Determination of growth rate and yield of nitrifying bacteria by measuring carbon dioxide uptake rate.

Nitrifier growth parameters--the maximum growth rate (microAmax) and yield (YA)--were estimated by measuring the rate of carbon dioxide uptake and additional rates of oxygen uptake and ammonia (or nitrite) use. Batch tests in a combined titrimetric and offgas analyzer with enriched Nitrobacter and Nitrosomonas cultures and an activated sludge sample were performed. The measured microAmax values for the Nitrobacter and Nitrosomonas cultures were 0.67 +/- 0.03 day(-1) and 0.54 +/- 0.09 day(-1), while the YA values were 0.072 +/- 0.01 g volatile suspended solids (VSS) x g nitrogen (N)(-1) and 0.14 +/- 0.02 gVSS x gN(-1), respectively. For the activated sludge sample, microAmax was observed to increase with pH (microAmax = 0.72 x 3.3(pH-7.1)) over the range 6.8 to 7.1. All microAmax and YA values determined by this method were similar to those previously reported. Compared with other microAmax and YA estimation methods, this method allows for unique microAmax and YA estimations for given conditions from a single experiment.

Kinetic characterisation of an enriched Nitrospira culture with comparison to Nitrobacter.

Nitrospira and Nitrobacter are nitrite-oxidising bacteria commonly identified in nitrogen removal wastewater treatment plants. Little is known about the growth parameters of Nitrospira or the effects of environmental conditions or inhibitory compounds on Nitrospira activity. These bacterial properties were investigated using an enriched Nitrospira culture and an enriched Nitrobacter culture or Nitrobacter literature values. Compared to Nitrobacter, Nitrospira was found to have a comparable optimal pH range (8.0-8.3); similar normalised activity-temperature relationship (0.44e(0.055(T-15))) for temperatures between 15 and 30 degrees C and a similar oxygen half-saturation constant, K(O) (0.54+/-0.14 mgL(-1)). The major differences identified were that Nitrospira had a lower nitrite half-saturation constant, K(S) (0.9+/-0.07 mgNO(2)-NL(-1)); lower inhibition threshold concentrations for free ammonia (between 0.04 and 0.08 mg NH(3)-NL(-1)) and free nitrous acid (less than 0.03 mg HNO(2)-NL(-1)) and a higher yield (0.15+/-0.04 g VSS g N(-1)). Therefore, Nitrospira is more likely to dominate nitrite oxidation under conditions with low ammonium and nitrite concentrations, which would provide an advantage to them due to their lower K(S) value while avoiding any free ammonia or free nitrous acid inhibition.

Effect of free ammonia on the respiration and growth processes of an enriched Nitrobacter culture.

The inhibitory effect of free ammonia (FA;NH(3)) on the metabolism of Nitrobacter is investigated using a method that allows decoupling energy generation from growth processes. A lab-scale sequencing batch reactor (SBR) was operated for the enrichment of Nitrobacter. Fluorescent in situ hybridization (FISH) analysis showed that 73% of the bacterial population in the reactor was Nitrobacter, while no Nitrospira was detected. Batch tests were carried out to measure the oxygen uptake rate (OUR) by the culture at various FA levels, in the presence (OUR with CO(2)) or absence (OUR without CO(2)) of inorganic carbon (CO(2), HCO(3)(-) and CO(3)(2-)). The FA inhibition on the respiration initiated at below 1mgNH(3)-NL(-1) in both cases. OUR without CO(2) gradually decreased by 12% when the FA concentration increased from 0 to approximately 4mgNH(3)-NL(-1) and remained at the same level till an FA level of 9mgNH(3)-NL(-1) (the highest FA concentration applied in this study). This indicates that FA has a limited inhibitory effect on the respiratory capability of Nitrobacter. Starting from a level that is 15% higher than OUR without CO(2) when no FA was present, OUR with CO(2)decreased more rapidly than OUR without CO(2) reaching the same level as OUR without CO(2) when FA was between 6-9mgNH(3)-NL(-1). This implies that in this range of FA the presence of inorganic carbon did not cause any increase in the respiration activity of Nitrobacter. The results suggest that, while still oxidizing nitrite at approximately 75% of the non-inhibited rate, Nitrobacter likely ceased to grow at an FA level of above 6mgNH(3)-NL(-1). While the real mechanisms remain to be identified, this study indicates that the FA inhibition on Nitrobacter is likely much more serious than suggested by previous studies where OUR with CO(2) (or the equivalent nitrite oxidation rate) was used as the sole measure of the inhibitory effects.

Effect of free ammonia and free nitrous acid concentration on the anabolic and catabolic processes of an enriched Nitrosomonas culture.

