (STEs), the zones in which seawater and subsurface groundwater mix, are recognized as hotspots for biogeochemical reactions; however, little is known of the microbial communities that control many of those reactions. This study investigated the potential functions of microbes inhabiting a cenote and an offshore submarine spring (Pargos) in the near-coastal waters of the Yucatan Peninsula, Mexico. The inland cenote (Cenote Siete Bocas; C7B) is characterized by a chemocline that is host to an array of physicochemical gradients associated with microbial activities. The chemocline includes an increasing gradient in sulfide concentrations with depth and a decreasing gradient in nitrate concentrations. The microbial community within the chemocline was dominated by Sulfurimonas and Sulfurovum of the Campylobacteria, which are likely responsible for sulfide oxidation coupled with nitrate reduction. Although C7B has not been directly connected with Pargos Spring, water discharging from the spring has physicochemical characteristics and microbial community structures similar to C7B, strongly suggesting biogeochemical processing in the STE impacts groundwater composition prior to discharge. This work yields insight into the microbial communities and biogeochemical reactions in STEs in karstic aquifers and provides evidence for the importance of Campylobacteria in controlling nitrate concentrations exported to marine springs.

Azithromycin and Ciprofloxacin Can Promote Antibiotic Resistance in Biosolids and Biosolids-Amended Soils.

Spread of biosolids-borne antibiotic resistance is a growing public and environmental health concern. Herein, we conducted incubation experiments involving biosolids, which are byproducts of sewage treatment processes, and biosolids-amended soil. Quantitative reverse transcription-PCR (RT-qPCR) was employed to assess responses of select antibiotic resistance genes (ARGs) and mobile elements to environmentally relevant concentrations of two biosolids-borne antibiotics, azithromycin (AZ) and ciprofloxacin (CIP). Additionally, we examined sequence distribution of gyrA (encoding DNA gyrase; site of action of CIP) to assess potential shifts in genotype. Increasing antibiotic concentrations generally increased the transcriptional activities of qnrS (encoding CIP resistance) and ermB and mefE (encoding AZ resistance). The transcriptional activity of intl1, a marker of class 1 integrons, was unaffected by CIP or AZ concentrations, but biosolids amendment increased intl1 activity in the soil by 4 to 5 times, which persisted throughout incubation. While the dominant gyrA sequences found herein were unrelated to known CIP-resistant genotypes, the increasing CIP concentrations significantly decreased the diversity of genes encoding the DNA gyrase A subunit, suggesting changes in microbial community structures. This study suggests that biosolids harbor transcriptionally active ARGs and mobile elements that could survive and spread in biosolids-amended soils. However, more research is warranted to investigate these trends under field conditions. IMPORTANCE Although previous studies have indicated that biosolids may be important spreaders of antibiotics and antibiotic resistance genes (ARGs) in environments, the potential activities of ARGs or their responses to environmental parameters have been understudied. This study highlights that certain biosolids-borne antibiotics can induce transcriptional activities of ARGs and mobile genetic elements in biosolids and biosolids-amended soil, even when present at environmentally relevant concentrations. Furthermore, these antibiotics can alter the structure of microbial populations expressing ARGs. Our findings indicate the bioavailability of the antibiotics in biosolids and provide evidence that biosolids can promote the activities and dissemination of ARGs and mobile genes in biosolids and soils that receive contaminated biosolids, thus, underscoring the importance of investigating anthropogenically induced antibiotic resistance in the environment under real-world scenarios.

Periphyton and Flocculent Materials Are Important Ecological Compartments Supporting Abundant and Diverse Mercury Methylator Assemblages in the Florida Everglades.

