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.     

The influence of nitrate concentration upon the end-products of nitrate dissimilation by bacteria in anaerobic salt marsh sediment

Abstract Experiments were carried out with slurries of saltmarsh sediment to which varying concentrations of nitrate were added. The acetylene blocking technique was used to measure denitrification by accumulation of nitrous oxide, while reduction of nitrate to nitrite and ammonium was also measured. There was good recovery of reduced nitrate and at the smallest concentration of nitrate used (250 μM) there was approximately equal reduction to either ammonium or nitrous oxide (denitrification). Nitrite was only a minor end-product of nitrate reduction. As the nitrate concentration was increased the proportion of the nitrate which was denitrified to nitrous oxide increased, to 83% at the greatest nitrate concentration used (2 mM), while reduction to ammonium correspondingly decreased. This change was attributed either to a greater competitiveness by the denitrifiers for nitrate as the ratio of electron donor to electron acceptor decreased; or to the increased production of nitrite rather than ammonium by fermentative bacteria under high nitrate, the nitrite then being reduced to nitrous oxide by denitrifying bacteria.     

Interactions of light, temperature and inorganic nitrogen in controlling planktonic nitrogen utilisation in the Tagus estuary

The effect of light, temperature and ammonium on inorganic nitrogen uptake by phytoplankton was investigated from June 1994 through December 1995 at three sites in the Tagus estuary (Portugal), during high tide of neap tides. Ammonium concentrations higher than 10 M reduced nitrate uptake down to 24% but never prevented it. Below this threshold concentration, nitrate uptake was neither inhibited nor changed. Uptake of both nitrate and ammonium as a function of light intensity exhibited a saturation response. Uptake reduction occurred in the near bottom phytoplankton populations, particularly for nitrate. The ammonium uptake system was less limited by light than the nitrate uptake system, indicating the importance of ammonium as a nitrogen source for the phytoplankton which is likely to experience high changes in light in the well-mixed water column of this estuarine environment. Ammonium uptake was exponentially related to temperature in the upper estuary whereas in the mid and lower estuary this relationship was linear. The effect of temperature on nitrate uptake was linear but far less marked than for ammonium uptake.     

Nitrate, Ammonium and Kinetin Effects on Growth and Enzyme Activities of Paul's Scarlet Rose Callus

Factorial experiments using the three variables nitrate, ammonium, and kinetin at six different concentrations each (nitrate 4.64 to 215 mM; ammonium 2.15 to 100 mM; and kinetin 0.1 to 4.64 mg/l) were set up to measure the effects of each of these factors, and their interactions, on the fresh weight, protein, and enzyme activities of callus of Paul's Scarlet Rose. Optimum fresh weight values were obtained with nitrate at 46.4 mM. Ammonium inhibited growth at concentrations above 2.15 mM, and kinetin had no significant effect. Significant interaction between nitrate and ammonium effects on growth was found. Kinetin did not interact significantly with either nitrate or ammonium to influence the fresh weight. The specific activity of glutamate dehydrogenase (NAD) in the aminating reaction increased with increasing ammonium concentrations to 21.5; at higher concentrations the activity remained high. Glutamine synthetase specific activity was constant over a large range of nitrate and ammonium concentrations, increasing only when nitrate went from 46.4 to 100 mM. Glutamine synthetase was sensitive to the nitrate: ammonium interaction. Specific activity decreased at progressively higher ammonium levels when nitrate concentration increased. No glutamate synthase activity was detected at optimal nitrate concentrations.     

Presence of nitrate-accumulating sulfur bacteria and their influence on nitrogen cycling in a shallow coastal marine sediment