The effects of free ammonia (FA; NH(3)) and free nitrous acid (FNA; HNO(2)) concentrations on the metabolisms of an enriched ammonia oxidizing bacteria (AOB) culture were investigated using a method allowing the decoupling of growth and energy generation processes. A lab-scale sequencing batch reactor (SBR) was operated for the enrichment of an AOB culture. Fluorescent in-situ hybridization (FISH) analysis showed that 82% of the bacterial population in the SBR bound to the NEU probe specifically designed for Nitrosomonas europaea. Batch tests were carried out to measure the oxygen and ammonium consumption rates by the culture at various FA and FNA levels, in the presence or absence of inorganic carbon (CO(2), HCO(3) (-), and CO(3) (2-)). It was revealed that FA of up to 16.0 mgNH(3)-N . L(-1), which was the highest concentration used in this study, did not have any inhibitory effect on either the catabolic or anabolic processes of the Nitrosomonas culture. In contrast, FNA inhibited both the growth and energy production capabilities of the Nitrosomonas culture. The inhibition on growth initiated at approximately 0.10 mgHNO(2)-N . L(-1), and the data suggested that the biosynthesis was completely stopped at an FNA concentration of 0.40 mgHNO(2)-N . L(-1). The inhibition on energy generation initiated at a slightly lower level but the Nitrosomonas culture was still oxidizing ammonia at half of the maximum rate at an FNA concentration of 0.50-0.63 mgHNO(2)-N . L(-1). The affinity constant of the Nitrosomonas culture with respect to ammonia was determined to be 0.36 mgNH(3)-N . L(-1), independent of the presence or absence of inorganic carbon.

The inhibitory effects of free nitrous acid on the energy generation and growth processes of an enriched nitrobacter culture.

The inhibitory effects of nitrite (NO2-)/free nitrous acid (HNO2-FNA) on the metabolism of Nitrobacter were investigated using a method allowing the decoupling of the growth and energy generation processes. A lab-scale sequencing batch reactor was operated forthe enrichment of a Nitrobacter culture. Fluorescent in situ hybridization (FISH) analysis showed that 73% of the bacterial population was Nitrobacter. Batch tests were carried out to assess the oxygen and nitrite consumption rates of the enriched culture at low and high nitrite levels, in the presence or absence of inorganic carbon. It was observed that in the absence of CO2, the Nitrobacter culture was able to oxidize nitrite at a rate that is 76% of that in the presence of CO2, with an oxygen consumption rate that is 85% of that measured in the presence of CO2. This enabled the impacts of nitrite/FNA on the catabolic and anabolic processes of Nitrobacter to be assessed separately. FNA rather than nitrite was likely the actual inhibitor to the Nitrobacter metabolism. It was revealed that FNA of up to 0.05 mg HNO2-N x L(-1) (3.4 microM), which was the highest FNA concentration used in this study, did not have any inhibitory effect on the catabolic processes of Nitrobacter. However, FNA initiated its inhibition to the anabolic processes of Nitrobacterat approximately 0.011 mg HNO2-N x L(-1) (0.8 microM), and completely stopped biomass synthesis at a concentration of approximately 0.023 mg HNO2-N x L(-1) (1.6 microM). The inhibitory effect could be described by an empirical inhibitory model proposed in this paper, but the underlying mechanisms remain to be revealed.

Stoichiometric and kinetic characterisation of Nitrobacter in mixed culture by decoupling the growth and energy generation processes.

The growth, maintenance and lysis processes of Nitrobacter were characterised. A Nitrobacter culture was enriched in a sequencing batch reactor (SBR). Fluorescent in situ hybridisation showed that Nitrobacter constituted 73% of the bacterial population. Batch tests were carried out to measure the oxygen uptake rate and/or nitrite consumption rate when both nitrite and CO2 were in excess, and in the absence of either of these two substrates. The results obtained, along with the SBR performance data, allowed the determination of the maintenance coefficient and in situ cell lysis rate of Nitrobacter. Nitrobacter spends a significant amount of energy for maintenance, which varies considerably with the specific growth rate. At maximum growth, Nitrobacter consume nitrite at a rate of 0.042 mgN/mgCOD(biomass) . h for maintenance purposes, which increases more than threefold to 0.143 mgN/mgCOD(biomass) . h in the absence of growth. In the SBR, where Nitrobacter grew at 40% of its maximum growth rate, a maintenance coefficient of 0.113 mgN/mgCOD . h was found, resulting in 42% of the total amount of nitrite being consumed for maintenance. The above three maintenance coefficient values obtained at different growth rates appear to support the maintenance model proposed in Pirt (1982). The in situ lysis rate of Nitrobacter was determined to be 0.07/day under aerobic conditions at 22 degrees C and pH 7.3. Further, the maximum specific growth rate of Nitrobacter was estimated to be 0.02/h (0.48/day). The affinity constant of Nitrobacter with respect to nitrite was determined to be 1.50 mgNO2(-)-N/L, independent of the presence or absence of CO2.