Mercury (Hg) methylation in the Florida Everglades is of great environmental concern because of its adverse effects on human and wildlife health through biomagnification in aquatic food webs. Periphyton and flocculant materials (floc) overlaying peat soil are important ecological compartments producing methylmercury (MeHg) in this ecosystem. These compartments retain higher concentrations of MeHg than did soil at study sites across nutrient and/or sulfate gradient(s). To better understand what controls Hg methylation in these compartments, the present study explored the structures and abundances of Hg methylators using genes hgcAB as biomarkers. The hgcA sequences indicated that these compartments hosted a high diversity of Hg methylators, including Deltaproteobacteria, Chloroflexi, Firmicutes, and Methanomicrobia, with community compositions that differed between these habitats. The copy numbers of hgcAB quantified by quantitative PCR revealed that floc and soil supported higher numbers of Hg methylators than periphyton in the Everglades ecosystem. The abundance of Hg methylators was strongly positively correlated with concentrations of carbon and nutrients (e.g., phosphorus and nitrogen) according to redundancy analysis. Strong correlations were also observed among numbers of sulfate reducers, methanogens, and the dominant hgcAB-carrying groups, suggesting that hgcAB would spread primarily through the growth of those assemblages. The abundances of Hg methylators were weakly negatively correlated to MeHg concentrations, suggesting that the size of this population would not solely determine the final concentrations of MeHg in the ecological compartments studied. This study extends the knowledge regarding the distribution of diverse potential mercury methylators in different environmental compartments in a wetland of national concern.IMPORTANCE Methylmercury is a potent neurotoxin that impacts the health of humans and wildlife. Most mercury in wetlands such as the Florida Everglades enters as inorganic mercury via atmospheric deposition, some of which is transformed to the more toxic methylmercury through the activities of anaerobic microorganisms. We investigated the numbers and phylogenetic diversity of hgcAB, genes that are linked to mercury methylation, in the soil, floc, and periphyton in areas of the Everglades with different sulfate and nutrient concentrations. Soil harbored relatively high numbers of cells capable of methylating mercury; however, little detectable methylmercury was present in soil. The greatest concentrations of methylmercury were found in floc and periphyton. The dominant methylators in those compartments included methanogens and Syntrophobacteriales This work provides significant insight into the microbial processes that control methylation and form the basis for accumulation through the food chain in this important environment.

Methanogens Are Major Contributors to Nitrogen Fixation in Soils of the Florida Everglades.

The objective of this study was to investigate the interaction of the nitrogen (N) cycle with methane production in the Florida Everglades, a large freshwater wetland. This study provides an initial analysis of the distribution and expression of N-cycling genes in Water Conservation Area 2A (WCA-2A), a section of the marsh that underwent phosphorus (P) loading for many years due to runoff from upstream agricultural activities. The elevated P resulted in increased primary productivity and an N limitation in P-enriched areas. Results from quantitative real-time PCR (qPCR) analyses indicated that the N cycle in WCA-2A was dominated by nifH and nirK/S, with an increasing trend in copy numbers in P-impacted sites. Many nifH sequences (6 to 44% of the total) and nifH transcript sequences (2 to 49%) clustered with the methanogenic Euryarchaeota, in stark contrast to the proportion of core gene sequences representing Archaea (≤0.27% of SSU rRNA genes) for the WCA-2A microbiota. Notably, archaeal nifH gene transcripts were detected at all sites and comprised a significant proportion of total nifH transcripts obtained from the unimpacted site, indicating that methanogens are actively fixing N2 Laboratory incubations with soils taken from WCA-2A produced nifH transcripts with the production of methane from H2 plus CO2 and acetate as electron donors and carbon sources. Methanogenic N2 fixation is likely to be an important, although largely unrecognized, route through which fixed nitrogen enters the anoxic soils of the Everglades and may have significant relevance regarding methane production in wetlands.IMPORTANCE Wetlands are the most important natural sources of the greenhouse gas methane, and much of that methane emanates from (sub)tropical peatlands. Primary productivity in these peatlands is frequently limited by the availability of nitrogen or phosphorus; however, the response to nutrient limitations of microbial communities that control biogeochemical cycling critical to ecosystem function may be complex and may be associated with a range of processes, including methane production. We show that many, if not most, of the methanogens in the peatlands of the Florida Everglades possess the nifH gene and actively express it for N2 fixation coupled with methanogenesis. These findings indicate that archaeal N2 fixation would play crucial role in methane emissions and overall N cycle in subtropical wetlands suffering N limitation.

Rhizobacterial Community Structures Associated with Native Plants Grown in Chilean Extreme Environments.

Chile is topographically and climatically diverse, with a wide array of diverse undisturbed ecosystems that include native plants that are highly adapted to local conditions. However, our understanding of the diversity, activity, and role of rhizobacteria associated with natural vegetation in undisturbed Chilean extreme ecosystems is very poor. In the present study, the combination of denaturing gradient gel electrophoresis and 454-pyrosequencing approaches was used to describe the rhizobacterial community structures of native plants grown in three representative Chilean extreme environments: Atacama Desert (ATA), Andes Mountains (AND), and Antarctic (ANT). Both molecular approaches revealed the presence of Proteobacteria, Bacteroidetes, and Actinobacteria as the dominant phyla in the rhizospheres of native plants. Lower numbers of operational taxonomic units (OTUs) were observed in rhizosphere soils from ATA compared with AND and ANT. Both approaches also showed differences in rhizobacterial community structures between extreme environments and between plant species. The differences among plant species grown in the same environment were attributed to the higher relative abundance of classes Gammaproteobacteria and Alphaproteobacteria. However, further studies are needed to determine which environmental factors regulate the structures of rhizobacterial communities, and how (or if) specific bacterial groups may contribute to the growth and survival of native plants in each Chilean extreme environments.

