Seasonal variation in denitrification and dissimilatory nitrate reduction to ammonia process rates and corresponding key functional genes along an estuarine nitrate gradient.

This research investigated spatial-temporal variation in benthic bacterial community structure, rates of denitrification and dissimilatory nitrate reduction to ammonium (DNRA) processes and abundances of corresponding genes and transcripts at three sites-the estuary-head, mid-estuary and the estuary mouth (EM) along the nitrate gradient of the Colne estuary over an annual cycle. Denitrification rates declined down the estuary, while DNRA rates were higher at the estuary head and middle than the EM. In four out of the six 2-monthly time-points, rates of DNRA were greater than denitrification at each site. Abundance of gene markers for nitrate-reduction (nitrate reductase narG and napA), denitrification (nitrite reductase nirS) and DNRA (DNRA nitrite reductase nrfA) declined along the estuary with significant relationships between denitrification and nirS abundance, and DNRA and nrfA abundance. Spatially, rates of denitrification, DNRA and corresponding functional gene abundances decreased along the estuary. However, temporal correlations between rate processes and functional gene and transcript abundances were not observed.

amoA Gene abundances and nitrification potential rates suggest that benthic ammonia-oxidizing bacteria and not Archaea dominate N cycling in the Colne Estuary, United Kingdom.

Nitrification, mediated by ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA), is important in global nitrogen cycling. In estuaries where gradients of salinity and ammonia concentrations occur, there may be differential selections for ammonia-oxidizer populations. The aim of this study was to examine the activity, abundance, and diversity of AOA and AOB in surface oxic sediments of a highly nutrified estuary that exhibits gradients of salinity and ammonium. AOB and AOA communities were investigated by measuring ammonia monooxygenase (amoA) gene abundance and nitrification potentials both spatially and temporally. Nitrification potentials differed along the estuary and over time, with the greatest nitrification potentials occurring mid-estuary (8.2 µmol N grams dry weight [gdw](-1) day(-1) in June, increasing to 37.4 µmol N gdw(-1) day(-1) in January). At the estuary head, the nitrification potential was 4.3 µmol N gdw(-1) day(-1) in June, increasing to 11.7 µmol N gdw(-1) day(-1) in January. At the estuary head and mouth, nitrification potentials fluctuated throughout the year. AOB amoA gene abundances were significantly greater (by 100-fold) than those of AOA both spatially and temporally. Nitrosomonas spp. were detected along the estuary by denaturing gradient gel electrophoresis (DGGE) band sequence analysis. In conclusion, AOB dominated over AOA in the estuarine sediments, with the ratio of AOB/AOA amoA gene abundance increasing from the upper (freshwater) to lower (marine) regions of the Colne estuary. These findings suggest that in this nutrified estuary, AOB (possibly Nitrosomonas spp.) were of major significance in nitrification.

UK catchment nutrient loads 1993-2003, a new approach using harmonised monitoring scheme data: temporal changes, geographical distribution, limiting nutrients and loads to coastal waters.

The work provides robust estimates of nutrient loads (nitrate and phosphate) from all UK catchments: as required by the Water Framework Directive to monitor catchments' health, and to inform management of these environments. To calculate nutrient loads, data for nutrient concentrations and water flow are combined. In the UK, flow data are typically available at hourly intervals at more than 1300 gauging stations but concentration data are collected less frequently (roughly weekly) and at fewer locations (about 280). The sparseness of the concentration data limits the occasions for which load can be calculated, so a mathematical model was derived which was used to interpolate the concentrations between measurements. The model's parameters provide useful information about the annual nutrient concentration cycles within any catchment, and permitted improved estimates of both the annual loads of N and P, and of the N : P ratios, from mainland UK catchments. Data from 1993-2003 showed nitrate loads from UK catchments were generally constant, while orthophosphate loads generally declined. N : P ratios suggested that most catchments in the north and west of the UK were potentially P-limited although a few were potentially N-limited, while many in central and eastern UK oscillated seasonally between N and P limitation. Knowledge of the nutrient which is potentially limiting to biological productivity is a key factor for management of a catchment's nutrient loads. Calculations of nutrient export loads to coastal regions showed that UK catchments contributed only about 16.5% of total fluvial loads of nitrate to the North Sea, or about 3% of the total N loads when inputs from the Atlantic were included. Orthophosphate loads from the UK catchments into the North Sea were only 1.7% of the total P inputs from rivers and the Atlantic but did not include riverine inputs of P adsorbed to particles.

