Factors Affecting Oxidation of Thiosalts by Thiobacilli

The effects of temperature, initial pH, and the concentrations of ammonium, phosphate, and heavy metals on the oxidation of thiosalts by an authentic strain of Thiobacillus thiooxidans (ATCC 8085) and by a mixed culture isolated from a base metal-processing mill effluent pond were studied. The optimum temperature was 30°C and the optimum initial pH was 3.75 for both cultures using thiosulfate and for the mixed culture using tetrathionate. T. thiooxidans ATCC 8085 did not oxidize tetrathionate. For a thiosalt concentration of 2,000 ppm (2,000 mg/liter), maximal rates of destruction occurred at concentrations of ammonium ion above 2 mg/liter and in the presence of 1 mg of phosphate per liter. Under optimal conditions, the rate of thiosulfate oxidation by the pure culture was 55 ± 3 mg/liter per h; the mixed culture oxidized thiosulfate at the rate of 40 ± 1 mg/liter per h and tetrathionate at the rate of 50 ± 2 mg/liter per h. Metal ions caused normal inhibition kinetics in the oxidation of thiosulfate by T. thiooxidans ATCC 8085. K_i values were calculated for cadmium (16 mg/liter), copper (0.46 mg/liter), lead (2 mg/liter), silver (3.1 mg/liter), and zinc (33 mg/liter). Only a slight additive effect was apparent in the presence of all of these metal ions. The mixed culture of thiosalt-oxidizing bacteria was less sensitive to heavy metal inhibition; the order of inhibition of thiosulfate oxidation was Cd < Zn < Pb < Ag < Cu, and that of tetrathionate oxidation was Zn < Cd < Pb < Ag < Cu.     

Efficiency of bacterial protein synthesis during anaerobic degradation of cattle waste

The rate of [15N]ammonia (15NH3) uptake or incorporation into bacterial cells was studied, using stirred, 3-liter benchtop digestors fed on a semicontinuous basis with cattle waste. The fermentations were carried out at 40 and 60 degrees C and at four different loading rates (3, 6, 9, and 12 g of volatile solids per liter of reactor volume per day). The rate of NH3-N incorporation for the period 1 to 5 h after feeding at the four different loading rates was 0.49, 0.83, 1.05, and 1.08 mg/liter per h in the mesophilic digestor and 0.68, 1.07, 1.17, and 1.21 mg/liter per h in the thermophilic digestor. Values were lower 7 to 21 h after feeding in both digestors and were related to the rate of fermentation or CH4 production. In the mesophilic digestors, the rate of bacterial cell production ranged from 3.97 to 8.72 mg of dry cells per liter per h, 1 to 5 h after feeding at the different loading rates. Corresponding values for the thermophilic digestors ranged from 5.46 to 9.77 mg of dry cells per liter per h. Cell yield values ranged from 2.3 to 3.1 mg of dry cells per mol of CH4 produced in the mesophilic and thermophilic digestors at the two lower loading rates. The values were higher (2.8 to 3.4) in the mesophilic digestors at the two higher loading rates because of the accumulation of propionate and a consequent reduction in CH4 production. Low cell yields such as those measured in this study are characteristic of low-specific-growth rates under energy-limited conditions.     

Efficiency of bacterial protein synthesis during anaerobic degradation of cattle waste.

The rate of [15N]ammonia (15NH3) uptake or incorporation into bacterial cells was studied, using stirred, 3-liter benchtop digestors fed on a semicontinuous basis with cattle waste. The fermentations were carried out at 40 and 60 degrees C and at four different loading rates (3, 6, 9, and 12 g of volatile solids per liter of reactor volume per day). The rate of NH3-N incorporation for the period 1 to 5 h after feeding at the four different loading rates was 0.49, 0.83, 1.05, and 1.08 mg/liter per h in the mesophilic digestor and 0.68, 1.07, 1.17, and 1.21 mg/liter per h in the thermophilic digestor. Values were lower 7 to 21 h after feeding in both digestors and were related to the rate of fermentation or CH4 production. In the mesophilic digestors, the rate of bacterial cell production ranged from 3.97 to 8.72 mg of dry cells per liter per h, 1 to 5 h after feeding at the different loading rates. Corresponding values for the thermophilic digestors ranged from 5.46 to 9.77 mg of dry cells per liter per h. Cell yield values ranged from 2.3 to 3.1 mg of dry cells per mol of CH4 produced in the mesophilic and thermophilic digestors at the two lower loading rates. The values were higher (2.8 to 3.4) in the mesophilic digestors at the two higher loading rates because of the accumulation of propionate and a consequent reduction in CH4 production. Low cell yields such as those measured in this study are characteristic of low-specific-growth rates under energy-limited conditions.     

