ABSTRACT

1. ENRICHMENT OF ANAMMOX BACTERIA IN REACTOR PACKED WITH KALDNES RINGS Çiğdem Kalkan1, Kozet Yapsaklı1, Bülent Mertoğlu2 1Marmara University, Environmental Engineering Department, Faculty of Engineering, 34722 Istanbul,Turkey 2 Marmara University, Bioengineering Department, Faculty of Engineering, 34722 Istanbul,Turkey e-mail:cigdemkalkan@marun.edu.tr ,kozet.yapsakli@marmara.edu.tr,bulent.mertoglu@marmara.edu.tr ABSTRACT ABSTRACT The influent NO2-N/NH4–N concentration ratio was chosen to be 1:1. However, the average removal ratio was found to be 1.1:1 in our system. A stoichiometric molar ratio of nitrite to ammonium was proposed at 1.32 by Strous et al. (1998) and used ever since for Anammox reaction. In the literature, this ratio varies between 1.04 (Bettazzi et al, 2010) and 1.50 (Strous et al, 1997). Kartal et al. (2007) reported that this stoichiometric ratio also can change in different stressed conditions. The nitrite to ammonium nitrogen consumption ratio in our case lies within the interval proposed earlier by researchers. Nitrate is produced during an Anammox process and it indicates a good Anammox growth. NO3-N production was observed by measuring influent and effluent nitrate nitrogen concentrations (Figure 4). In this study, Anammox process operated in a lab-scale upflow biofilm reactor which was filled with Kaldnes Rings as a carrier material. A synthetic feed solution was used for the Anammox system which consists mainly of 1:1 ratio of NH4+-N and NO2--N concentration along with other trace elements needed for the growth of Anammox. Anammox biomass was enriched by increasing the influent ammonium and nitrite concentrations step by step. 90% ammonium and 95 % nitrite removal efficiency was observed in the reactor at steady state operation. It is considered that, Kaldnes Rings offered a suitable environment for the growth of Anammox biomass and prevented the washed out of the biomass from the system. Keywords: Anammox, Kaldnes Rings, Biological Treatment, Nitrogen Removal Kaldnes Rings INTRODUCTION The conventional nitrogen removal processes resulting in high cost requirements due to the need of an oxygen and a carbon source during the process. Nowadays, Anammox has been reported as an alternative to remove nitrogen compounds from high nitrogen loaded wastewater with low organic matter content and that its application for nitrogen removal could lead to significantly lower (90% reduction) operational cost (Jetten et al., 2001). The consumption of ammonium and nitrite and the production of nitrate are in a relation of 1:1.31±0.06:0.22±0.02 (Van de Graaf et al., 1996). The Anammox bacteria obviously grow very slowly and have a low biomass yield therefore, Anammox bacteria can only be cultivated with very efficient biomass retention. In addition solid support materials like glass beads, Kaldnes rings or non-woven biomass carriers are good alternatives to ensure that Anammox biomass is retained in the reactor systems. Biofilm processes are widely studied due to the many possible advantages, such as savings in space, high surface area with relevant organisms, possibilities to obtain high conversion capacity and less biomass production. In the case of using Kaldnes ring as a support material for Anammox process high efficiencies are expected during the startup period and operation. Figure 1. Lab-scale continuous flow Anammox reactor setup RESULTS AND DISCUSSION The results showed that high removal efficiencies were recorded from the first day of reactor operation. The average removal efficiencies for nitrite and ammonium nitrogen are observed to be 92% and 88%, respectively. Influent concentrations were increased step by step from 100 mg/l to 400mg/l in response to high removal efficiencies. A nitrite nitrogen removal efficiency of 96% and an ammonium nitrogen removal efficiency of 86% were seen at the highest influent concentrations. Influent and effluent nitrite and ammonium nitrogen concentrations were shown in Figure 2 and Figure 3. The influent nitrate-nitrogen concentration was constant and equal to 25 mg/L during the operational period. Nitrate is added to prevent any sulfate reduction (Dapena-Mora et al., 2007). Effluent nitrate concentration increased as a result of increase ammonium and nitrite in influent. The ratio of the produced nitrate to consumed nitrite was found to be 0.10. According to Anammox stoichiometry, the nitrate production should amount to up to 11% of the reduced nitrogen (Cema et al., 2007). CONCLUSION It is important to choose appropriate reactor configuration which prevent the biomass washout and allows sufficient time for the biomass. During the reactor operation high nitrite and ammonia removal efficiencies were obtained. Therefore, it is considered that, Kaldnes Rings offered an appropiate environment for the growth of Anammox biomass and prevented the washed out of the biomass from the system. MATERIALS AND METHOD The laboratory scale Anammox reactor was operated for 80 days. The process was performed at a temperature of 35°C, dissolved oxygen concentration of below 0.3 mg O2/l, average pH value between 7.5 and 8.0. The continuous flow lab-scale Anammox system were seeded with Anammox culture which was enriched before in the scope of another study.  