Cl-、SO42-和PO43-對(duì)異養(yǎng)反硝化污泥的脅迫效應(yīng)

王 茹,鄭 平,楊 程,許少怡,戴陳琳,Abbas Ghulam (浙江大學(xué)環(huán)境工程系,浙江 杭州 310058)

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Cl-、SO42-和PO43-對(duì)異養(yǎng)反硝化污泥的脅迫效應(yīng)

王 茹,鄭 平*,楊 程,許少怡,戴陳琳,Abbas Ghulam (浙江大學(xué)環(huán)境工程系,浙江 杭州 310058)

摘要：反硝化菌是反硝化作用的驅(qū)動(dòng)者,探明Cl-、S O42-和PO43-對(duì)異養(yǎng)反硝化污泥(HDS)的脅迫效應(yīng),有助于含鹽廢水生物脫氮技術(shù)的研發(fā)和優(yōu)化.選用硝酸鹽還原酶(周質(zhì)酶)和堿性磷酸酯酶(胞內(nèi)酶)作為指標(biāo),考察了不同Cl-、SO42-和PO43-濃度對(duì)HDS酶活的影響;通過(guò)觀測(cè)HDS中的活菌水平和細(xì)胞形態(tài),考察了不同Cl-、SO42-和PO43-濃度對(duì)HDS微生物細(xì)胞結(jié)構(gòu)的影響.結(jié)果表明,Cl-、SO42-和PO43-對(duì)HDS硝酸鹽還原酶的半抑制濃度分別為0.15、0.12和0.05mol/L,對(duì)堿性磷酸酯酶的半抑制濃度為1.14、0.75和0.49mol/L;高濃度Cl-、SO42-和PO43-導(dǎo)致HDS微生物細(xì)胞膜結(jié)構(gòu)破損,通透性增加,細(xì)胞物質(zhì)外泄.陰離子對(duì)HDS的脅迫可分為滲透脅迫和電荷脅迫,滲透脅迫造成HDS中功能酶失活,電荷脅迫造成HDS中細(xì)胞膜破損,細(xì)胞物質(zhì)外泄.

關(guān)鍵詞：陰離子；異養(yǎng)反硝化污泥；滲透脅迫；電荷脅迫

* 責(zé)任作者, 教授, pzheng@zju.edu.cn

生物反硝化是廢水生物脫氮的重要組成部分,廣泛應(yīng)用于廢水脫氮除碳處理.然而在工業(yè)廢水生物脫氮中,經(jīng)常面臨高鹽的困擾[1-3].印染、腌制、制藥、化工和農(nóng)藥等行業(yè),排放大量高鹽含氮有機(jī)廢水[4-6].據(jù)報(bào)道,某印染廠對(duì)氨基偶氮苯酸鹽生產(chǎn)廢水的Cl-濃度高達(dá)100g/L,某煉鋼廠酸洗廢水的SO42-濃度高達(dá)80g/L,某制藥廠咪唑醛水解廢水的PO43-達(dá)95g/L[7-9].高濃度陰離子的存在嚴(yán)重干擾了反硝化菌的功能,限制了高效反硝化工藝的應(yīng)用[10-11].

鹽離子對(duì)微生物的脅迫可從兩個(gè)方面考慮:一是滲透脅迫,二是電荷脅迫.Jin等[12]以滲透壓表征鹽離子,研究了氣升式反應(yīng)器中滲透壓對(duì)硝化作用的影響,發(fā)現(xiàn)當(dāng)滲透壓從4.3×105Pa升為19.2×105Pa時(shí),氨去除率從100%降為69.2%; Glass等[13]以離子強(qiáng)度表征反應(yīng)器進(jìn)水總?cè)芙恹}度,研究了含氮高鹽廢水中離子強(qiáng)度對(duì)反硝化作用的影響.鹽離子對(duì)生物的脅迫效應(yīng)并非單一的滲透脅迫或電荷脅迫,而是兩者的協(xié)同作用[14-15].但迄今為止,還未見(jiàn)有關(guān)陰離子對(duì)反硝化菌滲透脅迫與電荷脅迫的文獻(xiàn)報(bào)道.

本文以Cl-、SO42-和PO43-為廢水中常見(jiàn)的陰離子代表,在保證陽(yáng)離子濃度相同的情況下探索了不同陰離子濃度滲透脅迫對(duì)異養(yǎng)反硝化污泥(HDS)酶活性的影響,觀測(cè)了不同陰離子濃度電荷脅迫下HDS中活菌水平及細(xì)胞形態(tài)的變化,以期探明陰離子對(duì)HDS的脅迫效應(yīng)及其機(jī)理.