Key physiology of anaerobic ammonium oxidation.

The physiology of anaerobic ammonium oxidizing (anammox) aggregates grown in a sequencing batch reactor was investigated quantitatively. The physiological pH and temperature ranges were 6.7 to 8.3 and 20 to 43 degrees C, respectively. The affinity constants for the substrates ammonium and nitrite were each less than 0.1 mg of nitrogen per liter. The anammox process was completely inhibited by nitrite concentrations higher than 0.1 g of nitrogen per liter. Addition of trace amounts of either of the anammox intermediates (1. 4 mg of nitrogen per liter of hydrazine or 0.7 mg of nitrogen per liter of hydroxylamine) restored activity completely.     

Simultaneous oxidation of ammonium and p-cresol linked to nitrite reduction by denitrifying sludge

The metabolic capability of denitrifying sludge to oxidize ammonium and p-cresol was evaluated in batch cultures. Ammonium oxidation was studied in presence of nitrite and/or p-cresol by 55 h. At 50 mg/L NH₄⁺-N and 76 mg/L NO₂⁻-N, the substrates were consumed at 100% and 95%, respectively, being N₂ the product. At 50 mg/L NH₄⁺-N and 133 mg/L NO₂⁻-N, the consumption efficiencies decreased to 96% and 70%, respectively. The increase in nitrite concentration affected the ammonium oxidation rate. Nonetheless, the N₂ production rate did not change. In organotrophic denitrification, the p-cresol oxidation rate was slower than ammonium oxidation. In litho-organotrophic cultures, the p-cresol and ammonium oxidation rates were affected at 133 mg/L NO₂⁻-N. Nonetheless, at 76 mg/L NO₂⁻-N the denitrifying sludge oxidized ammonium and p-cresol, but at different rate. Finally, this is the first work reporting the simultaneous oxidation of ammonium and p-cresol with the production of N₂ from denitrifying sludge.     

Key Physiology of Anaerobic Ammonium Oxidation

The physiology of anaerobic ammonium oxidizing (anammox) aggregates grown in a sequencing batch reactor was investigated quantitatively. The physiological pH and temperature ranges were 6.7 to 8.3 and 20 to 43°C, respectively. The affinity constants for the substrates ammonium and nitrite were each less than 0.1 mg of nitrogen per liter. The anammox process was completely inhibited by nitrite concentrations higher than 0.1 g of nitrogen per liter. Addition of trace amounts of either of the anammox intermediates (1.4 mg of nitrogen per liter of hydrazine or 0.7 mg of nitrogen per liter of hydroxylamine) restored activity completely.     

Nitrite and nitrate synthesis from pyruvic-oxime by anAlcaligenes sp.

AnAlcaligenes sp. isolated from soil was characterized as to its ability to oxidize and grow on pyruvic-oxime. Abundant nitrification of pyruvic-oxime was demonstrated with maximal nitrite and nitrate production of 1867 mg NO₂ ^–-N per liter and 42 mg NO₃ ^–-N per liter. TheAlcaligenes sp. oxidized hydroxylamine and this metabolism was stimulated when either acetate or pyruvate was present. This organism was also capable of limited pyruvic-oxime oxidation when cultured in an acidic medium. The metabolism of pyruvic-oxime and nitrification by theAlcaligenes sp. in the environment are discussed.     