Figure 2. Influent and effluent NO2-N concentrations for the lab-scale Anammox biofilter. REFERENCES Bettazzi E, Caffaz S, Vannini C, Lubello C (2010) Nitrite inhibition and intermediates effects on Anammox bacteria: A batch-scale experimental study. Process Biochemistry, 45, 573-580. Cema, G., Wiszniowski, J., Zabczynski, S., Godlewska, E.Z., Raszka, A., Gorska, J.S., (2007) Biological nitrogen removal from landfill leachate by deammonification assisted by heterotrophic denitrification in a rotating biological contactor (RBC), Water Science and Technology, 55, (8-9), 35-42. Dapena-Mora, A., Fernandez, I., Campos, J.L., Mosquera-Corral, A., Mendez, R., Jetten, M.S.M. (2007) Evaluation of activity and inhibition effects on Anammox process by batch tests based on the nitrogen gas production. Enzyme MicrobiolTechnol 40,859–65. Jetten, M. S.M., Wagner, M., Fuerst, J., Loosdrecht, M., Kuenen, G. , Strous, M. (2001)Microbiology and application of the anaerobic ammonium oxidation (‘anammox’) process, Environmental biotechnology, 12,283–288. Kartal, B., Kuypers, M.M., Lavik, G., Schalk, J., Op den Camp, H.J.M, Jetten, M.S.M, Strous, M. (2007) Anammox bacteria disguised as denitrifiers: nitrate reduction to dinitrogen gas via nitrite and ammonium, Environ Microbiol 9,635–642. Strous, M., Gerven, E.V., Zheng, P., Kuenen, J.G., Jetten, M.S.M. (1997) Ammonium removal from concentrated waste streams with the anaerobic ammonium oxidation (ANAMMOX) process in different reactor configurations ,Water Res. 31, 1955–1962. Strous, M., Heijnen, J.J., Kuenen, J.G., Jetten, M.S.M.(1998) The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms, Appl. Microbiol. Biotechnol. 50, 589–596. van de Graaf, A.A., de Bruijin, P., Robertson, L.A., Jetten, M.S.M. and Kuenen, J.G. (1996).Autotrophic growth of anaerobic ammonium-oxidizing microorganisms in a fluidized bed reactor. Microbiology, 142, 2187–2196. Figure 3. Influent and effluent NH4-N concentrations for the lab-scale Anammox biofilter.

2. ENRICHMENT OF ANAMMOX BACTERIA IN REACTOR PACKED WITH KALDNES RINGS Çiğdem Kalkan1, Kozet Yapsaklı1, Bülent Mertoğlu2 1Marmara University, Environmental Engineering Department, Faculty of Engineering, 34722 Istanbul,Turkey 2 Marmara University, Bioengineering Department, Faculty of Engineering, 34722 Istanbul,Turkey e-mail: cigdemkalkan@marun.edu.tr , kozet.yapsakli@marmara.edu.tr, bulent.mertoglu@marmara.edu.tr ABSTRACT ABSTRACT The influent NO2-N/NH4–N concentration ratio was chosen to be 1:1. However, the average removal ratio was found to be 1.1:1 in our system. A stoichiometric molar ratio of nitrite to ammonium was proposed at 1.32 by Strous et al. (1998) and used ever since for Anammox reaction. In the literature, this ratio varies between 1.04 (Bettazzi et al, 2010) and 1.50 (Strous et al, 1997). Kartal et al. (2007) reported that this stoichiometric ratio also can change in different stressed conditions. The nitrite to ammonium nitrogen consumption ratio in our case lies within the interval proposed earlier by researchers. Nitrate is produced during an Anammox process and it indicates a good Anammox growth. NO3-N production was observed by measuring influent and effluent nitrate nitrogen concentrations (Figure 4). In this study, Anammox process operated in a lab-scale upflow biofilm reactor which was filled with Kaldnes Rings as a carrier material. A synthetic feed solution was used for the Anammox system which consists mainly of 1:1 ratio of NH4+-N and NO2--N concentration along with other trace elements needed for the growth of Anammox. Anammox biomass was enriched by increasing the influent ammonium and nitrite concentrations step by step. 90% ammonium and 95 % nitrite removal efficiency was observed in the reactor at steady state operation. It is considered that, Kaldnes Rings offered a suitable environment for the growth of Anammox biomass and prevented the washed out of the biomass from the system. Keywords: Anammox, Kaldnes Rings, Biological Treatment, Nitrogen Removal Kaldnes Rings INTRODUCTION The conventional nitrogen removal processes resulting in high cost requirements due to the need of an oxygen and a carbon source during the process. Nowadays, Anammox has been reported as an alternative to remove nitrogen compounds from high nitrogen loaded wastewater with low organic matter content and that its application for nitrogen removal could lead to significantly