1　材料與方法

1.1 異養(yǎng)反硝化污泥(HDS)

異養(yǎng)反硝化污泥(HDS)取自本實(shí)驗(yàn)室高效反硝化脫氮反應(yīng)器.脫氮反應(yīng)以乙酸鈉為電子供體、硝酸鹽為電子受體.HDS的總固體含量(Total solids, TS)和揮發(fā)性固體含量(volatile suspended solid, VSS)分別為136.68g/L和52.89g/L[16].試驗(yàn)所用HDS均經(jīng)0.9% NaCl溶液清洗3次.

1.2 模擬廢水

試驗(yàn)所用的無(wú)機(jī)鹽為NaCl、Na2SO4和Na3PO4.設(shè)置的Cl-梯度濃度為:0.10、0.24、0.34、0.68、1.03、1.37、1.71mol/L;SO42-梯度濃度為: 0.05、0.10、0.17、0.34、0.51、0.68、0.85mol/L;PO43-梯度濃度為:0.04、0.08、0.11、0.23、0.34、0.45、0.57mol/L.

試驗(yàn)所用的模擬廢水基礎(chǔ)組成為:NaNO37.14mmol/L, CH3COONa 7.80mmol/L, KH2PO30.37mmol/L, CaCl23.60mmol/L,MgSO4·7H2O 0.41mmol/L, 微量元素液1mL/L.

試驗(yàn)所用的微量元素液組成為:EDTA 17.12mmol/L, MnCl2·4H2O 25.25mmol/L, FeSO4· 7H2O 10.79mmol/L, CoCl2·6H2O 0.21mmol/L, NiCl2·6H2O 0.17mmol/L, H3BO30.32mmol/L, (NH4)6Mo7O2·4H2O 0.02mmol/L, CuSO4·5H2O 0.04mmol/L, ZnSO40.02mmol/L.

1.3 硝酸鹽還原酶活性

采用65mL厭氧血清瓶進(jìn)行批次試驗(yàn),測(cè)定電荷脅迫下HDS中硝酸鹽還原酶活性[17].稱取5g HDS于50mL一定濃度Cl-、SO42-或PO43-模擬廢水中,30℃、120rmp搖床培養(yǎng)5h,測(cè)定硝氮濃度.根據(jù)HDS中VSS含量,計(jì)算HDS的硝酸鹽還原酶活性(式1).每組試驗(yàn)設(shè)置兩個(gè)平行,CK對(duì)照組采用基礎(chǔ)模擬廢水,不額外添加鹽類.

其中,rs為硝酸鹽還原酶活性(g N/(L·g VSS·h),△c為硝氮濃度變化值(g N/L),m為VSS質(zhì)量(g),t為反應(yīng)時(shí)間(h).

1.4 活死細(xì)胞觀察

取批次培養(yǎng)后的HDS,經(jīng)研磨后以細(xì)胞破碎儀對(duì)其進(jìn)行超聲破碎(功率為50W,超聲時(shí)間為30s),獲得菌懸液.采用Live & Dead試劑盒(Molecular Probes, USA)中的核酸染料SYTO○R9 和PI對(duì)樣品進(jìn)行染色,在Leica熒光顯微鏡下觀察、拍照;利用Image-Pro Plus 6.0計(jì)數(shù)活菌、死菌,算出活細(xì)胞比例[18].

1.5 透射電鏡觀察

取適量批次培養(yǎng)后的HDS,浸泡于2.5%戊二醛溶液中4℃固定過(guò)夜;傾棄固定液,用0.1M,pH7.0的磷酸緩沖液漂洗3次,每次15min; 用1%鋨酸溶液固定樣品1～2h;傾棄鋨酸廢液,用0.1M、pH7.0磷酸緩沖液漂洗樣品3次,每次15min;用梯度濃度(30%、50%、70%、80%、90% 和95%)的乙醇溶液對(duì)樣品進(jìn)行脫水處理,每種濃度處理15min,再用100%乙醇處理20min,最后過(guò)渡到純丙酮處理20min;用包埋劑與丙酮的混合液(V/V=1/1)處理樣品1h;用包埋劑與丙酮的混合液(V/V=3/1)處理樣品3h;純包埋劑處理樣品過(guò)夜;LEICA EM UC7型超薄切片機(jī)中切片,經(jīng)檸檬酸鋁溶液和醋酸雙氧鈾50%乙醇飽和溶液各染色5～10min,即可用Hitachi H-7650型透射電鏡觀察.

