Nitrate reduction by denitrifying bacteria in single and two stage continuous flow reactors

Denitrification by a mixed bacterial population of medium containing 1000 mg NO₃⁻-N1⁻¹ and acetate as carbon source was studied in batch, a single stage continuous flow stirred reactor (CFSTR) and a two stage CFSTR at 30°C. The optimum pH for denitrification, nitratase, nitrite reductase activities and growth was found to be 7.5 in batch culture. A single stage CFSTR growth limited by nitrate had an optimum denitrification rate of 0.13 mg NO₃⁻-N mg⁻¹ cells h at a residence time of 8 h. The experimentally observed carbon to nitrate ratio (mg CH₃ COO⁻-C mg⁻¹ NO₃⁻-N)was 1.7 for the dilution rates of 0.02–0.18 h⁻¹. For the second stage CFSTR, bacteria growing at the maximum rate of 0.25 h⁻¹ and not limited by nitrate had a denitrification rate of 0.24 mg NO₃⁻-N mg⁻¹ h. Dissolved oxygen (up to 9.5 mg 1⁻¹) did not effect denitrification rates in the second stage CFSTR. As the second stage CFSTR runs progressed extensive wall growth occurred and concurrently the output gas contained increasing quantities of nitrous oxide. A development from this study would be a two stage CFSTR with wall growth in the second stage which would make an efficient nitrate removal process.     

An improved hydrazine reduction method for the automated determination of low nitrate levels in freshwater

An automated nitrate determination is described in which nitrate is reduced to nitrite with hydrazine sulphate under alkaline conditions in the presence of Cu²⁺ and Zn²⁺. Interferances encountered in natural water samples were eliminated by the addition of Zn²⁺ to the Cu²⁺ catalyst solution.The method is suitable for the determination of low NO₃−N concentrations and compares favourably with the manual copperised cadmium technique for freshwater samples containing 10–800 mg m⁻³ NO₃−N. The method is also linear at nitrate concentrations below 10 mg N m⁻³. The standard deviations (S.D.) of blanks and of samples containing 2 mg NO₃−N m⁻³ were 0.013 and 0.06 mg N m⁻³ respectively at an analysis rate of 30 samples h⁻¹.     

Factors affecting the denitrification rate in two water-sediment systems

Effects of temperature, oxygen and nitrate concentrations of the overlying water, and the thickness of the sediment layer on the rate of denitrification in the sediment were investigated in two water-sediment systems, A and B. At 4°C, denitrification started after a prolonged lag period in contrast to nitrification which did not occur significantly. At 15°C, and particularly at 25°C, both processes proceeded readily. The disappearance of NO₂⁻ - N from the overlying water was more rapidly than that of NO₃⁻ - N.The denitrification rate was slightly reduced by increasing the dissolved oxygen concentration in the overlying water from 0 to approximately 2 mgl⁻¹. A further rise of the dissolved oxygen concentration had no further decreasing effect on the denitrification rate.The denitrification rate in sediment was dependent on the nitrate concentration in the overlying water approximating first order kinetics at lower concentrations, gradually becoming independent of the nitrate concentration at higher nitrate contents (zero order kinetics).When starting with a nitrate-nitrogen concentration of 25.2 mgl⁻¹, a sediment layer of 7 mm with A and 14 mm with B was roughly found to be involved in denitrification.Denitrification rates found in the present laboratory experiments were supposed to be considerably lower than those occurring under natural conditions as additional mechanisms for the transport of nitrate into sediments occurred in natural environments.     

Gas production in aquatic sediments in the presence and absence of nitrate

In the overlying water of a water-sediment system the pH was controlled at 7.0, the nitrate-nitrogen concentration at 25.0 mg 1⁻¹ and the dissolved oxygen concentration above 6.1 mg t⁻¹. The temperature of the whole system was kept at 15°C. The average rate of nitrate removal from the system as a result of denitrification amounted to 160 mg NO₃⁻ -N m⁻² day⁻¹. By means of Eh measurements at various depths in the sediment, it was attempted to figure out the course of the penetration fronts of nitrate and oxygen in the sediment during 241 days of incubation. From these results the layer in which denitrification occurred was derived. The course of the denitrification zone was followed during the incubation period. As a result of the depletion of the available hydrogen donors in the sediment, oxygen reached the bottom of the sediment after 235 days of incubation.     

