土木与环境工程学报  2021, Vol. 43 Issue (1): 184-192   PDF    
Effect of free ammonia on nitrogen removal and extracellular polymeric substances in the suspended activated sludge system
CAI Chenjian 1, WU Changfeng 2, YANG Hao 1, CHEN Tongsheng 3, SUN Hongwei 1,4     
1. School of Environmental and Municipal Engineering, LanzhouJiaotong University, Lanzhou 730070, P. R. China;
2. Lanzhou City Water Supply Group Co., Ltd, Lanzhou 730060, P. R. China;
3. South China Institute of Environmental Science, MEE, Guangzhou 510655, P. R. China;
4. School of Environmental and Material Engineering, Yantai University, Yantai 264005, Shandong, P. R. China
Abstract: To investigate the influences of free ammonia on nitrogen removal, the contents of extracellular polymeric substances and the chemical composition (proteins, polysaccharides, and DNA), four laboratory-scale sequencing batch reactors fed with synthetic wastewater were operated at free ammonia concentrations of 0.5, 5, 10 and 15 mg/L (R0.5, R5, R10 and R15, respectively). Results showed that high nitrogen removal efficiencies (97.6%~99.4%) were achieved under four free ammonia concentrations. Free ammonia had a significant impact on the three kinds of extracellular polymeric substance (loosely bound extracellular polymeric substance, tightly bound extracellular polymeric substance and total extracellular polymeric substance, respectively) and their composition. Results showed that with the increased of initial free ammonia concentrations from 0.5 to 10 mg/L, the production of three kinds of extracellular polymeric substance and their components were significantly increased. Further increased of free ammonia concentrations to 15 mg/L caused a decreased trend of them. Moreover, proteins were the main component of loosely bound extracellular polymeric substance, while polysaccharides dominated in tightly bound extracellular polymeric substance and total extracellular polymeric substance under different free ammonia concentrations. It was found that the contents of three kinds of extracellular polymeric substance and their components showed a similar variation tendency to the NOx-—N contents during the whole cycle under four free ammonia concentrations.
Keywords: free ammonia    nitrogen removal    extracellular polymeric substance    proteins    polysaccharides    
游离氨对活性污泥系统中脱氮性能及胞外聚合物的影响
蔡晨健 1, 吴常峰 2, 杨浩 1, 陈桐生 3, 孙洪伟 1,4     
1. 兰州交通大学 环境与市政工程学院, 兰州 730070;
2. 兰州城市供水集团有限公司, 兰州 730060;
3. 生态环境部 华南环境科学研究所, 广州 510655;
4. 烟台大学 环境与材料工程学院, 山东 烟台 264005
摘要:以人工模拟废水为研究对象,采用4组SBR反应器(R0.5、R5、R10和R15),考察了4种游离氨浓度(0.5、5、10和15 mg/L)对生物脱氮效能、胞外聚合物含量及其组分(蛋白质(PN)、多糖(PS)和核酸(DNA))影响。结果表明,4种游离氨浓度条件下都实现了较高的脱氮效果(97.6%~99.4%)。游离氨对3种胞外聚合物(LB-EPS、TB-EPS、总EPS)及其组分有显著影响。当游离氨浓度从0.5 mg/L增加到10 mg/L时,LB-EPS、TB-EPS、总EPS的含量及其组分的含量都显著增加;当游离氨浓度进一步增加到15 mg/L时,LB-EPS、TB-EPS、总EPS的含量及其组分的含量都呈降低的趋势;在4个游离氨浓度下,PN是LB-EPS的主要成分,PS是TB-EPS和总EPS的主要成分;在4个游离氨浓度下的整个反应周期中,3种胞外聚合物及其组分的含量都跟NOx-—N有着相同的变化趋势。
关键词游离氨    脱氮    胞外聚合物    蛋白质    多糖    

1 Introduction

Extracellular polymeric substances(EPS) accumulating on the surface of activated sludge account for 80% of the activated sludge matrix, and play a significant role on wastewater treatment[1-2].

Ammonia nitrogenis the main pollutant in multiple wastewaters. Therefore, there must be a certain concentration of free ammonia(FA) in the presence of NH4+—N in any wastewater. The concentration of free ammonia mainly depends on the values of NH4+—N, temperature and pH of the wastewater[3]. In recent decades, increasing efforts have been devoted to investigating the effects and mechanisms of free ammonia on nitrification, denitrification, Anammox, phosphorus removal and anaerobic digestion processes[4-7]. But limited literature has reported the effects of free ammonia on extracellular polymeric substance and its components (including proteins, polysaccharides and DNA). Zhang et al.[8]reported that free ammonia pretreatment, with the concentration controlled at 176.5 mg/L, can facilitate the breakdown of extracellular polymeric substances and kill the living cells. But the effects of low concentrations of free ammonia on the production of extracellular polymeric substances and their components are still unknown.