Nitrate flux between sediment and water, nitrate concentration profile at the sediment-water interface, and in situ sediment denitrification activity were measured seasonally at the innermost part of Tokyo Bay, Japan. For the determination of sediment nitrate concentration, undisturbed sediment cores were sectioned into 5-mm depth intervals and each segment was stored frozen at -30 degrees C. The nitrate concentration was determined for the supernatants after centrifuging the frozen and thawed sediments. Nitrate in the uppermost sediment showed a remarkable seasonal change, and its seasonal maximum of up to 400 microM was found in October. The directions of the diffusive nitrate fluxes predicted from the interfacial concentration gradients were out of the sediment throughout the year. In contrast, the directions of the total nitrate fluxes measured by the whole-core incubation were into the sediment at all seasons. This contradiction between directions indicates that a large part of the nitrate pool extracted from the frozen surface sediments is not a pore water constituent, and preliminary examinations demonstrated that the nitrate was contained in the intracellular vacuoles of filamentous sulfur bacteria dwelling on or in the surface sediment. Based on the comparison between in situ sediment denitrification activity and total nitrate flux, it is suggested that intracellular nitrate cannot be directly utilized by sediment denitrification, and the probable fate of the intracellular nitrate is hypothesized to be dissimilatory reduction to ammonium. The presence of nitrate-accumulating sulfur bacteria therefore may lower nature's self-purification capacity (denitrification) and exacerbate eutrophication in shallow coastal marine environments.     

Presence of Nitrate-Accumulating Sulfur Bacteria and Their Influence on Nitrogen Cycling in a Shallow Coastal Marine Sediment

Nitrate flux between sediment and water, nitrate concentration profile at the sediment-water interface, and in situ sediment denitrification activity were measured seasonally at the innermost part of Tokyo Bay, Japan. For the determination of sediment nitrate concentration, undisturbed sediment cores were sectioned into 5-mm depth intervals and each segment was stored frozen at −30°C. The nitrate concentration was determined for the supernatants after centrifuging the frozen and thawed sediments. Nitrate in the uppermost sediment showed a remarkable seasonal change, and its seasonal maximum of up to 400 μM was found in October. The directions of the diffusive nitrate fluxes predicted from the interfacial concentration gradients were out of the sediment throughout the year. In contrast, the directions of the total nitrate fluxes measured by the whole-core incubation were into the sediment at all seasons. This contradiction between directions indicates that a large part of the nitrate pool extracted from the frozen surface sediments is not a pore water constituent, and preliminary examinations demonstrated that the nitrate was contained in the intracellular vacuoles of filamentous sulfur bacteria dwelling on or in the surface sediment. Based on the comparison between in situ sediment denitrification activity and total nitrate flux, it is suggested that intracellular nitrate cannot be directly utilized by sediment denitrification, and the probable fate of the intracellular nitrate is hypothesized to be dissimilatory reduction to ammonium. The presence of nitrate-accumulating sulfur bacteria therefore may lower nature's self-purification capacity (denitrification) and exacerbate eutrophication in shallow coastal marine environments.     

Factors controlling anaerobic ammonium oxidation with nitrite in marine sediments

Factors controlling the anaerobic oxidation of ammonium with nitrate and nitrite were explored in a marine sediment from the Skagerrak in the Baltic-North Sea transition. In anoxic incubations with the addition of nitrite, approximately 65% of the nitrogen gas formation was due to anaerobic ammonium oxidation with nitrite, with the remainder being produced by denitrification. Anaerobic ammonium oxidation with nitrite exhibited a biological temperature response, with a rate optimum at 15 degrees C and a maximum temperature of 37 degrees C. The biological nature of the process and a 1:1 stoichiometry for the reaction between nitrite and ammonium indicated that the transformations might be attributed to the anammox process. Attempts to find other anaerobic ammonium-oxidizing processes in this sediment failed. The apparent K(m) of nitrite consumption was less than 3 microM, and the relative importance of ammonium oxidation with nitrite and denitrification for the production of nitrogen gas was independent of nitrite concentration. Thus, the quantitative importance of ammonium oxidation with nitrite in the jar incubations at elevated nitrite concentrations probably represents the in situ situation. With the addition of nitrate, the production of nitrite from nitrate was four times faster than its consumption and therefore did not limit the rate of ammonium oxidation. Accordingly, the rate of this process was the same whether nitrate or nitrite was added as electron acceptor. The addition of organic matter did not stimulate denitrification, possibly because it was outcompeted by manganese reduction or because transport limitation was removed due to homogenization of the sediment.     