Settleability and kinetics of a nitrifying sludge in a sequencing batch reactor

A physiological study of a nitrifying sludge was carried out in a sequencing batch reactor (SBR). Pseudo steady-state nitrification conditions were obtained with an ammonium removal efficiency of 99% +/- 1% and 98% +/- 2% conversion of NH4+-N to NO3 - -N. The rate of biomass production was negligible (1.3 +/- 0.1 mg microbial protein-N.L(-1).d(-1)). The sludge presented good settling properties with sludge volume index values lower than 20 mL.g(-1) and an exopolymeric protein/carbohydrate ratio of 0.53 +/- 0.34. Kinetic results indicated that the nitrifying behavior of the sludge changed with the number of cycles. After 22 cycles, a decrease in the specific rate of NO3- -N production coupled with an increase in the NO2- -N accumulation were observed. These results showed that the activity of the nitrite oxidizing bacteria decreased at a longer operation time. Ammonia oxidizing bacteria were found to exhibit the best stability. After 4 months of operation, the specific rates of NH4+-N consumption and NO3- -N production were 1.72 NH4+-N per microbial protein-N per hour (g.g(-1).h(-1)) and 0.54 NO3- -N per microbial protein-N per hour (g.g(-1).h(-1)), respectively.     

Enhanced Alcaligenes faecalis Denitrification Rate with Electrodes as the Electron Donor

The utilization by Alcaligenes faecalis of electrodes as the electron donor for denitrification was investigated in this study. The denitrification rate of A. faecalis with a poised potential was greatly enhanced compared with that of the controls without poised potentials. For nitrate reduction, although A. faecalis could not reduce nitrate, at three poised potentials of +0.06, −0.06, and −0.15 V (versus normal hydrogen electrode [NHE]), the nitrate was partially reduced with −0.15- and −0.06-V potentials at rates of 17.3 and 28.5 mg/liter/day, respectively. The percentages of reduction for −0.15 and −0.06 V were 52.4 and 30.4%, respectively. Meanwhile, for nitrite reduction, the poised potentials greatly enhanced the nitrite reduction. The nitrite reduction rates for three poised potentials (−0.06, −0.15, and −0.30 V) were 1.98, 4.37, and 3.91 mg/liter/h, respectively. When the potentials were cut off, the nitrite reduction rate was maintained for 1.5 h (from 2.3 to 2.25 mg/liter/h) and then greatly decreased, and the reduction rate (0.38 mg/liter/h) was about 1/6 compared with the rate (2.3 mg/liter/h) when potential was on. Then the potentials resumed, but the reduction rate did not resume and was only 2 times higher than the rate when the potential was off.     

Rates of nitrification and carbon uptake in the Rhône River plume (northwestern Mediterranean Sea)

Nitrification rates were measured along a salinity gradient in the Rhône River estuary, using specific inhibitors (allylthiourea and chlorate) coupled with the measurement of change in nitrite concentration and inorganic carbon uptake by nitrifiers. Rates of ammonium and nitrite oxidation were similar up to 15 practical salinity units (from 1 to 2 mol N oxidized liter^-1 day^-1). For higher salinities, nitrite and ammonium oxidation rates were 0.14 and 0.23 mol N oxidized liter^-1 day^-1, respectively. Ammonium oxidizers assimilated 19–150 × 10–3 mol C liter^-1 day^-1, while nitrite oxidizers fixed 4.8–72.6 × 10–3 mol C liter^-1 day^-1. The amounts of nitrogen oxidized and C incorporated demonstrated a linear correlation (r ² > 0.99). The ratio of N oxidized to C incorporated ranged between 14.3 to 12.3 for ammonium oxidizers, and between 31.6 and 29 for nitrite oxidizers, the lower values being measured in seawater. Offprint requests to: M. Bianchi.     

