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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.

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In laboratory experiments, source-separated urine was stabilised with nitrification and denitrified via nitritation and anaerobic ammonium oxidation. The highest total ammonia concentration in the influent was 7,300 gN/m3, the maximum pH 9.2. In a moving bed biofilm reactor (MBBR) with Kaldnes biofilm carriers, we stabilised urine as a 1:1 ammonium nitrate solution. The maximum nitrification rate was 380 gN/m3/d corresponding to 1.7 gN/m2(biofilm)/d. Nitrite ammonium solutions were produced in a continuous flow stirred tank reactor (CSTR) with 4.8 days sludge retention time (SRT) at 30 degrees C and in a sequencing batch reactor (SBR) with more than 30 days SRT. Nitrate build-up was negligible in both reactors. Nitritation rates were 780 gN/m3/d in the CSTR and 280 gN/m3/d in the SBR, respectively. However, shortening the cycles would increase nitritation in the SBR. High concentrations of nitrous acid, salts, and presumably hydroxylamine suppressed nitrite oxidation in the nitritation reactors. In all three nitrification reactors, maximally 50% of the influent total ammonia was oxidised without pH control. None of the common inhibition or limitation approaches could explain why ammonia oxidation always stopped at pH values around 6. In a batch experiment, we showed that source-separated urine can be denitrified autotrophically by anammox bacteria.

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 significantly reduces the nitrogen load of the activated sludge system. Two biological applications are considered for nitrogen elimination: (i) classical autotrophic nitrification/heterotrophic denitrification and (ii) partial nitritation/autotrophic anaerobic ammonium oxidation (anammox). With both applications 85-90% nitrogen removal can be achieved, but there are considerable differences in terms of sustainability and costs. The final gaseous products for heterotrophic denitrification are generally not measured and are assumed to be nitrogen gas (N2). However, significant nitrous oxide (N2O) production can occur at elevated nitrite concentrations in the reactor. Denitrification via nitrite instead of nitrate has been promoted in recent years in order to reduce the oxygen and the organic carbon requirements. Obviously this "achievement" turns out to be rather disadvantageous from an overall environmental point of view. On the other hand no unfavorable intermediates are emitted during anaerobic ammonium oxidation. A cost estimate for both applications demonstrates that partial nitritation/anammox is also more economical than classical nitrification/denitrification. Therefore autotrophic nitrogen elimination should be used in future to treat ammonium-rich sludge liquors.

Separate biological elimination of nitrogen from the digester supernatant of a municipal wastewater treatment plant (WWTP) was investigated in pilot and full-scale plants. Denitrification mainly via nitrite was achieved in a sequencing batch reactor (SBR) and a continuous flow reactor (CSTR or SHARON). Suppression of nitrite oxidation in the SBR was feasible at short aerobic/anaerobic intervals allowing for immediate denitrification of the produced nitrite. Nitrate production could also be stopped by exposing the biomass to anaerobic conditions for 11 days. Temporarily high concentrations (up to 80 gNH3-Nm(-3)) of free ammonia could not be considered as the major reason for inhibiting nitrite oxidation. In a full-scale SBR plant 90% of the nitrogen load was denitrified in a total hydraulic retention time (HRT) of 1.6 days and with a sludge age between 15 and 20 days. Ethanol and methanol were used for denitrification. The specific average substrate consumption was 2.2 gCOD(dosed) g(-1)N(removed) with an effective biomass yield of 0.2 gCOD(biomass) g(-1)COD(dosed). No dosing with base was required. In the SHARON process full nitrogen elimination was achieved only with a total HRT greater than 4 days at 29 degrees C. The overall costs were estimated at 1.4 euros kg(-1)N(removed) for the SBR and 1.63 euros kg(-1)N(removed) in SHARON mode, respectively. The SHARON process is simple in operation (CSTR) but the tank volume has to be significantly greater than in SBR.

Nitrogen can be eliminated effectively from sludge digester effluents by anaerobic ammonium oxidation (anammox), but 55-60% of the ammonium must first be oxidized to nitrite. Although a continuous flow stirred tank reactor (CSTR) with suspended biomass could be used, its hydraulic dilution rate is limited to 0.8-1 d(-1) (30 degrees C). Higher specific nitrite production rates can be achieved by sludge retention, as shown here for a moving-bed biofilm reactor (MBBR) with Kaldnes carriers on laboratory and pilot scales. The maximum nitrite production rate amounted to 2.7 gNO2-Nm(-2)d(-1) (3 gO2m(-3)d(-1), 30.5 degrees C), thus doubling the dilution rate compared to CSTR operation with suspended biomass for a supernatant with 700 gNH4-Nm(-3). Whenever the available alkalinity was fully consumed, an optimal amount of nitrite was produced. However, a significant amount of nitrate was produced after 11 months of operation, making the effluent unsuitable for anaerobic ammonium oxidation. Because the sludge retention time (SRT) is relatively long in biofilm systems, slow growth of nitrite oxidizers occurs. None of the selection criteria applied - a high ammonium loading rate, high free ammonia or low oxygen concentration - led to selective suppression of nitrite oxidation. A CSTR or SBR with suspended biomass is consequently recommended for full-scale operation.

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.