The application of a laboratory apparatus for the study of nutrient fluxes between sediment and water

Sediments of the river Elbe estuary have been studied to assess their impact on the total nitrogen budget of the estuary. A new laboratory incubation apparatus was used to provide a means of regulating important parameters such as temperature and oxygen concentrations. With this apparatus sediment cores from a typical shallow water area with high organic carbon content were incubated under varying oxygen concentrations in the overlying water. Measurements of ammonium, nitrite, nitrate and nitrous oxide in the water phase were carried out and the fluxes between sediment and water phase calculated. During aerobic conditions in the water phase overall nitrate fluxes between + 4 and –3.5 mmol Nm^–2d^–1 across the sediment/water interface were observed. Under anaerobic conditions the fluxes increased up to –10 mmol Nm^–2 d^–1. Nitrous oxide was formed within the sediment under both aerobic and anaerobic conditions. Fluxes into the water phase were highest when the oxygen concentrations in the water phase were low (between 0.1 and 0.6 mg l^–1).     

Spatial heterogeneity of bacterial community structure in the sediments of the Pearl River estuary

Denaturing gradient gel electrophoresis (DGGE) and multivariate statistical analytical methods were applied to investigate the spatial variation of bacterial community structure in the Pearl River estuary sediment and to address the relationship between microbial community composition and bottom water chemistry in ten different stations. Preliminary results of sequencing analysis of the excised DGGE bands suggested that α-Proteobacteria, γ-Proteobacteria, Acidobacteria and Actinobacteria were the dominant bacterial groups in the Pearl River estuary sediment. Results of multidimensional scaling analysis of these field data suggested that the composition of bacterial communities varied with sampling sites. Finally, canonical correspondence analysis of the data of environmental variables and bacterial community suggested that bacterial community structure was significantly influenced by the change of environmental variables (total phosphorus, nitrite, ammonium, dissolved oxygen, pH and salinity).     

Factors Controlling Anaerobic Ammonium Oxidation with Nitrite in Marine Sediments

Factors controlling the anaerobic oxidation of ammonium with nitrate and nitrite were explored in a marine sediment from the Skagerrak in the Baltic-North Sea transition. In anoxic incubations with the addition of nitrite, approximately 65% of the nitrogen gas formation was due to anaerobic ammonium oxidation with nitrite, with the remainder being produced by denitrification. Anaerobic ammonium oxidation with nitrite exhibited a biological temperature response, with a rate optimum at 15°C and a maximum temperature of 37°C. The biological nature of the process and a 1:1 stoichiometry for the reaction between nitrite and ammonium indicated that the transformations might be attributed to the anammox process. Attempts to find other anaerobic ammonium-oxidizing processes in this sediment failed. The apparent K_m of nitrite consumption was less than 3 μM, and the relative importance of ammonium oxidation with nitrite and denitrification for the production of nitrogen gas was independent of nitrite concentration. Thus, the quantitative importance of ammonium oxidation with nitrite in the jar incubations at elevated nitrite concentrations probably represents the in situ situation. With the addition of nitrate, the production of nitrite from nitrate was four times faster than its consumption and therefore did not limit the rate of ammonium oxidation. Accordingly, the rate of this process was the same whether nitrate or nitrite was added as electron acceptor. The addition of organic matter did not stimulate denitrification, possibly because it was outcompeted by manganese reduction or because transport limitation was removed due to homogenization of the sediment.     

Temperature dependence of inorganic nitrogen uptake: reduced affinity for nitrate at suboptimal temperatures in both algae and bacteria.

Nitrate utilization and ammonium utilization were studied by using three algal isolates, six bacterial isolates, and a range of temperatures in chemostat and batch cultures. We quantified affinities for both substrates by determining specific affinities (specific affinity = maximum growth rate/half-saturation constant) based on estimates of kinetic parameters obtained from chemostat experiments. At suboptimal temperatures, the residual concentrations of nitrate in batch cultures and the steady-state concentrations of nitrate in chemostat cultures both increased. The specific affinity for nitrate was strongly dependent on temperature (Q10 approximately 3, where Q10 is the proportional change with a 10 degrees C temperature increase) and consistently decreased at temperatures below the optimum temperature. In contrast, the steady-state concentrations of ammonium remained relatively constant over the same temperature range, and the specific affinity for ammonium exhibited no clear temperature dependence. This is the first time that a consistent effect of low temperature on affinity for nitrate has been identified for psychrophilic, mesophilic, and thermophilic bacteria and algae. The different responses of nitrate uptake and ammonium uptake to temperature imply that there is increasing dependence on ammonium as an inorganic nitrogen source at low temperatures.     

