蔡晨健 吳常峰 楊浩 陳桐生 孫洪偉

摘 要：以人工模擬廢水為研究對象，采用4組SBR反應(yīng)器（R0.5、R5、R10和R15），考察了4種游離氨濃度（0.5、5、10和15 mg/L）對生物脫氮效能、胞外聚合物含量及其組分（蛋白質(zhì)（PN）、多糖（PS）和核酸（DNA））影響。結(jié)果表明，4種游離氨濃度條件下都實現(xiàn)了較高的脫氮效果（97.6%～99.4%）。游離氨對3種胞外聚合物（LB-EPS、TB-EPS、總EPS）及其組分有顯著影響。當(dāng)游離氨濃度從0.5 mg/L 增加到10 mg/L時，LB-EPS、TB-EPS、總EPS的含量及其組分的含量都顯著增加;當(dāng)游離氨濃度進一步增加到15 mg/L時，LB-EPS、TB-EPS、總EPS的含量及其組分的含量都呈降低的趨勢;在4個游離氨濃度下，PN是LB-EPS的主要成分，PS是TB-EPS和總EPS的主要成分;在4個游離氨濃度下的整個反應(yīng)周期中，3種胞外聚合物及其組分的含量都跟NO-x—N有著相同的變化趨勢。

關(guān)鍵詞：游離氨;脫氮;胞外聚合物;蛋白質(zhì);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 NH+4—N in any wastewater. The concentration of free ammonia mainly depends on the values of NH+4—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 froma 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.

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.

Fig.1（b） and （c） show the concentration and variation of NO-2-N and NO-3-N during the whole reaction cycle under different concentrations of free ammonia. The oxidization of NH+4—N to NO-2-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 （NH+4—N→NO-2-N→N2）.NH+4—N convert to NO-3-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 （NH+4—N→NO-2-N→NO-3-N→NO-2-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.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.

3.3 Variation of extracellular polymeric substance and nitrogen in a typical sequencing batch reactor cycle under different concentrations of free ammoniaTo 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 NH+4—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 NO-3-N value （R=0.645 to 0.699， p<0.05） in R0.5 and R5 and the NO-2—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 NH+4—N through electrostatic interaction. Hence， more proteins were produced to provide sites for the adsorption of NH+4—N. Then the NH-4—N was further degraded by nitrifying bacteria， resulting in the increase in NO-x—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 NO-x—N and the total extracellular polymeric substance and its components[24-25].

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 NH+4—N during nitrification， but it was consistent with the variation of NO-x—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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（編輯 胡英奎）