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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 contents during the whole cycle under four free ammonia concentrations.

Keywords:free ammonia; nitrogen removal; extracellular polymeric substance; proteins; polysaccharides

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].

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, R10and 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 KH2PO4and 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, CuSO40.25 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

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)).

100%

(1)

(2)

(3)

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

(4)

(5)

3 Results and discussion

3.1 Performance of sequencing batch reactors under different concentrations of free ammonia

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.

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

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

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(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.

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

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

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

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.

Acknowledgements

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