Diurnal patterns of denitrification, oxygen consumption and nitrous oxide production in rivers measured at the whole-reach scale

1. Denitrification, net oxygen consumption and net nitrous oxide flux to the atmosphere were measured in three small rivers (discharge approximately 2–27 m³ s^?1) at the whole reach scale during Spring and Summer, 2002. Two of these rivers (Iroquois River and Sugar Creek in north‐west Indiana – north‐east Illinois, U.S.A.) drained agricultural catchments and the other (Millstone River in central New Jersey, U.S.A.) drained a mixed suburban–agricultural catchment. 2. Denitrification, oxygen consumption and N₂O flux were measured based on net changes in dissolved gas concentrations (N₂, O₂, and N₂O) during riverine transport, correcting for atmospheric exchange. On each date, measurements were made during both light and dark periods. 3. Denitrification rates in these rivers ranged from 0.31 to 15.91 mmol N m^?2 h^?1, and rates within each river reach were consistently higher during the day than during the night. This diurnal pattern could be related to cyclic patterns of nitrification driven by diurnal variations in water column pH and temperature. 4. Oxygen consumption ranged from 2.56 to 241 mmol O₂ m^?2 h^?1. In contrast to denitrification, net oxygen consumption was generally higher during the night than during the day. 5. River water was consistently supersaturated with N₂O, ranging from 102 to 209% saturated. Net flux of N₂O to the atmosphere ranged from 0.4 to 60 μmol N m^?2 h^?1. Net flux of N₂O was generally higher at night than during the day. The high flux of N₂O from these rivers strengthens the argument that rivers are an important contributor to anthropogenic emissions of this greenhouse gas.     

Limitations to measuring riverine denitrification at the whole reach scale: effects of channel geometry,wind velocity,sampling interval,and temperature inputs of N<Subscript>2</Subscript>-enriched groundwater

A model-based approach was recently introduced for measuring riverine denitrification based on measured changes in dissolved N₂ concentration during riverine transport (Laursen & Seitzinger, 2002a). Inputs to the model, including water temperature, channel depth, wind velocity, and time-of-travel between sampling locations, vary greatly among natural systems. Simulations were run by varying the values of these inputs and determining rates of N₂ accumulation in river water and the detection limits for measuring denitrification using this method. Dinitrogen was found to accumulate most rapidly in streams that were shallow, particularly under conditions of low wind velocity. Dissolved N₂ concentrations, modeled in rivers with a diurnal temperature variation of 5 °C and under conditions of no denitrification or 1 mmol N m⁻² h⁻¹, showed that sensitivity of the method can vary as temperatures change. Under low wind conditions and in rivers <1m in depth, this method is capable of detecting denitrification rates as low as 30–100 μmol N m⁻² h⁻¹. This limit of detection should be adequate to measure in situ rates in many North American streams, particularly in agricultural watersheds. In deeper rivers N₂ accumulated more slowly and the method became less sensitive. The results of this study should guide decisions regarding the application of this method based on the specific characteristics of a study reach (channel geometry) and the physical conditions (i.e. wind velocity and water temperature) under which measurements are to be made. The input of N₂-enriched groundwater along a study reach can result in N₂ accumulation that could be misinterpreted as denitrification. Some knowledge of the inputs of groundwater along a reach should also guide decisions regarding the application of this method.     

Nitrate reduction in sediments of lowland tropical streams draining swamp forest in Costa Rica: An ecosystem perspective

Nitrate reduction and denitrification were measured in swamp forest streams draining lowland rain forest on Costa Rica's Atlantic slope foothills using the C₂H₂-block assay and sediment-water nutrient fluxes. Denitrification assays using the C₂H₂-block technique indicated that the full suite of denitrifying enzymes were present in the sediment but that only a small fraction of the functional activity could be expressed without adding NO₃ ^–. Under optimal conditions, denitrification enzyme activity averaged 15 nmoles cm^–3 sediment h^–1. Areal NO₃ ^– reduction rates measured from NO₃ ^– loss in the overlying water of sediment-water flux chambers ranged from 65 to 470 umoles m^–2 h^–1. Oxygen loss rates accompanying NO₃ ^– depletion averaged 750 umoles m^–2 h^–1. Corrected for denitrification of NO₃ ^– oxidized from NH₄ ⁺ in the sediment, gross NO₃ ^– reduction rates increase by 130 umoles m^–2 h^–1, indicating nitrification may be the predominant source of NO₃ ^– for NO₃ ^– reduction in swamp forest stream sediments. Under field conditions approximately 80% of the increase in inorganic N mass along a 1250-m reach of the Salto River was in the form of NO₃ ^– with the balance NH₄ ⁺ . Scrutiny of potential inorganic N sources suggested that mineralized N released from the streambed was a major source of the inorganic N increase. Despite significant NO₃ ^– reduction potential, swamp forest stream sediments appear to be a source of inorganic N to downstream communities.     

