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 (£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 (£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.

Estuarine systems are the major conduits for the transfer of nitrate from agricultural and other terrestrialanthropogenic 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.

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.

The effect of environmental temperature on the affinity of microorganisms for substrates is discussed in relation to measurements of affinity by either K(s) values or specific affinity (a(A)). It can be shown for psychrophiles, mesophiles and thermophiles that when a(A) is used as the measure of affinity, affinity decreases consistently as temperature drops below the optimum temperature for growth. This effect may be because of stiffening of the lipids of the membrane below the temperature optimum, leading to decreased efficiency of transport proteins embedded in the membrane. The lower temperature limit for growth is, therefore, that temperature at which an organism is no longer able to supply the maintenance requirement of the growth rate-limiting nutrient because of loss of affinity for that substrate. This linking of temperature and affinity for substrates taken up by active transport (a temperature-modulated substrate affinity model) includes uptake of both organic and inorganic substrates. This effect of decreased substrate affinity at low temperature may have profound implications on the availability of substrates in the natural environment as environmental temperatures change. At temperatures below their optimum for growth microorganisms will become increasingly unable to sequester substrates from their environment because of lowered affinity, exacerbating the anyway near-starvation conditions in many natural environments.

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 35) 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 β-subclass of the Proteobacteria. Analysis of AOB-selective 16S rDNA amplification products identifiedNitrosomonas 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 β-Proteobacteria and the order Cytophagales, which implicated these bacteria in the assimilation of reduced one-carbon compounds or in the assimilation of the byproducts 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.

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 ϳ30% of N 2 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.Estuaries are major conduits for the transport of anthropogenically derived nitrogen (e.g., from fertilizer runoff and from wastewater treatment plants) from land to sea (18,20). Estuarine sediments are now recognized as being an important location for the removal of inorganic nitrogen from this environment via benthic nitrate reduction to nitrogenous gases (21). Previously, we have utilized the isotope-pairing technique (22) to investigate rates of denitrification (DN) of nitrate to N 2 O and N 2 along the nitrate and salinity gradients in the hypernutrified Colne estuary, United Kingdom, and demonstrated that DN rates are highest at the estuary head, where nitrate levels are also at their highest (7, 9). However, DN represents only one of three key pathways relevant to nitrate reduction, with a recent review suggesting that the importance of DN in aquatic systems may be overstated (3). A substantial proportion of nitrate may alternatively be converted to ammonium (1, 16, 17) via dissimilatory nitrate reduction to ammonium (DNRA), in which case inorganic nitrogen is retained within the aquatic environment. Nitrite, usually derived from nitrate reduc...

No abstract

No abstract