In addition, with respect to the main components of extracellular polymeric substances, there are some different findings. Some studies have reported that polysaccharides(PS) were the predominant components of extracellular polymeric substances[9-10], while other studies have found that proteins(PN) were the main components[11-12]. Furthermore, it was reported that extracellular polymeric substances have some different vital functions as the main components in activated sludge differed. Higher tightly bound extracellular polymeric substance(TB-EPS) contents have been reported to noticeably enable the extracellular polymeric substance to form flocs in activated sludge[13]. Moreover, protein enrichment in the tightly bound extracellular polymeric substance has positive effects on the sedimentation and dehydration of the activated sludge[9, 14]. However, it was reported that excessive extracellular polymeric substance contents, especially polysaccharide enriched loosely bound extracellular polymeric substance(LB-EPS), can induce poor dewaterability and bioflocculation[9, 14]. The effects of the concentration of free ammonia on the predominant components of extracellular polymeric substance are still unknown. Hence, it is necessary to research the main components of extracellular polymeric substances under different concentratiosns of free ammonia.

Therefore, the objectives of this study were to elucidate the effects of free ammonia on the production of three kinds of extracellular polymeric substance (loosely bound extracellular polymeric substance, tightly bound extracellular polymeric substance and total extracellular polymeric substance) and their components and to build a correlation between nitrogen variation and the production of the three kinds of extracellular polymeric substances and their components under four different concentrations of free ammonia.

2 Material and methods
2.1 Experimental set-up and operational procedure

Four bench-scale sequencing batch reactors(SBR) made of plexiglass15 cm in diameter with 5 L working volume and 40 cm height were used in the present study. The sequencing batch reactors were operated at four different concentrations of free ammonia (0.5, 5, 10 and 15 mg/L, respectively). The long-term operations (lasted for 244 days) were carried out in four sequencing batch reactors fed with synthetic wastewater. The operational cycles of the four sequencing batch reactors with varying concentrations of free ammonia (R0.5, R5, R10 and R15) consisted of influent, aerobic reaction, anoxic reaction, settling, decanting and idle. Table 1 shows details of the operational conditions of the four sequencing batch reactors during the entire experimental cycle.

2.2 Inoculated sludge and influent contents

Inoculated activated sludge was collected from a plant mainly treating domestic and brewery wastewater (accounted for about 60%~70% and 30%~40%, respectively). The plant was located at Lanzhou, Gansu province in China, where the anaerobic-anoxic-oxic process was employed. The initial concentration of the mixed liquor suspended solids was 3 000 mg/L. The inoculated sludge was domesticated for 20 days and fed with synthetic wastewater with the following composition per liter: 115 mg of NH4Cl, 385 mg of CH3COONa, 26 mg of KH2PO4 and trace element solution. The trace element solution consisted of MgSO4·7H2O 5.07 mg/L, MnSO4·4H2O 0.31 mg/L, FeSO4·7H2O 2.49 mg/L, CuSO4 0.25 mg/L, Na2MoO4· 2H2O 1.26 mg/L, ZnSO4·7H2O 0.44 mg/L, NaCl 0.25 mg/L, CaSO4·2H2O 0.43 mg/L, CoCl2·6H2O 0.41 mg/L, EDTA 1.88 mg/L. Table 1 presents a summary of the characteristics of the influent of the four sequence batch reactors.

Table 1 Operating conditions of four sequence batch reactors

2.3 Extracellular polymeric substance extraction

With respect to extracellular polymeric substance extraction, it mainly included loosely bound extracellular polymeric substance and tightly bound extracellular polymeric substance extraction. The modified two-step thermal extraction method was used to extract loosely bound extracellular polymeric substance and tightly bound extracellular polymeric substance[15]. In the present study, the total extracellular polymeric substance content was regarded as the sum of the loosely bound extracellular polymeric substance and the tightly bound extracellular polymeric substance fractions.