Potential rates of nitrification and denitrification in an oligotrophic freshwater sediment system

Potential rates of nitrification and denitrification were measured in an oligotrophic sediment system. Nitrification potential was estimated using the CO oxidation technique, and potential denitrification was measured by the acetylene blockage technique. The sediments demonstrated both nitrifying and denitrifying activity. E_h, O₂, and organic C profiles showed two distinct types of sediment. One type was low in organic C, had high O₂ and E_h, and had rates of denitrification 1,000 times lower than the other which had high organic C, low O₂, and low E_h. Potential nitrification and denitrification rates were negatively correlated with E_h. This suggests that environmental heterogeneity in denitrifier and nitrifier populations in oligotrophic sediment systems may be assessed using E_h before sampling protocols for nitrification or denitrification rates are established. There was no correlation between denitrification and nitrification rates or between either of these processes and NH₄ ⁺ or NO₃ ^– concentrations. The maximum rate of denitrification was 0.969 nmole N cm^–3 hour^–1, and the maximum rate of nitrification was 23.6 nmole cm^–3 hour^–1, suggesting nitrification does not limit denitrification in these oligotrophic sediments. Some sediment cores had mean concentrations of 6.0 mg O₂/liter and still showed both nitrification and denitrification activity.     

Denitrification in the water column of an intermittently anoxic fjord

Denitrification was studied in the water column in the Bunnefjord, inner part of the Oslofjord in southern Norway, using a ¹⁵N-technique (the isotope pairing method). The fjord is 150 m deep and during our surveys in September–December 1998 hydrogen sulphide was present in the deep water below 80 m. No significant denitrification was found in water samples from the surface layer (4 m depth), but high rates were observed within a deep density gradient between 62 and 78 m depth. Oxygen concentration within this layer was low (<21 mmol m^–3), and the concentration of NO₃ decreased from ca. 15 mmolm^–3 at 62 m depth to not detectable below 78 m. Pronounced peaks of NO₂ up to 4.4 mmol m^–3 were observed at 70–78 m depth. The maximum denitrification rate of 1.5 mmol N m^–3 d^–1 was observed at 70 m depth. Integrated for the whole layer, the denitrification rate was 13 mmol N m^–2 d^–1. A significant linear correlation was found between the denitrification rate and the ambient nitrate concentration which indicated that the rate was primarily controlled by the availability of nitrate in the O₂-poor water. Compared to rates reported for coastal water, denitrification in the water column in the Bunnefjord was high and the process appears to be a major sink of bioavailable nitrogen in the fjord.     

Measurement of Denitrification in Two Freshwater Sediments by an In Situ Acetylene Inhibition Method

An acetylene inhibition method was satisfactorily used for the in situ measurement of denitrification in two sediment-water systems incubated for not more than 22 h. In the presence of added nitrate, denitrification acted as a source of nitrous oxide in a drainage pond, but acted as a sink in its absence. The averaged rates of nitrous oxide accumulation with nitrate enrichment in the absence and presence of acetylene were 0.15 and 0.30 mg of N m⁻²h⁻¹, respectively. Acetylene reduction at an average rate of 0.07 mmol of C₂H₄ formed m⁻²h⁻¹ was simultaneously measured in the absence of added nitrate. In a small eutrophic lake where nitrogen was nonlimiting, the in situ rates of sediment denitrification were 0.09 and 0.11 mg of N m⁻²h⁻¹ in the presence and absence of macrophytes, respectively, and no acetylene reduction activity was found.     

