Effect of hydraulic residence time on microbial sulfide production in an upflow sludge blanket denitrification reactor fed with methanol

Summary In the combined ion exchange/biological denitrification process for nitrate removal from ground water anion exchange resins are regenerated in a closed circuit by way of an upflow sludge blanket denitrification reactor. The regenerant (a concentrated sodium bicarbonate solution) is recirculated through the ion exchanger in the r generation mode and the denitrification reactor. In the closed system sulfate accumulates to very high concentrations. For that reason it was examined under what process conditions sulfate reduction occurs in an upflow sludge blanket denitrification reactor, when the influent contains high sulfate concentrations (5.45 g SO ₄ ^2- /l) and high sodium bicarbonate concentrations (19.8 g NaHCO₃/l) in addition to nitrate and methanol. It appeared that at a hydraulic residence time of 5 h sulfide production started, when the nitrate loading rate was 20% of the denitrification reactor capacity and methanol was added in excess. The excess of methanol was converted into acetate after nitrate was depleted. Conversion of methanol into acetate was a function of the hydraulic residence time. At hydraulic residence times above 8 h this conversion was complete. Also in batch experiments it was observed that excess of methanol was converted into acetate, and that sulfate reduction started when nitrate was depleted. From all experiments it is clear that, provided that methanol is added in good relation to the quantity of nitrate that has to be denitrified, acetate will not be produced and sulfate reduction will not occur in the denitrification reactor, even in the presence of very high sulfate concentrations.     

Nitrite reduction by a mixed culture under conditions relevant to shortcut biological nitrogen removal

Dissimilative reduction of nitrite by nitrite-acclimated cellswas investigated in a batch reactor under various environmental conditions that can beencountered in shortcut biological nitrogen removal (SBNR: ammonia to nitrite andnitrite to nitrogen gas). The maximum specific nitrite reduction rate was as much as 4.3 times faster than the rate of nitrate reduction when individually tested, but the reaction was inhibited in the presence of nitrate when the initial nitrate concentration was greater than approximately 25 mg-N/l or the initialNO ₃ ^- N/NO ₂ ^- N ratio was larger than 0.5. Nitrite reduction was also inhibited by nitrite itself when theconcentration was higher than that to which the cells had been acclimated. Therefore, it was desirable to avoid excessively high nitrite and nitrate concentrations in a denitrification reactor. Nitrite reduction, however, was not affected by an alkaline pH (in the range of 7–9) or a high concentration of FA (in the range of 16–39 mg/l), which can be common in SBNR processes. The chemical oxygen demand (COD) requirement for nitrite reduction was approximately 22–38% lower than that for nitrate reduction, demonstrating that the SBNR process can be economical. The specific consumption,measured as the ratio of COD consumed to nitrogen removed, was affected by the availability of COD and the physiological state of the cells. The ratio increased when the cells grew rapidly and were storing carbon and electrons.     

Nitrate removal from the effluent of a fertilizer industry using a bioreactor packed with immobilized cells of <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis>and<Emphasis Type="Italic">Comamonas testosteroni</Emphasis>

A bioreactor for the removal of nitrate nitrogen (NO₃-N) from industrial effluent is described which is comprised of a glass column (60 cm × 6 cm) packed with alginate beads containing denitrifying organisms Pseudomonas stutzeri and Comamonas testosteroni. The effluent containing high concentrations of nitrate (600–950 mg l^–1) from the fertilizer industry and fusel oil (methanol as a major component) as organic carbon were used in the process. The reactor is operated in the continuous mode by injecting the pretreated nitrate-containing effluent at the top of the column. The Hydraulic retention time (HRT) was adjusted by changing the flow rates. When nitrate-containing wastewater was treated with immobilized cells, the nitrate removal rate reached a maximum 1.66 ± 0.07 Kg NO₃-N m^–3d^–1 at an influent NO₃-N concentration of 850 mg NO_3M-N l^–1within 12 h. The denitrification activity of the immobilized cells was compared with that of the free cells.     

