不同碳源条件下功能菌共代谢降解典型PPCPs的效能与机理
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摘要
药品及个人护理用品(pharmaceuticals and personal care products, PPCPs)是一类新兴痕量有机污染物,在人类的日常生活中被频繁使用并通过污水排放、地表径流等途径进入自然环境,于土壤、地下水和地表水中均有检出。PPCPs通过食物链富集后可能对人类健康产生潜在影响,因此为广大研究者所重视,并采取各种手段对环境中的PPCPs进行处理。已有研究表明,共代谢是难降解有机污染物的重要去除途径之一,此方法在PPCPs的处理中同样有效,然而共代谢的作用基础是以外加碳源作为生长基质给微生物提供能量来源后,微生物再进一步降解以非生长基质形式存在的有机污染物,因此外加碳源对共代谢过程十分重要。本研究的目的就在于讨论宏观和微观程度上不同的碳源对典型PPCPs共代谢过程中功能菌的影响。本研究选取碘普罗胺(iopromide, IOP)、碘美普尔(iomeprol, IOM)和苯扎贝特(bezafibrate, BZF)三种药品作为典型PPCPs,利用课题组前期分离得到的可分别高效降解IOP和BZF的功能菌Pseudomonas SP.I-24(I-24)和Pseudomonas putida B-31(B-31)进行研究,分析了不同碳源对共代谢过程中多因素的作用,研究了酶学调控机制,建立了酶活力表征方法,并以此为基础,探讨降解酶的最佳作用条件及其各项性质,同时通过蛋白质差异表达的测定明确不同碳源对降解酶诱导过程的影响,并将功能菌和降解酶应用于曝气生物滤池(biological aerated filter, BAF)中,观察PPCPs和常规污染指标的去除情况,对比静态实验效果,验证本研究的实际应用价值。研究结果如下：
(1)探明了外加碳源与目标污染物的共同降解过程以及该过程中功能菌生长和代谢活性的变化趋势
结合已有的碳源检测方法,建立了葡萄糖、麦芽糖、淀粉和甘油在本实验过程中的优化浓度测定方法。分析了不同碳源条件下,功能菌I-24对IOP和IOM以及功能菌B-31对BZF的共代谢特性,发现淀粉和葡萄糖分别为功能菌I-24和B-31的最佳碳源。以淀粉为外加碳源时,功能菌I-24对IOP和IOM的最高降解率分别为92.70%和38.43%；以葡萄糖为外加碳源时,功能菌B-31对BZF的最高降解率为76.98%,然而功能菌I-24和B-31分别在葡萄糖和淀粉条件下生长情况最佳,故表观生长情况并非影响目标污染物降解率的决定性因素,但仍可作为降解率的指示因子之一。通过对不同碳源下功能菌电子传递系统活性(electron transport system activity, ETSA)的研究,得出麦芽糖对功能菌ETSA的促进效果最强,在IOP、IOM和BZF条件下,培养第一日分别为32.12μg/(g·h)、100.92μg/(g·h)和215.54μg/(g·h),显著高于无外加碳源的样品ETSA值,推测功能菌的表面需要先被外加碳源的电子活化后才会易于与目标污染物接触,因此ETSA可作为外加碳源参与共代谢过程中释放电子效率高低的表征数值。
(2)建立了便捷而有效的酶提取及活力表征方法
建立并优化功能菌I-24和B-31所分泌的降解酶活力表征方法,得出超声破碎运行时间20min,超声功率150W时,工作3s,休息2s和工作3s,休息1s分别可从功能菌B-31和I-24中提取最高浓度的降解酶。同时明确降解酶属于胞内酶,它们的测定条件为：反应时间2h,培养温度30℃,缓冲液pH=7,IOP降解酶和IOM降解酶浓度80～100mg/L,灭活温度80℃,BZF降解酶浓度90mg/L,灭活温度100℃。
(3)探讨了环境影响因子对酶促反应过程的作用
针对从I-24和B-31两种功能菌中所提取降解酶的性质进行了研究和测定,得出IOP降解酶在pH7-8、10-40℃,IOM降解酶在pH6-8、0-60℃,BZF降解酶在pH6-7、10～40℃时具有较好的pH和温度稳定性,IOP降解酶、IOM降解酶和BZF降解酶的米氏常数Km和最大反应速度Vm分别为136.70μmol/L、91.08μmol/L、41.85μmol/L和0.05μmol/(L·min)、0.04μmol/(L·min)、0.074gmol/(L·min).虽然IOP、IOM和BZF并非功能菌正常生长的必须因子,却是与PPCPs相关的降解酶的诱导因子之一。无外加碳源的条件下,降解酶通过内源呼吸保证菌体活性,诱导过程受到强烈抑制而导致其活力较低。不同外加碳源与PPCPs共同诱导的降解酶有活力差异,其中淀粉作为外加碳源时,IOP降解酶和IOM降解酶活力最高,分别为0.182mU和0.143mU,葡萄糖作为外加碳源时,BZF降解酶活力最高,达到0.188mU,但是培养3d后三种降解酶的活力都会逐渐减小,其原因可能包括代谢产物毒性累积和酶的自身老化。无机盐培养基中淀粉和葡萄糖的最佳投加浓度为1g/L和3g/L,淀粉浓度过高将与目标污染物产生竞争性抑制,减少降解酶的诱导量。虽然在1-3g/L的葡萄糖范围内其竞争性抑制尚未发生,但不代表更高浓度的葡萄糖不会抑制酶活力。对降解酶进行双底物条件的活力测定表明反应时间2h内IOP降解酶、IOM降解酶和BZF降解酶均无法测得酶活力,说明共代谢降解酶具有非专一特性,在反应体系中存在两种底物时,将优先降解结构简单的底物。
(4)分析了功能菌I-24蛋白质受碳源影响所产生的差异表达
碳源对功能菌及降解酶共代谢过程和降解特性的表观影响已有所研究,但其对功能菌蛋白质的影响尚未明确,因此本研究进一步着眼于功能菌I-24蛋白质受碳源影响所产生的差异表达,通过对碳源条件分别为IOP、IOP+淀粉和IOP+葡萄糖这三组不同培养基条件下的功能菌蛋白质进行双向电泳测试,发现在只有IOP的样品中蛋白质等电点在4.5-6.0之间,其他两种蛋白质为4.5-8.5,三组蛋白质分子量均位于25kDa和45kDa之间。对淀粉和葡萄糖条件下的蛋白质进行差异点分析,得出28个差异点,相对于葡萄糖条件下的蛋白质,淀粉条件下的蛋白质共有23个点位上调表达,5个点位下调表达。MALDI-TOF MS成功鉴定了其中24个点位,经过分析得出淀粉通过ATP合成、蛋白质转录和运输等重要生理环节提高了降解酶的诱导水平,它对于谷氨酸盐利用效率高于葡萄糖,由此获得更为显著的ATP合成作用,与此同时,在细胞蛋白代谢过程和超氧化物代谢水平上淀粉都有所提升。
