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.     

Partial nitrification under limited dissolved oxygen conditions

Partial nitrification to nitrite is technically feasible and economically favourable, especially when wastewaters contained high ammonium concentrations or low C/N ratios. Partial nitrification can be obtained by selectively inhibiting nitrite-oxidizing bacteria (NOB) through appropriate regulation of the pH, temperature and dissolved oxygen (DO) concentrations. The effect of pH, DO levels and temperature on ammonia oxidation rate and nitrite accumulation was investigated in order to determine the optimal conditions for partial nitrification of synthetic wastewater with high ammonia concentration. The experiments performed at low DO levels to lower the total oxygen needed in the nitrification step, which means great saving in aeration. During the start-up stage pH and DO were set at 7.0–7.4 and 0.5 mg/l, respectively. The reactor was operated until complete partial nitrification was achieved. The effect of pH, DO on partial nitrification was studied, as pH was kept at 6.5, 7.5, 8.5, 9.5 and DO at 0.5±0.2, 1.5±0.2 and 2.5±0.2 mg/l, and temperature at 30 °C. The influence of temperature on k_a value was studied by keeping pH=7.5, DO=1.5 mg/l and temperature was controlled at 12, 20 and 30 °C, respectively. The results showed that partial nitrification to nitrite was steadily obtained and the optimal operational parameters were pH=7.5, DO=1.5 mg/l, T=30 °C based on ammonia oxidation rate and nitrite accumulation rate. The maximum k_a was achieved and to be 115.1×10⁻³ mg NH₄⁺–N (mg VSS h)⁻¹ under this condition.     

Anaerobic metabolism of Nitrosomonas europaea

Nitrosomonas europaea is capable of maintaining an anaerobic metabolism, using pyruvate as an electron donor and nitrite as an electron acceptor; utilization of nitrite depends upon supply of both pyruvate and ammonia. The role of ammonia in this reaction was not determined. N europaea also assimilates CO₂ anaerobically into cell material in the presence of nitrite (0.5–1.0 mM), pyruvate and ammonia. This reaction was partially inhibited by nitrite which apparently competed with CO₂ for reducing power. Anaerobic nitrite respiration is sensitive to ionophores, FCCP being the most effective.Non-standard-abbreviations TCA trichloroacetic acid - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazon     

Anaerobic ammonia oxidation by cell-free extracts of Nitrosomonas eutropha

Cell-free extracts of Nitrosomonas eutropha oxidized ammonia to nitrite with NO₂ (N₂O₄) as electron acceptor. The ammonia oxidation activity was shown to be sensitive against oxygen. In the absence of oxygen ammonia and NO₂ were consumed in a ratio of approximately 1:2 and hydroxylamine occurred as an intermediate. NO was released in amounts equimolar to the consumption of NO₂. After passing the cell suspension through a French pressure cell and fractionating it by density gradient centrifugation using a linear sucrose gradient, two soluble and two membrane fractions were detectable. Highest ammonia oxidation activity was measured in the membrane fractions and highest hydroxylamine oxidation activity in the soluble fractions. The K_S values of the ammonia oxidizing system in cell-free extracts was about 20 m NH₃ and remained unchanged between pH 7.25 to 8.25.     

Effects of temperature on simultaneous nitrification and denitrification via nitrite in a sequencing batch biofilm reactor

A laboratory scale experiment was described in this paper to enhance biological nitrogen removal by simultaneous nitrification and denitrification (SND) via nitrite with a sequencing batch biofilm reactor (SBBR). Under conditions of total nitrogen (TN) about 30 mg/L and pH ranged 7.15–7.62, synthetic wastewater was cyclically operated within the reactor for 110 days. Optimal operation conditions were established to obtain consistently high TN removal rate and nitrite accumulation ratio, which included an optimal temperature of 31 °C and an aeration time of 5 h under the air flow of 50 L/h. Stable nitrite accumulation could be realized under different temperatures and the nitrite accumulation ratio increased with an increase of temperature from 15 to 35 °C. The highest TN removal rate (91.9%) was at 31 °C with DO ranged 3–4 mg/L. Process control could be achieved by observing changes in DO and pH to judge the end-point of oxidation of ammonia and SND.     

