Shortcut Nitrification Treatment of High Strength Ammonia Nitrogen Wastewater by Aerobic Granular Sludge

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1 ENVIRONMENTAL Yuan Ren et al., J.Chem.Soc.Pak., Vol. 38, No. 06, Shortcut Nitrification Treatment of High Strength Ammonia Nitrogen Wastewater by Aerobic Granular Sludge 1 Yuan Ren*, 2 Lilong Yan**, 2 Guoxin Hao, 2 Xiaolei Zhang, 2 Yan Wen, 2 Yihan Guo and 1 Zhonglin Chen 1 School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin , China. 2 School of Resource and Environment, Northeast Agricultural University, Harbin China. renyuanhit@163.com*, yanll98@163.com** (Received on 29 th September 2015, accepted in revised form 10 th February 2016) Summary: This study aims to evaluate the feasibility of treating wastewater with high strength ammonia nitrogen with aerobic granular sludge for the long term. The ratio of ammonia nitrogen removal and the stability of granular sludge were investigated over a period of 150 days by gradually increasing the ammonia nitrogen concentration in the influent. Results showed that when the mean concentration of the ammonia nitrogen in the influent increased from mg/l to mg/l, the mean ratio of ammonia nitrogen removal and the nitrite accumulation ratio were stable at more than 90% and more than 80%, respectively. The content and composition of extracellular polymeric substances (EPSs) were influenced by the ammonia nitrogen concentration in the influent. When this concentration increased, the concentration of polysaccharides (PS) decreased by 41.21%, the content of protein (PN) increased by 24.36%, and the PN/PS ratio increased twofold. Thus, a large number of EPSs and a high PN/PS ratio comprise a suitable condition for the stable, long-term use of aerobic granular sludge. Keywords:Aerobic granular sludge; High strength ammonia nitrogen; Shortcut nitrification; EPSs Introduction With the aggravation of eutrophication and the implementation of strict sewage discharge standards, the task of ammonia nitrogen removal from wastewater is important and urgent. This reduction, especially of high-strength ammonia nitrogen, is an objective of relieving the degree of eutrophication. Ammonia nitrogen in wastewater can be removed by physicochemical and biological processes. In particular, biological processes have been widely used to treat the wastewater containing ammonia nitrogen because of their advantages in terms of low energy consumption and no secondary pollution. Many industrial and agricultural activities, such as petroleum refining, landfilling, and livestock farming, generate are reduced large amounts of wastewater containing high ammonia nitrogen [1]. The biological treatment of such wastewater often induces biological inhibition due to the high concentration of free ammonia (FA); as a result, ammonia nitrogen removal is inhibited [2]. This nitrogen is converted into a substrate and also acts as an inhibitor of the reaction process at the same time. Thus, an increase in the concentration of ammonia nitrogen in influent is reduced effluent quality. Extensive research has been conducted on the inhibitory effect of FA on the activities and thresholds of nitrite-oxidizing bacteria (NOB) and ammonia-oxidizing bacteria (AOB) [2-4]. Although the results reported in different studies vary, the conclusion that FA inhibits the activities of NOB and AOB is generally accepted. The concentration of ammonia nitrogen is effected the efficiency of removal and the existing form of the substrate, such as nitrite nitrogen. This form is the main product generated during the biological nitrification of ammonia nitrogen, i.e., shortcut nitrification. In the stable, long term period, the shortcut nitrification process is transformed into the entire nitrification process through the adaptation of microorganisms to the high concentration of ammonia nitrogen in wastewater [5, 6]. Aerobic granular sludge technology is regarded as a promising wastewater treatment process for ammonia nitrogen removal [1]. Granular sludge exhibits desirable characteristics, such as a good sedimentation property, high processing efficiency, high sludge concentration, and a strong resistance to shock loads [7, 8]. In addition, granular sludge is commonly used to treat all types of wastewater, especially toxic and harmful wastewater [9]. Nonetheless, granular sludge technology is subject to shortcomings such as the considerable time required for sludge granulation and the instability of granular sludge [1]. Moreover, the high FA concentration is effected hydrophilicity, a specific * To whom all correspondence should be addressed.