Nitrate reduction functional genes and nitrate reduction potentials persist in deeper estuarine sediments. Why?

Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) are processes occurring simultaneously under oxygen-limited or anaerobic conditions, where both compete for nitrate and organic carbon. Despite their ecological importance, there has been little investigation of how denitrification and DNRA potentials and related functional genes vary vertically with sediment depth. Nitrate reduction potentials measured in sediment depth profiles along the Colne estuary were in the upper range of nitrate reduction rates reported from other sediments and showed the existence of strong decreasing trends both with increasing depth and along the estuary. Denitrification potential decreased along the estuary, decreasing more rapidly with depth towards the estuary mouth. In contrast, DNRA potential increased along the estuary. Significant decreases in copy numbers of 16S rRNA and nitrate reducing genes were observed along the estuary and from surface to deeper sediments. Both metabolic potentials and functional genes persisted at sediment depths where porewater nitrate was absent. Transport of nitrate by bioturbation, based on macrofauna distributions, could only account for the upper 10 cm depth of sediment. A several fold higher combined freeze-lysable KCl-extractable nitrate pool compared to porewater nitrate was detected. We hypothesised that his could be attributed to intracellular nitrate pools from nitrate accumulating microorganisms like Thioploca or Beggiatoa. However, pyrosequencing analysis did not detect any such organisms, leaving other bacteria, microbenthic algae, or foraminiferans which have also been shown to accumulate nitrate, as possible candidates. The importance and bioavailability of a KCl-extractable nitrate sediment pool remains to be tested. The significant variation in the vertical pattern and abundance of the various nitrate reducing genes phylotypes reasonably suggests differences in their activity throughout the sediment column. This raises interesting questions as to what the alternative metabolic roles for the various nitrate reductases could be, analogous to the alternative metabolic roles found for nitrite reductases.

Changes in benthic denitrification, nitrate ammonification, and anammox process rates and nitrate and nitrite reductase gene abundances along an estuarine nutrient gradient (the Colne estuary, United Kingdom).

Estuarine sediments are the location for significant bacterial removal of anthropogenically derived inorganic nitrogen, in particular nitrate, from the aquatic environment. In this study, rates of benthic denitrification (DN), dissimilatory nitrate reduction to ammonium (DNRA), and anammox (AN) at three sites along a nitrate concentration gradient in the Colne estuary, United Kingdom, were determined, and the numbers of functional genes (narG, napA, nirS, and nrfA) and corresponding transcripts encoding enzymes mediating nitrate reduction were determined by reverse transcription-quantitative PCR. In situ rates of DN and DNRA decreased toward the estuary mouth, with the findings from slurry experiments suggesting that the potential for DNRA increased while the DN potential decreased as nitrate concentrations declined. AN was detected only at the estuary head, accounting for approximately 30% of N2 formation, with 16S rRNA genes from anammox-related bacteria also detected only at this site. Numbers of narG genes declined along the estuary, while napA gene numbers were stable, suggesting that NAP-mediated nitrate reduction remained important at low nitrate concentrations. nirS gene numbers (as indicators of DN) also decreased along the estuary, whereas nrfA (an indicator for DNRA) was detected only at the two uppermost sites. Similarly, nitrate and nitrite reductase gene transcripts were detected only at the top two sites. A regression analysis of log(n + 1) process rate data and log(n + 1) mean gene abundances showed significant relationships between DN and nirS and between DNRA and nrfA. Although these log-log relationships indicate an underlying relationship between the genetic potential for nitrate reduction and the corresponding process activity, fine-scale environmentally induced changes in rates of nitrate reduction are likely to be controlled at cellular and protein levels.