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

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.

Diversity and distribution of actinobacterial aromatic ring oxygenase genes across contrasting soil properties.

The diversity of a gene family encoding Actinobacterial aromatic ring oxygenases (AAROs) was detected by the PCR-cloning approach using a newly designed PCR primer set. The distribution of AAROs was investigated in 11 soils representing different land management and vegetation zones and was correlated with several geochemical parameters including pH, organic matter (OM), total Kjeldahl nitrogen (TKN), and nitrogen oxides (NO(x)-N: mostly NO3(-)-N). The distribution of individual clades encoding enzymes with potentially different substrates were correlated with different environmental factors, suggesting differential environmental controls on the distribution of specific enzymes as well as sequence diversity. For example, individual clades associated with phthalate dioxygenases were either strongly negatively correlated with pH, or not correlated with pH but showed strong positive correlation with organic carbon content. A large number of clones clustering in a clade related to PAH oxygenases were positively correlated with pH and nitrogen, but not with organic matter. This analysis may yield insight into the ecological forces driving the distribution of these catabolic genes.

Syntrophs dominate sequences associated with the mercury methylation-related gene hgcA in the water conservation areas of the Florida Everglades.

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.

Stimulation of anaerobic biodegradation of DDT and its metabolites in a muck soil: laboratory microcosm and mesocosm studies.

The aim of this study was to evaluate the impact of selected electron donors and electron acceptors on the anaerobic biodegradation of DDT and its major metabolites in a muck soil with a long history of exposure to the pesticide. Loss of DDT was measured in anaerobic microcosms supplemented with H2, lactate, and acetate. The greatest loss of DDT (approximately 87 %) was observed in microcosms amended with lactate and no additional electron acceptor compared to the no additional electron donor or acceptor sets. An increase in measureable concentrations of DDx was observed in un-amended microcosms. In larger scale mesocosms, significant increases in dissolved organic carbon (DOC) corresponded with low redox potentials. Increases in DOC corresponded with sharp increases in measured concentrations of DDx, followed by a decrease in measured DDT concentrations in lactate-amended mesocosms. Our studies indicate that sorbed DDx is released upon anaerobic incubation, and that indigenous microorganisms capable of DDx degradation respond to lactate additions. Both the potential for release of sorbed DDx and the potential for biodegradation of DDx should be considered during remediation of DDx-contaminated organic soils at low redox potentials.

Methyl coenzyme M reductase A (mcrA) gene-based investigation of methanogens in the mudflat sediments of Yangtze River estuary, China.

Methanogen populations of an intertidal mudflat in the Yangtze River estuary of China were investigated based on the methyl coenzyme M reductase A (mcrA) gene using 454-pyrosequencing and quantitative real-time polymerase chain reaction (qPCR). Samples were collected at six depths from three locations. In the qPCR analyses, a mean depth-wise change of mcrA gene abundance was observed from (1.23 ± 0.13) × 10(7) to (1.16 ± 0.29) × 10(8) per g dried soil, which was inversely correlated with the depletion of sulfate (R(2) = 0.74; α = 0.05) and salinity (R (2) = 0.66; α = 0.05). The copy numbers of mcrA was at least 1 order of magnitude higher than dissimilatory sulfate reductase B (dsrB) genes, likely indicating the importance of methanogenesis at the mudflat. Sequences related to the orders Methanomicrobiales, Methanosarcinales, Methanobacteriales, Methanococcales and the uncultured methanogens; Rice Cluster I (RC-I), Zoige cluster I (ZC-I) and anaerobic methane oxidizing archaeal lineage-1 (ANME-1) were detected. Methanomicrobiales and Methanosarcinales dominated the entire sediment layers, but detectable changes of proportions were observed with depth. The hydrogenotrophic methanogens Methanomicrobiales slightly increased with depth while Methanosarcinales showed the reverse. Chao1 and ACE richness estimators revealed higher diversity of methanogens near the surface (0-10 cm) when compared with the bottom sediments. The near-surface sediments were mainly dominated by the family Methanosarcinaceae (45 %), which has members that can utilize substrates that cannot be used by sulfate-reducing bacteria. Overall, current data indicate that Methanosarcinales and Methanomicrobiales are the most dominant methanogens within the entire depth profile down to 100 cm, with higher abundance and diversity of methanogens in the deeper and upper sediment layers, respectively.