Detoxification of seawater used for gas scrubbing in the steel industry

Cyanide ion present in seawater after scrubbing blast furnace and coke ovens gases can be removed by sedimentation of hexacyanoferrate complexes followed by oxidation of residual cyanide with Caro's acid. Zinc ion is removed at the same time by adsorption on the hexacyanoferrate/hydrous ferric oxide precipitate.Sulphide is precipitated as ferrous sulphide, then oxidised by atmospheric oxygen. At 25°C and using an Fe/CN ratio of 1·00, initial concentrations of 50 mg l⁻¹ of CN⁻ and 10 mg l⁻¹ of Zn²⁺ in seawater are reduced to 5–7 mg l⁻¹ and 0·1 mg l⁻¹. Subsequent treatment with H₂SO₅/CN = 1·2 reduces the [CN⁻] to 0·1 mg l⁻¹.Treatment of a combined blast furnace/coke ovens effluent ([CN⁻] = 24 mgl⁻¹, [Zn²⁺] = 4·0 mgl⁻¹) with Fe/CN = 1·5 reduced [CN⁻] to 0·2 mg l⁻¹ and [Zn²⁺] to <0·1 mgl⁻¹. Subsequent treatment with H₂SO₅/CN = 2·0 reduced [CN⁻] to 0·2 mg l⁻¹. The process operates best in the pH range 7–9 and so is not affected by the buffer characteristics of seawater.     

Water quality criteria for European freshwater fish: Report on ammonia and inland fisheries

In establishing water quality criteria for European inland fisheries, the effect of ammonia is an important factor to be considered. Sewage effluent, effluents from certain industries and from agriculture are common sources of ammonia in water.The harmful effects of ammonia on fish are related to the pH value and the temperature of the water due to the fact that only the un-ionized fraction of ammonia is poisonous. The un-ionized fraction increases with rising pH value, and with rising temperature.Fish differ slightly in their tolerance to ammonia depending on species. The difference in tolerance being more significant for short periods of exposure. The difference in tolerance is, however, not great enough to justify different criteria for different species.The lowest toxic concentration found for salmonids is 0·2 mg NH₃ 1⁻¹ (un-ionized), but other adverse effects caused by prolonged exposure are only absent at concentrations lower than 0·025 mg NH₃ 1⁻¹ (un-ionized). Concentrations of total ammonia which contain this amount of un-ionized ammonia vary from 19·6 mg 1⁻¹ (pH 7·0, 5°C) to 0·12 mg 1⁻¹ (pH 8·5, 30°C).The criterion should not be applied to temperatures below 5°C or to pH values above 8·5 when other factors have to be taken into consideration.     

Effect of copper and hexavalent chromium on the specific growth rate of ciliata isolated from activated-sludge

After growing mass and monoxenic cultures of three species of Ciliata (Vorticella microstoma, Colpidium campylum, and Opercularia sp.) with Alcaligenes faecalis as the sole protozoan food, and confirming that copper and hexavalent chromium did not affect the bacterial growth, the effect of these metals on the specific growth rate of each protozoa was examined. The term IL_m (median inhibitory limit) was used to define the metal concentration required to reduce the specific growth rate of protozoa to one-half of that of a control. The IL_m for copper was: 0·25 mg 1⁻¹ (V. microstoma), 0·32 mg 1⁻¹ (C. campylum), and 0·27 mg 1⁻¹ (Opercularia sp.). Regarding hexavalent chromium, IL_m for V. microstoma, C. campylum, and Opercularia sp. were 0·53, 12·9 and 20·2 mg 1⁻¹, respectively. Acclimation of each protozoa to these metals for 96 h resulted in IL_m enhanced values of 1·2–2·2 times as large as that for the control.     

