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PubMed2001

Development and evaluation of functional gene arrays for detection of selected genes in the environment.

To determine the potential of DNA array technology for assessing functional gene diversity and distribution, a prototype microarray was constructed with genes involved in nitrogen cycling: nitrite reductase (nirS and nirK) genes, ammonia mono-oxygenase (amoA) genes, and methane mono-oxygenase (pmoA) genes from pure cultures and those cloned from marine sediments. In experiments using glass slide microarrays, genes possessing less than 80 to 85% sequence identity were differentiated under hybridization conditions of high stringency (65 degrees C). The detection limit for nirS genes was approximately 1 ng of pure genomic DNA and 25 ng of soil community DNA using our optimized protocol. A linear quantitative relationship (r(2) = 0.89 to 0.94) was observed between signal intensity and target DNA concentration over a range of 1 to 100 ng for genomic DNA (or genomic DNA equivalent) from both pure cultures and mixed communities. However, the quantitative capacity of microarrays for measuring the relative abundance of targeted genes in complex environmental samples is less clear due to divergent target sequences. Sequence divergence and probe length affected hybridization signal intensity within a certain range of sequence identity and size, respectively. This prototype functional gene array did reveal differences in the apparent distribution of nir and amoA and pmoA gene families in sediment and soil samples. Our results indicate that glass-based microarray hybridization has potential as a tool for revealing functional gene composition in natural microbial communities; however, more work is needed to improve sensitivity and quantitation and to understand the associated issue of specificity.

Bacteria↗

PubMed2024

Effectiveness of stabilization methods for the immediate and short-term preservation of bovine fecal and upper respiratory tract genomic DNA.

Previous research on stabilization methods for microbiome investigations has largely focused on human fecal samples. There are a few studies using feces from other species, but no published studies investigating preservation of samples collected from cattle. Given that microbial taxa are differentially impacted during storage it is warranted to study impacts of preservation methods on microbial communities found in samples outside of human fecal samples. Here we tested methods of preserving bovine fecal respiratory specimens for up to 2 weeks at four temperatures (room temperature, 4°C, -20°C, and -80°C) by comparing microbial diversity and community composition to samples extracted immediately after collection. Importantly, fecal specimens preserved and analyzed were technical replicates, providing a look at the effects of preservation method in the absence of biological variation. We found that preservation with the OMNIgene®•GUT kit resulted in community structure most like that of fresh samples extracted immediately, even when stored at room temperature (~20°C). Samples that were flash-frozen without added preservation solution were the next most representative of original communities, while samples preserved with ethanol were the least representative. These results contradict previous reports that ethanol is effective in preserving fecal communities and suggest for studies investigating cattle either flash-freezing of samples without preservative or preservation with OMNIgene®•GUT will yield more representative microbial communities.

Cattle↗

PubMed2006

Influence of lead acetate on soil microbial biomass and community structure in two different soils with the growth of Chinese cabbage (Brassica chinensis).

A greenhouse pot experiment was conducted to evaluate the impact of different concentrations of lead acetate on soil microbial biomass and community structure during growth of Chinese cabbage (Brassica chinensis) in two different soils. The field soils were used for a small pot, short-term 60-day growth chamber study. The soils were amended with different Pb concentrations, ranging from 0 to 900mgkg(-1) soil. The experimental design was a 2 soilx2 vegetation/non-vegetationx6 treatments (Pb)x3 replicate factorial experiment. At 60 days the study was terminated and soils were analyzed for microbial parameters, namely, microbial biomass, basal respiration and PLFAs. The results indicated that the application of Pb at lower concentrations (100 and 300mgkg(-1)) as lead acetate resulted in a slight increase in soil microbial biomass, whereas Pb concentrations >500mgkg(-1) caused an immediate gradual significant decline in biomass. However, the degree of impact on soil microbial biomass and basal respiration by Pb was related to management (plant vegetation) or the contents of clay and organic matter in soils. The profiles of 21 phospholipid fatty acids (PLFAs) were used to assess whether observed changes in functional microbial parameters were accompanied by changes in the composition of the microbial communities after Pb application at 0, 300 and 900mg Pbkg(-1) soil. The results of principal component analyses (PCA) indicated that there were significant increases in fungi biomarkers of 18:3omega6c, 18:1omega9c and a decrease in cy17:0, which is an indicator of gram-negative bacteria for the high levels of Pb treatments In a word, soil microbial biomass and community structure, therefore, may be sensitive indicators reflecting environmental stress in soil-Pb-plant system. However, further studies will be needed to better understand how these changes in microbial community structure might actually impact soil microbial community function.