The influence of nitrate on the phosphorus flux to and from oxygen depleted lake sediments

Addition of nitrate to an oxygen depleted sediment leads to a stimulation of the mineralization process if a major part of the nitrate is dissimilatorily reduced. This may cause an increased release of phosphate from some sediments. Nitrate, however, maintains a high redox potential at the sediment surface and thus prevents a release of iron-bound phosphorus. These two counteracting effects of nitrate addition to sediment-water systems were demonstrated in laboratory experiments. A high supply of nitrate to a phosphorus-saturated sediment caused an increased release of phosphate and ammonium once the nitrate had been used up. However, from other sediments there was no or very little release of phosphate but an increased release of ammonium caused by high nitrate dissimilation, probably due to the very high capacity of these sediments to bind liberated phosphate or due to storage of phosphorus in an increased bacterial biomass. Phosphorus fractionation, before and after the experiments with the sediments, provided information on transfers to, from and within the sediments.     

Diversity, abundance, and potential activity of nitrifying and nitrate-reducing microbial assemblages in a subglacial ecosystem

Subglacial sediments sampled from beneath Robertson Glacier (RG), Alberta, Canada, were shown to harbor diverse assemblages of potential nitrifiers, nitrate reducers, and diazotrophs, as assessed by amoA, narG, and nifH gene biomarker diversity. Although archaeal amoA genes were detected, they were less abundant and less diverse than bacterial amoA, suggesting that bacteria are the predominant nitrifiers in RG sediments. Maximum nitrification and nitrate reduction rates in microcosms incubated at 4°C were 280 and 18.5 nmol of N per g of dry weight sediment per day, respectively, indicating the potential for these processes to occur in situ. Geochemical analyses of subglacial sediment pore waters and bulk subglacial meltwaters revealed low concentrations of inorganic and organic nitrogen compounds. These data, when coupled with a C/N atomic ratio of dissolved organic matter in subglacial pore waters of ~210, indicate that the sediment communities are N limited. This may reflect the combined biological activities of organic N mineralization, nitrification, and nitrate reduction. Despite evidence of N limitation and the detection of nifH, we were unable to detect biological nitrogen fixation activity in subglacial sediments. Collectively, the results presented here suggest a role for nitrification and nitrate reduction in sustaining microbial life in subglacial environments. Considering that ice currently covers 11% of the terrestrial landmass and has covered significantly greater portions of Earth at times in the past, the demonstration of nitrification and nitrate reduction in subglacial environments furthers our understanding of the potential for these environments to contribute to global biogeochemical cycles on glacial-interglacial timescales.     

Application of Molecular Biological Techniques to a Seasonal Study of Ammonia Oxidation in a Eutrophic Freshwater Lake

The autotrophic ammonia-oxidizing bacteria in a eutrophic freshwater lake were studied over a 12-month period. Numbers of ammonia oxidisers in the lakewater were small throughout the year, and tangential-flow concentration was required to obtain meaningful estimates of most probable numbers. Sediments from littoral and profundal sites supported comparatively large populations of these bacteria, and the nitrification potential was high, particularly in summer samples from the littoral sediment surface. In enrichment cultures, lakewater samples nitrified at low (0.67 mM) ammonium concentrations only whereas sediment samples exhibited nitrification at high (12.5 mM) ammonium concentrations also. Enrichments at low ammonium concentration did not nitrify when inoculated into high-ammonium medium, but the converse was not true. This suggests that the water column contains a population of ammonia oxidizers that is sensitive to high ammonium concentrations. The observation of nitrification at high ammonium concentration by isolates from some winter lakewater samples, identified as nitrosospiras by 16S rRNA probing, is consistent with the hypothesis that sediment ammonia oxidizers enter the water column at overturn. With only one exception, nested PCR amplification enabled the detection of Nitrosospira 16S rDNA in all samples, but Nitrosomonas (N. europaea-eutropha lineage) 16S rDNA was never obtained. However, the latter were part of the sediment and water column communities, because their 16S rRNA could be detected by specific oligonucleotide probing of enrichment cultures. Furthermore, a specific PCR amplification regime for the Nitrosomonas europaea ammonia monooxygenase gene (amoA) yielded positive results when applied directly to sediment and lakewater samples. Patterns of Nitrosospira and Nitrosomonas detection by 16S rRNA oligonucleotide probing of sediment enrichment cultures were complex, but lakewater enrichments at low ammonium concentration were positive for nitrosomonads and not nitrosospiras. Analysis of enrichment cultures has therefore provided evidence for the existence of subpopulations within the lake ammonia-oxidizing community distinguishable on the basis of ammonium tolerance and possibly showing a seasonal distribution between the sediment and water column.     