pH adjustment in anaerobic digestion

In the pH range 6·0–7·5, the pH in anaerobic processes is controlled by the interaction of the carbonic system and a net strong base. The acid-base state of a digestor can be monitored by only measuring pH and CO₂ partial pressure. Shock doses of strong bases and carbonates causes temporary undersaturated CO₂ conditions and excessively high pH. Bicarbonate dosing leaves the CO₂ solubility equilibrium unchanged. In the absence of a CaCO₃ precipitation inhibiting agent. CaCO₃ solubility limits the pH, and Ca(OH)₂ dosing is unable to raise the pH significantly. Orthophosphates inhibit CaCO₃ precipitation. With [PO₄] > 1·0 × 10⁻³mole·1⁻¹. CaCO₃. precipitation is partially inhibited. Ca(OH)₂ dosing being approximately 45 per cent effective for doses up to 15000 mg 1⁻¹ as CaCO₃. At [PO₄] < 1·0 × 10⁻³moles·1⁻¹ orthophosphates eventually precipitate out during Ca(OH)₂ dosing, thus removing the inhibition mechanism: pH is then limited by the CaCO₃ solubility. Most wastes contain [PO₄] > 2·0 × 10⁻³moles·1⁻¹ making pH adjustment with Ca(OH)₂ possible to a pH of about 7·2 although the dosages will be very high. The pH changes in a process following dosing can be predicted by the graphical representation of the carbonic and net strong base systems.     

High nitrate denitrification in continuous flow-stirred reactors

Continuous flow stirred reactors were used to evaluate the maximum denitrification specific removal rates for influent solutions made from NH₄NO₃, CaNO₃, KNO₃ and UO₂ fuel fabrication waste water. Nitrate substrate concentrations ranged from 0.01 to 20 kg NO₃/m³. Values for U_max (maximum specific substrate removal rate per unit mass of microorganisms per unit time, days⁻¹) were determined using graphical solutions to the Lineweaver-Burk equations. For NH₄NO₃ solutions at nitrate substrate concentrations <6 kg NO₃/m³ the value for U_max was found to be 1.73 days⁻¹. At nitrate substrate concentrations >6 kg NO₃/m³ a nonlinear relationship was observed in the Lineweaver-Burk plots indicating nitrate substrate inhibition. Specific removal rates at nitrate concentrations >6 kg NO₃/m³ averaged <1.0 days⁻¹. Ammonia toxicity may also have occurred as the pH of the mixed liquor was near 8. Methanol concentrations as high as 11.6 kg CH₃OH/m³ did not inhibit denitrification rates. The highest specific removal rates recorded (3.13 ± 0.56 days⁻¹) were with influents made from UO₂ fuel fabrication waste water.     

The occurrence and removal of nitrogen in subsurface agricultural drainage from the San Joaquin Valley, California

During the years 1967–1973 there have been extensive studies of subsurface agricultural drainage in the San Joaquin Valley of California. These studies, by cooperating state and federal agencies, were to determine the composition and quantity of drainage waters produced from irrigated agriculture, to evaluate possible methods of removing problem constituents (mainly nitrogen) from these waters, and to obtain an idea of the effectiveness of the treatment methods studied for reducing the waters biostimulatory content with respect to potential receiving waters. The results of the studies indicated that on an annual average, the drainage waters will probably contain about 20 mg NO₃-N I⁻¹ even after 50 years of leaching and that most of the nitrogen is derived from native soil nitrogen. Treatment studies demonstrated that the nitrogen could be reduced from 20 to 3–5 mg N I⁻¹ by any one of several biological treatment processes including bacterial denitrification (filter and pond), algae growth and harvesting, and by a combination plant growth—bacterial denitrification (“symbiotic”) process. Cost estimates for the processes studied ranged from $10 to $36 1000⁻¹ m³ (1969 dollars). Laboratory algal assays demonstrated that the nitrogen removal systems studied effectively reduced the drainage waters biostimulatory content.     