Biomass↗

PubMed2006

Molecular mechanisms of bacterial resistance to antimicrobial peptides.

Cationic antimicrobial peptides (CAMPs) are integral compounds of the antimicrobial arsenals in virtually all kinds of organisms, with important roles in microbial ecology and higher organisms' host defense. Many bacteria have developed countermeasures to limit the efficacy of CAMPs such as defensins, cathelicidins, kinocidins, or bacteriocins. The best-studied bacterial CAMP resistance mechanisms involve electrostatic repulsion of CAMPs by modification of cell envelope molecules, proteolytic cleavage of CAMPs, production of CAMP-trapping proteins, or extrusion of CAMPs by energy-dependent efflux pumps. The repertoire of CAMPs produced by a given host organism and the efficiency of microbial CAMP resistance mechanisms appear to be crucial in host-pathogen interactions, governing the composition of commensal microbial communities and the virulence of bacterial pathogens. However, all CAMP resistance mechanisms have limitations and bacteria have never succeeded in becoming fully insensitive to a broad range of CAMPs. CAMPs or conserved CAMP resistance factors are discussed as new mediators and targets, respectively, of novel and sustainable anti-infective strategies.

Animals↗

PubMed1999

Differential enumeration and in situ localization of microorganisms in the hindgut of the lower termite mastotermes darwiniensis by hybridization with rRNA-targeted probes

We examined the abundance and spatial distribution of major phylogenetic groups of the domain Bacteria in hindguts of the Australian lower termite Mastotermes darwiniensis by using in situ hybridization with group-specific, fluorescently labeled, rRNA-targeted oligonucleotide probes. Between 32.0 +/- 7.2% and 52.3 +/- 8.2% of the DAPI-stained cells in different hindgut fractions were detected with probe EUB338, specific for members of the domain Bacteria. About 85% of the prokaryotic cells were associated with the flagellates of the thin-walled anterior region (P3a) and the thick wall of the posterior region (P3b/P4) of the hindgut, as shown by DAPI staining. At most, half of the EUB338-detected cells hybridized with one of the other probes that targeted a smaller assemblage within the bacterial domain. In most fractions, cells were found in varying numbers with probe ALF1b, which targeted members of the alpha-Proteobacteria, whereas substantial amounts of sulfate-reducing bacteria, gram-positive bacteria with a high DNA G+C content and members of the Cytophaga-Flavobacterium cluster of the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum could be detected only in the wall fraction of P3b/P4. This clearly indicates that the hindgut microhabitats differ in the composition of their microbial community. In situ hybridization of cryosections through the hindgut showed only low numbers of bacteria attached to the P3a wall. In contrast, the wall of P3b was densely colonized by rod- and coccus-shaped bacteria, which could be assigned to the Cytophaga-Flavobacterium cluster of the CFB phylum and to the group of gram-positive bacteria with a high DNA G+C content, respectively. Oxygen concentration profiles determined with microelectrodes revealed steep oxygen gradients both in P3a and P3b. Oxygen was consumed within 100 &mgr;m below the gut surface, and anoxic conditions prevailed in the central portions of both gut regions, indicating that oxygen consumption in the hindgut does not depend on the presence of a biofilm on the hindgut wall.

Journal Article↗

PubMed2005

Comparison of cellulose solubilisation rates in rumen and landfill leachate inoculated reactors.

The aim of this study was to conduct a number of controlled digestions to obtain easily comparable cellulose solubilisation rates and to compare these rates to those found in the literature to see which operational differences were significant in affecting cellulose degradation during anaerobic digestion. The results suggested that differences in volumetric cellulose solubilisation rates were not indicative of the true performance of cellulose digestion systems. When cellulose solubilisation rates were normalised by the mass of cellulose in the reactor at each time step, the comparison of the rates became more meaningful. Cellulose solubilisation was surface area limited. Therefore, changes in the loading rate of cellulose to the reactor altered the volumetric solubilisation rate without changing the mass normalised rate. Comparison of mass normalised solubilisation rates from this study and the literature demonstrated that differences in reactor configuration and operational conditions did not significantly impact on the solubilisation rate whereas the difference in composition of the microbial communities showed a marked effect. This work highlights the importance of using appropriately normalised data when making comparisons between systems with differing operational conditions.

