The synthesis of the dissimilatory nitrate reductase under aerobic conditions in a number of denitrifying bacteria, isolated from activated sludge and drinking water

A number of denitrifying bacteria were isolated from activated sludge and drinking water. These bacteria were tested for the synthesis of the dissimilatory nitrate reductase under aerobic conditions (dissolved oxygen concentration above 4 mg · l⁻¹). The synthesis of this enzyme varied from total repression by oxygen in some bacteria, especially those isolated from drinking water, until a nearly non oxygen-repressed synthesis in other bacteria (strains 15 and N4). The effect of the dissolved oxygen concentration during growth of the bacteria on the synthesis of the dissimilatory nitrate reductase in cells of strain 15 was studied more extensively. A considerable repression of the enzyme synthesis was obtained when the dissolved oxygen concentration was relatively high (approx 15 mg·l⁻¹). Addition of chlorate to the growth medium of strain 15 (using NH⁺₄-N as nitrogen source) also resulted in a serious repression of the nitrate reductase synthesis during aerobic growth (dissolved oxygen above 4 mg·l⁻¹). The dissimilatory nitrate reductase of aerobically grown cells of strains 15 and N4 was found to be mainly localized in the membrane fraction.     

Denitrification with a bacterial disc unit

An enclosed rotating disc unit was operated anaerobically as a denitrifying system, with methanol as the hydrogen donor. As the bacterial population became established, denitrification rate increased by 1·5 mg NO₃⁻—N reduced m⁻² h⁻², to a maximum rate of 260 mg NO₃⁻—N reduced m⁻²h⁻¹. The C:N ratio necessary for complete denitrification was found to be 2·6:1. Optimum pH for denitrification lay in the range between pH 7·0 and 8·5. Q₁₀ values were 1·38 between 10 and 30°C, −2·66 above 30°C and 13·06 below 10°C.     

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.     

A pilot-plant scale evaluation of potential precipitants in the secondary precipitation process

Chemical precipitation of presettled domestic wastewater (secondary precipitation) was studied in three pilot-plants operating in parallel with alum, ferric iron and lime as precipitants. With all precipitants phosphates were effectively removed down to less than 0·1 mg P l⁻¹. Total phosphorus removals were closely connected to the settling properties of the flocs. The factor that seemed to influence settling properties most was pH. In a favourable pH-range it was possible to achieve total phosphorus concentrations in the effluent of less than 0·5 mg P l⁻¹. The COD removal was at an average 70, 60 and 55% with alum, ferric iron and lime as precipitants.     

Concurrent denitrification and oxygen uptake in microbial films

Mixed microbial films were grown on the inner walls of a tubular reactor with recirculation of the reactor contents and continuous flow-through of nutrient solution. The loss of total oxidized nitrogen was correlated to the film population, the nitrite concentration and the dissolved oxygen concentration in the reactor. When film population was greater than 0.5 × 10⁹ cells cm⁻², reactor dissolved oxygen concentration greater than 1 mg l⁻¹ had little effect on the nitrate loss. Nitrate loss declined for film populations greater than 2 × 10⁹ cells cm⁻². Models based on Monod and zero-order microbial kinetics were calibrated using these data and a nonlinear least squares method. There was little difference in the residual errors with these methods.     

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 determination of chemical oxygen demand in water and wastes by a simplified acid dichromate digestion

A simplified and improved chemical oxygen demand (COD) procedure is suitable for water and waste water samples containing up to 1500 mg Cl l⁻¹. Samples are digested with sulfuric acid, potassium dichromate, silver sulfate, mercury(II) sulfate and sulfamic acid in open glass tubes for 2 h at 140 ± 2°C without boiling, and excess dichromate is determined colorimetrically at 440 nm. The use of 49-position digestion racks, a large oven and a spectrophotometer fitted with a 1-cm flow cell allow analysis of ca. 12 samples h⁻¹. A correction for chloride interference is not required in the 0–500 mg Cl l⁻¹ range. The detection limit is 3 mg l⁻¹, and the relative standard deviation at the 112 mg COD l⁻¹ level is 4.3%. Thirty-five waste water samples were analyzed by the standard dichromate reflux method and by the proposed procedure. There was no significant difference between the two sets of data (P > 0.25). Recovery data for 15 major water pollutants including benzene, toluene and pyridine are presented.     

Effluent denitrification with anaerobic filters

Experiments with two pilot-scale anaerobic filters were conducted to investigate their efficiencies in denitrifying a wastewater effluent under ambient, tropical conditions. It was found that these anaerobic filters could effectively denitrify a wide range of influent nitrate concentrations from about 20 to 410 mg l⁻¹ (corresponding to the nitrate and nitrite loadings of 50–550 g m⁻³ d⁻¹). The denitrification efficiencies achieved were between 89–98%, and a straight-line relationship between the nitrate and nitrite loadings vs the denitrification rates was observed. The specific surface areas of the filter medium employed in this study did not exhibit any significant effects upon the anaerobic filter performance. Neither filter became clogged during the 8-month operational period.     