Simultaneous nitrification and denitrification in a mixed methanotrophic culture

Simultaneous nitrification and denitrification using a mixed methanotrophic culture was investigated. When both NO₃ ^–-N (108 mg l^–1) and NH₃-N (59 mg l^–1) were added into batch reactors, nitrate removal was complete within 10 h at the rate of 47 mg NO₃ ^–-N g VSS^–1 day^–1 when dissolved oxygen (DO) concentration was maintained at 2 mg DO l^–1. Ammonia removal started simultaneously with nitrate removal at a slower rate of 14 NH₃-N g VSS^–1 day^–1. No significant accumulation of nitrite or nitrate during ammonia utilization suggested the occurrence of simultaneous nitrification and denitrification.     

Use of an industrial effluent as a carbon source for denitrification of a high-strength wastewater

Denitrification of a high-strength synthetic wastewater (150 g NO^- ₃ l^-1) was carried out using a wine distillery effluent as an example of an industrial carbon source (22.7 g chemical oxygen demand l^-1). Two configurations were tested: one consisted of an acidogenesis reactor followed by a denitrifying reactor and the other was a single reactor directly fed with the raw effluents. In both cases, denitrification was achieved at a nitrate load of 9.54 g NO^- ₃ l^-1 day^-1 (2.19 g N as NO^- ₃ l^-1 day^-1) with good specific reduction rates: 32.6 mg and 35.2 mg N as NO _x  g volatile suspended solids h^-1, calculated on a single day, for the two-step and the one-step process respectively. Dissimilatory nitrate reduction to ammonium did not occur, even in the one-step process. Received: 26 October 1995/Received revision: 15 February 1996/Accepted: 20 February 1996     

Kinetics of a fluidized-bed reactor for ground-water denitrification

A fluidized-bed reactor, with sand as the carrier and ethanol as the carbon and electron source, was investigated for the biological denitrification of ground water. The paper concentrates on the reactor's kinetics, with special emphasis on nitrite as the intermediate product. Intrinsic zero-order kinetic parameters for both nitrate and nitrite were determined by batch and continuous experiments. Values for the maximum specific nitrate and nitrite removal rates of 11 g and 6 g NO _inf3 ^sup– (g volatile suspended solids)^–1 day^–1, respectively, were obtained. These values were used to interpret nitrate and nitrate concentration profiles in an experimental fluidized-bed reactor operating at different conditions of hydraulic loading and retention time.     

Autotrophic denitrification by Thiobacillus denitrificans in a packed bed reactor

Summary An upflow packed bed reactor with lava stones as support for the microbial growth proved to be very useful for the denitrification of industrial waste water by Thiobacillus denitrificans. The application of the plug flow principle allowed higher concentrations of nitrate to be employed than in a stirred tank reactor because inhibitory concentrations of sulfate from thiosulfate oxidation built up only in the upper part of the column — if at all. In experiments with synthetic media nitrate solutions of different strength (NO ₃ ^– g/l: 1.8; 3.0; 4.3; 6.1) were tested, each at 5 different residence times (5; 3.3; 2.5; 2.0; 1.7 h). The combination of the two parameters which still allowed 95% denitrification was 3 g NO ₃ ^- /l and 2.5 h residence time; this corresponded to a volumetric nitrate loading of about 25 kg/m³·d. Higher nitrate loadings led to incomplete denitrification coupled with the occurence of nitrite in the outflow. Below the critical loading rate nitrite accumulated only in the lower part of the column and was then gradually reduced. Experiments with simulated middle active waste from processing nuclear fuel which contained numerous heavy metals yielded similar results. — Although pure inorganic media were fed into the reactor the microflora developing as a dense layer covering the lava stones consisted not only of T. denitrificans but also of heterotrophic denitrifiers, mainly Pseudomonas aeruginosa.     

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.     