Diversity and abundance of nitrate reductase genes (narG and napA), nitrite reductase genes (nirS and nrfA), and their transcripts in estuarine sediments.

Estuarine systems are the major conduits for the transfer of nitrate from agricultural and other terrestrial-anthropogenic sources into marine ecosystems. Within estuarine sediments some microbially driven processes (denitrification and anammox) result in the net removal of nitrogen from the environment, while others (dissimilatory nitrate reduction to ammonium) do not. In this study, molecular approaches have been used to investigate the diversity, abundance, and activity of the nitrate-reducing communities in sediments from the hypernutrified Colne estuary, United Kingdom, via analysis of nitrate and nitrite reductase genes and transcripts. Sequence analysis of cloned PCR-amplified narG, napA, and nrfA gene sequences showed the indigenous nitrate-reducing communities to be both phylogenetically diverse and also divergent from previously characterized nitrate reduction sequences in soils and offshore marine sediments and from cultured nitrate reducers. In both the narG and nrfA libraries, the majority of clones (48% and 50%, respectively) were related to corresponding sequences from delta-proteobacteria. A suite of quantitative PCR primers and TaqMan probes was then developed to quantify phylotype-specific nitrate (narG and napA) and nitrite reductase (nirS and nrfA) gene and transcript numbers in sediments from three sites along the estuarine nitrate gradient. In general, both nitrate and nitrite reductase gene copy numbers were found to decline significantly (P < 0.05) from the estuary head towards the estuary mouth. The development and application, for the first time, of quantitative reverse transcription-PCR assays to quantify mRNA sequences in sediments revealed that transcript numbers for three of the five phylotypes quantified were greatest at the estuary head.

Evaluation of quantitative polymerase chain reaction-based approaches for determining gene copy and gene transcript numbers in environmental samples.

Quantitative polymerase chain reaction (Q-PCR) amplification is widely applied for determining gene and transcript numbers within environmental samples. This research evaluated Q-PCR reproducibility via TaqMan assays quantifying 16S rRNA gene and transcript numbers in sediments, within and between replicate Q-PCR assays. Intra-assay variation in 16S rRNA gene numbers in replicate DNA samples was low (coefficients of variation; CV from 3.2 to 5.2%). However, variability increased using replicated standard curves within separate Q-PCR assays (CV from 11.2% to 26%), indicating absolute comparison of gene numbers between Q-PCR assays was less reliable. 16S rRNA transcript quantification was evaluated using standard curves of diluted RNA or cDNA (before, or following, reverse transcription). These standard curves were statistically different with cDNA-derived curves giving higher r(2) values and Q-PCR efficiencies. Template concentrations used in Q-PCR also affected 16S rRNA gene and transcript numbers. For DNA, 10(-3) dilutions yielded higher gene numbers than 10(-1) and 10(-2) dilutions. Conversely, RNA template dilution reduced numbers of transcripts detected. Finally, different nucleic acid isolation methods also resulted in gene and transcript number variability. This research demonstrates Q-PCR determination of absolute numbers of genes and transcripts using environmental nucleic acids should be treated cautiously.

Use of chlorate as a selective inhibitor to distinguish membrane-bound nitrate reductase (Nar) and periplasmic nitrate reductase (Nap) of dissimilative nitrate reducing bacteria in sediment.

