Publications (2)3.05 Total impact
Article: Stoichiometric and kinetic characterisation of Nitrosomonas sp. in mixed culture by decoupling the growth and energy generation processes.[show abstract] [hide abstract]
ABSTRACT: A novel method that relies on the decoupling of the energy production and biosynthesis processes was used to characterise the maintenance, cell lysis and growth processes of Nitrosomonas sp. A Nitrosomonas culture was enriched in a sequencing batch reactor (SBR) with ammonium as the sole energy source. Fluorescent in situ hybridization (FISH) showed that Nitrosomonas bound to the NEU probe constituted 82% of the bacterial population, while no other known ammonium or nitrite oxidizing bacteria were detected. Batch tests were carried out under conditions that both ammonium and CO2 were in excess, and in the absence of one of these two substrates. The oxygen uptake rate and nitrite production rate were measured during these batch tests. The results obtained from these batch tests, along with the SBR performance data, allowed the determination of the maintenance coefficient and the in situ cell lysis rate, as well as the maximum specific growth rate of the Nitrosomonas culture. It is shown that, during normal growth, the Nitrosomonas culture spends approximately 65% of the energy generated for maintenance. The maintenance coefficient was determined to be 0.14-0.16 mgN mgCOD(biomass)(-1)h(-1), and was shown to be independent of the specific growth rate. The in situ lysis rate and the maximum specific growth rate of the Nitrosomonas culture were determined to be 0.26 and 1.0 day(-1) (0.043 h(-1)), respectively, under aerobic conditions at 30 degrees C and pH 7.Journal of Biotechnology 12/2006; 126(3):342-56. · 3.05 Impact Factor
Article: KINETIC AND STOICHIOMETRIC CHARACTERISATION OF ENRICHED NITROSOMONAS AND NITROBACTER CULTURES BY DECOUPLING THE GROWTH AND ENERGY GENERATION PROCESSES[show abstract] [hide abstract]
ABSTRACT: Biological nitrification and denitrification is the most economical and environmentally friendly technology for removing nitrogenous compounds from wastewater. Nitrification consists of two steps involving two groups of microorganisms collectively known as nitrifiers. The first step of nitrification, the oxidation of ammonium to nitrite, is carried out by ammonia oxidizing bacteria (AOB), while the second step, the oxidation of nitrite to nitrate is catalyzed by nitrite oxidizing bacteria (NOB). The understanding of the biochemistry and physiological behaviors of these organisms is highly beneficial for the design and optimization of biological nitrogen removal systems. The project aims to fill in the following knowledge gaps concerning the metabolism of both AOB and NOB: (i) the maintenance energy requirements and their dependency on the bacterial specific growth rates, (ii) the in-situ lysis rates, and (iii) the mechanism involved in the free ammonia and free nitrous acid inhibition on the metabolic processes of these microorganisms. This study uses enriched mixed cultures of ammonia and nitrite oxidizers, rather than pure cultures, to better represent the typical nitrifiers in treatment systems. Two labscale sequencing batch reactors were operated for the enrichment of AOB and NOB. Fluorescent In-Situ Hybridization (FISH) analysis showed that the reactors are 82% and 73% enriched with Nitrosomonas and Nitrobacter, respectively. The characteristics of Nitrosomonas and Nitrobacter were determined using the information obtained from batch tests carried out using the Titration and Off-Gas Analysis (TOGA) sensor, which allowed to investigate the energy production and growth processes separately through controlling the CO2 supply to the biomass. The kinetic parameters determined include the specific maintenance energy consumption rates of both Nitrosomonas and Nitrobacter, as a function of specific growth rate, the in-situ lysis rates of these bacteria, as well as their maximum specific growth rates and affinity constants with respect to the key substrates. Utilizing the same method, the inhibitory effects of free ammonia (FA) and free nitrous acid (FNA) on the catabolic and anabolic mechanisms of both Nitrosomonas and Nitrobacter were evaluated separately. The main contributions of this thesis are as follows. Both Nitrosomonas and Nitrobacter spend significant amount of energy (about 65% for Nitrosomonas and 20- 75% for Nitrobacter) on maintenance processes, which may be partially responsible for the low cell growth yield often observed for these organisms. Further, it was observed that the maintenance energy demand of Nitrosomonas is likely independent of its specific growth rate, while that of Nitrobacter varies considerably with the specific growth rate and the dependency appears to be well described by the Pirt maintenance energy model. The in-situ lysis rates of both the Nitrosomonas and Nitrobacter cultures were determined as 0.26 d-1 (300C) and 0.07 d-1 (200C), respectively. These values appear to be lower than the aerobic lysis rates of nitrifiers (0.15-0.43 d-1 at 20-300C) commonly reported in literature using the starvation method. This is to our best knowledge the first time the in-situ lysis rates were directly determined from the activity of heterotrophic bacteria, which would not be possible with pure culture studies and/or with the starvation method. The inhibition studies conducted in this research demonstrated that FNA and FA rather than nitrite and ammonium are the actual inhibitors. FNA and FA were found to have much stronger inhibitory effect on the biosynthesis compared to the catabolic processes of nitrifiers. Further, it was revealed that Nitrobacter and Nitrosomonas possess different level of tolerance to FNA and FA. Both FA and FNA were found to have strong inhibition on the anabolic processes of Nitrobacter, but limited inhibitory effect on the catabolism of this culture. The biosynthesis of Nitrobacter was totally inhibited at an FA level of 6.0 mgNH3-N.L-1 (or above) or an FNA level of 0.02 mgHNO2-N.L-1 (or above). At the same level of FA, the energy production capability of Nitrobacter was only inhibited by 12%, whereas an FNA level of up to 0.05 mgHNO2-N.L-1 did not show any inhibition on the energy production of Nitrobacter. FA up to 16.0 mgNH3-N.L-1 was found to not have any inhibitory effect on either the catabolic or anabolic processes of the Nitrosomonas culture, but both these processes were inhibited by FNA. While an FNA level of 0.40-0.63 mgHNO2-N.L-1 inhibited the energy production capability of Nitrosomonas by 50%, the growth process of the culture was completely inhibited by an FNA concentration of 0.40 mgHNO2-N.L-1. The results obtained in this Ph.D. research demonstrated that the energy decoupling method is a useful tool to determine the kinetic parameters of Nitrobacter and Nitrosomonas, and possibly other microorganisms that use different substrates as carbon and energy sources. Further, the data gained from the inhibition studies suggest that the different level of tolerance to FNA and FA inhibition by Nitrobacter and Nitrosomonas likely contribute to the elimination of nitrite oxidizers from the systems that treat high nitrogen load wastewater through partial nitrification. Moreover, the different inhibitory effect of FNA and FA on the anabolism and catabolism of these bacteria suggest that the inhibition on catabolic and anabolic processes should be investigated separately.