Whole-system estimation of denitrification in a plains river: a comparison of two methods

Whole-system denitrification in the South Platte River was measured over a 13-month period using an open-channel N₂ method and mass-balance measurements. Concentrations of dissolved N₂ were measured with high precision by membrane-inlet mass spectrometry and estimates of denitrification were based on the mass flux of N₂, after correction for reaeration and groundwater flux. Open-channel estimates of denitrification ranged from 0 to 3.08 g N m^–2 d^–1 and the mean annual rate was 1.62 g N m^–2 d^–1, which corresponds to removal of approximately 34% of the nitrate transported by the river over a distance of 18.5 km. Over the same period of time, estimates of denitrification based on mass-balance measurements ranged from 0.29 to 5.25 g N m^–2 d^–1 and the mean annual rate was 2.11 g N m^–2 d^–1. The two methods revealed similar seasonal patterns of denitrification the highest rates were measured from late April to August and the lowest rates were in winter. Both methods provide whole-system estimates of denitrification in running waters; where reaeration rate coefficients are low and flux of groundwater is well quantified, the open-channel method has fewer sources of uncertainty and is easier to implement.     

Nitrogen fluxes and budget seasonality in the Ria Vigo [2pt] (NW Iberian Peninsula)

Inorganic and organic nitrogen fluxes in the Ria Vigo have been quantified in order to recognise the contrasting nitrogen budget scenarios and understand the biogeochemical response to eutrophication events. According to the nitrogen biogeochemical pathways of the ria reservoir (photosynthesis, remineralization, denitrification, PON rain rate and sedimentation), three main seasonal behavioural trends are emphasised: (1) low inorganic nitrogen inputs and low organic nitrogen fluxes, (2) high inorganic nitrogen input and output, (3) high inorganic nitrogen input and high organic nitrogen output. The first scenario occurs in late spring and in summer during non-upwelling situations. The consumption of inorganic nitrogen by net photosynthesis is approximately 2 mol N s^–1 and the ria is oligotrophic (12 mgC m^–2 h^–1). The outgoing estuarine residual current transports phytoplanktonic material towards the mouth of the ria whereupon it sediments and is remineralized as it falls to the lower water layers and the incoming residual current. The regenerated nitrogen is reintroduced to the photic ria layer which leads to the greatest reduction in dissolved oxygen concentration (50% of saturation). Recycled nutrients play an important role in primary production during this oligotrophic state of the ria. Thus, approximately half of the inorganic nitrogen utilised by photosynthesis is ammonium. The majority of PON is deposited inside the ria (0.8 mmol N m^–2 d^–1) and the denitrification rate is 0.3 mmol N₂ m^–2 d^–1. The other two cases occur in winter and spring–summer with upwelling. In winter, estuarine circulation and freshwater contributions control the nitrogen cycle. The ria mainly exports nitrate (up to 14 mol N s^–1) and so there is fertilisation but no eutrophication. In spring and summer, the nitrogen cycle is controlled by upwelling circulation. The inorganic nitrogen consumption by net photosynthesis is high, 7–14 mmol N m^–2 d^–1, and the ria is a natural eutrophic system (70 mgC m^–2 h^–1). Accordingly, 90% of organic nitrogen is synthesised from nitrate and the upwelling-increased circulation exports 6.5 mol N s^–1 of organic nitrogen.     

Seasonal denitrification in flooded and exposed sediments from the Amazon floodplain at Lago Camaleão

Denitrification processes were measured by the acetylene-blockage technique under changing flood conditions along the aquatic/terrestrial transition zone on the Amazon floodplain at Lago Camaleão, near Manaus, Brazil. In flooded sediments, denitrification was recorded after the amendment with NO ₃ ^– (100 mol liter^–1) throughout the whole study period from August 1992 to February 1993. It ranged from 192.3 to 640.7 mol N m^–2 h^–1 in the 0- to 5-cm sediment layer. Without substrate amendment, denitrification was detected only during low water in November and December 1992, when it occurred at a rate of up to 12.2 mol N m^–2 h^–1 Higher rates of denitrification at an average rate of 73.3 mol N m^–2 h^–1 were measured in sediments from the shallow lake basin that were exposed to air at low water. N₂O evolution was never detected in flooded sediments, but in exposed sediments, it was detected at an average rate of 28.3 mol N m^–2 h^–1 during the low-water period. The results indicate that under natural conditions there is denitrification and hence a loss in nitrogen from the Amazon floodplain to the atmosphere. Rates of denitrification in flooded sediments were one to two orders of magnitude smaller than in temperate regions. However, the nitrogen removal of exposed sediments exceeded that of undisturbed wetland soils of temperate regions, indicating a considerable impact of the flood pulse on the gaseous turnover of nitrogen in the Amazon floodplain.     