2 Yuan Ren et al., J.Chem.Soc.Pak., Vol. 38, No. 06, degradation ratio, and the production of extracellular polymeric substances (EPSs). These products affect the stability of granular sludge [10, 11]. According to existing reports, granular sludge has been utilized to treat wastewater containing low concentration of ammonia nitrogen or under a short operational time [12, 13]. Studies are still rarely conducted on the long-term treatment of wastewater containing much ammonia nitrogen with the use of granular sludge. High FA concentrations can cause particles to disintegrate and then destroy the granular sludge treatment system. Thus, the feasibility of the long-term treatment of wastewater containing much ammonia nitrogen using granular sludge must be discussed further along with the stability of this sludge. In the present study, the practicability of the long-run treatment of wastewater containing high concentration ammonia nitrogen via the utilization of aerobic granular sludge is determined via granular sludge cultured through nitrification [14]. The effect of ammonia nitrogen concentration on the purification effect of granular sludge is investigated, and the cause of nitrite accumulation is analyzed. The composition and content of EPSs are determined to lay a foundation for the practical application of granular sludge technology to the treatment of wastewater containing high concentration of ammonia nitrogen. Experimental Experiment Process A sequencing batch reactor (SBR) with a plexiglass column was used in the experimental setup. The reactor had a height of 30 cm, an inner diameter of 26 cm, and a working volume of 13 L. Air was circulated by an air pump and aerator, and a mass flow controller system was employed to keep the airflow constant. A time controller was used at each stage. The reactor operated at three cycles daily, and each cycle was composed of the following phases: 2 min. of feeding, 360 min. of aeration, 5 min. of settling, 5 min. of effluent withdrawal, and 108 min. of stewing. The control parameters were as follows: the initial mixed liquid suspended solid (MLSS) was 3684 mg/l, the temperature was room temperature, and the dissolved oxygen (DO) concentration was maintained at above 4 mg/l. The experiment was divided into five stages, and the ratio of ammonia nitrogen removal with aerobic granular sludge was measured by gradually increasing the concentration of ammonia nitrogen in the influent. The main experimental conditions during each operational stage of the reactor are shown in Table-1. Experimental Wastewater Synthetic wastewater was employed in this experiment. Glucose and sodium acetate (with a chemical oxygen demand (COD) mass ratio of 1:1) acted as carbon sources, and ammonium chloride served as a nitrogen source. The synthetic wastewater was composed of the following: mg/l COD, mg/l NH 4 + -N, mg/l PO 4 3 -P, mg/l CaCl 2 2H 2 O, and mg/l MgSO 4 7H 2 O. Trace elements were added to the wastewater at a total volumetric concentration of 0.5 ml/l to supply the necessary elements for microbial growth. The components were 1.50 g/l FeCl 3 6H 2 O, 0.15 g/l H 3 BO 3, 0.03 g/l CuSO 4 5H 2 O, 0.18 g/l KI, 0.12 g/l MnCl 2 4H 2 O, 0.06 g/l Na 2 MoO 4 2H 2 O, 0.12 g/l ZnSO 4 7H 2 O, 0.15 g/l CoCl 2 6H 2 O, and g/l ethylene diamine tetraacetic acid [14]. Analysis Methods COD, NH + 4 -N, NO - 2 -N, NO - 3 -N, MLSS, and the sludge volume index (SVI) were measured in accordance with standard methods [15]. DO concentration was determined with a DO meter (FG4-FK, Mettler Toledo Co., Ltd, Switzerland). ph level was determined with a phs-3c precision ph meter (PHS-3C, Shanghai Precision and Scientific Instrument Co., Ltd., China). EPSs were extracted through a heating method [16]. Polysaccharide (PS) concentration was determined with the Anthrone method [17]. Protein (PN) concentration was measured with the modified Lowry method [18]. Sludge morphology was observed through scanning electron microscopy (SEM). Results and Discussion Effect of Initial Ammonia Nitrogen Concentration on Removal Efficiency When all other conditions remain stable, an increase in the ammonia nitrogen concentration of influent may increase the concentration in the effluent. The increase in ammonia nitrogen concentration in the influent may also exert an inhibitory effect on microbes; consequently, the effect of sewage treatment is weakened further. The results of the influence of the initial ammonia nitrogen concentration on removal efficiency are depicted in Fig. 1.