The use of chlorate as a selective inhibitor of dissimilative nitrate reduction was studied using pure cultures of Comamonas testosteroni (a denitrifier) and Klebsiella pneumoniae (a nitrate-ammonifier) isolated from estuarine sediment, and in sediment slurry. Pure culture experiments demonstrated that chlorate selectively inhibited membrane-bound nitrate reductase (Nar) activity, probably by blocking nitrate transporters (NarK). Sediment slurry experiments showed that chlorate inhibited nitrate reduction and N(2)O formation, but did not inhibit nitrite reduction and its N(2)O formation, indicating that chlorate selectively inhibited only the first step of nitrate reduction. Chlorite chemically oxidized nitrite to nitrate and could not be used as a selective inhibitor of nitrite metabolism, although chlorite apparently selectively inhibited formation of N(2)O from nitrite. Chlorate can be used as a specific inhibitor to distinguish between nitrate reduction by Nap or Nar in natural communities of microorganisms.

Environmental costs of freshwater eutrophication in England and Wales.

Eutrophication has many known consequences, but there are few data on the environmental and health costs. We developed a new framework of cost categories that assess both social and ecological damage costs and policy response costs. These findings indicate the severe effects of nutrient enrichment and eutrophication on many sectors of the economy. We estimate the damage costs of freshwater eutrophication in England and Wales to be $105-160 million yr(-1) (pound 75.0-114.3 m). The policy response costs are a measure of how much is being spent to address this damage, and these amount to $77 million yr(-1) pound 54.8 m). The damage costs are dominated by seven items each with costs of $15 million yr(-1) or more: reduced value of waterfront dwellings, drinking water treatment costs for nitrogen removal, reduced recreational and amenity value of water bodies, drinking water treatment costs for removal of algal toxins and decomposition products, reduced value of nonpolluted atmosphere, negative ecological effects on biota, and net economic losses from the tourist industry. In common with other environmental problems, it would represent net value (or cost reduction) if damage was prevented at source. A variety of effective economic, regulatory, and administrative policy instruments are available for internalizing these costs.

Detection and diversity of expressed denitrification genes in estuarine sediments after reverse transcription-PCR amplification from mRNA.

The expression of five denitrification genes coding for two nitrate reductases (narG and napA), two nitrite reductases (nirS and nirK), and nitrous oxide reductase (nosZ) was analyzed by reverse transcription (RT)-PCR of mRNA extracted from two sediment samples obtained in the River Colne estuary (United Kingdom), which receives high nitrogen inputs and for which high denitrification rates have been observed. The presence of all five genes in both sediment samples was confirmed by PCR amplification from extracted DNA prior to analysis of gene expression. Only nirS and nosZ mRNAs were detected; nirS was detected directly as an RT-PCR amplification product, and nosZ was detected following Southern blot hybridization. This indicated that active expression of at least the nirS and nosZ genes was occurring in the sediments at the time of sampling. Amplified nirS RT-PCR products were cloned and analyzed by sequencing, and they were compared with amplified nirS gene sequences from isolates obtained from the same sediments. A high diversity of nirS sequences was observed. Most of the cloned nirS sequences retrieved were specific to one site or the other, which underlines differences in the compositions of the bacterial communities involved in denitrifrification in the two sediments analyzed.

Identification of active methylotroph populations in an acidic forest soil by stable-isotope probing.

Stable-isotope probing (SIP) is a culture-independent technique that enables the isolation of DNA from micro-organisms that are actively involved in a specific metabolic process. In this study, SIP was used to characterize the active methylotroph populations in forest soil (pH 3.5) microcosms that were exposed to (13)CH(3)OH or (13)CH(4). Distinct (13)C-labelled DNA ((13)C-DNA) fractions were resolved from total community DNA by CsCl density-gradient centrifugation. Analysis of 16S rDNA sequences amplified from the (13)C-DNA revealed that bacteria related to the genera Methylocella, Methylocapsa, Methylocystis and Rhodoblastus had assimilated the (13)C-labelled substrates, which suggested that moderately acidophilic methylotroph populations were active in the microcosms. Enrichments targeted towards the active proteobacterial CH(3)OH utilizers were successful, although none of these bacteria were isolated into pure culture. A parallel analysis of genes encoding the key enzymes methanol dehydrogenase and particulate methane monooxygenase reflected the 16S rDNA analysis, but unexpectedly revealed sequences related to the ammonia monooxygenase of ammonia-oxidizing bacteria (AOB) from the beta-subclass of the PROTEOBACTERIA: Analysis of AOB-selective 16S rDNA amplification products identified Nitrosomonas and Nitrosospira sequences in the (13)C-DNA fractions, suggesting certain AOB assimilated a significant proportion of (13)CO(2), possibly through a close physical and/or nutritional association with the active methylotrophs. Other sequences retrieved from the (13)C-DNA were related to the 16S rDNA sequences of members of the Acidobacterium division, the beta-Proteobacteria and the order Cytophagales, which implicated these bacteria in the assimilation of reduced one-carbon compounds or in the assimilation of the by-products of methylotrophic carbon metabolism. Results from the (13)CH(3)OH and (13)CH(4) SIP experiments thus provide a rational basis for further investigations into the ecology of methylotroph populations in situ.