Denitrification potential of a river floodplain during flooding with nitrate-rich water: grasslands versus reedbeds

Denitrification is a major mechanism for nitrogen removal from nitrogen-rich waters, but it requires oxygen-poor conditions. We assessed denitrification rates in nitrate-rich but also oxygen-rich river water during its stay in a floodplain. We measured diurnal oxygen fluctuations in floodwater along the river Rhine, and carried out an experiment to assess denitrification rates during day, evening and night. Denitrification in floodwater and flooded sediment were measured, comparing activity of periphyton and sediment from agricultural grasslands and reedbeds. Floodwater along the river Rhine was oxygen-saturated (> 10 mg O₂/L) during the day, but oxygen largely disappeared during the night (0.4–0.8 mg O₂/L). Independent of oxygen concentrations, denitrification in surface water alone hardly occurred. In flooded sediments, however, denitrification rates were much higher (1.1–1.5 mg N m^–2 h^–1), particularly at dark and oxygen-poor conditions (nighttime). In the experimental jars, reedbed-periphyton bacteria achieved similar denitrification rates as bacteria in sediment, but overall periphyton denitrification was of minor importance when calculated per square meter. Apart from oxygen levels, maximum denitrification appeared to be regulated by nitrate diffusion from water into the sediment, as the maximum quantity of N denitrified in the sediment equalled the quantity of N lossed from the surface water. Assessed 24-hr denitrification rates in the flooded floodplains (c. 15 mg N m^–2 d^–1) were similar in grasslands and reedbeds, and were rather low compared to rates in other floodplains.     

Periphyton production in an Appalachian river

Periphyton primary production was measured by ¹⁴C uptake on natural substrates in two sections of the New River, Virginia, U.S.A. Production ranged from 6.71 ± 0.43 mg C g^–1 h^–1 in summer to 1.47 ± 0.22 mg C g^–1 h^–1 in late autumn in the hardwater reach and from l.90 ± 0.10 mg C g^–1 h^–1 to 0.12 ± 0.08 mg C g^–1 h^–1 in the softwater reach. Production in the hardwater reach was 3–5 times greater than in the softwater reach and significantly correlated with dissolved inorganic carbon (DIC) concentration (r² = 0.506). No significant correlation was found between periphyton production and photosynthetically active radiation (PhAR). Extrapolation of periphyton production to a 135 km reach of the New River yielded an estimated annual input of 2 252 T AFDW from this source. Estimates of allochthonous (excluding upstream contributions) and aquatic macrophyte inputs to this same reach were 64 T AFDW and 2 001 T AFDW, respectively. While periphyton is not a large source of organic matter, its high food quality and digestibility make it an important component of the New River energy dynamics.     

Estimating denitrification rates in estuarine sediments: A comparison of stoichiometric and acetylene based methods

We compared denitrification rates obtained using an adaptation of the acetylene block technique to rates estimated from benthic flux nutrient stoichiometry in the subtidal sediments of Tomales Bay, California (USA). By amending whole cores with acetylene and saturating nitrate concentrations, we obtained potential denitrification rates, which ranged between 4 and 30 mmol N m^–2 d^–1. We determined the apparent Michaelis constant (K_app) and the maximum potential rate (V_mp) of the denitrifying community and used these constants in a rectangular hyperbola to estimatein situ denitrification rates. Both the K_app and V_mp of the denitrifying community exhibited significant variation over both depth in the sediment column and time of sampling.Estimates ofin situ denitrification obtained using our kinetic-fix adaptation of the acetylene block ranged between 1.8 (March) and 9 (Sept.) mmol N m^–1 d^–1. Denitrification rates obtained using benthic flux stoichiometry ranged between 0.7 and 4.1 mmol N m^–2 d^–1. Average denitrification rates obtained using the kinetic-fix acetylene block approach exceeded those obtained from net benthic flux stoichiometry; however, these differences were not significant. We conclude that our kinetic-fix adaptation of the acetylene block technique provides realistic estimates of denitrification in sediments, even when pore water nitrate concentrations are low and nitrification and denitrification are closely coupled.     