3 Yuan Ren et al., J.Chem.Soc.Pak., Vol. 38, No. 06, Table-1: Main experimental conditions during different operational stages of the reactor. Stage Time(d) Influent ammonia nitrogen concentration(mg/l) Influent COD concentration(mg/l) ph I II III IV V Fig. 1: Effect of initial ammonia nitrogen concentration on removal efficiency. The initial concentration of ammonia nitrogen increases overall removal efficiency. In the first three stages, the average concentrations of ammonia nitrogen in the effluent were 1.64, 0.67, and 6.31 mg/l; the corresponding removal ratios were 98.27%, 99.59%, and 97.47%. Thus, the removal efficiency was high. When ammonia nitrogen concentration increased further to 400 mg/l, the removal ratio of ammonia decreased by up to 71.33%. Under certain conditions of ammonia nitrogen removal capacity, an increase in the ammonia nitrogen concentration in the influent gradually reduced the efficiency of removing this nitrogen. The ph level of the effluent was between 6.0 and 6.1 at this point. The insufficient alkalinity in the system affected the efficiency of ammonia nitrogen removal. When other conditions were stable, the ph level of the influent was controlled at Furthermore, the alkalinity of the influent was increased to meet the nitrification demand. As a result, the removal ratio of ammonia nitrogen was stable at approximately 90%; this percentage corresponded to a satisfactory efficiency for such a removal process. Aerobic granular sludge technology that utilizes nitrifying bacteria as the dominant flora exhibited a desirable capability for ammonia nitrogen removal and a certain degree of resistance to shock loads. Furthermore, much ammonia nitrogen in wastewater can be removed efficiently with this technology. In fact, a few scholars have studied the treatment of wastewater containing much ammonia nitrogen with the application of aerobic granular sludge technology. The efficiency of aerobic granular sludge in terms of ammonia nitrogen removal was investigated by Cydzik-Kwiatkowska et al. during an 8 h operation cycle; when the ammonia nitrogen concentration in the influent was 500 mg/l, the ratio of removal reached 99% [19]. The effect of an aerobic up-flow fluidized bed reactor on the removal of wastewater with a high concentration of ammonia nitrogen (500 mg/l) was investigated by Tsuneda et al. [20], who used a pure oxygen supply; the removal ratio of ammonia nitrogen was more than 80%. Landfill leachate was also treated with aerobic granular sludge technology by Wei et al.; when the ammonia nitrogen concentration in the influent was 366 mg/l, the removal ratio of ammonia nitrogen was 92.3% [1]. Wastewater containing high concentration ammonia nitrogen was treated by Yu et al. under a 12 h operation cycle; when the ammonia nitrogen concentration in the influent was 600 mg/l, the ammonia removal ratio was 71.3% [1]. The effects of aerobic granular sludge technology on ammonia nitrogen removal were analyzed by different researchers given the variations in aeration times, influent qualities, operating conditions, and reactor types. Effect of Initial Ammonia Nitrogen Concentration on the Characteristics of Shortcut Nitrification Ammonia nitrogen was transformed into nitrite nitrogen and nitrate nitrogen by AOB and NOB, respectively. In the five stages, the nitrite nitrogen and nitrate nitrogen concentrations in the effluent were monitored, and the results are shown in Fig. 2. In all five stages, the respective concentrations of nitrite nitrogen were 41.07, 79.67, , , and , whereas those of nitrate nitrogen were 7.71, 11.13, 20.01, 29.48, and The corresponding nitrite accumulation ratios [NO 2 - -N 100/ (NO 2 - -N+ NO 3 - -N)] were 83.58%, 88.10%, 83.03%, 84.42%, and 84.66%. A stable shortcut nitrification process was observed in these stages. Many factors affect the shortcut nitrification process, which is often performed under the following conditions: higher ph level, higher temperature, lower DO concentration, and higher ammonia nitrogen concentration [21].