Effect of temperature and free ammonia on nitrification and nitrite accumulation in landfill leachate and analysis of its nitrifying bacterial community by FISH

The cause of seasonal failure of a nitrifying municipal landfill leachate treatment plant utilizing a fixed biofilm was investigated by wastewater analyses and batch respirometric tests at every treatment stage. Nitrification of the leachate treatment plant was severely affected by the seasonal temperature variation. High free ammonia (NH3-N) inhibited not only nitrite oxidizing bacteria (NOB) but also ammonia oxidizing bacteria (AOB). In addition, high pH also increased free ammonia concentration to inhibit nitrifying activity especially when the NH4-N level was high. The effects of temperature and free ammonia of landfill leachate on nitrification and nitrite accumulation were investigated with a semi-pilot scale biofilm airlift reactor. Nitrification rate of landfill leachate increased with temperature when free ammonia in the reactor was below the inhibition level for nitrifiers. Leachate was completely nitrified up to a load of 1.5 kg NH4-N m(-3)d(-1) at 28 degrees C. The activity of NOB was inhibited by NH3-N resulting in accumulation of nitrite. NOB activity decreased more than 50% at 0.7 mg NH3-N L(-1). Fluorescence in situ hybridization (FISH) was carried out to analyze the population of AOB and NOB in the nitrite accumulating nitrifying biofilm. NOB were located close to AOB by forming small clusters. A significant fraction of AOB identified by probe Nso1225 specifically also hybridized with the Nitrosomonas specific probe Nsm156. The main NOB were Nitrobacter and Nitrospira which were present in almost equal amounts in the biofilm as identified by simultaneous hybridization with Nitrobacter specific probe Nit3 and Nitrospira specific probe Ntspa662.     

Simultaneous determinations of nitrification and nitrate reduction in coastal sediments by a 15N dilution technique.

Nitrification, the oxidation of ammonia to nitrite and nitrate, and nitrate reduction by bacteria in coastal sediments of Mangoku-Ura and Odawa Bay were simultaneously determined by a 15N dilution technique. In muddy sediments of Mangoku-Ura, nitrate reduction proceeded at a rate of 10(-2) to 10 X 10(-2) microgram-atoms of N/g per h. Nitrification was far less intensive. Denitrification, or N2 production from nitrate, accounted for about 30% of the nitrate reduction. A simultaneous occurrence of nitrification and nitrate reduction with a similar rate of 10(-2) microgram-atoms of N/g per h was demonstrated in sandy sediment collected from a Zostera bed of Odawa Bay.     

Ammonium uptake in alpine streams in the High Tatra Mountains (Slovakia)

Uptake of NH _inf4 ^sup+ -N by streambed biota of mountain brooks was studied in the alpine zone of the High Tatra Mountains. Experiments were performed involving in situ dosing of ammonium directly to the acidified stream and incubations of ammonium and streambed bryophytes in enclosures within a range of pH from 4.45 to 8.10.NH _inf4 ^sup+ -N uptake length decreased with decreasing stream discharge, while comparable values of discharge-normalized uptake lengths were found during two in situ experiments.Maximum uptake rates of NH _inf4 ^sup+ -N obtained during the incubation of bryophytes (6 to 11 mg m^–2 h^–1) were comparable with results of two in situ experiments (8 and 12 mg m^–2 h^–1). The average NH _inf4 ^sup+ -N uptake rates observed during incubations lasting 3 to 5 hours (4.3 mg m^–2 h^–1) were not related to the pH of stream water. Nitrification of about 50% of the NH _inf4 ^sup+ -N added was observed in non-acidified streams, but was negligible in acidified streams. Significant photoinhibition of nitrification was observed in non-acidified streams during enclosure experiments.     

Biological treatment of ammonium-rich wastewater by partial nitritation and subsequent anaerobic ammonium oxidation (anammox) in a pilot plant