Nitrification kinetics in activated sludge at various temperatures and dissolved oxygen concentrations

Activated sludge from a domestic sewage works was enriched with nitrifying bacteria by running a laboratory fermenter on ammonia-supplemented sewage. This enriched culture was used to determine respirometrically the kinetics of microbial nitrification. It was demonstrated that the reaction fits the Michaelis-Menten model for temperatures from 10 to 35°C, having a temperature optimum at 15°C (K₃ 0.72 mg 1⁻¹ NH₃). Nitrification is unaffected by high dissolved oxygen concentration 38 mg 1⁻¹ O₂ at 30°C) after acclimatisation. Nitrite concentrations > 20 mg 1⁻¹ are inhibitory to the reaction.     

Semi-automated ammonia probe determination of Kjeldahl nitrogen in freshwaters

A commercially available ammonia probe was used in an automated continuous flow-through system to determine the ammonia-N concentration in Kjeldahl digests of fresh waters. The method is suitable for samples with Kjeldahl-N concentrations greater than 0·1 mg N.1⁻¹. In the 0·1–1·0 mg N.1⁻¹ range 10 digests.h⁻¹ can be analysed. Only micro-digestion equipment and sample volumes of 25 ml are required. The ammonia probe with modified filling solution and polytetafluoroethylene membrane is sufficiently stable for continuous use throughout a working day. Analyses of Kjeldahl digests of freshwater samples by this method and the distillation-Nesslerisation procedure showed good agreement. More precise results were obtained by the ammonia probe method. The ammonia probe method obviates the steam-distillation operation which is time-consuming and during which errors can be easily introduced by loss or gain of ammonia.     

Acute toxicity and behavioral responses of coho salmon (Oncorhynchus kisutch) and shiner perch (Cymatogaster aggregata) to chlorine in heated sea-water

The acute toxicity and behavioral response to chlorinated and heated sea-water was determined for coho salmon smolts and 1–3 month old shiner perch. LC₅₀'s were determined for 7.5, 15, 30 and 60 min exposure times; 13, 16 and 20°C (Δt = 0, 3, 7°C) temperatures and total residual oxidant (TRO) concentrations ranging from 0.077 to 1.035 mg l⁻¹. The mean 60 min LC₅₀ for shiner perch was significantly reduced (P ≤ 0.05) from 308 μg l⁻¹ TRO at 13°C to 230 μg l⁻¹ TRO at 20°C. The 60 min LC₅₀ for coho salmon decreased from 208 μg l⁻¹ TRO at 13°C to 130 μg l⁻¹ at 20°C. The LC₅₀'s for coho salmon in chlorinated sea-water averaged 55% of those for shiner perch. The relationship between TRO concentration, exposure time, and percent survival in chlorinated sea-water at 13°C is presented for both species.A significant (P ≤ 0.01) avoidance threshold for coho salmon occurred at 2 μg l⁻¹ TRO and was reinforced with increasing temperature. A significant (P ≤ 0.01) avoidance threshold for shiner perch occurred at 175 μg l⁻¹ TRO, while a significant preference (P ≤ 0.05 or 0.01) response at 16°C and 20°C occurred at 10, 25, 50 and 100 μg l⁻¹ TRO. The ecological implications of the toxicity tests and the behavioral responses are discussed.     