2.3.1 Loosely bound extracellular polymeric substance fraction extraction

In brief: well-mixed sludge water was centrifuged at 2 100 g and 4 ℃ for 10 min to separate the supernatant from the solids, then the obtained supernatant was filtered by a 0.45 μm microporous membrane for analysis, with the collected supernatant regarded as the loosely bound extracellular polymeric substance fraction.

2.3.2 Tightly bound extracellular polymeric substance fraction extraction

Then Ringer solution was added to the residual activated sludge and the mixture was heated at 80 ℃ for 60 min in a constant temperature water bath and subsequently centrifuged again at 12 000 g and 4 ℃ for 10 min. Finally, the obtained supernatant was filtered again by a 0.45 μm microporous membrane for analysis, with the collected supernatant regarded as the tightly bound extracellular polymeric substance fraction.

2.4 Analytical methods
2.4.1 Extracellular polymeric substance quantification

The protein and polysaccharide contents of the loosely bound extracellular polymeric substance and the tightly bound extracellular polymeric substance were measured using the Lowry method and the phenol-sulphuric acid method, with bovine serum albumin and glucose, respectively, used as the standards[16-17]. DNA was determined by means of the ultraviolet absorption method[18]. The sums of the protein, polysaccharide and DNA fractions in the loosely bound extracellular polymeric substance and the tightly bound extracellular polymeric substance were regarded as the content of the loosely bound extracellular polymeric substance and the tightly bound extracellular polymeric substance, respectively.

2.4.2 Conventional analytical

COD, NH4+—N, NO2-—N, NO3-—N and mixed liquor suspended solids were analyzed in accordance with the standard methods[19]. Temperature, pH and dissolved oxygen values were monitored by using on-line probes (WTW Multi 3420, Germany).

The ammonia removal rate, the nitrite accumulation rate and the nitrate accumulation rate were calculated according to the following equation (Eq. (1), (2) and (3)).

$ \begin{array}{l} {\rm{ARE}}\left( \% \right) = \frac{{{{\left[ {{\rm{NH}}_4^ + - {\rm{N}}} \right]}_{{\rm{influent}}}} - {{\left[ {{\rm{NH}}_4^ + - {\rm{N}}} \right]}_{{\rm{effluent}}}}}}{{{{\left[ {{\rm{NH}}_4^ + - {\rm{N}}} \right]}_{{\rm{influent}}}}}} \times \\ \;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;100\% \end{array} $ (1)
$ {\rm{NiAR}}\left( \% \right) = \frac{{{\rm{NO}}_2^ - - {\rm{N}}}}{{\left[ {{\rm{NO}}_2^ - - {\rm{N}}} \right] + \left[ {{\rm{NO}}_3^ - - {\rm{N}}} \right]}} \times 100\% $ (2)
$ {\rm{NaAR}}\left( \% \right) = \frac{{{\rm{NO}}_3^ - - {\rm{N}}}}{{\left[ {{\rm{NO}}_2^ - } \right] + \left[ {{\rm{NO}}_3^ - - {\rm{N}}} \right]}} $ (3)

The nitrogen consumption and free ammonia concentration were calculated according to the following equation (Eq.(4) and (5)).

$ \begin{array}{l} \;\;\;\;\;\;\;\;\;\;{\rm{NC}}\left( {{\rm{mg}}/{\rm{L}}} \right) = {\left[ {{\rm{NH}}_4^ + - {\rm{N}}} \right]_{{\rm{influent}}}} - \\ {\left\{ {\left[ {{\rm{NH}}_4^ + - {\rm{N}}} \right] + \left[ {{\rm{NO}}_2^ - - {\rm{N}}} \right] + \left[ {{\rm{NO}}_3^ - -{\rm{N}}} \right]} \right\}_{{\rm{effluent}}}} \end{array} $ (4)
$ {\rm{FA}}\left( {{\rm{mg}}/{\rm{L}}} \right) = \frac{{17}}{{14}} \times \frac{{\left[ {{\rm{NH}}_4^ + - {\rm{N}}} \right] \times {{10}^{{\rm{pH}}}}}}{{{\rm{exp}}\left( {\frac{{6\;334}}{{273 + T}}} \right)}} + {10^{{\rm{pH}}}} $ (5)
3 Results and discussion
3.1 Performance of sequencing batch reactors under different concentrations of free ammonia

The NH4+—N concentration in the influent and effluent under four different concentrations of free ammonia are shown in Fig. 1(a). Although the influent NH4+—N concentration was controlled at 30.7~45.3 mg/L(R0.5), 76.0~99.2 mg/L(R5), 112.4~139.4 mg/L(R10) and 42.1~60.9 mg/L(R15), respectively, the effluent NH4+—N concentration was lower than 1.2 mg/L with NH4+—N removal efficiency maintained at 97.6%~ 99.4%. Hence, the present results show that the increase of free ammonia and NH4+—N concentration had no adverse effect on the nitrogen removal of the systems. Xu et al.[20]reported that free ammonia pretreatment (below 44.5 mg/L) can improve NH4+—N release, and thereby enhance NH4+—N removal.