Release of fossil methane from mineral soil particles, and its implication for estimation of methane oxidation in a mineral subsoil

In a preliminary experiment we found that methane evolved from a sandy subsoil during aerobic incubation of shaken soil slurries. In the study presented here the methane was found to be released from the sand particles by mechanical weathering, caused by the grinding effect of the shaking. Large amounts of gas (about 0.5 ml gas g^–1 soil) were extracted by intense grinding of the soil in gas tight serum vials. Methane was the main hydrocarbon in the emitted gas, but also a considerable amount of ethane was present, as well as minor amounts of heavier hydrocarbons (up to C₆). The ¹³C-values of the emitted methane and ethane were –33 and –29 , respectively. Together these results demonstrate a thermogenic origin of the gas. This paper also reports the results of an incubation experiment where possible methane oxidation was looked for. If a possible release of methane is not accounted for, methane oxidation may be overlooked, as illustrated in this paper. Methane consumption was detected only in soil from 40 cm, in contrast to soil sampled at 100 cm and deeper where a slight production was measured. When methane oxidation was inhibited by dimethyl-ether, a significant release of methane was seen. The release was probably caused by chemical weathering. When this methane release was taken into account, methane oxidation was found to be present at all measured depths (40 to 200 cm). Fertilization with urea inhibited the methane oxidation at 40 cm but not at deeper layers. It is hypothesized that ammonia oxidizing bacteria were the main methane oxidizers in this mineral subsoil (deeper than 1 m), and that oxidation of methane might be a survival mechanism for ammonia oxidizers in ammonia limited environments.     

Oxygen consumption and ammonia accumulation in the hypolimnion of Walker Lake, Nevada

Walker Lake (area = 140 km², Z _mean = 19.3 m) is a large, terminal lake in western Nevada. As a result of anthropogenic desiccation, the lake has decreased in volume by 75% since the 1880s. The hypolimnion of the lake, now too small to meet the oxygen demand exerted by decaying matter, rapidly goes anoxic after thermal stratification. Field and laboratory studies were conducted to examine the feasibility of using oxygenation to avoid hypolimnetic anoxia and subsequent accumulation of ammonia in the hypolimnion, and to estimate the required DO capacity of an oxygenation system for the lake. The accumulation of inorganic nitrogen in water overlaying sediment was measured in laboratory chambers under various DO levels. Rates of ammonia accumulation ranged from 16.8 to 23.5 mg-N m^–2 d^–1 in chambers with 0, 2.5 and 4.8 mg L^–1 DO, and ammonia release was not significantly different between treatments. Beggiatoa sp. on the sediment surface of the moderately aerated chambers (2.5 and 4.8 mg L^–1 DO) indicated that oxygen penetration into sediment was minimal. In contrast, ammonia accumulation was reversed in chambers with 10 mg L^–1 DO, where oxygen penetration into sediment stimulated nitrification and denitrification. Ammonia accumulation in anoxic chambers (18.1 and 20.6 mg-N m^–2 d^–1) was similar to ammonia accumulation in the hypolimnion from July through September of 1998 (16.5 mg-N m^–2 d^–1). Areal hypolimnetic oxygen demand averaged 1.2 g O₂ m^–2 d^–1 for 1994–1996 and 1998. Sediment oxygen demand (SOD) determined in experimental chambers averaged approximately 0.14 g O₂ m^–2 d^–1. Continuous water currents at the sediment-water interface of 5–6 cm s^–1 resulted in a substantial increase in SOD (0.38 g O₂ m^–2 d^–1). The recommended oxygen delivery capacity of an oxygenation system, taking into account increased SOD due to mixing in the hypolimnion after system start-up, is 215 Mg d^–1. Experimental results suggest that the system should maintain high levels of DO at the sediment-water interface (10 mg L^–1) to insure adequate oxygen penetration into the sediments, and a subsequent inhibition of ammonia accumulation in the hypolimnion of the lake.     