Animals↗

PubMed2001

Influence of sediment and pore-water composition on nitrite accumulation in a nitrate-perfused freshwater sediment.

Anaerobic nitrate consumption was accompanied by the accumulation of nitrite in the range of 3-52 micromoll(-1) in laboratory sediment cores continuously perfused with nitrate from below. Highest interstitial nitrite concentrations were observed in organically poor sediment cores with a low extractable protein content. Supplemented with an additional source of fixed nitrogen (ammonium), the nitrate-perfused sediments exhibited considerably lower nitrite concentrations than the respective control treatments. The simultaneous addition of ammonium and chloramphenicol, however, resulted in high nitrite concentrations. This suggests that in the absence of chloramphenicol an ammonium-fuelled bacterial protein de novo synthesis prevented the accumulation of nitrite in the course of anaerobic nitrate consumption. Additional DOC (glucose, methanol) generally increased the anaerobic nitrate consumption rate and the interstitial nitrite concentrations. The different scales of these increases were probably related to the degradability of the respective DOC compound and to the taxonomic composition of the microbial community evolving under different feeding regimes.

Anaerobiosis↗

PubMed2003

Importance of atmospherically deposited nitrogen to the annual nitrogen budget of the Neuse River estuary, North Carolina.

Wet deposition of nitrogen, as NH(4)(+), NO(3)(-), and organic N, contributes up to 50% of the total externally supplied or 'new' N flux to the Neuse River Estuary (North Carolina). Excessive nitrogen (N) loading to N-sensitive waters such as the Neuse River Estuary has been linked to changes in microbial and algal community composition and function (harmful algal blooms), hypoxia/anoxia, and fish kills. In a 4-year study from July 1996 to July 2000, the weekly wet deposition of NH(4)(+), NO(3)(-), and dissolved organic N was calculated, based on concentration and precipitation measurements, at 11 sites on a northwest-southeast transect in the watershed. Data from this period indicate that the annual mean total wet atmospherically deposited (AD)-N flux was 11 kg ha(-1) year(-1). Deposition was fairly evenly distributed between nitrate, ammonium, and organics (32%, 32%, and 36%, respectively). Seasonally, the summer (June-August) months contained the highest weekly wet total N deposition; this trend was not driven by precipitation amount. Estimates of watershed N retention and in-stream riverine processing revealed that the AD-N flux contributed an estimated 20% (range of 15-51%) of the total 'new' N flux to the estuary, with direct deposition of N to the estuary surface accounting for 6% of the total 'new' N flux. This study did not measure the dry depositional flux, which may double the contribution of AD-N to the estuary. The AD-N is an important source of 'new' N to the Neuse River Estuary as well as other estuarine and coastal ecosystems downwind of major emission sources. As such, AD-N should be included in effective nutrient mitigation and management efforts for these N-sensitive waters.

Air Pollutants↗

PubMed2006

Which are the polyphosphate accumulating organisms in full-scale activated sludge enhanced biological phosphate removal systems in Australia?

AIMS: To see if the compositions of the microbial communities in full scale enhanced biological phosphorus removal activated sludge systems were the same as those from laboratory scale sequencing batch reactors fed a synthetic sewage. METHODS: Biomass samples taken from nine full scale enhanced biological phosphate removal (EBPR) activated sludge plants in the eastern states of Australia were analysed for their populations of polyphosphate (polyP)-accumulating organisms (PAO) using semi-quantitative fluorescence in situ hybridization (FISH) in combination with DAPI (4'-6-diamidino-2-phenylindole) staining for polyP. RESULTS: Very few betaproteobacterial Rhodocyclus related organisms could be detected by FISH in most of the plants examined, and even where present, not all these cells even within a single cluster, stained positively for polyP with DAPI. In some plants in samples from aerobic reactors the Actinobacteria dominated populations containing polyP. CONCLUSIONS: The PAO populations in full-scale EBPR systems often differ to those seen in laboratory scale reactors fed artificial sewage, and Rhodocyclus related organisms, dominating these latter communities may not be as important in full-scale systems. Instead Actinobacteria may be the major PAO. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings illustrate how little is still known about the microbial ecology of EBPR processes and that more emphasis should now be placed on analysis of full-scale plants if microbiological methods are to be applied to monitoring their performances.