Application of N-15 dilution for simultaneous estimation of nitrification and nitrate reduction in soil-water columns

Nitrification and denitrification rates were estimated simultaneously in soil-floodwater columns of a Crowley silt loam (Typic Albaqualfs) rice soil by an¹⁵N isotopic dilution technique. Labeled NO ₃ ^– was added to the floodwater of soil-water columns, half were treated with urea fertilizer. The (NO ₃ ^– +NO ₂ ^– )–N and (NO ₃ ^– +NO ₂ ^– )–N concentrations in the floodwater were measured over time and production and reduction rates for NO ₃ ^– calculated. Nitrate reduction in the urea amended columns averaged 515 mol N m^–2h^–1 and nitrification averaged 395 mol N m^–2h^–1 over the 35–153 d incubation. The nitrification rate for 4–19 d sampling period (1,560 mol N m^–2h^–1) in the urea amended columns was almost 9 times greater than the reduction rate (175 mol N m^–2h^–1) over the same period. Without the addition of urea the NO ₃ ^– production rate averaged 32 mol N m^–2h^–1 and reduction 101 mol N m^–2h^–1.     

Benithic-pelagic coupling of nutrient metabolism along an estuarine eutrophication gradient

The shallow, brackish (11–18% salinity) Roskilde Fjord represents a eutrophication gradient with annual averages of chlorophyll, ranging from 3 to 25 mg chl a m^–3. Nutrient loadings in 1985 were 11.3–62.4 g N m^–2 yr^–1 and 0.4–7.3 g P m^–2 yr^–1. A simple one-layer advection-diffusion model was used to calculate mass balances for 7 boxes in the fjord. Net loss rates varied from –32.2 to 17.9 g P m^–2 yr^–1 and from –3.3 to 66.8 g N m^–2, corresponding to 74% of the external P-loading and 88% of the external N-loading to the entire estuary.Gross sedimentation rates measured by sediment traps were between 7 and 52 g p m^–2 yr^–1 and 50 and 426 g N M^–2 yr^–1, respectively. Exchangeable sediment phosphorus varied in annual average between 2.0 and 4.8 g P m^–2 and exchangeable sediment nitrogen varied from 1.9 to 33.1 g N m–1. Amplitudes in the exchangeable pools followed sedimentation peaks with delays corresponding to settling rates of 0.3 m d^–1. Short term nutrient exchange experiments performed in the laboratory with simultaneous measurements of sediment oxygen uptake showed a release pattern following the oxygen uptake, the changes in the exchangeable pools and the sedimentation peaks.The close benthic-pelagic coupling also exists for the denitrification with maxima during spring of 5 to 20 mmol N m^–2 d–1. Denitrification during the nitrogen-limited summer period suggests dependence on nitrification. Comparisons with denitrification from other shallow estuaries indicate a maximum for denitrification in estuaries of about 250 µmol N m^–2 h^–2 achieved at loading rates of about 25–125 g N m^–2 yr^–1.     

Rapid Nitrate Loss and Denitrification in a Temperate River Floodplain

Nitrogen (N) pollution is a problem in many large temperate zone rivers, and N retention in river channels is often small in these systems. To determine the potential for floodplains to act as N sinks during overbank flooding, we combined monitoring, denitrification assays, and experimental nitrate (NO₃⁻ -N) additions to determine how the amount and form of N changed during flooding and the processes responsible for these changes in the Wisconsin River floodplain (USA). Spring flooding increased N concentrations in the floodplain to levels equal to the river. As discharge declined and connectivity between the river and floodplain was disrupted, total dissolved N decreased over 75% from 1.41 mg l⁻¹, equivalent to source water in the Wisconsin River on 14 April 2001, to 0.34 mg l⁻¹ on 22 April 2001. Simultaneously NO₃⁻ -N was attenuated almost 100% from 1.09 to <0.002 mg l⁻¹. Unamended sediment denitrification rates were moderate (0–483 μg m⁻² h⁻¹) and seasonally variable, and activity was limited by the availability of NO ₃⁻ -N on all dates. Two experimental NO₃⁻ -N pulse additions to floodplain water bodies confirmed rapid NO₃⁻ -N depletion. Over 80% of the observed NO ₃⁻ -N decline was caused by hydrologic export for addition #1 but only 22% in addition #2. During the second addition, a significant fraction (>60%) of NO₃⁻ -N mass loss was not attributable to hydrologic losses or conversion to other forms of N, suggesting that denitrification was likely responsible for most of the NO₃⁻ -N disappearance. Floodplain capacity to decrease the dominant fraction of river borne N within days of inundation demonstrates that the Wisconsin River floodplain was an active N sink, that denitrification often drives N losses, and that enhancing connections between rivers and their floodplains may enhance overall retention and reduce N exports from large basins.     