1.6 堿性磷酸酯酶活性

取批次培養(yǎng)后的HDS適量,將其懸浮于1mL pH 8.0的PBS溶液.經(jīng)研磨后采用細(xì)胞破碎儀破碎(功率為100W,超聲時(shí)間為1min),獲得ALP提取液.以對(duì)硝基鄰苯酸二鈉(pNPP)為底物,測(cè)定單位時(shí)間內(nèi)pN的生成量[19].計(jì)算方法如式2所示.

其中:EALP為堿性磷酸酯酶活性,μmol/(gVSS?min);V為反應(yīng)體系體積,mL;△cpN為pN的摩爾濃度增量,μmol/mL;mVSS為HDS樣品中VSS質(zhì)量,g;t為反應(yīng)時(shí)間,min.

1.7 分析項(xiàng)目及方法

硝氮(NO3--N):紫外分光光度法[20];二價(jià)鐵:1,10-啉菲啰啉分光光度法;總鐵:1,10-啉菲啰啉分光光度法;pH:pHS-9V型酸度計(jì);TS和VSS:重量法.

2　結(jié)果與討論

2.1 陰離子脅迫下的硝酸鹽還原酶活性

鎖定溶液中陽(yáng)離子濃度,采用批次試驗(yàn)考察了不同Cl-、SO42-和PO43-濃度對(duì)HDS硝酸鹽還原酶活性的脅迫效應(yīng),結(jié)果如圖1所示.3種陰離子影響HDS硝酸鹽還原酶活性的趨勢(shì)一致,均經(jīng)歷快速下降區(qū)段和緩慢下降區(qū)段.在快速下降區(qū)段,陰離子濃度與HDS硝酸鹽還原酶活性下降呈線性相關(guān),而在緩慢下降區(qū)段,HDS硝酸鹽還原酶活性隨陰離子濃度的升高而趨緩下降.

圖1　不同陰離子濃度脅迫下硝酸鹽還原酶活性Fig.1　Nitrate removal rate of HDS versus concentration of anion

采用式(3)[21]對(duì)圖1數(shù)據(jù)進(jìn)行擬合.

式中: rs為硝酸鹽還原速率,g N/(g VSS·h);KI為半抑制濃度, mol/L;k為基質(zhì)反應(yīng)常數(shù);SI為抑制物濃度,mol/L.

根據(jù)擬合結(jié)果,Cl-、SO42-和PO43-對(duì)HDS硝酸鹽還原酶活性的半抑制濃度分別為0.15、0.12 和0.05mol/L,與文獻(xiàn)報(bào)道值相當(dāng)[22-23];相應(yīng)的抑制強(qiáng)度依次為PO43->SO42->Cl-,與陰離子Kosmotropic序列相符[24].

2.2 陰離子脅迫下的堿性磷酸酯酶活性

以堿性磷酸酯酶(參與蛋白磷酸化和去磷酸化調(diào)控)作為胞內(nèi)酶代表,研究了不同Cl-、SO42-和PO43-濃度對(duì)HDS堿性磷酸酯酶的脅迫效應(yīng).對(duì)照組中,HDS堿性磷酸酯酶活性為1.23μmol/(g VSS?min),以該值為參比,計(jì)算獲得各試驗(yàn)組的相對(duì)堿性磷酸酯酶活性,如圖2所示.在測(cè)試范圍內(nèi), 3種陰離子對(duì)HDS堿性磷酸酯酶活性的脅迫效應(yīng)隨離子濃度的增加呈線性增強(qiáng).

對(duì)3組數(shù)據(jù)分別進(jìn)行線性擬合,相關(guān)性良好(R2>0.9784).計(jì)算獲得Cl-、SO42-和PO43-對(duì)HDS堿

性磷酸酯酶活性的半抑制濃度依次為1.14、0.75和0.49mol/L,相應(yīng)的抑制強(qiáng)度依次為PO43-> SO42-> Cl-.