4 Yuan Ren et al., J.Chem.Soc.Pak., Vol. 38, No. 06, significantly affected and the ratio of nitrite accumulation was not reduced by such activities. Thus, the results of the present study are inconsistent with those reported in literature [2]. Fig. 2: Effect of initial ammonia nitrogen concentration on nitrite nitrogen and nitrate nitrogen concentrations in the effluent and the nitrite accumulation ratio. This experiment was conducted at room temperature, DO above 4 mg/l and the ph level of the influent remained stable in the 7 8 range. During the different stages, the average FA concentrations in the influent were analyzed and calculated as follows: 2.09, 2.49, 6.88, 8.15, and mg/l. Inhibition concentrations of FA for NOB and AOB were mg/l and mg/l, respectively [2]. The growth of NOB was completely inhibited when the FA concentration reached 6 mg/l. The complete inhibition concentrations of free nitrous acid (FNA) for NOB and AOB were 0.02 and 0.40 mg/l, respectively [22-25]. In the experiment, the minimum concentration of FA in the influent was 2.09 mg/l; this value is higher than the thresholds mentioned in literature [2]. Thus, the nitrite accumulation in this experiment was attributed to the inhibition by high FA concentration as a result of high ammonia nitrogen concentration in the influent. According to literature, however, NOB adapt to high FA concentration gradually; thus, the shortcut nitrification process is difficult to achieve in the long run under high ammonia nitrogen concentration [26, 27]. In the fifth stage of the experiment, the average FA concentration in the influent was mg/l; this value is higher than the thresholds of NOB and AOB as reported in literature [2]. As per previous studies, the activities of NOB and AOB are inhibited by FA; this finding is directly reflected in the reduced ammonia removal efficiency and the effect on the shortcut nitrification process. Based on Fig. 1 and 2, however, ammonia nitrogen removal was not Sludge flocs and the pure culture of bacteria have been the main objects of research in previous studies. Efficient granular sludge characterized by shortcut nitrification after long-term cultivation was used in this research. As the dominant flora in the SBR, AOB contribute to the efficient removal of ammonia nitrogen. Shortcut nitrification is generally easier to realize under high FA concentration; furthermore, the layered structure of granular sludge is beneficial to this process [28, 29]. AOB were distributed outside this sludge, whereas NOB was distributed within. First, ammonia nitrogen was transformed by the AOB, which are the dominant flora. The NOB experienced twofold inhibition due to the high FA and FNA concentrations; moreover, the bacteria inside the granular sludge were inhibited by the low DO concentration. The diffusion limitation of DO was increased because of the sludge particles were enlarged; as a result, nitrite was accumulated [30]. As depicted in Fig. 3, the ph level in the effluent decreased gradually when the ammonia nitrogen concentration in the influent increased. The ph levels in the influent were 8.67, 8.25, 6.77, 6.25, and 7.99 in the five stages; the corresponding levels in the effluent were 7.56, 7.59, 7.42, 7.32, and During the nitrification process, acid is produced through alkali consumption; 7.14 mg of alkali must be consumed to remove 1 mg of ammonia nitrogen. The ammonia nitrogen content in the influent was transformed in the experiment; therefore, the ph level of the effluent was influenced by the quantities of transformed organics and ammonia nitrogen under the condition of stable influent alkalinity and aeration ratio. The removal ratios for the organics were close in the five stages (no shown). Thus, the quantity of transformed ammonia nitrogen influences the ph level of the effluent: the higher the quantity of transformed ammonia nitrogen, the lower the ph value of the effluent is. As illustrated in Fig. 3, the influent alkalinities in the first two stages were sufficient. The CO 2 produced in the transformation process of organics was blown out of the reactor through aeration. Given that sodium acetate caused the ph level in the effluent to increase, this level was higher than that of the influent at this point [31]. A large amount of alkali was consumed when the ammonia nitrogen concentration in the influent

5 Yuan Ren et al., J.Chem.Soc.Pak., Vol. 38, No. 06, increased and a massive amount of ammonia nitrogen was transformed. This phenomenon contributed to the decline in the ph level of the effluent. Fig. 3: Fig. 4: Changes in the ph levels of the influent and the effluent. Changes in the concentrations of PN and PS as well as PN/PS ratio over time. Effect of Initial Ammonia Nitrogen Concentration on EPSs EPSs are often detected in biological treatment systems as a type of biological secretion. These substances play an important role in maintaining the stability of granular sludge. The concentration of PN and PS which are the main components of EPSs, were analyzed in different stages, and the results are shown in Fig. 4. As presented in Fig. 4, PS concentration gradually decreased with an increase in the ammonia nitrogen concentration in the influent; both PN concentration and the PN/PS ratio tended to increase. When the average concentration of ammonia nitrogen in the influent increased from mg/l to mg/l, PS content decreased from mg/g MLSS to mg/g MLSS. PN concentration and the PN/PS ratio increased from mg/g MLSS to mg/g MLSS and from 2.29 to 4.84, respectively. The large amounts of PN and the gradual increase in the PN/PS ratio were therefore