Composition of methane-oxidizing bacterial communities as a function of nutrient loading in the Florida everglades.

Agricultural runoff of phosphorus (P) in the northern Florida Everglades has resulted in several ecosystem level changes, including shifts in the microbial ecology of carbon cycling, with significantly higher methane being produced in the nutrient-enriched soils. Little is, however, known of the structure and activities of methane-oxidizing bacteria (MOB) in these environments. To address this, 0 to 10 cm plant-associated soil cores were collected from nutrient-impacted (F1), transition (F4), and unimpacted (U3) areas, sectioned in 2-cm increments, and methane oxidation rates were measured. F1 soils consumed approximately two-fold higher methane than U3 soils; additionally, most probable numbers of methanotrophs were 4-log higher in F1 than U3 soils. Metabolically active MOB containing pmoA sequences were characterized by stable-isotope probing using 10 % (v/v) (13)CH(4). pmoA sequences, encoding the alpha subunit of methane monooxygenase and related to type I methanotrophs, were identified from both impacted and unimpacted soils. Additionally, impacted soils also harbored type II methanotrophs, which have been shown to exhibit preferences for high methane concentrations. Additionally, across all soils, novel pmoA-type sequences were also detected, indicating presence of MOB specific to the Everglades. Multivariate statistical analyses confirmed that eutrophic soils consisted of metabolically distinct MOB community that is likely driven by nutrient enrichment. This study enhances our understanding on the biological fate of methane being produced in productive wetland soils of the Florida Everglades and how nutrient-enrichment affects the composition of methanotroph bacterial communities.

Bioavailability of biosolids-borne ciprofloxacin and azithromycin to terrestrial organisms: Microbial toxicity and earthworm responses.

Information on bioavailability of two antibiotic TOrCs, ciprofloxacin (CIP) and azithromycin (AZ), to terrestrial organisms is severely limited, especially in the biosolids context. Responses of two terrestrial organisms, earthworms and microbes, to a range of environmentally relevant concentrations of biosolids-borne CIP and AZ were assessed in laboratory incubation studies involving 3H-labeled compounds. Earthworm assessments were based on the Earthworm Sub-chronic Toxicity Test (OCSPP 850.3100). Microbial impacts were assessed using respiration and reverse transcriptase-quantitative PCR (mRNA) analyses of nutrient (N and P) cycling genes as toxicity markers. Antibiotic extractability and stability during incubations were assessed using sequential extractions with CaCl2, methanol:water, and accelerated solvent extraction and analyses using thin layer chromatography. Subsample combustion, in addition to sequential extraction, recovered nearly 100% of the added antibiotic. The two compounds persisted (estimated half-lives ≥ 3 y), but extractable fractions (especially of CIP) decreased over time. Neither biosolids-borne antibiotic significantly impacted overall respiration or N and P cycling. Microbial toxicity responses were minimal; complementary DNA (cDNA) concentrations of ammonia oxidizing bacterial genes were affected, but only initially. Similarly, earthworms showed no apparent response related to toxicity to environmentally relevant (and much greater) concentrations of biosolids-borne CIP and AZ. Earthworms, however, accumulated both compounds, and the bioaccumulation factor (BAF) values (dry weight basis) were ~4 (CIP) and ~7 (AZ) in depurated worms and ~20 (CIP and AZ) in un-depurated worms. The microbial and earthworm responses strongly to moderately correlated with "bioaccessible" fractions of the target TOrCs. The results suggest that biosolids-borne CIP and AZ toxicity to terrestrial microbes and earthworms is minimal, but there is a potential for target TOrC entry into ecological food web.

Genetic and functional variation in denitrifier populations along a short-term restoration chronosequence.

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.

Response of microbial populations regulating nutrient biogeochemical cycles to oiling of coastal saltmarshes from the Deepwater Horizon oil spill.