Multi‐species nitrifying biofilm model (MSNBM) including free ammonia and free nitrous acid inhibition and oxygen limitation

A multi‐species nitrifying biofilm model (MSNBM) is developed to describe nitrite accumulation by simultaneous free ammonia (FA) and free nitrous acid (FNA) inhibition, direct pH inhibition, and oxygen limitation in a biofilm. The MSNBM addresses the spatial gradient of pH with biofilm depth and how it induces changes of FA and FNA speciation and inhibition. Simulations using the MSNBM in a completely mixed biofilm reactor show that influent total ammonia nitrogen (TAN) concentration, bulk dissolved oxygen (DO) concentration, and buffer concentration exert significant control on the suppression of nitrite‐oxidizing bacteria (NOB) and shortcut biological nitrogen removal (SBNR), but the pH in the bulk liquid has a weaker influence. Ammonium oxidation increases the nitrite concentration and decreases the pH, which together can increase FNA inhibition of NOB in the biofilm. Thus, a low buffer concentration can accentuate SBNR. DO and influent TAN concentrations are efficient means to enhance DO limitation, which affects NOB more than ammonia‐oxidizing bacteria (AOB) inside the biofilm. With high influent TAN concentration, FA inhibition is dominant at an early phase, but finally DO limitation becomes more important as TAN degradation and biofilm growth proceed. MSNBM results indicate that oxygen depletion and FNA inhibition throughout the biofilm continuously suppress the growth of NOB, which helps achieve SBNR with a lower TAN concentration than in systems without concentration gradients. Biotechnol. Bioeng. 2010;105: 1115–1130. © 2009 Wiley Periodicals, Inc.     

Modeling the partial nitrification in sequencing batch reactor for biomass adapted to high ammonia concentrations

Partial nitrification has proven to be an economic way for treatment of industrial N-rich effluent, reducing oxygen and external COD requirements during nitrification/denitrification process. One of the key issues of this system is the intermediate nitrite accumulation stability. This work presents a control strategy and a modeling tool for maintaining nitrite build-up. Partial nitrification process has been carried out in a sequencing batch reactor at 30 degrees C, maintaining strong changing ammonia concentration in the reactor (sequencing feed). Stable nitrite accumulation has been obtained with the help of an on-line oxygen uptake rate (OUR)-based control system, with removal rate of 2 kg NH4 (+)-N x m(-3)/day and 90%-95% of conversion of ammonium into nitrite. A mathematical model, identified through the occurring biological reactions, is proposed to optimize the process (preventing nitrate production). Most of the kinetic parameters have been estimated from specific respirometric tests on biomass and validated on pilot-scale experiments of one-cycle duration. Comparison of dynamic data at different pH confirms that NH3 and NO2- should be considered as the true substrate of nitritation and nitratation, respectively. The proposed model represents major features: the inhibition of ammonia-oxidizing bacteria by its substrate (NH3) and product (HNO2), the inhibition of nitrite-oxidizing bacteria by free ammonia (NH3), the INFluence of pH. It appears that the model correctly describes the short-term dynamics of nitrogenous compounds in SBR, when both ammonia oxidizers and nitrite oxidizers are present and active in the reactor. The model proposed represents a useful tool for process design and optimization.     

Utilization of formate as an additional energy source by glucose-limited chemostat cultures ofCandida utilis CBS 621 andSaccharomyces cerevisiae CBS 8066

The oxidation of benzene to phenol by whole cells of Nitrosomonas europaea is catalysed by ammonia monooxygenase, and therefore requires a source of reducing power. Endogenous substrates, hydrazine, hydroxylamine and ammonium ions were compared as reductants. The highest rates of benzene oxidation were obtained with 4 mM benzene and hydrazine as reductant, and equalled 6 mol· h^-1·mg protein^-1. The specificity of ammonia monooxygenase for benzene as a substrate was determined by measuring k _cat/K _m for benzene relative to k _cat/K _m for uncharged ammonia, a value of 0.4 being obtained. Phenol was found to be further hydroxylated to yield hydroquinone. This reaction, like benzene oxidation, was sensitive to the ammonia monooxygenase inhibitor allylthiourea. Catechol and resorcinol were not detected as products of phenol oxidation, implying that at least 88% of the hydroxylation is para-directed.     

三种不同小球藻去除亚硝态氮和氨氮能力的研究

为了研究小球藻在不同质量浓度的氨氮和亚硝态氮环境下的去除能力与生长效果,从实验室挑选3株不同的小球藻CV315-1(Chlorella sp.)、CV315-2(Chlorella sorokiniana)和CV315-3(Chlorella pyrenoidosa)分别置于不同质量浓度的氨氮和亚硝态氮模拟污水中,在温...     