Actinobacteria↗

PubMed1991

16S rRNA sequences of uncultivated hot spring cyanobacterial mat inhabitants retrieved as randomly primed cDNA.

Cloning and analysis of cDNAs synthesized from rRNAs is one approach to assess the species composition of natural microbial communities. In some earlier attempts to synthesize cDNA from 16S rRNA (16S rcDNA) from the Octopus Spring cyanobacterial mat, a dominance of short 16S rcDNAs was observed, which appear to have originated only from certain organisms. Priming of cDNA synthesis from small ribosomal subunit RNA with random deoxyhexanucleotides can retrieve longer sequences, more suitable for phylogenetic analysis. Here we report the retrieval of 16S rRNA sequences from three formerly uncultured community members. One sequence type, which was retrieved three times from a total of five sequences analyzed, can be placed in the cyanobacterial phylum. A second sequence type is related to 16S rRNAs from green nonsulfur bacteria. The third sequence type may represent a novel phylogenetic type.

Base Sequence↗

PubMed1999

Automated approach for ribosomal intergenic spacer analysis of microbial diversity and its application to freshwater bacterial communities.

An automated method of ribosomal intergenic spacer analysis (ARISA) was developed for the rapid estimation of microbial diversity and community composition in freshwater environments. Following isolation of total community DNA, PCR amplification of the 16S-23S intergenic spacer region in the rRNA operon was performed with a fluorescence-labeled forward primer. ARISA-PCR fragments ranging in size from 400 to 1,200 bp were next discriminated and measured by using an automated electrophoresis system. Database information on the 16S-23S intergenic spacer was also examined, to understand the potential biases in diversity estimates provided by ARISA. In the analysis of three natural freshwater bacterial communities, ARISA was rapid and sensitive and provided highly reproducible community-specific profiles at all levels of replication tested. The ARISA profiles of the freshwater communities were quantitatively compared in terms of both their relative diversity and similarity level. The three communities had distinctly different profiles but were similar in their total number of fragments (range, 34 to 41). In addition, the pattern of major amplification products in representative profiles was not significantly altered when the PCR cycle number was reduced from 30 to 15, but the number of minor products (near the limit of detection) was sensitive to changes in cycling parameters. Overall, the results suggest that ARISA is a rapid and effective community analysis technique that can be used in conjunction with more accurate but labor-intensive methods (e.g., 16S rRNA gene cloning and sequencing) when fine-scale spatial and temporal resolution is needed.

Bacteria↗

PubMed2001

Bacteria and Archaea physically associated with Gulf of Mexico gas hydrates.

Although there is significant interest in the potential interactions of microbes with gas hydrate, no direct physical association between them has been demonstrated. We examined several intact samples of naturally occurring gas hydrate from the Gulf of Mexico for evidence of microbes. All samples were collected from anaerobic hemipelagic mud within the gas hydrate stability zone, at water depths in the ca. 540- to 2,000-m range. The delta(13)C of hydrate-bound methane varied from -45.1 per thousand Peedee belemnite (PDB) to -74.7 per thousand PDB, reflecting different gas origins. Stable isotope composition data indicated microbial consumption of methane or propane in some of the samples. Evidence of the presence of microbes was initially determined by 4,6-diamidino 2-phenylindole dihydrochloride (DAPI) total direct counts of hydrate-associated sediments (mean = 1.5 x 10(9) cells g(-1)) and gas hydrate (mean = 1.0 x 10(6) cells ml(-1)). Small-subunit rRNA phylogenetic characterization was performed to assess the composition of the microbial community in one gas hydrate sample (AT425) that had no detectable associated sediment and showed evidence of microbial methane consumption. Bacteria were moderately diverse within AT425 and were dominated by gene sequences related to several groups of Proteobacteria, as well as Actinobacteria and low-G + C Firmicutes. In contrast, there was low diversity of Archaea, nearly all of which were related to methanogenic Archaea, with the majority specifically related to Methanosaeta spp. The results of this study suggest that there is a direct association between microbes and gas hydrate, a finding that may have significance for hydrocarbon flux into the Gulf of Mexico and for life in extreme environments.

Archaea↗

PubMed2003

Stimulating the in situ activity of Geobacter species to remove uranium from the groundwater of a uranium-contaminated aquifer.