Improvement of the culture stability of non-anchorage-dependent animal cells grown in serum-free media through immobilization

A murine hybridoma cell line producing a monoclonal antibody against penicillin-G-amidase and a murine transfectoma cell line secreting a monovalent chimeric human/mouse Fab-antibody fragment were cultivated in three different media (serum-containing, low protein serum-free, and iron-rich protein-free) in flask cultures, stirred reactors and a fixed bed reactor. In static batch cultures in flasks both cell lines showed similar good growth in all three media.In suspension in a stirred reactor, the hybridoma cell line could be cultivated satisfactory only in serum-containing medium. In low protein serum-free medium, Pluronic F68 had to be added to protect the hybridoma cells against shear stress. But even with this supplement only batch, not chemostat mode was possible. In iron-rich protein-free medium the hybridoma cells grew also in continuous chemostat mode, but the stability of the culture was low. The transfectoma cell line did not grow in stirred reactors in any of the three media.Good results with both cell lines were obtained in fixed bed experiments, where the cells were immobilized in macroporous Siran^®-carriers. The media, which were optimized in flask cultures, could be used without any further adaptation in the fixed bed reactor. Immobilization improved the stability and reliability of cultures of non-adherent animal cells in serum-free media tremendously compared to suspension cultures in stirred reactors. The volume-specific glucose uptake rate, an, indicator of the activity of the immobilized cells, was similar in all three media. Deviations in the metabolism of immobilized and suspended cells seem to be mainly due to low oxygen concentrations within the macroporous carriers, where the cells are supplied with oxygen only by diffusion.List of symbols c substrate or product concentration mmol l^–1 - c₀ substrate or product concentration in the feed mmol l^–1 - c_Glc glucose concentration mmol l^–1 - c_Gln glutamine concentration mmol l^–1 - c_Amm ammonia concentration mmol l^–1 - c_Lac lactate concentration mmol l^–1 - c_FAB concentration of Fab# 10 antibody fragment g l^–1 - c_MAb monoclonal antibody concentration mg l^–1 - D dilution rate d^–1 - q cell-specific substrate uptake or metabolite production rate mmol cell^–1 h^–1 - q_Glc cell-specific glucose uptake rate mmol cell^–1 h^–1 - q_Gln cell-specific glutamine uptake rate mmol cell^–1 h^–1 - q_MAb cell-specific MAb production rate mg cell^–1 h^–1 - q^* volume-specific substrate uptake or metabolite production rate mmol l^–1 h^–1 - q^*FB volume-specific substrate uptake or metabolite production rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - q^*FB,Glc volume-specific glucose uptake rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - q^*FB,Gln volume-specific glutamine uptake rate related to the fixed volume mmol l_FB ^–1 h^–1 - q^*FB,MAb volume-specific MAb production rate related to the fixed volume mg l_FB ^–1 h^–1 - q^*FB,02 volume-specific oxygen uptake rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - t time h - U superficial flow velocity mm s^–1 - V medium volume in the conditioning vessel of the fixed bed reactor l - V_FB volume of the fixed bed l - x_v viable cell concentration cells ml^–1 - y_Amm,Gln yield of Ammonia from glutamine - y_Lac,Glc yield of lactate from glucose - specific growth rate h^–1 - _d specific death rate h^–1     