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.     

Lethal response by atlantic salmon parr to some polyoxyethylated cationic and nonionic surfactants

Lethal response of Atlantic salmon parr, as 96-h LC50, is semi-logarithmically related to the number of moles of ethylene oxide in the polyoxyethylated surfactant. 96-h LC50 of polyoxyethylene (10) monolaurate = 7.5 mg l¹, polyoxyethylene (10) lauryl ether = 3·5 mg l⁻¹, and polyoxyethylene (10) octadecyl amine = 0·2 mg l⁻¹.Evidence is presented which suggests that polyoxyethylene esters with up to 18–20 moles of ethylene oxide are partially detoxified in the animal, resulting in changes in lethal response. Possible physiological explanations for the relationship between polyoxyethylene chain length and lethality involve uptake rates and attainment of a critical concentration of surfactant at the unknown active site.     

The effect of dissolved oxygen concentration on nitrification

The effect of dissolved oxygen concentration on the rate of nitrification has been investigated by a number of researchers using both pure and mixed cultures, and cultures found in wastewater treatment systems. The maximum growth rate of both nitrification reactions are reported to be affected by dissolved oxygen concentration over the range of 0.3 mg l⁻¹ to as much as 4.0 mg l⁻¹. In some instances, it has been reported that a dissolved oxygen concentration in excess of 4.0 mg l⁻¹ is required to achieve maximum nitrification rates, while other investigators have found that only 0.5 to 1.0 mg l⁻¹ is required.It has been proposed that several factors are responsible for the wide range of reported nitrification rates with varying dissolved oxygen concentrations. Among these factors are the effects of oxygen diffusion in flocs, variation between measured results due to steady-state and dynamic measuring techniques, and double-substrate limited kinetics. This paper reviews the nitrification literature with respect to the effects of dissolved oxygen concentration, and shows that double-substrate limiting kinetics could account for the variation in the reported results.     

Modified curcumin and dianthrimide methods for the determination of boron in the presence of high levels of nitrate and organics

Modifications have been made to the curcumin and 1,1′-dianthrimide methods for boron analysis so that they may be used in the presence of up to 700 mg l⁻¹NO⁻₃ and 500 mg l⁻¹ organic carbon. In the curcumin method, nitrate is removed by alkaline reduction using a slurry of aluminum powder. In the dianthrimide method, nitrate up to 1000 mg l⁻¹NO⁻₃ is removed in the dehydration-with-sulfuric-acid step; organics are most conveniently removed by treatment of the dehydrated sample with solid potassium persulfate. With dianthrimide, an automated procedure is used for the colour formation and measurement steps.     

Activity of Zoogloea ramigera growing in flocs and in suspension

A comparison was made between cells of Zoogloea ramigera occurring in flocs and in suspension. Suspended cells were obtained by adding cellulose to the growth medium, which prevented floc formation without damaging the cells. Differences in protein synthesis and poly-β-hydroxybutyrate production of cells in suspension and cells in flocs were only found between slowly shaken cultures. Increasing the oxygen content of such cultures did not raise the growth limitation of the cells in flocs. The oxygen uptake of the suspended cells was found to represent a zero order reaction at an oxygen concentration of the culture above 0.1 mg/l. and that of cells in flocs above 1.5 mg/l. Below 20 mg/ml glucose the glucose respiration rate of cells in flocs was lower than that of suspended cells. Above approx 20 mg/l. glucose, the glucose respiration of both types of cells was almost a zero order reaction.     

Assessing unrecorded organic pollution from agricultural, urban, and wooded lands

An investigation was conducted of the organic pollution for seven small New Jersey watersheds representing agricultural, urban, and wooded lands. The 5-day biochemical oxygen demand (BOD) was used as a main index of organic pollution. Data obtained for a 2·5 yr period indicate background BOD concentrations averaging from 0·5 to 2·0 mg l⁻¹ in all streams, except that a value of 9·0 mg l⁻¹ was obtained for a residential-commercial-industrial area. During or after rainstorms, the BOD loadings, in pounds per day per unit area, averagely became more than ten times as the background amount for all streams. No significant correlation was found between BOD concentration and flow rate, but good correlations were obtained between BOD loadings and flows. The seasonal pattern of BOD loading distribution was examined. Also the frequency distribution of BOD concentrations and loadings were obtained.     

