Enzyme and Microbial Technology 40 (2007) 859���865 Evaluation of activity and inhibition effects on Anammox process by batch tests based on the nitrogen gas production A. Dapena-Mora a, I. Fernandez �� a, J.L. Campos a, A. Mosquera-Corral a,���, R. Mendez �� a, M.S.M. Jetten b a Department of Chemical Engineering, Institute of Technological Research, School of Engineering, University of Santiago de Compostela, Rua Lope G�� omez de Marzoa s/n, E-15782 Santiago de Compostela, Spain b Department of Microbiology, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands Received 17 April 2006 received in revised form 23 June 2006 accepted 27 June 2006 Abstract In order to assess the applicability at industrial-scale of the anaerobic ammonium oxidation (Anammox) process it is necessary to study the toxic effects on the maximum specific Anammox activity (SAA) of different compounds commonly present in industrial effluents. The present study was focused on the application of batch tests to determine the maximum SAA in different conditions. The batch tests were based on the measurement of nitrogen gas production. The initial conditions for the tests established to obtain the maximum value of the measured SAA were: 30 ���C, pH 7.8, shaking speed 150 rpm, and biomass concentration 1 g VSS L���1. The accuracy of the method was evaluated by mass balances of the nitrogen compounds and the obtained errors were smaller than 7%. Neither the initial biomass concentrations tested (0.5���2.0 g VSS L���1) nor the S0/X0 ratio between 0.018 and 0.140 g NO2���-N g VSS���1 had significant influence on the estimated SAA. The addition of a second feeding tended to increase it around 20 �� 10%. The developed method is afterwards applied to the study of the inhibition caused on the Anammox process by different compounds (NH4+, NO2���, NO3���, NaCl, SO42���, S2���, flocculant, etc.). The effects of chloramphenicol (inhibitor of the denitrifying process) and allylthiourea (inhibitor of the nitrifying process) were tested in order to be used to distinguish between the Anammox activity from nitrifying and denitrifying activities. The developed batch experiments were found suitable to establish not only the maximum SAA of certain sludge but also the inhibitory effects of certain tested compounds. �� 2006 Elsevier Inc. All rights reserved. Keywords: Anammox Batch test Inhibition Specific Anammox activity (SAA) 1. Introduction The existence of the anaerobic ammonium oxidation pro- cess has been initially predicted from calculations based on the Redfield ratio in marine ecosystems and from thermodynamic calculations [1]. Nevertheless, it was not until 1999 that Strous et al. [2] identified the organism involved in the process as a Planc- tomycete type bacterium. This microorganism combines ammo- niumandnitriteunderanoxicconditionstogeneratenitrogengas according to the stoichiometry shown in the following equation: NH4+ + 1.32NO2��� + 0.066HCO3��� + 0.13H+ ��� 1.02N2 + 0.256NO3��� + 0.066CH2O0.5N0.15 + 2.03H2O (1) ��� Corresponding author. Tel.: +34 981 563100x16779 fax: +34 981 528050. E-mail address: eqanusmc@usc.es (A. Mosquera-Corral). Nowadays, bacteria able to carry out the Anammox process are being found in natural water sources and wastewater treatment plants spread all over the world [3]. This process is increasing in importance at global level due to its contribution to oceanic nitrogen loss and its advantages compared to the nitrification and denitrification processes for nutrient removal from wastew- ater [4]. Its importance in the nitrogen cycle of the oceans has been studied [5] and it has been recently demonstrated that in continental shelf sediments, up to 67% of the nitrogen gas for- mation is due to anaerobic ammonium oxidation and only 33% due to denitrification. Up to now, the found bacteria performing the Anammox process belong to three main genera: Candidatus Brocadia, Candidatus Kuenenia and Candidatus Scalindua. From the engineering point of view, its importance is more related to its application for nitrogen removal from wastewaters. It is known that the use of Anammox process combined with partial nitrification would lead to an impor- tant reduction of operational costs compared to conventional 0141-0229/$ ��� see front matter �� 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.06.018

860 A. Dapena-Mora et al. / Enzyme and Microbial Technology 40 (2007) 859���865 nitrification���denitrification processes [1]. The combined partial nitrification���Anammox process is being currently implemented in the WWTP Rotterdam [1] to treat sludge digester effluents. This process is applicable to wastewaters characterized by low carbon to nitrogen ratio content and high ammonia concentra- tions [6]. Furthermore, it is common that these streams contain also compounds that inhibit the Anammox process such as chlo- ride, sulphide, sulphate [7���10]. Therefore, studies to test the possible toxicity of the wastewater are necessary to know the feasibility of this treatment. The influence of toxic compounds on biomass activities is usually researched by means of batch experiments with addi- tion of the studied inhibitory compound, which give important information to be translated to continuous operation. In the case of the Anammox process only a few studies have been focussed on this subject [11]. Further research is needed to develop a sim- ple method to estimate the Anammox activity and establish the effects of different toxic compounds. The present work is focused on the stablishment of the opti- mal conditions for the determination of the maximum specific Anammox activity (SAA) using batch tests based on the mon- itoring of nitrogen gas production. Once these conditions were determined, the effects on the maximum SAA of different com- pounds usually present in wastewaters (NH4+, NO2���, NO3���, NaCl, PO43���, SO42���, S2���, acetate, flocculants, allylthiurea, chloramphenicol) are studied. 