Estimating denitrification in North Atlantic continental shelf sediments

A model of coupled nitrification/denitrification was developed for continental shelf sediments to estimate the spatial distribution of denitrification throughout shelf regions in the North Atlantic basin. Using data from a wide range of continental shelf regions, we found a linear relationship between denitrification and sediment oxygen uptake. This relationship was applied to specific continental shelf regions by combining it with a second regression relating sediment oxygen uptake to primary production in the overlying water. The combined equation was: denitrification (mmol N m^–2 d^–1)=0.019^* phytoplankton production (mmol C m^–2 d^–1). This relationship suggests that approximately 13% of the N incorporated into phytoplankton in shelf waters is eventually denitrified in the sediments via coupled nitrification/denitrification, assuming a C:N ratio of 6.625:1 for phytoplankton. The model calculated denitrification rates compare favorably with rates reported for several shelf regions in the North Atlantic.The model-predicted average denitrification rate for continental shelf sediments in the North Atlantic Basin is 0.69 mmol N m^{– 2} d^–1. Denitrification rates (per unit area) predicted by the model are highest for the continental shelf region in the western North Atlantic between Cape Hatteras and South Florida and lowest for Hudson Bay, the Baffin Island region, and Greenland. Within latitudinal belts, average denitrification rates were lowest in the high latitudes, intermediate in the tropics and highest in the mid-latitudes. Although denitrification rates per unit area are lowest in the high latitudes, the total N removal by denitrification (53 × 10¹⁰ mol N y^–1) is similar to that in the mid-latitudes (60 × 10¹⁰ mol N y^–1) due to the large area of continental shelf in the high latitudes. The Gulf of St. Lawrence/Grand Banks area and the North Sea are responsible for seventy-five percent of the denitrification in the high latitude region. N removal by denitrification in the western North Atlantic (96 × 10¹⁰ mol N y^–1) is two times greater than in the eastern North Atlantic (47 × 10¹⁰ mol N y^–1). This is primarily due to differences in the area of continental shelf in the two regions, as the average denitrification rate per unit area is similar in the western and eastern North Atlantic.We calculate that a total of 143 × 10¹⁰ mol N y^–1 is removed via coupled nitrification/denitrification on the North Atlantic continental shelf. This estimate is expected to underestimate total sediment denitrification because it does not include direct denitrification of nitrate from the overlying water. The rate of coupled nitrification/denitrification calculated is greater than the nitrogen inputs from atmospheric deposition and river sources combined, and suggests that onwelling of nutrient rich slope water is a major source of N for denitrification in shelf regions. For the two regions where N inputs to a shelf region from onwelling have been measured, onwelling appears to be able to balance the denitrification loss.     

Improvement of a mammalian cell culture process by adaptive,model-based dialysis fed-batch cultivation and suppression of apoptosis