Nitrous oxide formation in the Colne estuary, England: the central role of nitrite.

Nitrate and nitrite concentrations in the water and nitrous oxide and nitrite fluxes across the sediment-water interface were measured monthly in the River Colne estuary, England, from December 1996 to March 1998. Water column concentrations of N(2)O in the Colne were supersaturated with respect to air, indicating that the estuary was a source of N(2)O for the atmosphere. At the freshwater end of the estuary, nitrous oxide effluxes from the sediment were closely correlated with the nitrite concentrations in the overlying water and with the nitrite influx into the sediment. Increases in N(2)O production from sediments were about 10 times greater with the addition of nitrite than with the addition of nitrate. Rates of denitrification were stimulated to a larger extent by enhanced nitrite than by nitrate concentrations. At 550 microM nitrite or nitrate (the highest concentration used), the rates of denitrification were 600 micromol N.m(-2).h(-1) with nitrite but only 180 micromol N.m(-2).h(-1) with nitrate. The ratios of rates of nitrous oxide production and denitrification (N(2)O/N(2) x 100) were significantly higher with the addition of nitrite (7 to 13% of denitrification) than with nitrate (2 to 4% of denitrification). The results suggested that in addition to anaerobic bacteria, which possess the complete denitrification pathway for N(2) formation in the estuarine sediments, there may be two other groups of bacteria: nitrite denitrifiers, which reduce nitrite to N(2) via N(2)O, and obligate nitrite-denitrifying bacteria, which reduce nitrite to N(2)O as the end product. Consideration of free-energy changes during N(2)O formation led to the conclusion that N(2)O formation using nitrite as the electron acceptor is favored in the Colne estuary and may be a critical factor regulating the formation of N(2)O in high-nutrient-load estuaries.

Comparison of the molecular diversity of the methanogenic community at the brackish and marine ends of a UK estuary.

Abstract The 16S rRNA sequence diversity of the euryarchaeal community in a predominately freshwater sediment at East Hill Bridge (EHB) on the River Colne estuary, Essex, UK was investigated and compared to that from marine sediments at the mouth of the river (Colne Point). The East Hill Bridge sediments appear to support the full range of methanogen phenotypes with some genotypes similar to those previously detected at Colne Point. However, no Marine Benthic Group D or halophilic archaeal genotypes, both abundant in gene libraries at Colne Point, were detected at East Hill Bridge. Clones related to Methanosarcina and Methanocorpusculum were detected only at East Hill Bridge while clones closely related to Methanoculleus and Methanococcoides were detected only at Colne Point. The most common clones in the East Hill Bridge library were closely related to the obligate acetate-utilising Methanosaeta concilii, suggesting they may be important methanogens in these sediments. Clones that group closely with M. concilii appear to be ubiquitous in freshwater sediments and we suggest that they are prime candidates for a globally important acetoclastic methanogenic group. The distribution of clones in the East Hill Bridge and Colne Point libraries implies that certain methanogen groups are generalists, adapted to the range of conditions within an estuarine environment (e.g. Methanogenium) while others are more specialist (e.g. Methanosaeta).