2. Materials and methods 2.1. Batch tests procedure The assays were performed in vials with a total volume of 38 mL and a vol- ume of liquid of 25 mL, each closed with a gas-tight coated septum capable of withstanding about 2 bars of pressure. The vials were inoculated with Anam- mox biomass enriched in bacteria belonging to the specie Candidatus Kuenenia stuttgartiensis [12], washed and re-suspended in phosphate buffer (0.14 g L���1 KH2PO4 and 0.75 g L���1 K2HPO4). The headspace and liquid phase were gasi- fied with argon to remove the oxygen. The initial pH value was fixed at 7.8. The vials were placed in a thermostatic shaker, at 150 rpm and 30 ���C until stable conditions were reached. Then the substrates ((NH4)2SO4 and NaNO2) were added and pressure was equalized to the atmospheric one. The production of N2 gas was tracked by measuring the overpressure in the headspace with a time frequency depending on the biomass activity in each vial test. The final pH value was always measured in order to check that it was maintained in the opti- mal range for the Anammox activity. This methodology has been developed for the determination of denitrifying activities [13]. When an inhibitory compound was tested the corresponding amounts were added together with the substrates [14]. 2.2. Performed experiments A first run of assays, performed in triplicate, was carried out to assess the accuracy of the method to estimate the specific Anammox activity (SAA). In this run, activity tests, using different initial concentrations of substrates of 42, 56 and 70 mg N L���1 for both ammonia and nitrite, and an initial biomass concentration of 1 g VSS L���1, were performed to evaluate the consumption and production of nitrogenous compounds in the liquid and gas phases, respectively. A second run of assays was carried out to study the effects of the initial sub- strate (NO2���-N) to biomass concentrations ratio (S0/X0) and the initial biomass concentration (X0) on SAA. To test the effect of the S0/X0 ratio, different activ- ity tests with constant biomass concentration of 1 g VSS L���1 were performed using several S0/X0 ratios between 0.018 and 0.14 g NO2���-N (g VSS)���1. The effect of initial biomass concentration was checked at a constant S0/X0 ratio of 0.07 g NO2���-N (g VSS)���1 using several X0 of 0.25, 0.5, 1.0 and 2.0 g VSS L���1. A third run of assays, performed in duplicate, was used to determine the influence of a second feeding. In this case initial concentrations of ammonia and nitrite were fixed at 70 mg N L���1, respectively, while the initial biomass concentration was 1 g VSS L���1. Finally, in a fourth run, the toxic effects of compounds present in some indus- trial effluents were tested (inhibition assays). The batch experiment procedure was similar to that previously described in the third run, these toxic compounds being added to the vials at different concentrations together with the substrates. 2.3. Calculations 2.3.1. Accuracy of the method The total amount of N2 gas produced was calculated from the overpressure measured in the headspace of each vial at the end of the assay by using the ideal gas law equation. The amount of nitrogen removed from the liquid phase was also calculated by measuring the ammonium, nitrite and nitrate concentrations at the beginning and the end of the experiment and taking into account the volume of the liquid phase. The relative error of the method was calculated based on the difference between both amounts. 2.3.2. Specific Anammox activity (SAA) The N2 gas production rate was calculated from the maximum slope of the curve describing the pressure increase in the vial along the time (��) (Eq. (2)): dN2 dt = �� VG RT , mol N2 min���1 (2) where VG is the volume of the headspace (L), R the ideal gas coefficient and T the temperature (K). The SAA is calculated from the N2 gas production rate divided by the biomass concentration in the vial X (g VSS L���1) (Eq. (3)): SAA = dN2/dt XVL 28 g N mol N2 1440 min d , g N2-N (g VSS)���1d���1 (3) where VL is the volume of the liquid phase (L). Since the values of the affinity constant of the Anammox bacteria for ammo- nia and nitrite were lower than 10 and 5 M, respectively [11], it can be considered that the activity measured is the maximum activity for the range of nitrite and ammonia concentrations used. 2.3.3. Activity percentage and IC50 The percentage of activity maintained when inhibitory compounds were tested was calculated as (Eq. (4)) SAA(%) = SAA SAA0 �� 100 (4) where SAA0 is the maximum specific activity on the control assay (no presence of toxicants) and SAA the maximum specific activity of the tests with inhibitory compounds. The IC50 is the concentration of the tested compound, which corresponds to a percentage of SAA of 50% compared to that obtained without the presence of this compound. 2.4. Analytical methods Ammonium was analysed by the phenol���hypochloride method [15]. Nitrite and nitrate were analysed by spectrophotometry [16]. Biomass concentration was measured as volatile suspended solids (g VSS L���1), according to standard methods [16]. The pH value was measured using a selective electrode Ingold model U-455 connected to a pH/mV measurer Crison 506. Theoverpressureintheheadspacewasmeasuredusingadifferentialpressure transducer 0���5 psi, linearity 0.5% of full-scale, Centerpoint Electronics. The biogas composition was analysed with a gas chromatograph Hewlett Packard 5890 Series II.