Fig. 1 Long-term performance of SBR system under four different concentrations of free ammonia

Fig. 1(b) and (c) show the concentration and variation of NO2--N and NO3--N during the whole reaction cycle under different concentrations of free ammonia. The oxidization of NH4+—N to NO2--N was mainly achieved at R10 and R15 with the nitrite accumulation rate stable in 96.6%~99.3% (since 80 cycles), which shows that partial nitrification was achieved at R10 and R15 (NH4+—N→NO2--N→N2).NH4+—N convert to NO3--N was achieved at R0.5 and R5 with the nitrite accumulation lower than 6.3% during the whole reaction, suggesting that R0.5 and R5 were during the full nitrification process (NH4+—N→NO2--N→NO3--N→NO2--N→N2). This shows that high free ammonia concentration was beneficial to the formation of partial nitrification. Liu et al.[21]found that higher free ammonia concentration was conducive to the realization of partial nitrification as the free ammonia exerted a stronger inhibition on nitrite oxidation than on ammonium oxidation.

The correlation between free ammonia concentration and nitrogen consumption during the nitrification process was studied (data not shown). Nitrogen consumption increased from 7.0 mg/L at 0.5 mg/L free ammonia to 31.5 mg/L at 10 mg/L free ammonia, and then subsequently decreased to 14.2 mg/L at 15 mg/L free ammonia, which indicated that high nitrogen removal efficiency was available through added free ammonia concentration. Wang et al.[22] found that the nitrogen removal was significantly improved after free ammonia pretreatment in sequencing batch reactors treating synthetic wastewater.

3.2 Production of three kinds of extracellular polymeric substance and their components under different concentrations of free ammonia

Fig. 2 shows the comparison of the total contents of extracellular polymeric substance, tightly bound extracellular polymeric substance and loosely bound extracellular polymeric substance under different concentrations of free ammonia. Although the contents of total extracellular polymeric substance, tightly bound extracellular polymeric substance and loosely bound extracellular polymeric substance all showed upward trend with the free ammonia concentrations increased from 0.5 to 10 mg/L, they all showed downward trend when free ammonia concentrations continue increased to 15 mg/L, indicating that the free ammonia concentration was certainly correlated with extracellular polymeric substance production. The mechanism of this phenomenon was that microorganism would produce amount of extracellular polymeric substance to ensure microorganism can survival in toxic environmental condition. However, the contents of total extracellular polymeric substance, tightly bound extracellular polymeric substance and loosely bound extracellular polymeric substance all decreased at 15 mg/L free ammonia by comparison with the 10 mg/L of free ammonia, which showed that the auto-protection ability of the microorganism was limited, so that the higher free ammonia concentrations can cause cell inactivation due to the biocidal impact of free ammonia, which triggers the reduction of metabolites, resulting in extracellular polymeric substance reduction.

Fig. 2 Effect of the FA concentrations of the influent on the contents of LB-EPS, TB-EPS and EPS

Fig. 3 (a), (b) and (c) show the effects of different concentrations of free ammonia on proteins, polysaccharides and DNA in total extracellular polymeric substance, tightly bound extracellular polymeric substance and loosely bound extracellular polymeric substance. The protein and polysaccharide contents in the three fractions of extracellular polymeric substance all showed an upward trend as the free ammonia concentrations was increased from 0.5 to 10 mg/L. However, when the free ammonia concentrations further increased to 15 mg/L, the protein and polysaccharide contents in the three fractions extracellular polymeric substance all showed a downward trend. The DNA content gradually increased as the free ammonia increased, which shows the concentration of free ammonia can cause cell inactivation. Generally, the contents of the three kinds of extracellular polymeric substances and their components increased as the free ammonia concentration increased, triggered by the denser layers of loosely bound extracellular polymeric substance and tightly bound extracellular polymeric substance, which can stop free ammonia from diffusing into the interior of the activated sludge and reduce the free ammonia toxicity to bacteria[23].