Microbial communities play vital roles in the biogeochemistry of nutrients in coastal saltmarshes, ultimately controlling water quality, nutrient cycling, and detoxification. We determined the structure of microbial populations inhabiting coastal saltmarsh sediments from northern Barataria Bay, Louisiana, USA to gain insight into impacts on the biogeochemical cycles affected by Macondo oil from the 2010 Deepwater Horizon well blowout two years after the accident. Quantitative PCR directed toward specific functional genes revealed that oiled marshes were greatly diminished in the population sizes of diazotrophs, denitrifiers, nitrate-reducers to ammonia, methanogens, sulfate-reducers and anaerobic aromatic degraders, and harbored elevated numbers of alkane-degraders. Illumina 16S rRNA gene sequencing indicated that oiling greatly changed the structure of the microbial communities, including significant decreases in diversity. Oil-driven changes were also demonstrated in the structure of two functional populations, denitrifying and sulfate reducing prokaryotes, using nirS and dsrB as biomarkers, respectively. Collectively, the results from 16S rRNA and functional genes indicated that oiling not only markedly altered the microbial community structures, but also the sizes and structures of populations involved in (or regulating) a number of important nutrient biogeochemical cycles in the saltmarshes. Alterations such as these are associated with potential deterioration of ecological services, and further studies are necessary to assess the trajectory of recovery of microbial-mediated ecosystem functions over time in oiled saltmarsh sediment.

Cellulolytic, fermentative, and methanogenic guilds in benthic periphyton mats from the Florida Everglades.

Phosphorus enrichment caused by runoff from agricultural areas has resulted in ecosystem-level changes in the northern Florida Everglades, including a loss of periphyton mats from nutrient-impacted areas. The potential for methanogenesis resulting from the anaerobic decomposition of cellulose and fermentation products, and the microorganisms responsible for these processes, were studied in mats from a region not impacted by nutrient enrichment. Methane was produced from periphyton incubated with cellulose, propionate, butyrate, and formate, with an accumulation of fatty acids in incubations. The accumulation of fatty acids may have been caused by the inhibition of syntrophic oxidation, a potentially significant route for methane production in soils. Sequence analysis of 16S rRNA genes characteristic of Clostridium, the primary genus responsible for anaerobic decomposition and fermentation in soils of the area, indicated that Clostridium Cluster I assemblages present in the mat differed from those in the soils of the area. Significantly, sequences characteristic of the Clostridium group that dominates the soils of the area, group XIV, were not detected in the mat. These results indicate that benthic periphyton is probably a significant source of methane in the Everglades, and the responsible microorganisms differ significantly from those in the soils of the area.

Cellulolytic and fermentative guilds in eutrophic soils of the Florida Everglades.

The northern Florida Everglades has been subject to eutrophication in recent years, resulting in well-documented changes in microbial ecology and a shift in the dominant plant species. This study investigated effects of plant quality and eutrophication on activities and composition of cellulolytic and fermentative guilds in soils. Most probable numbers of cellulolytic bacteria in eutrophic (F1) and transition (F4) soils were 10-fold higher than in oligotrophic soils (U3). Higher potential methanogenesis was observed from cellulose in microcosms with soils from F1 and F4, compared to U3 soils. Nutrient status of soil, rather than plant type, was the major factor controlling methanogenesis rates, although numbers of fermentative bacteria were higher in microcosms supplemented with ground cattail (dominant in F1 and F4) than with sawgrass (dominant component of soil in U3), regardless of soil origin. DNA sequence analysis indicated Clostridium assemblage composition correlates with soil nutrient status.

Distributions, abundances and activities of microbes associated with the nitrogen cycle in riparian and stream sediments of a river tributary.

River tributaries are ecologically important environments that function as sinks of inorganic nitrogen. To gain greater insight into the nitrogen cycle (N-cycle) in these environments, the distributions and activities of microbial populations involved in the N-cycle were studied in riparian and stream sediments of the Santa Fe River (SFR) tributaries located in northern Florida, USA. Riparian sediments were characterized by much higher organic matter content, and extracellular enzyme activities, including cellobiohydrolase, ß-d-glucosidase, and phenol oxidase than stream sediments. Compared with stream sediments, riparian sediments exhibited significantly higher activities of nitrification, denitrification, dissimilatory nitrate reduction to ammonia (DNRA) and anaerobic ammonia oxidation; correspondingly, with higher copies of amoA (a biomarker for enumerating nitrifiers), nirS and nirK (for denitrifiers), and nrfA (for DNRA bacteria). Among N-cycle processes, denitrification showed the highest activities and the highest concentrations of the corresponding gene (nirK and nirS) copy numbers. In riparian sediments, substantial nitrification activities (6.3 mg-N kg soil-1d-1 average) and numbers of amoA copies (7.3 × 107 copies g soil-1 average) were observed, and nitrification rates correlate with denitrification rates. The guild structures of denitrifiers and nitrifiers in riparian sediments differed significantly from those found in stream sediments, as revealed by analysis of nirS and archaeal amoA sequences. This study shows that riparian sediments serve as sinks for inorganic nitrogen loads from non-point sources of agricultural runoff, with nitrification and denitrification associated with elevated levels of carbon and nitrogen contents and extracellular enzyme activities.