圖2　不同陰離子濃度脅迫下堿性磷酸酯酶活性Fig.2　Relative ALP activity of HDS versus concentration of anion

2.3 陰離子脅迫下的活菌水平

采用Live/Dead試劑盒對(duì)HDS中微生物細(xì)胞進(jìn)行染色,在熒光顯微鏡下觀察了Cl-、SO42-和PO43-對(duì)HDS中活菌水平的影響,熒光顯微照片如圖3所示.對(duì)照組中,顯微視野下大多微生物為活菌,少量死菌;而暴露于高陰離子濃度(1.71mol/L Cl-、0.85mol/L SO42-或0.57mol/L PO43-)中5h后,顯微視野下大多微生物為死菌,少量活菌.結(jié)果表明,高濃度陰離子脅迫下HDS中活菌水平顯著下降.

圖3　熒光顯微鏡下HDS活菌/死菌照片F(xiàn)ig.3　Fluorescence microscopic images of microorganisms in HDSA:對(duì)照組;B: 1.71mol/L Cl-; C: 0.85mol/L S O42-; D: 0.57mol/L PO43-.紅色為活細(xì)胞,藍(lán)色為死細(xì)胞.標(biāo)尺為200μm

表1　不同Cl-、SO42-和PO43-濃度對(duì)HDS中活菌水平的影響Table 1　Live/Dead cell versus different salt concentrations

采用Image-Pro Plus 6.0對(duì)熒光顯微照片中活菌和死菌進(jìn)行計(jì)數(shù),對(duì)照組HDS活菌個(gè)數(shù)比例為99.60%,試驗(yàn)組結(jié)果如表1所示.

2.4 陰離子脅迫下的細(xì)胞形態(tài)

采用透射電鏡觀察Cl-、SO42-和PO43-脅迫下HDS中微生物細(xì)胞形態(tài)結(jié)構(gòu)的變化,結(jié)果如圖4所示.在對(duì)照組中,細(xì)胞呈規(guī)則球形,擬核處于細(xì)胞中部,細(xì)胞物質(zhì)分散均勻,細(xì)胞壁和細(xì)胞膜結(jié)構(gòu)完整(圖4A);暴露于高陰離子濃度(1.71mol/L Cl-、0.85mol/L SO42-或0.57mol/L PO43-)中5h后,細(xì)胞形態(tài)改變,細(xì)胞脫水致使原生質(zhì)體聚縮,質(zhì)壁分離,甚至細(xì)胞破裂,細(xì)胞物質(zhì)(包括擬核)外泄(圖4B、C、D). 2.5

圖4　透射電鏡下HDS細(xì)胞形態(tài)Fig.4　Transmission electron microscopy (TEM) of microorganism in HDSA:對(duì)照組B: 1.71mol/L Cl-; C: 0.85mol/L SO42-; D: 0.57mol/L PO43-紅色箭頭為細(xì)胞壁,藍(lán)色箭頭為細(xì)胞膜,白色箭頭為核酸

陰離子對(duì)HDS的脅迫機(jī)理

陰離子對(duì)微生物細(xì)胞的脅迫效應(yīng)主要包括滲透脅迫作用和電荷脅迫作用.

在陰離子滲透脅迫下,HDS細(xì)胞周質(zhì)酶和胞內(nèi)酶均失活.硝酸鹽還原酶結(jié)合在細(xì)胞膜上,但活性中心朝向細(xì)胞周質(zhì)空間,可作為周質(zhì)酶的代表;堿性磷酸酯酶位于細(xì)胞內(nèi),可作為胞內(nèi)酶的代表.以硝酸鹽還原酶和堿性磷酸酯酶為指標(biāo),考察了不同Cl-、SO42-和PO43-濃度對(duì)HDS酶活的脅迫效應(yīng),如3.1和3.2所示.結(jié)果表明,陰離子脅迫下HDS中的硝酸鹽還原酶和堿性磷酸酯酶活性顯著下降.據(jù)此認(rèn)為,陰離子不僅直接影響膜外酶活性,也可進(jìn)入細(xì)胞而間接影響胞內(nèi)酶活性.溶液中帶電陰離子可結(jié)合自由水而降低水活度,并通過(guò)滲透作用而減少細(xì)胞內(nèi)自由水,致使胞內(nèi)酶及核酸分子由溶膠態(tài)轉(zhuǎn)變?yōu)槟z態(tài),喪失活性[25-26].