The potential for removing uranium from contaminated groundwater by stimulating the in situ activity of dissimilatory metal-reducing microorganisms was evaluated in a uranium-contaminated aquifer located in Rifle, Colo. Acetate (1 to 3 mM) was injected into the subsurface over a 3-month period via an injection gallery composed of 20 injection wells, which was installed upgradient from a series of 15 monitoring wells. U(VI) concentrations decreased in as little as 9 days after acetate injection was initiated, and within 50 days uranium had declined below the prescribed treatment level of 0.18 micro M in some of the monitoring wells. Analysis of 16S ribosomal DNA (rDNA) sequences and phospholipid fatty acid profiles demonstrated that the initial loss of uranium from the groundwater was associated with an enrichment of Geobacter species in the treatment zone. Fe(II) in the groundwater also increased during this period, suggesting that U(VI) reduction was coincident with Fe(III) reduction. As the acetate injection continued over 50 days there was a loss of sulfate from the groundwater and an accumulation of sulfide and the composition of the microbial community changed. Organisms with 16S rDNA sequences most closely related to those of sulfate reducers became predominant, and Geobacter species became a minor component of the community. This apparent switch from Fe(III) reduction to sulfate reduction as the terminal electron accepting process for the oxidation of the injected acetate was associated with an increase in uranium concentration in the groundwater. These results demonstrate that in situ bioremediation of uranium-contaminated groundwater is feasible but suggest that the strategy should be optimized to better maintain long-term activity of Geobacter species.

Acetates↗

PubMed2003

Microbial ecology of an extreme acidic environment, the Tinto River.

The Tinto River (Huelva, southwestern Spain) is an extreme environment with a rather constant acidic pH along the entire river and a high concentration of heavy metals. The extreme conditions of the Tinto ecosystem are generated by the metabolic activity of chemolithotrophic microorganisms thriving in the rich complex sulfides of the Iberian Pyrite Belt. Molecular ecology techniques were used to analyze the diversity of this microbial community. The community's composition was studied by denaturing gradient gel electrophoresis (DGGE) using 16S rRNA and by 16S rRNA gene amplification. A good correlation between the two approaches was found. Comparative sequence analysis of DGGE bands showed the presence of organisms related to Leptospirillum spp., Acidithiobacillus ferrooxidans, Acidiphilium spp., "Ferrimicrobium acidiphilum," Ferroplasma acidiphilum, and Thermoplasma acidophilum. The different phylogenetic groups were quantified by fluorescent in situ hybridization with a set of rRNA-targeted oligonucleotide probes. More than 80% of the cells were affiliated with the domain Bacteria, with only a minor fraction corresponding to Archaea. Members of Leptospirillum ferrooxidans, Acidithiobacillus ferrooxidans, and Acidiphilium spp., all related to the iron cycle, accounted for most of the prokaryotic microorganisms detected. Different isolates of these microorganisms were obtained from the Tinto ecosystem, and their physiological properties were determined. Given the physicochemical characteristics of the habitat and the physiological properties and relative concentrations of the different prokaryotes found in the river, a model for the Tinto ecosystem based on the iron cycle is suggested.

DNA Fingerprinting↗

PubMed2005

Biomass production and assimilation of dissolved organic matter by SAR11 bacteria in the Northwest Atlantic Ocean.

Members of the SAR11 clade often dominate the composition of marine microbial communities, yet their contribution to biomass production and the flux of dissolved organic matter (DOM) is unclear. In addition, little is known about the specific components of the DOM pool utilized by SAR11 bacteria. To better understand the role of SAR11 bacteria in the flux of DOM, we examined the assimilation of leucine (a measure of biomass production), as well as free amino acids, protein, and glucose, by SAR11 bacteria in the Northwest Atlantic Ocean. We found that when SAR11 bacteria were >25% of total prokaryotes, they accounted for about 30 to 50% of leucine incorporation, suggesting that SAR11 bacteria were major contributors to bacterial biomass production and the DOM flux. Specific growth rates of SAR11 bacteria either equaled or exceeded growth rates for the total prokaryotic community. In addition, SAR11 bacteria were typically responsible for a greater portion of amino acid assimilation (34 to 61%) and glucose assimilation (45 to 57%) than of protein assimilation (< or = 34%). These data suggest that SAR11 bacteria do not utilize various components of the DOM pool equally and may be more important to the flux of low-molecular-weight monomers than to that of high-molecular-weight polymers.

Alphaproteobacteria↗

PubMed2006

Genetic profiling of the oral microbiota associated with severe early-childhood caries.