Low temperature removal of nitrate by bacterial denitrification

The efficiency of two denitrifying sludges enriched at 5 and 20°C were compared using methanol as an electron donor. Both sludges were exposed to the same hydraulic and chemical conditions using an influent containing methanol and mineral salts. The low temperature sludge seemed to have several advantages over the sludge selected for at the higher temperature. In the range 0–17°C, the specific denitrification rate was 1.5–4 times the rate for the high temperature sludge, temperatures below 8°C being the most favourable. At 2°C, under nitrate limiting conditions, 98% nitrate reduction was obtained at a hydraulic residence time of 3.5 h, with an effluent concentration of 0.8 mg NO₃---Nl⁻¹. Sedimentation characteristics were always better for the low temperature sludge, and the utilization of methanol equally good as the high temperature sludge. The low temperature sludge appeared to be biochemically and microbiologically stable to temperature changes within the range 0–17°C, the latter temperature being close to the limit for maintaining the psychrophilic characteristics of the sludge. Studies on pure culture isolates of the denitrifying bacteria showed >90% dominance of one bacterial strain in both sludges. Studies of the isolates also showed that the low-temperature sludge consisted predominantly of psychrotrophs/psychrophiles, and not well-adapted mesophiles, which were only present in low concentrations. The dominant strain in both sludges was unable to grow on methanol in pure culture without access to nutrient growth factors. Only a few minor strains were obligate methylotrophs.Low temperature sludges were tested in a 3-stage biological process receiving domestic sewage. Each stage; carbon oxidation, nitrification and denitrification had separate sludge recycle, and methanol was added to the denitrification stage. These sludges were grown and selected for at temperatures 5°C. At 5°C the laboratory scale process gave 90% removal of total nitrogen at hydraulic residence times of 1.5, 9 and 4 h for the two aeration stages and the anaerobic stage respectively. Overall nitrification/denitrification was 95%, while denitrification separately was 98%. The effluent contained 0.4 mg NO₃---Nl⁻¹. The critical step in the process was unquestionably nitrification. Oxidation of ammonium was satisfactory at low temperature, but the reaction was somewhat vulnerable to changes in external conditions. The low temperature denitrifying sludge was originally enriched on synthetic waste but did not appear to change its microbial composition or characteristics by exposure to municipal wastewater.     

A modified version of the sodium salicylate method for analysis of wastewater nitrates

The German sodium salicylate method for nitrate determination has been modified and improved by utilizing flocculating effect of the preservative HgCl₂ (1000 mg 1⁻¹). The method gives highly reproducible results in the range of 0·2–16 mg 1⁻¹ nitrate nitrogen and is applicable for routine analysis of untreated sewage. Interfering substances are chloride ions above 1500 mg 1⁻¹ and nitrite ions above 10 mg 1⁻¹N. The yellow color produced by sodium salicylate with nitrate ions obeys Beer's law and remains stable for several hours.     

The influence of substrate, pH, diet and temperature upon cadmium accumulation in the asiatic clam (Corbicula fluminea) in laboratory artificial streams

The influence of substrate, pH, diet and temperature upon the accumulation of cadmium (0.05 mg l⁻¹ dose < 0.001 mg l⁻¹ control) in the visceral mass of the Asiatic clam Corbicula fluminea was studied in laboratory artificial stream systems at intervals of 0, 3, 5, 7, 10 and 14-day exposures. Four substrate conditions, sand; sand, silt and clay (SSC); sand, clay and organic matter (SCO); and no substrate (NoS), were considered. The greatest tissue accumulation of cadmium in C. fluminea occurred at 0.05 mg l⁻¹ Cd in NoS and the lowest in clams occupying SCO. Complexation of available metals, lower clam filtering rates and physical protection by the substrate were attributed to the depressed cadmium accumulation of clams exposed in the SCO substrate. Lower pH exposures (5.0 vs 7.8) significantly (P 0.05 level) reduced cadmium uptake at 21°C but had little effect at 9°C. Uptake was higher in clams fed with cadmium-exposed Chlamydomonas reinhardt at 21°C but not at 9°C. In temperature exposures alone at 0.05 mg l⁻¹ Cd, accumulation was significantly higher in C. fluminea exposed at 21°C than at 9°C. The results are discussed relative to the importance of standardized laboratory protocol and the use of C. fluminea as a bioindicator of heavy metal stress.     