Fig. 3 Effect of the FA concentrations of the influent on the contents of DNA, PS and PN ina)LB-EPS b)TB-EPS c)total EPS d)Effect of the FA concentrations on the percentage of DAN, PS and PN in the three different EPS fractions

Fig. 3(d) shows the percentage of the protein polysaccharide and DNA contents in the three assessed extracellular polymeric substance fractions under four different free ammonia conditions. It can be seen that polysaccharides accounted for between 47.1% and 55.8% of the tightly bound extracellular polymeric substance and total extracellular polymeric substance under four concentrations of free ammonia, followed by proteins (39.8%~46.2%) and DNA (4.2%~7.1%), indicating that polysaccharides were the main component at these four free ammonia conditions. Especially, the protein contents and DNA of the tightly bound extracellular polymeric substance and total extracellular polymeric substance was augmented, while the polysaccharide content decreased as the concentration of free ammonia increased from 0.5 to 15 mg/L.

However, a discrepancy was observed in loosely bound extracellular polymeric substance. Proteins were the dominant component of the loosely bound extracellular polymeric substance (44.3%~64.2%), while polysaccharides was the second largest component (32.8%~53.3%) and DNA was the smallest component (2.4%~3.1%). Furthermore, we found that the percentage of polysaccharides of the loosely bound extracellular polymeric substance was augmented with the increase in the concentration of free ammonia from 0.5 to 10 mg/L, while the percentage of polysaccharides decreased when the concentration of free ammonia was increased to 15 mg/L. An opposite trend was observed in the percentage of proteins of the loosely bound extracellular polymeric substances.

3.3 Variation of extracellular polymeric substance and nitrogen in a typical sequencing batch reactor cycle under different concentrations of free ammonia

To better evaluate the reactor performance, a typical sequencing batch reactors cycle (201 cycle) was examined during nitrification and dentrification under four different concentrations of free ammonia (Fig. 4). Three kinds of extracellular polymeric substance and their components showed an upward trend during the nitrification process, then a downward trend during the denitrification process under the four different concentrations of free ammonia, except for the DNA content. The trend of the total extracellular polymeric substance and its components was negatively correlated with the NH4+—N value (R=-0.787 to -0.856, p < 0.05) in the four kinds of free ammonia conditions during the nitrification process, but was positively correlated with the NO3--N value (R=0.645 to 0.699, p < 0.05) in R0.5 and R5 and the NO2-—N value (R=0.654 to 0.701, p < 0.05) in R10 and R15 during the whole reaction cycle, suggesting that certain correlations were stored intracellularly. Sheng et al.[1] believed that the negatively charged proteins are always binding with the positively charged NH4+—N through electrostatic interaction. Hence, more proteins were produced to provide sites for the adsorption of NH4+—N. Then the NH4-—N was further degraded by nitrifying bacteria, resulting in the increase in NOx-—N and the protein content during the nitrification process, while the polysaccharide content was reduced during the subsequent denitrification process. The main reason for this was that the extracellular polymeric substance served as an energy source and was utilized by denitrifying bacteria to perform denitrification, which led to the reduction of NOx-—N and the total extracellular polymeric substance and its components[24-25].

Fig. 4 Variation between nitrogen and EPS and its components during a typical cycle under four different concentrations of FA

4 Conclusions

This paper has presented a comparative study of three kinds of extracellular polymeric substances and their components, as well as the nitrogen removal performance of the influent at different concentrations of free ammonia. The following conclusions may be drawn from this study:

1) Free ammonia concentration at less than 15 mg/L had no obvious effect on the nitrogen removal performance of the sequencing batch reactors.

2) Free ammonia promoted the production of loosely bound extracellular polymeric substance, tightly bound extracellular polymeric substance and their components when its concentration increased from 0.5 to 10 mg/L. However, an opposite result was observed when its concentration was increased to 15 mg/L.

3) Polysaccharides dominated in the tightly bound extracellular polymeric substance and total extracellular polymeric substance fractions, while proteins dominated in the loosely bound extracellular polymeric substance fraction under four different concentrations of free ammonia.

4) The variation of three kinds of extracellular polymeric substance and their components was completely opposite the variation of NH4+—N during nitrification, but it was consistent with the variation of NOx-—N during the whole reaction cycle under the four different concentrations of free ammonia.

Acknowledgements: The authors would like to acknowledge the financial support from the National Natural Science Foundation of China (Grant No. 51668031).
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