In wastewater treatment plants with anaerobic sludge digestion, 15-20% of the nitrogen load is recirculated to the main stream with the return liquors from dewatering. Separate treatment of this ammonium-rich digester supernatant would significantly reduce the nitrogen load of the activated sludge system. Some years ago, a novel biological process was discovered in which ammonium is converted to nitrogen gas under anoxic conditions with nitrite as the electron acceptor (anaerobic ammonium oxidation, anammox). Compared to conventional nitrification and denitrification, the aeration and carbon-source demand is reduced by over 50 and 100%, respectively. The combination of partial nitritation to produce nitrite in a first step and subsequent anaerobic ammonium oxidation in a second reactor was successfully tested on a pilot scale (3.6 m(3)) for over half a year. This report focuses on the feasibility of nitrogen removal from digester effluents from two different wastewater treatment plants (WWTPs) with the combined partial nitritation/anammox process. Nitritation was performed in a continuously stirred tank reactor (V=2.0 m(3)) without sludge retention. Some 58% of the ammonium in the supernatant was converted to nitrite. At 30 degrees C the maximum dilution rate D(x) was 0.85 d(-1), resulting in nitrite production of 0.35 kg NO(2)-N m(-3)(reactor) d(-1). The nitrate production was marginal. The anaerobic ammonium oxidation was carried out in a sequencing batch reactor (SBR, V=1.6 m(3)) with a nitrogen elimination rate of 2.4 kg N m(-3)(reactor) d(-1) during the nitrite-containing periods of the SBR cycle. Over 90% of the inlet nitrogen load to the anammox reactor was removed and the sludge production was negligible. The nitritation efficiency of the first reactor limited the overall maximum rate of nitrogen elimination.     

Role of agitation conditions in nitrification

The objective of this study was to determine the role of agitation conditions in the oxidation of nitrite ions by Nitrobacter. Batch reaction kinetic experiments were conducted in baffled stirred tanks. The range of agitation conditions studied was 6200 ? 95700 ergs/cm³ sec. This power input corresponds to 3.2 ? 45.6 hp/ 1000 gal, or a “hem Scale” of 3 ? 9. After a lag phase, the reaction kinetics were found to be zero order with respect to nitrite over a concentration range of 590 to 10 mg/liter nitrite nitrogen (NO₂^?-N). The zero-order rate constants were found to significantly decrease with increasing impeller power input per volume of liquid (P / V).     

Efficacy of copper and silver ions and reduced levels of free chlorine in inactivation of Legionella pneumophila.

Water disinfection systems utilizing electrolytically generated copper and silver ions (200 and 20, 400 and 40, or 800 and 80 micrograms/liter) and low levels of free chlorine (0.1 to 0.4 mg/liter) were evaluated at room (21 to 23 degrees C) and elevated (39 to 40 degrees C) temperatures in filtered well water (pH 7.3) for their efficacy in inactivating Legionella pneumophila (ATCC 33155). At room temperature, a contact time of at least 24 h was necessary for copper and silver (400 and 40 micrograms/liter) to achieve a 3-log10 reduction in bacterial numbers. As the copper and silver concentration increased to 800 and 80 micrograms/liter, the inactivation rate significantly (P less than or equal to 0.05) increased from K = 2.87 x 10(-3) to K = 7.50 x 10(-3) (log10 reduction per minute). In water systems with and without copper and silver (400 and 40 micrograms/liter), the inactivation rates significantly increased as the free chlorine concentration increased from 0.1 mg/liter (K = 0.397 log10 reduction per min) to 0.4 mg/liter (K = 1.047 log10 reduction per min). Compared to room temperature, no significant differences were observed when 0.2 mg of free chlorine per liter with and without 400 and 40 micrograms of copper and silver per liter was tested at 39 to 40 degrees C. All disinfection systems, regardless of temperature or free chlorine concentration, showed increase inactivation rates when 400 and 40 micrograms of copper and silver per liter was added; however, this trend was significant only at 0.4 mg of free chlorine per liter.     

Nitrification Rates in the Baltic Sea: Comparison of Three Isotope Techniques

Simultaneous measurements of nitrification in the Baltic Sea were made at 10- to 30-m intervals in the months of June and November by three isotope techniques: [¹⁵N]nitrate dilution, N-serve sensitive [¹⁴C]bicarbonate incorporation, and [¹⁵N]ammonium oxidation to nitrite and nitrate. Nitrification rates of 1 to 280 nmol liter⁻¹ day⁻¹ were recorded, and each method showed that the highest rates of nitrification occurred below the halocline. Even in the presence of ammonium, dark incubations of mixed layer (above ca. 50 m) waters never yielded nitrification rates exceeding 45 nmol liter⁻¹ day⁻¹. The rates measured by the ammonium oxidation method were two- to sevenfold greater than those obtained by ¹⁴C incorporation or ¹⁵N dilution. The merits of each technique are discussed, and it is suggested that the [¹⁵N]ammonium oxidation method should be used in conjunction with the [¹⁴C]bicarbonate incorporation method.     