(5)检测了功能菌和降解酶在曝气生物滤池反应器中的处理效果
本研究采用实验室设计的小型BAF装置,选择两种微污染水进行小试研究,将两种功能菌及其提取的降解酶投加到两组滤池中,与一组只接种活性污泥的滤池对照,分析比较常规工况和碳源种类对三组滤池(空白,投加功能菌,投加降解酶)中常规污染指标和三种PPCPs的处理情况,分析功能菌和降解酶的实际应用效能。结果显示功能菌和降解酶对CODMn、氨氮、UV254等常规污染指标的去除无明显提升作用,但是对于IOP、IOM和BZF则体现出了较好的去除效果和抗负荷性能。降解酶由于存在酶的流失现象,因此其处理效率会产生较大的波动。相对于复杂的自然水体,模拟污水条件下,滤池对各种污染物指标的去除更加有效,水力负荷越低,PPCPs与生物膜及滤料的接触机会越多；溶解氧在一定范围内逐渐增高可促进PPCPs去除,但若持续增高,将可能破坏生物膜,影响去除效果；碳源种类同样影响滤池运行状况,且最佳碳源与静态实验一致,表明在复杂的环境中,功能菌和降解酶同样。IOP和IOM的最佳处理条件为：水力负荷0.08m3/m2-h,DO9.5mg/L,碳源淀粉,BZF最佳处理条件为：水力负荷0.08m3/m2-h,DO7.5mg/L,碳源葡萄糖,三组滤池的PPCPs平均去除率分别为IOP：97.21%、98.12%和98.68%,IOM：97.15%、96.53%和98.50%,BZF：90.04%、90.26%和93.08%。自然水体下,三组滤池的常规污染指标和PPCPs的去除率均低于模拟污水,但是向其投加碳源后,各项PPCPs去除率均得到提升,接近模拟污水的去除水平。由于曝气生物滤池反冲洗会影响装置运行效果,因此对反冲洗方式进行了选择,其最适运行方式如下：以模拟污水作为来源时,反冲洗周期为10d,运行方式为气冲,时间1min,强度9-12L/(m2·s),间歇时间1min,频率3次。20d后改为气水联合反冲,时间2min,气体强度3-4L/(m2.s),水力强度7-8L/(m2·s)。以自然水体作为来源时,反冲洗周期为5d,运行方式为气水联合反冲,时间2min,气体强度4-5L/(m2·s),水力强度7-8L/(m2.s)。
Pharmaceuticals and personal care products (PPCPs) are one kind of emerging micropollutant, which are used frequently in daily life and discharged into the environment via sewage and surface runoff. In recent years, many PPCPs were detected in soil, ground water, surface water, etc. In fact, PPCPs can be concentrated through food chains, potentially threatening human health. As a result, various methods have been used to remove PPCPs from different environmental media. The existing studies reported that co-metabolism was one of the most important removal pathways for the persistent organic pollutants and PPCPs as well. However, co-metabolism relied on the additional carbon sources for providing energy to microorganisms as growth substrate to degrade the organic pollutants. Therefore, this research aimed at both the macroscopic and microcosmic influences of different carbon sources on the typical PPCPs co-metabolic processes. Iopromide (IOP), iomeprol (IOM) and bezafibrate (BZF) were chosen as the target PPCPs and two pre-isolated functional strains named Pseudomonas SP.1-24(1-24) and Pseudomonas putida B-31(B-31), were investigated. The effects of different carbon sources on the co-metabolic processes were studied. Also, the enzymology regulatory mechanism was investigated and the enzymatic activity detection methods were established. The optimal reactive conditions and characteristics were discussed. The differential expression of proteome studies revealed inducement of various proteins by different carbon sources. In order to verify the practical applicability of the functional strains and degradation enzymes, biological aerated filters (BAF) were used to compare their performance for the removal of PPCPs and conventional pollution indicators. The results are shown as follows:
(1) The investigation of degradation processes of additional carbon sources and target pollutants, as well as the tendency of growth and metabolic activity of functional strains.
Methods for the detection of glucose, malt sugar, starch and glycerol were optimized according to the established pathways. The tests of co-metabolism process using different carbon sources indicated that starch and glucose were the most suitable carbon sources for I-24and B-31, respectively. Removal efficiencies of92.70%and38.43%for IOP and IOM by I-24, respectively, were obtained by using starch. While using glucose, B-31degraded BZF by76.98%. However, I-24grew best under glucose condition, indicating that growth condition did not determine degradation efficiency, but it could still exist as one of indicative factors for degradation efficiency. The observation of electron transport system activity (ETSA) of functional strains suggested that malt sugar promoted ETSA mostly. ETSA values of I-24in IOP and IOM, and B-31in BZF were32.12μg/(g·h),100.92μg/(g·h) and215.54μg/(g·h) in the first cultivation day, respectively. From the comparison between non-growth substrate and growth substrate, we guess that the functional strains can hardly get in touch with target pollutants until their surface are activated by carbon electron. As a result, ETSA was useful for evaluating electron releasing efficiency of additional carbon sources in co-metabolism conditon.
(2) Establishment of effective and easy detection methods for enzymatic activity
The degradation enzyme excreted by I-24and B-31were defined as IOP enzyme, IOM enzyme and BZF enzyme according to their substrate. The optimum conditions for IOP enzyme and IOM enzyme extraction were:ultrasonic power of150W, running time of20min, working time of3s and resting time of1s. The optimum conditions for BZF enzyme extraction were: ultrasonic power of150W, running time of20min, working time of3s and resting time of2s. The detection methods for enzymatic activity were established and optimized as pH7, reaction temperature of30℃, reaction time of2h and enzyme concentration of80～100mg/L for both IOP and IOM enzymes, and90mg/L for BZF enzyme. The inactivation temperature for IOP and IOM enzymes was80℃, while for BZF enzyme was100℃. Based on the activity tests, the degradation enzymes were found to be intracellular enzyme.