Relationships between DOC bioavailability and nitrate removal in an upland stream: An experimental approach

The Catskill Mountains of southeastern New York State have among thehighest rates of atmospheric nitrogen deposition in the United States. Somestreams draining Catskill catchments have shown dramatic increases in nitrateconcentrations while others have maintained low nitrate concentrations. Streamsin which exchange occurs between surface and subsurface (i.e. hyporheic) watersare thought to be conducive to nitrate removal via microbial assimilationand/ordenitrification. Hyporheic exchange was documented in the Neversink River inthesouthern Catskill Mountains, but dissolved organic carbon (DOC) and nitrate(NO₃ ^–) losses along hyporheic flowpaths werenegligible. In this study, Neversink River water was amended with natural,bioavailable dissolved organic carbon (BDOC) (leaf leachate) in a series ofexperimental mesocosms that simulated hyporheic flowpaths. DOC and N dynamicswere examined before and throughout a three week BDOC amendment. In addition,bacterial production, dissolved oxygen demand, denitrification, and sixextracellular enzyme activities were measured to arrive at a mechanisticunderstanding of potential DOC and NO₃ ^– removalalong hyporheic flowpaths. There were marked declines in DOC and completeremoval of nitrate in the BDOC amended mesocosms. Independent approaches wereused to partition NO₃ ^– loss into two fractions:denitrification and assimilation. Microbial assimilation appears to be thepredominant process explaining N loss. These results suggest that variabilityinBDOC may contribute to temporal differences in NO₃ ^–export from streams in the Catskill Mountains.     

Denitrification of a landfill leachate with high nitrate concentration in an anoxic rotating biological contactor

The denitrification performance of a lab-scale anoxic rotating biological contactor (RBC) using landfill leachate with high nitrate concentration was evaluated. Under a carbon to nitrogen ratio (C/N) of 2, the reactor achieved N-NO₃ ⁻ removal efficiencies above 95% for concentrations up to 100 mg N-NO₃ ⁻ l⁻¹. The highest observed denitrification rate was 55 mg N-NO₃ ⁻ l⁻¹ h⁻¹ (15 g N-NO₃ ⁻ m⁻² d⁻¹) at a nitrate concentration of 560 mg N-NO₃ ⁻ l⁻¹. Although the reactor has revealed a very good performance in terms of denitrification, effluent chemical oxygen demand (COD) concentrations were still high for direct discharge. The results obtained in a subsequent experiment at constant nitrate concentration (220 mg N-NO₃ ⁻ l⁻¹) and lower C/N ratios (1.2 and 1.5) evidenced that the organic matter present in the leachate was non-biodegradable. A phosphorus concentration of 10 mg P-PO₄ ³⁻ l⁻¹ promoted autotrophic denitrification, revealing the importance of phosphorus concentration on biological denitrification processes.     

Denitrification of nitrate and nitric acid with methanol as carbon source

Summary A methanol/nitrate-medium and anaerobic conditions yielded an enrichment culture which consisted ofHyphomicrobium andParacoccus. This mixed culture proved to be very effective in denitrification of solutions containing high concentrations of nitrate and free nitric acid when grown in a chemostat (D=0.04 h^-1). With 0.1 mol/l nitric acid solution as feed medium the pH in the culture vessel adjusted itself to 5.8. For the reduction of 1 g NO₃–N 2.6 g methanol were consumed and 0.56 g cells were produced.     

Microelectrode Measurements of Nitrate Gradients in the Littoral and Profundal Sediments of a Meso-Eutrophic Lake (Lake Vechten, The Netherlands)

NO₃⁻ concentration profiles were measured in the sediments of a meso-eutrophic lake with a newly developed microelectrode. The depth of penetration of NO₃⁻ varied from only 1.3 mm in organic-rich profundal silty sediments to 5 mm in organic-poor littoral sandy sediments. The thickness of the zone of denitrification in the organic-rich sediments was <500 μm. Oxygen profiles measured simultaneously revealed that the zone of denitrification was directly adjacent to the aerobic zone. The results demonstrate high denitrification rates (0.26 to 1.31 mmol m⁻² day⁻¹) at in situ nitrate concentrations in the overlying water (0.030 mmol liter⁻¹) and limitation of denitrification by nitrate availability.     