Degradation of n-hexadecane and its metabolites by Pseudomonas aeruginosa under microaerobic and anaerobic denitrifying conditions

A strategy for sequential hydrocarbon bioremediation is proposed. The initial O(2)-requiring transformation is effected by aerobic resting cells, thus avoiding a high oxygen demand. The oxygenated metabolites can then be degraded even under anaerobic conditions when supplemented with a highly water-soluble alternative electron acceptor, such as nitrate. To develop the new strategy, some phenomena were studied by examining Pseudomonas aeruginosa fermentation. The effects of dissolved oxygen (DO) concentration on n-hexadecane biodegradation were investigated first. Under microaerobic conditions, the denitrification rate decreased as the DO concentration decreased, implying that the O(2)-requiring reactions were rate limiting. The effects of different nitrate and nitrite concentrations were examined next. When cultivated aerobically in tryptic soy broth supplemented with 0 to 0.35 g of NO(2)(-)-N per liter, cells grew in all systems, but the lag phase was longer in the presence of higher nitrite concentrations. However, under anaerobic denitrifying conditions, even 0.1 g of NO(2)(-)-N per liter totally inhibited cell growth. Growth was also inhibited by high nitrate concentrations (>1 g of NO(3)(-)-N per liter). Cells were found to be more sensitive to nitrate or nitrite inhibition under denitrifying conditions than under aerobic conditions. Sequential hexadecane biodegradation by P. aeruginosa was then investigated. The initial fermentation was aerobic for cell growth and hydrocarbon oxidation to oxygenated metabolites, as confirmed by increasing dissolved total organic carbon (TOC) concentrations. The culture was then supplemented with nitrate and purged with nitrogen (N(2)). Nitrate was consumed rapidly initially. The live cell concentration, however, also decreased. The aqueous-phase TOC level decreased by about 40% during the initial active period but remained high after this period. Additional experiments confirmed that only about one-half of the derived TOC was readily consumable under anaerobic denitrifying conditions.     

Nitrification and mineralization potentials in a limed Ultisol in the humid tropics

Summary We studied the effect of liming on the rates of mineralization and nitrification in a coarse-textured kaolinitic Ultisol. Soil samples were taken from field plots which received lime rates from 0 to 4mt/ha three years prior to the study. The pH of the soil samples varied from 4.2 to 6.1. Ammonification of soil organic N and added urea source proceeded readily and was not affected by lime rate. Nitrification occurred in both limed and unlimed soils but the rate of nitrification depended upon the rate of lime application. Soil pH, exchangeable Ca and exchangeable A1 were significantly correlated with the amount of NO₃-N accumulated at the end of the 65 days incubation period. Nitrification of NH₄-N from ammonium sulfate was absent in soils receiving lower rates of lime which gave pH values ranging from 4.2 to 4.8. Added ammonium source was nitrified readily after a 3-week delay period in the soil (pH 6.1) which received a higher rate of lime (4 mt/ha).     

Nitrogen removal from wastewaters at low C/N ratios with ammonium and acetate as electron donors

A denitrifying upflow anaerobic sludge blanket (UASB) reactor was operated at different nitrate loading rates at a C/N ratio of 1.2, with acetate as an electron donor. This resulted in an increase in the accumulation of nitrite. After this, the UASB reactor was supplemented with 100 mg NH4+-Nl(-1) d(-1), while acetate was gradually limited in the medium. This prevented nitrite accumulation at a C/N ratio of 0.6 due to an enhanced nitrite reduction rate achieved in the reactor. An increasing amount of ammonium was consumed when the C/N ratio was lowered in the medium. This suggested that ammonium was used as an alternative electron donor during denitrification, which is supported by nitrogen balances. Nitrite was shown to be toxic for the nitrogen removal process at 200-400 mg NO2--N(l(-1) when the C/N ratio was decreased to 0.4 leading to formation of ammonium. The present study showed that addition of ammonium as an alternative electron donor for denitrification achieved a nitrogen removal process with negligible accumulation of undesirable intermediates.