Sediment-water fluxes of nutrients in an Antarctic coastal environment: influence of bioturbation

Rates of exchanges of nitrate and ammonium across the sediment-water interface were measured in an inshore marine environment at Signy Island, South Orkney Islands, Antarctica, over 6 months from August 1991 to February 1992. The sediment was a source of ammonium to the water column but a sink of nitrate, although nitrate exchange rates were very variable. Concentration profiles of nitrate and ammonium in the sediment porewater corroborated the measured vertical exchanges. Bioturbation, by a largely amphipod benthic infauna which was confined to the top 2 cm of sediment, was investigated experimentally. Removal of bioturbation depressed sedimentary O₂ uptake by 33% and sedimentary release of NH₄ ⁺ by 50%. In contrast, in the absence of bioturbation, the removal of NO₃ ^– from the water column by the sediment increased in rate. The measured fluxes of ammonium and nitrate from the sediment did not match with the amount of nitrogen mineralised within the sediment, and urea may account for the difference. It is suggested that the export of nitrogen from the bottom sediment may be significant in sustaining primary production in the Antarctic inshore environment. Ammonium and urea are preferred to nitrate as a nitrogen source by phytoplankton. The nitrate concentrations in the sediment porewater were low, but a large pool of nitrate was identified in the top 0–2 cm layer, which was released by KCl extraction or by freezing of the sediment. This extractable pool of nitrate did not equilibrate with the soluble nitrate pool in the sediment, but seemed to be released from components of the benthic infauna, which were also largely confined to the top 0–2 cm. The physiological role of this nitrate is unknown.     

Community Composition and Abundance of Ammonia-Oxidizing Archaea in Sediments from the Changjiang Estuary and its Adjacent Area in the East China Sea

Community composition and abundance of ammonia-oxidizing archaea (AOA) were investigated using ammonia monooxygenase α subunit (amoA) in sediments from the Changjiang estuary and its adjacent area in the East China Sea (ECS). Real-time quantitative polymerase chain reaction (qPCR), clone libraries and sequencing were performed to characterize the AOA community. Clone libraries analysis showed that the majority of amoA sequences fell within the Nitrosopumilus cluster. Correlation analysis showed that AOA diversity was closely related to the nitrite concentration, which was consistent with the canonical correspondence analysis (CCA) where a significant association between nitrite and AOA community composition was observed. The qPCR results were found to be significantly correlated with the environmental parameters. In the gravity cores, a significant positive correlation was found between ammonium concentrations and amoA gene copy numbers from different sediment depths at station S31. At station S33, however, ammonium concentration had a negative correlation and nitrite concentration had a positive correlation with amoA gene copy numbers. In the surface sediments, chlorophyll a concentration had a negative correlation and nitrate concentration had a positive correlation with amoA gene copy numbers. Compared amoA gene copy numbers from AOA with those from ammonia-oxidizing β-proteobacteria (β-AOB) in the same studied areas, the amoA gene copy ratio of β-AOB to AOA was negatively correlated with the phosphate concentration and dissolved oxygen concentration, but was not significantly correlated with either ammonium concentrations or salinity. Our data provided valuable information to achieve a better understanding of the potential role of ammonia oxidizers at the interface between terrestrial and marine environments.     