conducive to granular sludge stability; this finding is consistent with the results described in literature [11, 30]. When the FA concentration reached mg/l, the PN/PS ratio decreased from 1.69 to 7.26 and PS concentration decreased significantly. Furthermore, the production of PS in bacteria was inhibited by FA [11]. This result agrees with that obtained by Yang et al., the PS/PN ratio decreased gradually and stabilized with an increase in FA [31]. This ratio dropped by 4.5 times before stabilizing. The differences between the concentrations of PN and PS were caused by the quality of the experimental water and the varying EPS extraction methods. The concentration of organics in the wastewater reached 1000 mg/l, according to Yang et al. [31]. By contrast, the concentration of the organics in the present study was approximately 200 mg/l. The high concentration of organics indicated that heterotrophic microbes were found in granular sludge as the dominant bacterial community. The species, quantity, and distribution of the flora in the granular sludge vary, and these bacteria can secrete more PS than nitrifying bacteria can [32, 33]. In the process, the yields and compositions of EPSs are affected. When the FA concentration in the influent increased and other conditions remained unchanged, a high concentration inhibited the growth of heterotrophic and autotrophic bacteria. As the product of the metabolism synthesis of microbial cells (mainly heterotrophic bacteria), the content of PS in EPSs is significantly affected by the quantity and activity of heterotrophic bacteria. When the FA concentration in the influent increased, the activity of heterotrophic bacteria declined and the proportion of heterotrophic bacteria in the granular sludge decreased. Consequently, the PS concentration in the reaction system exhibited a significant downtrend. As the product of intracellular polymer secretion or of cytolysis [32, 33], the increase in PN content was attributed to the low concentration of influent organics. Easily biodegradable organics were rapidly degraded and consumed, thereby resulting in the endogenous metabolism of heterotrophic bacteria and cytolysis. The PS stored in the EPSs were degraded and transformed to maintain cellular metabolism, thus prompting the microbes to secrete large amounts of enzymatic PN substances to increase the PN content in EPSs and the PN/PS ratio. The Evaluation of Aerobic Granular Sludge

6 Yuan Ren et al., J.Chem.Soc.Pak., Vol. 38, No. 06, As per the observation of granular sludge during the stable operation, the mature granular sludge was tawny in color (Fig. 5a), and the outline of the granules was either spheroidic or round with a clear boundary. The weight percentages of the particles larger than 0.85 and 1.40 mm were 97% and 77%, respectively. a b Fig. 5: Images of the granular sludge on day 110. Digital image of the granules (a) and scanning electron microscopy images of granules (b) on day 110. Based on SEM observations (Fig. 5b), the granular sludge was aggregated by many tiny polymers. A large number of pores were detected between the interior and the surface of these polymers; these pores facilitated the passage of nutrients, gas transmission, and product output. Numerous cocci and a small number of bacilli were distributed on the surface of the granular sludge, and this distribution laid a foundation for the efficient removal of ammonia nitrogen in wastewater. Conclusions Aerobic granular sludge can tolerate higher biological inhibition by FA than traditional activated sludge can; moreover, the former can facilitate the effective and stable long-term treatment of wastewater containing high concentration of ammonia nitrogen. In particular, stable nitrite accumulation can be realized and a foundation laid for the application of a new, high-efficiency, and low-consumption denitrification process for the treatment of wastewater with a high concentration of ammonia nitrogen, such as anammox, as well as for shortcut nitrification denitrification. When the mean concentration of the ammonia nitrogen in the influent increases from mg/l to mg/l, the removal ratio of ammonia nitrogen and the nitrite accumulation ratio are stable more than 90% and 80%, respectively. The concentration of PS decreases and the PN/PS ratio increases gradually when the ammonia nitrogen concentration in the influent increases. The high concentration of PN and the gradual increase in PN/PS are conducive to the stability of aerobic granular sludge. The test conducted in this study investigated the feasibility of treating wastewater with a high ammonia nitrogen concentration through aerobic granular sludge with the use of synthetic wastewater. The optimization of process parameters must be researched further, and additional information must be obtained on microbiological aspects. In addition, the influence of other components in sewage, such as poisonous and harmful materials and metal elements, on the treatment effects must be discussed further. Acknowledgments Yuan Ren and Lilong Yan contributed equally to this work. This work was supported by National Natural Science Foundation of China (NO ); Academic Backbone Support Project of Northeast Agricultural University (15XG07). References 1. X. C. Yu, Z. Wan, X. Lei, Y. Liu, J. H. Tay and D. J. Lee, Use of Aerobic Granules for Treating Synthetic High-Strength Ammonium Wastewater. Environ. Technol., 35, 1785 (2014). 2. A. C. Anthonisen, R. C. Loehr, T. B. S. Prakasam and G. Srinath, Inhibition of Nitrification by Ammonia and Nitrous Acid, J.

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