在陰離子脅迫下,HDS細(xì)胞結(jié)構(gòu)發(fā)生變化.Live/Dead試劑盒鑒定活/死細(xì)胞的原理基于細(xì)胞膜通透性的改變,活細(xì)胞具有完整細(xì)胞膜,物質(zhì)通透性穩(wěn)定;死細(xì)胞細(xì)胞膜受到損傷,物質(zhì)通透性顯著增大.如圖3和圖4所示,在高陰離子濃度(1.71mol/L Cl-、0.85mol/L SO42-或0.57mol/L PO43-)脅迫下,HDS中活細(xì)胞比例下降,細(xì)胞結(jié)構(gòu)嚴(yán)重破損.據(jù)此認(rèn)為,陰離子不僅可致細(xì)胞膜通透性增強(qiáng),甚至導(dǎo)致細(xì)胞死亡.溶液中帶電陰離子可產(chǎn)生靜電力,破壞原有的細(xì)胞電荷平衡,造成細(xì)胞膜破損,甚至破裂[27-28].

圖5　陰離子對(duì)HDS脅迫機(jī)理Fig.5　Mechanism of the stress by anion on HDS

綜合滲透脅迫和電荷脅迫,提出陰離子對(duì)HDS的脅迫機(jī)理如圖5所示.正常條件下,HDS細(xì)胞結(jié)構(gòu)完整,活性較高(圖5A);置于陰離子脅迫環(huán)境中,陰離子可進(jìn)入細(xì)胞周質(zhì)空間抑制硝酸鹽還原酶活性,進(jìn)入細(xì)胞抑制堿性磷酸酯酶活性(圖5B);高濃度陰離子還可通過(guò)滲透壓導(dǎo)致細(xì)胞原生質(zhì)體失水,質(zhì)膜皺縮(圖5C);高濃度陰離子積累負(fù)電荷,則可產(chǎn)生強(qiáng)靜電力,致使細(xì)胞膜破損(圖5D),細(xì)胞內(nèi)含物(包括核酸和胞內(nèi)酶)外泄(圖5E),造成細(xì)胞死亡.

3　結(jié)論

Cl-、SO42-和PO43-對(duì)HDS的脅迫效應(yīng)可分為滲透脅迫作用和電荷脅迫作用.滲透脅迫作用可抑制HDS硝酸鹽還原酶和堿性磷酸酯酶活性,電荷脅迫作用可引起HDS中微生物細(xì)胞膜通透性增加,甚至導(dǎo)致細(xì)胞膜破裂,細(xì)胞質(zhì)外泄.

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Stress of Cl-, SO42-and PO43-on the heterotrophic denitrifying sludge.

WANG Ru, ZHANG Ping*, YANG Cheng, XU Shao-yi, DAI Chen-lin, Abbas Ghulam (Department of Environmental Engineering, Zhejiang University, Hangzhou 310058, China). China Environmental Science, 2016,36(4)：1039～1044

Abstract：As the functional part of denitrification, denitrifier was obviously stressed by anions. To investigate the effects and the mechanism of anions on heterotrophic denitrifying sludge (HDS) are of great significance for the practical application of high-rate denitrification in saline nitrogen-rich wastewater treatments. Nitrate reductase and alkaline phosphatase were chosen as the indices to determine the effects of Cl-, SO42-and PO43-on the enzyme activities in HDS, while the ratios of live/dead cells and cellular morphology were examined and observed to characterise the effects of Cl-, SO42-and PO43-on the cell structures in HDS. Results showed that, the IC50values of Cl-, SO42-and PO43-for nitrate reductase were 0.15, 0.12 and 0.05mol/L respectively, while the IC50values of Cl-, SO42-and PO43-for alkaline phosphatase were 1.14, 0.75 and 0.49mol/L, respectively. Anions with high concentrations (1.71mol/L Cl-, 0.85mol/L SO42-, 0.57mol/L PO43-) resulted in the damage of cell membranes, and caused the leakage of cell inclusion. In conclusion, the effects of anions on HDS were attributed to the osmotic stresses which affected the enzyme activities, and the ion stresses which caused the damage of cell membranes.

Key words：anion；heterotrophic denitrifying sludge；osmotic stresses；ion stresses

作者簡(jiǎn)介：王 茹(1989-),女,山西長(zhǎng)治人,浙江大學(xué)博士生在讀,主要從事廢水生物處理技術(shù)研究.

基金項(xiàng)目：國(guó)家自然科學(xué)基金(51278457);國(guó)家科技支撐項(xiàng)目(2013BAD21B04);浙江省創(chuàng)新團(tuán)隊(duì)(2013TD12).

收稿日期：2015-09-23

中圖分類號(hào)：X703

文獻(xiàn)標(biāo)識(shí)碼：A

文章編號(hào)：1000-6923(2016)04-1039-06