Assessing Nitrification and Denitrification in the Seine River and Estuary Using Chemical and Isotopic Techniques

Downstream from metropolitan Paris (France), a large amount of ammonium is discharged into the Seine River by the effluents of the wastewater treatment plant at Achères. To assess the extent of nitrification and denitrification in the water column, concentrations and isotopic compositions of ammonium (δ¹⁵N–NH₄⁺) and nitrate (δ¹⁵N–NO₃⁻, δ¹⁸O–NO₃⁻) were measured during summer low-flow conditions along the lower Seine and its estuary. The results indicated that most of the ammonium released from the wastewater treatment plant is nitrified in the lower Seine River and its upper estuary, but there was no evidence for water-column denitrification. In the lower part of the estuary, however, concentration and isotopic data for nitrate were not consistent with simple mixing between riverine and marine nitrate. A significant departure of the nitrate isotopic composition from what would be expected from simple mixing of freshwater and marine nitrates suggested coupled nitrification and denitrification in the water, in spite of the apparent conservative behavior of nitrate. Denitrification rates of approximately 0.02 mg N/L/h were estimated for this part of the estuary.     

Relative influence of temperature and electron donor and electron acceptor concentrations on bacterial sulfate reduction in saltmarsh sediment

The relative importance of three environmental variables known to influence the rate of bacterial sulfate reduction was examined using sediment from a saltmarsh pan. The variables investigated were temperature, electron donor concentration, and electron acceptor concentration. Their relative influence on the rate of bacterial sulfate reduction was examined with multiple replicate sediment samples in which the variables were experimentally adjusted. Sulfate reduction rates were measured with³⁵SO ₄ ^2– .The relative importance of each variable to sulfate reduction rate was assessed with multiple regression analysis by calculating the standardized partial regression coefficients, and the results were compared with the ranges of the three variables encountered in the natural sediment. Temperature proved to have the greatest influence, followed by electron donor and electron acceptor concentrations, in that order. The sulfate concentration was shown to have little influence on sulfate reduction rate at seawater concentrations of sulfate, but its effect increased if sulfate concentrations were diminished compared to those of seawater.     

Nitrate Regeneration Coupled to Degradation of Different Size Fractions of DON by the Picoplankton in the Elbe Estuary

Bacterial conversion of high (HMW > 3,000 Da) and low (LMW < 3,000 Da) molecular weight DON (dissolved organic nitrogen) was studied along the freshwater section of the Elbe estuary during the summer of 1997. Indigenous populations of picoplankton were incubated in a flow-through chamber that allowed a constant exchange of sterile, filtered Elbe water as the culture medium for the microorganisms, which remained within the chamber. Nitrogen conversion was followed by changes in the concentrations of total and low molecular weight DON and dissolved inorganic nitrogen compounds, the uptake of O2, and bacterial growth. Along the Elbe estuary, total DON concentrations varied between 0.69 and 1.1 mg N L-1, of which between 64 and 79% was LMW-DON. Ammonium was a minor nutrient present in the Elbe at concentrations below 0.26 mg N L-1. During incubation in the laboratory between 27 and 64% of the LMW-DON was consumed at rates ranging from 24 to 51 g N L-1 h-1. HMW-DON was used only when the degradable LMW-DON pool became exhausted and accounted for between 60 and 100% of the HMW-DON. This produced an increase in the DON consumption rate between 43 and 79 g N L-1 h-1. Nitrification rates were independent of the external ammonium concentration until it decreased to below 1 g N-NH4 L-1. Most of the N in the nitrification process originated as NH4 regenerated from DON. Between 75 and 100% of the LMW-DON and ammonium consumed was rapidly converted to nitrate. This amount decreased to between 65 and 85% when HMW-DON was consumed in addition to the LMW-DON. DON and ammonium consumption supported nitrification rates up to 71 g N L-1 h-1. The amount of DOC (dissolved organic carbon) degraded was not equivalent to the C:N ratio of the total dissolved organic matter. Calculations based on oxygen consumption for respiration and ammonium regeneration revealed that the substrates used during the incubations contained C:N at ratios of about 3:1. These results suggest that the nitrogen-rich compounds had been removed from the dissolved organic matter and subsequently consumed by bacteria, while the carbon skeleton remained mostly unaffected by the degradation processes.