Ozonation of water containing chlorine or chloramines. Reaction products and kinetics

Ozone reacts with free aqueous chlorine when present as hypochlorite ion (OCl⁻) with a second order rate constant of 120 ± 15 M⁻¹ s⁻¹ at 20°C. About 77% of the chlorine reacts to produce Cl⁻ and 23% is oxidized to ClO⁻₃. No ClO⁻₄ is formed. Conversion of chlorine to monochloramine reduces the ozone reaction rate to 26 ± 4 M⁻¹ s⁻¹, independent of pH, NH₂Cl is transformed quantitatively to NO⁻₃ and Cl⁻ by O₃. Rate data for other chloramines are also presented. The direct reaction of ozone with chlorine accounts for a significant amount of the chlorine and ozone demand found when the two oxidants are used in combination under water works conditions.     

Nitrification in porous media during rapid,unsaturated water flow

A substantial nitrification in rapid infiltration (RI) systems for wastewater treatment is a prerequisite for obtaining good N removal by denitrification. The purpose of this study is to investigate nitrification in porous media at conditions corresponding to RI treatment systems. Nitrification in six 50-cm porous media columns (98% weathered granite or sand and 2% field soil) during unsaturated leaching at constant flow rates of synthetic wastewater was investigated. Concentrations of NH₄-N between 20 and 60 mg l⁻¹ were applied and vertical concentration profiles of NO₃-N, NO₂-N and NH₄-N were measured for 54 d at 30°C (three columns) and for 140 d at 10°C (three columns). A time lag in nitrification of 20 d was found at 10°C. Complete nitrification was obtained after 3–5 at 30°C and after approximately 50 d at 10°C. Assuming first-order nitrification at steady-state, the corresponding first order reaction rate coefficients (k₁) for NO₃ production in the columns were estimated to be between 0.4 and 1 h⁻¹ at 10°C and between 6 and 9 h⁻¹ at 30°C. Steady-state NO₃ profiles were obtained between 1.5 and up to 9 weeks after the experiments were started. At the actual soil-air contents (0.10 cm³ air phase cm⁻³ soil), oxygen limitations were not observed during the experiments. Nitrogen loadings (water flow times N concentration) above 100 mg N 1⁻¹ cm h⁻¹ (1 g N m⁻² h⁻¹) caused NH₄ accumulation in the columns at 10°C and should probably be avoided during operation of RI system.     

Enhancement of Nitrobacter activity by heterotrophic bacteria

The present work shows that a supply which is not sterile and which contains organic matter does not interfere with the performance of a column filled with a support previously inoculated with Nitrobacter winogradskyi serotype agilis.Measurements of oxidation rates give results higher than those obtained under axenic conditions (maximum oxidation rate: 220 mg NO₂⁻ h⁻¹ l⁻¹ of reactor volume under non-sterile conditions: 160 mg NO₂⁻ h⁻¹ l⁻¹ of reactor volume under axenic conditions).This finding has concentrated our work on the effect of heterotrophic bacteria and of organic matter on the growth of Nitrobacter. We show that a fermentation filtrate of a heterotrophic bacterium (Pseudomonas sp.) added to a fermenter culture of Nitrobacter produces an increase of activity. Experimental results indicate an appreciable reduction in the latence period (15-0 h) and a considerable increase in the rate of growth of Nitrobacter. (Maximum growth rate with 10% of heterotrophic filtrate: 0.044 h⁻¹; without filtrate: 0.032 h⁻¹.)     

Rising sludge in secondary settlers due to denitrification

High suspended solids concentrations in settler effluents can be caused by rising sludge, which is the effect of flotation of solids by nitrogen gas resulting from biological denitrification. Many factors influence the nitrogen gas bubble evolution. The most important factor is the rate of biological denitrification. Factors like nitrogen gas solubility and oxygen concentration in settler influent only play a minor role. The hydraulic retention time in the bottom part of the settler is, for all practical purposes, so high that sufficient nitrogen gas will be generated at temperatures above 20°C, if the nitrate content in the influent to the settler is above the critical one. For temperatures around 20°C the critical nitrate-nitrogen concentration is 6–8 g NO₃⁻-N/m³. The best measure in order to avoid rising sludge is to denitrify the wastewater in the treatment process ahead of the settler.