Determination of the decay rate of nitrifying bacteria

The growth and decay of nitrifying organisms determines the amount of nitrifying bacteria in activated sludge systems. The growth rate of the nitrifying organisms is reasonable, well defined, and studied, while the decay rate is still rather uncertain. Experiments in previous studies were over periods up to 14 days and obtained results were not confirmed. Contradicting decay rates of nitrifiers in different bacterial communities is reported. No differentiation between ammonia and nitrite oxidizers was made. Therefore, in this studyper day the decay rate of the nitrifying organisms was studied. The starvation condition (aerobic, anoxic, or anaerobic), temperature, type of bacterial community, and the presence of higher organisms are the main aspects that were investigated. A simple and reliable method (adapted from previous studies) for determining the decay rate of nitrifying organisms under different starvation conditions and different temperatures was developed. The test procedure has been used for determining the decay rate of ammonium and nitrite oxidizing bacteria in an enriched nitrifying culture and in activated sludge. The test was successfully applied at starvation periods up to 30 days. The decay rate of the enriched culture of nitrifiers was very low compared to values for nitrifiers in activated sludge. The decay rate of the nitrifiers in activated sludge was found to be to 0.2, 0.1, and 0.06 per day for aerobic, anoxic, and anaerobic conditions, respectively. The decay rate of ammonia oxidizers and nitrite oxidizers was the same at the corresponding conditions.     

Nitrous oxide production by ammonia oxidizers: Physiological diversity,niche differentiation and potential mitigation strategies

Oxidation of ammonia to nitrite by bacteria and archaea is responsible for global emissions of nitrous oxide directly and indirectly through provision of nitrite and, after further oxidation, nitrate to denitrifiers. Their contributions to increasing N₂O emissions are greatest in terrestrial environments, due to the dramatic and continuing increases in use of ammonia‐based fertilizers, which have been driven by requirement for increased food production, but which also provide a source of energy for ammonia oxidizers (AO), leading to an imbalance in the terrestrial nitrogen cycle. Direct N₂O production by AO results from several metabolic processes, sometimes combined with abiotic reactions. Physiological characteristics, including mechanisms for N₂O production, vary within and between ammonia‐oxidizing archaea (AOA) and bacteria (AOB) and comammox bacteria and N₂O yield of AOB is higher than in the other two groups. There is also strong evidence for niche differentiation between AOA and AOB with respect to environmental conditions in natural and engineered environments. In particular, AOA are favored by low soil pH and AOA and AOB are, respectively, favored by low rates of ammonium supply, equivalent to application of slow‐release fertilizer, or high rates of supply, equivalent to addition of high concentrations of inorganic ammonium or urea. These differences between AOA and AOB provide the potential for better fertilization strategies that could both increase fertilizer use efficiency and reduce N₂O emissions from agricultural soils. This article reviews research on the biochemistry, physiology and ecology of AO and discusses the consequences for AO communities subjected to different agricultural practices and the ways in which this knowledge, coupled with improved methods for characterizing communities, might lead to improved fertilizer use efficiency and mitigation of N₂O emissions.     

Kinetic studies on ammonia and methane oxidation by Nitrosococcus oceanus

The kinetics of ammonia oxidation and the ability of a marine ammonia-oxidizing bacterium, Nitrosococcus oceanus, to metabolize methane were investigated in semicontinuous batch culture. The effects of inhibitors (acetylene and nitrapyrin) and coreactants were determined in order to elucidate the behavior of the ammonia oxygenase enzyme in N. oceanus. Acetylene and nitrapyrin were potent inhibitors and their effects were not mitigated by increased ammonia concentrations. Oxygen concentration had the effect of a mixed-type inhibitor; reduced oxygen inhibited the rate or ammonia oxidation at high substrate concentration but may enhance the rate at low substrate concentrations. Substrate affinity in terms of NH ₄ ⁺ increased (K _m decreased) with increasing pH. Optimal pH was about 8. Methane inhibited ammonia oxidation; the interaction was not simple competitive inhibition and the presence of multiple active sites on the enzyme was indicated by the behaviour of the inhibited treatments. Half-saturation constants for methane (K _i=6.6 M) and ammonia (K _m=8.1 M) were similar. N. oceanus oxidized methanol and methane linearly over time, with CO₂ and cell material being produced at approximately equal rates.     