Both conventional and genetic engineering techniques can significantly improve the performance of animal cell cultures for the large-scale production of pharmaceutical products. In this paper, the effect of such techniques on cell yield and antibody production of two NS0 cell lines is presented. On the one hand, the effect of fed-batch cultivation using dialysis is compared to cultivation without dialysis. Maximum cell density could be increased by a factor of ~5–7 by dialysis fed-batch cultivation. On the other hand, suppression of apoptosis in the NS0 cell line 6A1 bcl-2 resulted in a prolonged growth phase and a higher viability and maximum cell density in fed-batch cultivation in contrast to the control cell line 6A1 (100)3. These factors resulted in more product formation (by a factor ~2). Finally, the adaptive model-based OLFO controller, developed as a general tool for cell culture fed-batch processes, was able to control the fed-batch and dialysis fed-batch cultivations of both cell lines.Abbreviations A membrane area (dm²) - c _Glc,F glucose concentration in nutrient feed (mmol L^–1) - c _Glc,FD glucose concentration in dialysis feed (mmol L^–1) - c _Glc,i glucose concentration in inner reactor chamber (mmol L^–1) - c _Glc,o glucose concentration in outer reactor chamber (dialysis chamber) (mmol L^–1) - c _Lac,FD lactate concentration in dialysis feed (mmol L^–1) - c _Lac,i lactate concentration in inner reactor chamber (mmol L^–1) - c _Lac,o lactate concentration in outer reactor chamber (dialysis chamber) (mmol L^–1) - c _LS,FD limiting substrate concentration in dialysis feed (mmol L^–1) - c _LS,i limiting substrate concentration in inner reactor chamber (mmol L^–1) - c _LS,o limiting substrate concentration in outer reactor chamber (dialysis chamber) (mmol L^–1) - c _Mab monoclonal antibody concentration (mg L^–1) - F _D feed rate of dialysis feed (L h^–1) - F _Glc feed rate of nutrient concentrate feed (L h^–1) - K _d maximum death constant (h^–1) - k _d,LS death rate constant for limiting substrate (mmol L^–1) - k _Glc monod kinetic constant for glucose uptake (mmol L^–1) - k _Lac monod kinetic constant for lactate uptake (mmol L^–1) - k _LS monod kinetic constant for limiting substrate uptake (mmol L^–1) - K _Lys cell lysis constant (h^–1) - K _S,Glc monod kinetic constant for glucose (mmol L^–1) - K _S,LS monod kinetic constant for limiting substrate (mmol L^–1) - µ cell-specific growth rate (h^–1) - µ _d cell-specific death rate (h^–1) - µ _d,min minimum cell-specific death rate (h^–1) - µ _max maximum cell-specific growth rate (h^–1) - P _Glc membrane permeation coefficient for glucose (dm h^–1) - P _Lac membrane permeation coefficient for lactate (dm h^–1) - P _LS membrane permeation coefficient for limiting substrate (dm h^–1) - q _Glc cell-specific glucose uptake rate (mmol cell^–1 h^–1) - q _Glc,max maximum cell-specific glucose uptake rate (mmol cell^–1 h^–1) - q _Lac cell-specific lactate uptake/production rate (mmol cell^–1 h^–1) - q _Lac,max maximum cell-specific lactate uptake rate (mmol cell^–1 h^–1) - q _LS cell-specific limiting substrate uptake rate (mmol cell^–1 h^–1) - q _LS,max maximum cell-specific limiting substrate uptake rate (mmol cell ^–1 h^–1) - q _Mab cell-specific antibody production rate (mg cell^–1 h^–1) - q _MAb,max maximum cell-specific antibody production rate (mg cell^–1 h^–1) - t time (h) - V _i volume of inner reactor chamber (culture chamber) (L) - V _o volume of outer reactor chamber (dialysis chamber) (L) - X _t total cell concentration (cells L^–1) - X viable cell concentration (cells L^–1) - Y _Lac/Glc kinetic production constant (stoichiometric ratio of lactate production and glucose uptake) (–)     

Effect of benzoic acid on the ammonium transport system of Zygosaccharomyces bailii

Summary A study of the ammonium transport system of Zygosaccharomyces bailii was carried out using methylammonium as a non-metabolizable analogue. Benzoic acid in the growth medium decreased the capacity of the transport system from 1.46 ± 0.11 mmol.g^–1.h^–1 to 0.41±0.04 mmol.g^–1.h^–1, while the affinity for ammonium was not significatively affected. Although ammonium uptake was inhibited by benzoic acid, the ammonium transport system was still active at preservative concentrations which fully inhibited growth suggesting that inhibition of growth was not governed by the uptake of this nutrient.     

Biological denitrification of water in a two-chambered immobilized-cell bioreactor

Summary A double-chambered bioreactor based on a composite immobilized-cell gel layer/microporous membrane structure was applied to the continuous denitrification of high-nitrate water. Immobilized denitrifying bacteria (Pseudomonas denitrificans) were provided with separate flows of nitrate and carbon (C) nutrient, with no contamination of the treated water by cell leakage from the gel. Using acetate (7.5 mm) as a C source and a C/N ratio of 3 (mol/mol), specific denitrification rates ranging from 15 to 25 g NO _inf3 ^sup– · h^–1 · – cm^–2 membrane surface (50–85 g NO _inf3 ^sup– · h^–1 · cm^–3 gel) were obtained. The denitrifying activity remained stable for several months. At the flow rate used (10 cm³ · h^–1), the effluents contained noticeable amounts of NO _inf2 ^sup– ions but the treated water remained uncontaminated by the carbon nutrient. Most NO _inf2 ^sup– ions disappeared from the treated water in a second reactor connected in series. When fed with an unchlorinated sludge supernatant as C nutrient, immobilized bacteria performed efficient denitrification of water for only 3 weeks. Diffusion experiments showed that acetate ions diffused much less rapidly than NO _inf3 ^sup– or NO _inf2 ^sup– ions through the composite structure. Further developments of the system are considered.