Pore water nutrient profiles and dynamics in soft bottoms of the northern Baltic Sea

Chemical profiles of nutrients at the sediment–water interface were measured in the northern Baltic Sea. A whole core squeezer technique capable of mm-scale resolution was used to obtain the vertical profiles of NO₃ ^–, NO₂ ^–, o-P, NH₄ ⁺ and Si in the soft bottom sediments. The profiles were compared with nutrient flux and denitrification measurements. In the Gulf of Finland, the profiles revealed a marked chemical zonation in NO₃ ^– and NO₂ ^– distribution indicating strong potential of nitrification just under the sediment surface followed by a layer of denitrification down to a depth of 30 mm. Below the depth of 20 mm NO₃ ^– was usually absent, whereas other nutrients were increasing steadily in concentration. A distinct minimum of NO₃ ^– was observed at the sediment–water interface, suggesting NO₃ ^– uptake by a microbial biofilm and/or active denitrification at the suboxic microniches usually present in organic-rich sediments. At the deep stations in the Baltic Proper, the NO₃ ^– concentration in pore water, as well as denitrification, were very low. The concentrations of NH₄ ⁺, o-P and Si were usually increasing steadily with depth.     

Physiology and kinetics of autotrophic denitrification by Thiobacillus denitrificans

Summary A strain of Thiobacillus denitrificans was isolated after enrichment under anaerobic conditions by the continuous culture technique using thiosulfate as energy source and nitrate as electron acceptor and nitrogen source. The isolate was an active denitrifyer, the optimal conditions being 30°C and pH 7.5–8.0. Denitrification was inhibited by sulfate (the reaction product) above 5 g SO ₄ ⁼ /l, whereas high concentrations of the substrates nitrate and thiosulfate were less harmful; nitrite affected denitrification above 0.2 g NO ₂ ^– /l. During the time course of denitrification in a batch culture growth and substrate consumption slowed down already after only half the substrate was utilized due to product inhibition. The following parameters were determined in continuous culture under nitrate limitation: _max=0.11 h^–1, K _S=0.2 mg NO ₃ ^– /l, maximum denitrification rate=0.78 g NO ₃ ^– /g cells·h, g cells/g NO ₃ ^– , g cells/g S₂O ₃ ⁼ . Nitrite did not accumulate during steady state denitrification; the denitrification gas was almost pure N₂. The concentrations of N₂O and NO were below 1 ppm.     

Denitrification kinetics of simulated fish processing wastewater at different ratios of nitrate to biomass

Denitrification was studied in anoxic batch cultures of a simulated fish processing wastewater at 37 r C and pH 7.5, using a denitrifying enrichment culture from fishery wastewater. Different initial nitrate to biomass ratios (So/Xo) were used: nitrate and biomass varied from 7.5 to 94.7 mg NO₃-N l^–1, and from 20 to 4300 mg volatile suspended solids l^–1, respectively. The specific maximum denitrification rate (r _m) and the cell yield (Y _{X / S}) depended on the So/Xo ratio under anoxic conditions: r _m increased from 1.2 to 1584 mg NO₃-N g^–1 VSS h^–1 and Y _{X / S} decreased from 42 to 0.03 mg VSS mg^–1 NO₃-N when So/Xo varied from 5.5 10^{– 3} to 9.3 mg NO₃-N/mg VSS. Nomenclature C_{NO3 – N} nitrate concentration, mg NO₃-N l^–1 K _S saturation constant, mg NO₃-N l^–1 r _m specific maximum denitrification rate, mg NO₃-N g^–1 VSS h^–1 So initial substrate concentration, mg l^–1 t time, h TOC total organic carbon VSS volatile suspended solids x biomass concentration, g VSS l^–1 Xo initial biomass concentration, g VSS l^–1 Y _X/S substrate to biomass cell yield, mg VSS/mg N Greek symbols: _m maximum specific growth rate of the anoxic microbial population, 1 h^–1     

Denitrification of high nitrate wastewater in a cloth strip bioreactor with immobilized sludge

Denitrification of synthetic high nitrate wastewater containing 40,000?ppm NO₃ (9,032?ppm NO₃-N) was achieved using immobilized activated sludge in a column reactor. Active anoxic sludge adsorbed onto Terry cloth was used in the denitrification of high nitrate wastewater. The operational stability of the immobilized sludge system was studied both in a batch reactor and in a continuous reactor. The immobilized sludge showed complete degradation of different concentrations of NO₃-N (1,129, 1,693, 3,387, 6,774, and 9,032?ppm) in a batch process. The reactors were successfully run for 90?days without any loss in activity. The immobilized cell process has yielded promising results in attaining high denitrifying efficiency.     