Denitrification en continu a l'aide de microorganismes immobilises sur des supports solides

In this paper, we describe a study of biological denitrification by immobilized cells. Nitrates are reduced in sterile solutions by Pseudomonas aeruginosa immobilized in a fixed bed reactor, and in synthetic waste water by mixed cultures immobilized into a fluidized bed reactor.The fixed bed reactor is a Plexiglas column filled with corn stovers (Table 1). It is 0.05 m in diameter and 0.55 in height, its volume being approx. 11. The fresh medium is injected at the base of the column and the liquid level is regulated by an overflow weir. Reactor and carrier are sterilized with ethylene-oxide. After sterilization 1 l. of a growing batch culture of Pseudomonas aeruginosa is introduced aseptically and the reactor is then fed continuously (45 ml h⁻¹) with fresh medium (N---NO₃ = 40 mg l⁻¹) until the first steady state is reached.Nitrates and nitrites are determinated by means of a colorimetric method.Reactor efficiency remains constant for over 40 days. Nitrates and nitrites concentrations are measured inside the reactor for flow varying from 2 to 16 ml min⁻¹ (Fig. 2). Reductions of nitrates and nitrites seem to be two first-order reactions (Fig. 3 and Table 2) and constant rate increases with flow rate (Fig. 4). Until nitrate concentration reaches 960 mg/l⁻¹ (N---NO₃) degradation is correct (Figs 5 and 6), beyond nitrites, which have been formed, seem to be inhibitor.Using this reactor, 50 mg N---NO₃ have been reduced per hour and per liter of empty reactor, but it may be possible to reduce 140 mg N---NO₃ l⁻¹ h⁻¹ if fresh medium contains 200 mg N---NO₃ l⁻¹.The fluidized bed reactor is a Plexiglas column filled with earthenware. It is 0.05 m in diameter and 3.15 m in height, its volume being approx. 6.201. Fresh medium is injected at the base of the column and the liquid level is regulated by an overflow weir. Figure 7 shows the retention time of the liquid in the reactor in relation to flow. The first steady state has been reached after 2 weeks, and it has not been possible to know half life time of the column.Four experiments were conducted (Table 3) and, for each nitrate, nitrite and methanol concentrations in the reactor were measured (Fig. 8). So, it appears that reduction of nitrates and nitrites are two first-order reactions (Table 4) and that constant rate values, which are higher than in fixed bed reactor, increase with flow.The reactor is more affected by a flow shift than by a nitrate concentration shift in fresh medium, and biomass linked onto carrier is about 76 mg of dry matter g⁻¹ of earthenware.So, our fluidized bed column is able to reduce 560 mg N---NO₃ h⁻¹ l⁻¹ of empty reactor, then retention time of liquid is less than 3 min.     

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.     

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.     

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.     

Nitrate determination in fresh and some estuarine waters by ultraviolet light absorption: A new proposed method

The new proposed u.v./resin technique for nitrate determination is either not affected by, or can allow for, the following interfering chemicals at levels occurring in natural polluted or unpolluted waters; chloride, phosphate, sulphate, carbonate/bicarbonate, bromide, nitrite, coloured metal complexes, humic acids, ammonium, dyes, detergents, phenol and other u.v. absorbing organics. The method is quick and has an accuracy of ±3%. Concentrations of NO₃.N in the range 0.1–3.0 mg l⁻¹ can be determined in fresh water. Concentration of the sample to determine lower levels by evaporation is feasible with certain upland waters but should not be attempted if the sample has a high humic acid concentration. The technique can only be used in nitrate rich estuarine and coastal waters because the lower limit of detection is raised to 0.5 mg l⁻¹ when the sample is diluted to remove bromide interference.     

Detergent phosphorus and algal growth

The total biomass of Chlorella pyrenoidosa (two strains) Chlamydomas reinhardtii. Euglena gracilis. Anabaena flos-aquae and Plectonenema boryanum was determined after the algae were grown in waters from Sylvan, Pleasant and Pidgeon Lakes (all in northeastern Indiana) that had been supplemented with 0.1, 1 or 10% sewage effluents (Indianapolis and Crawsfordsville, Indiana). Biomass was found not to be significantly decreased when the total phosphorus was reduced by alkaline treatment from 7.20-3.50 mg l⁻¹ (50 per cent reduction) for the Crawfordsville effluents. In another series of experiments Chlorella pyrenoidosa was grown in Sugar Creek water (west central Indiana) to which had been added 0.1. 1 or 10% sewage effluents that originated from a motel treatment system. Reactive sewage phosphorus was reduced from 15.4 to 7.44 mg l⁻¹ (57 per cent reduction) by supplying the motel with non-phosphorus cleaning products. No significant reduction in algal growth was observed. Only when effluents were advanced treated so that reactive phosphorus levels were below 1.2 mg l⁻¹ (92 per cent reduction) was algal growth significantly decreased.