Ammonia Formation By The Reduction Of Nitrite/Nitrate By Fes: Ammonia Formation Under Acidic Conditions

One issue for the origin of life under a non-reducing atmosphere is the availability of the reduced nitrogen necessary for amino acids, nucleic acids, etc. One possible source of this nitrogen is the formation of ammonia from the reduction of nitrates and nitrites produced by the shock heating of the atmosphere and subsequent chemistry. Ferrous ions will reduce these species to ammonium, but not under acidic conditions. We wish to report results on the reduction of nitrite and nitrate by another source of iron (II), ferrous sulfide, FeS. FeS reduces nitrite to ammonia at lower pHs than the corresponding reduction by aqueous Fe^{+ 2}. The reduction follows a first order decay, in nitrite concentration, with a half-life of about 150 min (room temperature, CO₂, pH 6.25). The highest product yield of ammonia measured was 53%. Under CO₂, the product yield decreases from pH 5.0 to pH 6.9. The increasing concentration of bicarbonate, at higher pH, interferes with the reaction. Comparing experiments under N₂ CO₂ shows the interference of bicarbonate. The reaction proceeds well in the presence of such species as chloride, sulfate, and phosphate, though the yield drops significantly with phosphate. FeS also reduces nitrate and, unlike with Fe^{+ 2}, the reduction shows more reproducibility. Again, the product yield decreases with increasing pH, from 7% at pH 4.7 to 0% at pH 6.9. It appears that nitrate is much more sensitive to the presence of added species, perhaps not competing as well for binding sites on the FeS surface. This may be the cause of the lack of reproducibility of nitrate reduction by Fe^{+ 2} (which also can be sensitive to binding by certain species)     

Evidence for the nitrate assimilation-dependent nitrite excretion in cyanobacterium Nostoc MAC

Nitrate uptake and nitrite efflux patterns in Nostoc MAC showed a rapid phase followed by their saturation. Nitrite efflux was maximum in nitrate medium whereas the cells incubated in N₂ and NH ₄ ⁺ media exhibited a decreased nitrite efflux activity. The simultaneous presence of NH ₄ ⁺ and nitrate significantly decreased nitrite efflux. L-Methionine-Dl-sulphoximine (MSX) prevented inhibition of nitrite efflux by NH ₄ ⁺ . In the dark there was negligible nitrite efflux, whereas illumination increased the rate of nitrite efflux significantly. The nitrite efflux system was maximally operative at pH 8.0, 30°C and a photon fluence rate of 50 mol m^-2. s^-1. These results confirm that (i) the nitrite efflux system in Nostoc MAC is dependent upon nitrate uptake and assimilation and is repressible by NH ₄ ⁺ ; (ii) NH ₄ ⁺ itself is not the actual repressor of nitrite efflux; a product of NH ₄ ⁺ assimilation via glutamine synthetase (GS) is required for repression to occur; (iii) the catalytic function of GS does not appear to be involved in nitrate assimilation-dependent nitrite efflux, and (iv) the optimum pH, temperature and illumination for maximum nitrite efflux were found to be 8.0, 30°C and 50mol m^-2. s respectively.B.B. Singh, P.K. Pandey and P.S. Bisen are with the Department of Microbiology, Barkatullah University. Bhopal 462026, India. S.Singh is with the Department of Microbiology, School of Life Sciences, North Maharashtra University, Jalgaon, India     

Some observations on free ammonia inhibition to Nitrobacter in nitrifying biofilm reactor

Summary Free ammonia inhibition to Nitrobacter population in a nitrifying biofilm was investigated. It was found that when the tree ammonia concentration is greater than 0.1 mgN/l the oxidative activity of Nitrobacter was significantly inhibited, and resulting in a transient accumulation of nitrite ions. The results show that Nitrobacter population is capable of rapidly recovering its lost metabolic activity once the free ammonia concentration becomes less than 0.1 mgN/l, and a complete oxidation of nitrite to nitrate was achieved.     

