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)

Abstract

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...

Deep-sea hydrothermal ventEpsilonproteobacteriaencode a conserved and widespread nitrate reduction pathway (Nap)

“…The saturation constants (K s ) for nitrate for the two strains were estimated to be 15 and 50 mM for Nap and Nar, respectively (Potter et al, 1999). These observations, also supported by a study where NapA-mediated nitrate reduction in estuarine sediments remained important at low nitrate concentrations (Dong et al, 2009), imply that between the two enzymes NapA is better adapted to function in nitrate-depleted environments. Epsilonproteobacteria include very different organisms, such as deep-sea vent chemolithoautotrophs (for example, Caminibacter and Sulfurimonas spp.)…”

“…This is consistent with data showing that ammonification is more prevalent than denitrification in nitrate-reducing thermophiles (Blochl et al, 1997;Vetriani et al, 2004;Perez-Rodriguez et al, 2012), and may be related to a possible decrease in nitrate concentrations as the hydrothermal fluid temperature increases. In line with this hypothesis, an increase in the significance of nitrate ammonification relative to denitrification as the concentration of nitrate decreased was observed in estuarine sediments (Dong et al, 2009). Phylogenetic analyses of the denitrification or ammonification pathways in these bacteria should provide further clues on their respective evolutionary histories.…”

“…Although surveys of genes involved in nitrate reduction have been carried out in soils and estuarine environments (Bru et al, 2007;Smith et al, 2007;Dong et al, 2009), the distribution and diversity of the nitrate reductase in natural microbial communities from deep-sea geothermal environments are scarce. Recently, Perez-Rodriguez et al (2013) identified Gamma-and Alphaproteobacteriarelated, membrane-bound nitrate reductases, NarG, in vent microbial communities.…”

Lineage-specific molecular probing reveals novel diversity and ecological partitioning of haplosporidians

“…Sixteen B2 g sediment samples were collected from each site in July and October. To provide a wider geographic and ecological representation, five further sample sets were analysed: (1) South Africa (SA): 120 water (B2 l) and sediment (B1-2 g) samples collected in December 2011 from freshwater and marine sites in South Africa, (2) BioMarKs (BM): matched DNA and cDNA from water column and sediment samples from eight coastal marine sites from the North Sea, the Mediterranean, the Baltic and the Black Sea (collected as part of the BioMarKs consortium, http:// www.biomarks.eu) (Bass et al, 2012;Logares et al, 2012;Pawlowski et al, 2013), (3) Exeter (EXE): 118 DNA extractions enriched in small metazoans (by filter fractionation, picking of specimens and so on) from freshwater and marine water samples from southern England and the Mediterranean (K Hamilton and B Williams, personal communication), (4) Estuarine gradient (EST): DNA from 32 marine-tobrackish sediment samples (B1-2 g), collected along the River Colne estuary in southeast England (Hawkins and Purdy, 2007;Dong et al, 2009) and (5) Panama (PAN): 12 marine sediment samples (B1-2 g) from sandy beach sites on the Pacific and Caribbean coasts around Panama City and Portobelo (Coló n). All the samples tested are listed in Supplementary Material 1.…”

Activity and abundance of denitrifying bacteria in the subsurface biosphere of diffuse hydrothermal vents of the Juan de Fuca Ridge

“…NO − 2 and NO − 3 supply have been shown to stimulate denitrification and influence the distribution of denitrifying genes in marine, estuarine (e.g., Jayakumar et al, 2004;Dong et al, 2009) and terrestrial (e.g., Bradley et al, 1992;Smith et al, 2006;Opdyke et al, 2007;Zhong et al, 2010) environments. The significant negative relationship observed between denitrification rates and in situ NO We also found a significant relationship between anammox rates and the DIN deficit, but this relationship should be interpreted with caution because of the limited number of measurements available for comparison (Fig.…”