The determination of the composition of the microbial community in the oral cavity is usually based on cultivation methods; however, nearly half of the bacteria in the saliva and the dental plaque are not cultivable. In this study, we evaluated the difference in oral microbial diversity between children with severe early-childhood caries (S-ECC) and caries-free (CF) controls by means of a cultivation-independent approach called denaturing gradient gel electrophoresis (DGGE). Pooled dental plaque samples were collected from 20 children aged 2 to 8 years. Total microbial genomic DNA was isolated from those subjects, and a portion of the 16S rRNA gene locus was PCR amplified by using universal primers. We observed that the mean species richness of the bacterial population was greater in the CF children (n = 12) (42 +/- 3.7) than in the S-ECC children (n = 8) (35 +/- 4.3); the difference was statistically significant (P = 0.005). The overall diversity of plaque samples as measured by the Shannon index was 3.5 for the S-ECC group and 3.7 for the CF group (P = 0.004). Differences in DGGE profiles were distinguished on the basis of a cluster analysis. Sequence analysis of excised DGGE bands consisted of 2.7 phylotypes, on average. After adjusting for the number of observed bands, we estimated that the S-ECC group exhibited 94.5 total phylotypes and that the CF group exhibited 113.4. These results suggest that the microbial diversity and complexity of the microbial biota in dental plaque are significantly less in S-ECC children than in CF children.

Bacterial Typing Techniques↗

PubMed2006

Decomposers (Lumbricidae, Collembola) affect plant performance in model grasslands of different diversity.

Decomposer invertebrates influence soil structure and nutrient mineralization as well as the activity and composition of the microbial community in soil and therefore likely affect plant performance and plant competition. We established model grassland communities in a greenhouse to study the interrelationship between two different functional groups of decomposer invertebrates, Lumbricidae and Collembola, and their effect on plant performance and plant nitrogen uptake in a plant diversity gradient. Common plant species of Central European Arrhenatherion grasslands were transplanted into microcosms with numbers of plant species varying from one to eight and plant functional groups varying from one to four. Separate and combined treatments with earthworms and collembolans were set up. Microcosms contained 15N labeled litter to track N fluxes into plant shoots. Presence of decomposers strongly increased total plant and plant shoot biomass. Root biomass decreased in the presence of collembolans and even more in the presence of earthworms. However, it increased when both animal groups were present. Also, presence of decomposers increased total N concentration and 15N enrichment of grasses, legumes, and small herbs. Small herbs were at a maximum in the combined treatment with earthworms and collembolans. The impact of earthworms and collembolans on plant performance strongly varied with plant functional group identity and plant species diversity and was modified when both decomposers were present. Both decomposer groups generally increased aboveground plant productivity through effects on litter decomposition and nutrient mineralization leading to an increased plant nutrient acquisition. The non-uniform effects of earthworms and collembolans suggest that functional diversity of soil decomposer animals matters and that the interactions between soil animal functional groups affect the structure of plant communities.

Animals↗

PubMedDate not supplied

Spatiotemporal variability of wet atmospheric nitrogen deposition to the Neuse River Estuary, North Carolina.

Excessive nitrogen (N) loading to N-sensitive waters such as the Neuse River estuary (North Carolina) has been shown to promote changes in microbial and algal community composition and function (harmful algal blooms), hypoxia and anoxia, and fish kills. Previous studies have estimated that wet atmospheric deposition of nitrogen (WAD-N), as deposition of dissolved inorganic nitrogen (DIN: NO3-, NH3/NH4+) and dissolved organic nitrogen, may contribute at least 15% of the total externally supplied or "new" N flux to the coastal waters of North Carolina. In a 3-yr study from June 1996 to June 1999, we calculated the weekly wet deposition of inorganic and organic N at eleven sites on a northwest-southeast transect in the watershed. The annual mean total (wet DIN + wet organics) WAD-N flux for the Neuse River watershed was calculated to be 956 mg N/m2/yr (15026 Mg N/yr). Seasonally, the spring (March-May) and summer (June-August) months contain the highest total weekly N deposition; this pattern appears to be driven by N concentration in precipitation. There is also spatial variability in WAD-N deposition; in general, the upper portion of the watershed receives the lowest annual deposition and the middle portion of the watershed receives the highest deposition. Based on a range of watershed N retention and in-stream riverine processing values, we estimate that this flux contributes approximately 24% of the total "new" N flux to the estuary.

Agriculture↗