Cooxidation of naphthalene and other polycyclic aromatic hydrocarbons by the nitrifying bacterium,Nitrosomonas europaea

The soil nitrifying bacterium Nitrosomonas europaea has shown the ability to transform cometabolically naphthalene as well as other 2- and 3-ringed polycyclic aromatic hydrocarbons (PAHs) to more oxidized products. All of the observed enzymatic reactions were inhibited by acetylene, a selective inhibitor of ammonia monooxygenase (AMO). A strong inhibitory effect of naphthalene on ammonia oxidation by N. europaea was observed. Naphthalene was readily oxidized by N. europaea and 2-naphthol was detected as a major product (85%) of naphthalene oxidation. The maximum naphthol production rate was 1.65 nmole/mg protein-min in the presence of 240 M naphthalene and 10 mM NH₄ ⁺. Our results demonstrate that the oxidation between ammonia and naphthalene showed a partial competitive inhibition. The relative ratio of naphthalene and ammonia oxidation, depending on naphthalene concentrations, demonstrated that the naphthalene was oxidized 2200-fold slower than ammonia at lower concentration of naphthalene (15 M) whereas naphthalene was oxidized only 100-fold slower than ammonia oxidation. NH₄ ⁺- and N₂H₄-dependent O₂ uptake measurement demonstrated irreversible inhibitory effects of the naphthalene and subsequent oxidation products on AMO and HAO activity.     

Short-cut nitrification of domestic wastewater in a pilot-scale A/O nitrogen removal plant

The feasibility of nitrite accumulation in a pilot-scale A/O (anoxic/oxic) nitrogen removal plant treating domestic wastewater was investigated at various dissolved oxygen (DO) concentrations and pH levels. The results showed that the pH was not a useful operational parameter to realize nitrite accumulation. Significant nitrite accumulation was observed at the low DO concentration range of 0.3–0.8 mg/l and the maximum nitrite accumulation ratio of about 90% occurred at a DO concentration of 0.6 mg/l. This suggests a reduction of 22% in the oxygen consumption, and therefore a considerable saving in aeration. However, the nitrite accumulation was destroyed at the high DO concentration and the resumption was very slow. In addition, the average ammonia removal efficiency reached as high as 93% at the low DO level. Moreover, experimental results indicated that nitrogen could be removed by simultaneous nitrification and denitrification (SND) via nitrite in the aerobic zones at the low DO concentration, with the efficiency of 6–12%.     

Adaptation of hybridoma cells to higher ammonia concentration

Using two mouse-mouse hybridoma cell lines, the response to ammonia step and serial changes was investigated in batch and continuous cultures with serum-free medium. The inhibitory effect of ammonia on cell growth depended on the cultivation mode, and differed markedly between cell lines. The cell line, 4C10B6 producing IgG monoclonal antibody against Pseudomonas, showed a high adaptation ability to ammonia. The 4C10B6 cells could grow under ammonia concentration as high as 21 mmol/l NH₄Cl with a viability of 80% in the continuous culture with serial increase in ammonia concentration. Whereas, in the batch culture with ammonia step change the cell growth completely ceased at 12 mmol/l NH₄Cl. The other cell line, TO-405 producing IgG monoclonal antibody against hepatitis B surface antigen, could not adapt to ammonia, and the cell growth did not occur at 9 mmol/l NH₄Cl even under the ammonia serial change.List of symbols D_Feed d^-1 Dilution rate of fresh feed medium (=F_o/V) - D_Out d^-1 Dilution rate of cell suspension (=F₁/V) - F₁ ml·d^-1 Volumetric discharge rate of cell suspension - F₀ ml·d^-1 Volumetric flow rate of fresh feed medium - k_D h^-1 Specific death rate - P mmol·l^-1 Product concentration - S mmol·l^-1 Substrate concentration in culture broth - S₀ mmol·l^-1 Substrate concentration in feed medium - t d Cultivation time - V ml Working volume of reactor - X₀ cells·ml^-1 Total cell density - X_V cells·ml^-1 Viable cell density - Y_P/S mmol·mmol^-1 Yield of product from substrate - Y_X/S cells·mmol^-1 Yield of cells from substrate - mmol·cell^-1·h^-1 Specific production rate - h^-1 Specific growth rate - mmol·cell^-1·h^-1 Specific consumption rate of substrate