lower (90% reduction) operational cost (Jetten et al., 2001). The consumption of ammonium and nitrite and the production of nitrate are in a relation of 1:1.31±0.06:0.22±0.02 (Van de Graaf et al., 1996). The Anammox bacteria obviously grow very slowly and have a low biomass yield therefore, Anammox bacteria can only be cultivated with very efficient biomass retention. In addition solid support materials like glass beads, Kaldnes rings or non-woven biomass carriers are good alternatives to ensure that Anammox biomass is retained in the reactor systems. Biofilm processes are widely studied due to the many possible advantages, such as savings in space, high surface area with relevant organisms, possibilities to obtain high conversion capacity and less biomass production. In the case of using Kaldnes ring as a support material for Anammox process high efficiencies are expected during the startup period and operation. Figure 1. Lab-scale continuous flow Anammox reactor setup RESULTS AND DISCUSSION The results showed that high removal efficiencies were recorded from the first day of reactor operation. The average removal efficiencies for nitrite and ammonium nitrogen are observed to be 92% and 88%, respectively. Influent concentrations were increased step by step from 100 mg/l to 400mg/l in response to high removal efficiencies. A nitrite nitrogen removal efficiency of 96% and an ammonium nitrogen removal efficiency of 86% were seen at the highest influent concentrations. Influent and effluent nitrite and ammonium nitrogen concentrations were shown in Figure 2 and Figure 3. The influent nitrate-nitrogen concentration was constant and equal to 25 mg/L during the operational period. Nitrate is added to prevent any sulfate reduction (Dapena-Mora et al., 2007). Effluent nitrate concentration increased as a result of increase ammonium and nitrite in influent. The ratio of the produced nitrate to consumed nitrite was found to be 0.10. According to Anammox stoichiometry, the nitrate production should amount to up to 11% of the reduced nitrogen (Cema et al., 2007). CONCLUSION It is important to choose appropiate reactor configuration which prevent the biomass washout and allows sufficient time for the biomass. During the reactor operation high nitrite and ammonia removal efficiencies were obtained. Therefore, it is considered that, Kaldnes Rings offered an appropiate environment for the growth of Anammox biomass and prevented the washed out of the biomass from the system. MATERIALS AND METHOD The laboratory scale Anammox reactor was operated for 80 days. The process was performed at a temperature of 35°C, dissolved oxygen concentration of below 0.3 mg O2/l, average pH value between 7.5 and 8.0. The continuous flow lab-scale Anammox system were seeded with Anammox culture which was enriched before in the scope of another study.  Figure 2. Influent and effluent NO2-N concentrations for the lab-scale Anammoxbiofilter. REFERENCES Bettazzi E, Caffaz S, Vannini C, Lubello C (2010) Nitrite inhibition and intermediates effects on Anammox bacteria: A batch-scale experimental study. Process Biochemistry, 45, 573-580. Cema, G., Wiszniowski, J., Zabczynski, S., Godlewska, E.Z., Raszka, A., Gorska, J.S., (2007) Biological nitrogen removal from landfill leachate by deammonification assisted by heterotrophic denitrification in a rotating biological contactor (RBC), Water Science and Technology, 55, (8-9), 35-42. Dapena-Mora, A., Fernandez, I., Campos, J.L., Mosquera-Corral, A., Mendez, R., Jetten, M.S.M. (2007) Evaluation of activity and inhibition effects on Anammox process by batch tests based on the nitrogen gas production. Enzyme MicrobiolTechnol 40,859–65. Jetten, M. S.M., Wagner, M., Fuerst, J., Loosdrecht, M., Kuenen, G. , Strous, M. (2001)Microbiology and application of the anaerobic ammonium oxidation (‘anammox’) process, Environmental biotechnology, 12,283–288. Kartal, B., Kuypers, M.M., Lavik, G., Schalk, J., Op den Camp, H.J.M, Jetten, M.S.M, Strous, M. (2007) Anammox bacteria disguised as denitrifiers: nitrate reduction to dinitrogen gas via nitrite and ammonium, Environ Microbiol 9,635–642. Strous, M., Gerven, E.V., Zheng, P., Kuenen, J.G., Jetten, M.S.M. (1997) Ammonium removal from concentrated waste streams with the anaerobic ammonium oxidation (ANAMMOX) process in different reactor configurations ,Water Res. 31, 1955–1962. Strous, M., Heijnen, J.J., Kuenen, J.G., Jetten, M.S.M.(1998) The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms, Appl. Microbiol. Biotechnol. 50, 589–596. van de Graaf, A.A., de Bruijin, P., Robertson, L.A., Jetten, M.S.M. and Kuenen, J.G. (1996).Autotrophic growth of anaerobic ammonium-oxidizing microorganisms in a fluidized bed reactor. Microbiology, 142, 2187–2196. Figure 3. Influent and effluent NH4-N concentrations for the lab-scale Anammoxbiofilter.