Effect of high nitrate concentrations on growth and nitrate uptake by free-living and immobilizedChlorella vulgaris cells

Growth and nitrate uptake were studied on free-living and immobilizedChlorella vulgaris cells cultivated in medium containing different nitrate concentrations. First, the effect of nitrate concentrations on growth indicated that cells can live in the presence of high concentrations as high as 97 mM. Although no lethal effect on cells was observed such concentration a slow down in growth and a decrease in biomass produced was observed. The rate of nitrate uptake increased with the nitrate concentration in the medium. The maximum uptake rate was reached in first days of culture in both free-living and immobilized cells. The rate dropped more rapidly for cells growing in 2 mM nitrate than for cells growing in higher nitrate concentration. The maximum rate was very much the same for free-living and immobilized and was within the order of 0.45 to 0.57 g NO₃ h^–1 10^–6 cells. Immobilization modified the changes of nitrate uptake rate for concentration higher than 2 mM.     

Influence of bed media characteristics on ammonia and nitrate removal in shallow horizontal subsurface flow constructed wetlands

Two bed media were tested (gravel and Filtralite) in shallow horizontal subsurface flow (HSSF) constructed wetlands in order to evaluate the removal of ammonia and nitrate for different types of wastewater (acetate-based and domestic wastewater) and different COD/N ratios. The use of Filtralite allowed both higher mass removal rates (1.1 g NH₄–N m⁻² d⁻¹ and 3 g NO₃–N m⁻² d⁻¹) and removal efficiencies (>62% for ammonia, 90–100% for nitrate), in less than 2 weeks, when compared to the ones observed with gravel. The COD/N ratio seems to have no significant influence on nitrate removal and the removal of both ammonia and nitrate seems to have involved not only the conventional pathways of nitrification–denitrification. The nitrogen loading rate of both ammonia (0.8–2.4 g NH₄–N m⁻² d⁻¹) and nitrate (0.6–3.2 g NO₃–N m⁻² d⁻¹) seem to have influenced the respective removal rates.     

Culture of the astaxanthin-producing green algaHaematococcus pluvialis 1. Effects of nutrients on growth and cell type

The freshwater green algaHaematococcus pluvialis (Strain Vischer 1923/2) grows best at high nitrate concentrations (about 0.5 to 1.0 g 1^–1 KNO₃), intermediate phosphate concentration (about 0.1 g 1^–1 K₂HPO₄) and over a wide range of Fe concentrations. Low nitrate or high phosphate induce the formation of reddish palmella cells and aplanospores. Mixotrophic growth with acetate improves growth rate and final cell yield, and also stimulates the formation of the astaxanthin-containing palmella cells and aplanospores.H. pluvialis cannot grow above about 28 °C, or above a salinity of approximately 1% w/v NaCl. An increase in temperature or the addition of NaCl also stimulates the formation of palmella cells and aplanospores.     

Nitrate removal with bacterial cells attached to quartz sand and zeolite from salty wastewaters

A mixed bacterial culture was acclimated to the removal of high nitrate-N concentrations (100–750 mg NO₃ ⁻-N L⁻¹) from salty wastewaters. The experiments were carried out under anoxic conditions in the presence of 0.5, 1.5 and 3% (w/v) NaCl at different temperatures. The acclimated mixed bacterial culture was attached to quartz sand and zeolite. Denitrification was monitored in a continuous-flow bioreactor at different hydraulic retention times (HRT). Nitrate removal with cells attached to quartz sand and zeolite was completed at HRT of 167 h and 25 h respectively. Then brine denitrification with bacterial cells attached to zeolite was monitored for 85 days. Under the increased nitrate loading rate, nitrate removal was above 90%. Furthermore, during denitrification, not more than 0.5 mg NO₂ ⁻-N L⁻¹ could be produced. It can be concluded that nitrate removal with the cells attached to zeolite is economically and operationally a promising solution to denitrification of brine wastewaters.