(3) Influence of environmental factors on enzyme reactive processes
The characteristics of IOP enzyme, IOM enzyme and BZF enzyme were studied as follows: the pH stability ranges were7～8for IOP enzyme,6～8for IOM enzyme and6～7for BZF enzyme, the temperature stability ranges were10～40℃for IOP enzyme,0～60℃for IOM enzyme and10～40℃for BZF enzyme. Michaelis constant tests indicated that Km of IOP enzyme, IOM enzyme and BZF enzyme were136.70μmol/L,91.08μmol/L and41.85μmol/L, respectively, while the Vm were0.05μmol/(L·min),0.04μmol/(L·min) and0.074μmol/(L·min), respectively. Though IOP, IOM and BZF were not the limiting factors of strain growth, they were still found to be one of inductors of the degradation enzymes. Once there was other carbon source, both of them would induce degradation enzyme in union. However, enzyme inducement was supposed to be strongly restrained in poor energy environment. Enzymatic activities induced by different additional carbon sources verified that starch accelerated the activities of IOP enzyme and IOM enzyme to0.182mU and0.143mU, while glucose accelerated BZF enzyme activity to0.188mU. Due to the accumulation of intermediate products and enzyme aging, their activities decreased. The suitable dosage of starch and glucose were determined as1g/L and3g/L. Excessive starch might result in competitive inhibition with target pollutants and reduced enzyme inducement. Though there was no circumstance suggesting glucose in the concentration between1and3g/L induced inhibition, it did not represent that no inhibition would occur by high concentration of glucose. Double-substrate enzyme reaction method was also established, which showed no IOP, IOM and BZF enzyme activity during2h reaction time, demonstrating the non-specific characteristic of co-metabolic enzymes. Once there was two substrates, the substrate with simple structure would be superior to be degraded.
(4) The differential expression of functional strain1-24influenced by verified carbon sources
In order to see the influence of verified carbon sources (IOP, IOP+starch and IOP+glucose) on strain, the differential expression of1-24was studied. By means of two-dimensional gel electrophoresis tests, the isoelectric point (pI) of protein in IOP condition was suggested to be between4.5and6.0, while the pIs of the other two proteins were both between4.5and8.5. Nevertheless, the molecular mass ranges were all between25kDa and45kDa. The analysis of protein pages between IOP+starch and IOP+glucose samples pointed out28different spots, of which23spots up regulated and5spots down regulated in IOP+starch sample according to IOP+glucose sample. MALDI-TOF MS analysis of these spots showed that ATP synthesis, protein transcription and transportation were involved in carbon influence. Starch utilized glutamate more efficiency than glucose, therefore, ATP synthesis process was much more outstanding. In the mean time, starch was superior in cell protein metabolism and superoxide metabolism processes.
(5) The application of functional strains and degradation enzyme in BAFs
The functional strains and degradation enzymes were applied to two BAF separately to compare the removal efficiencies of conventional pollution indicators and PPCPs under different operating conditions and carbon sources. The results showed that the functional strains and degradation enzymes did not exhibit any acceleration on CODMn, NH3-N and UV254removal, but it showed favorable removal efficiency and load resistance of IOP, IOM and BZF. The BAF exhibited fluctuant removal efficiencies because degradation enzymes could be washed away easily. Compared to complex natural water, simulated micro-polluted water was easier to treat. Carbon sources affected PPCPs removal in accordance with static tests. The lower the hydraulic load was, the contact opportunity the PPCPs would get with biological membrane and filter materials. However, if the DO was too high, the biological membrane might be destroyed and the removal efficiency would be restrained as well. Under the optimum treatment conditions of hydraulic load of0.08m3/m2-h, DO of9.5mg/L and carbon source of starch for IOP and IOM, in simulated micro-polluted water, the IOP and IOM removal rates of three BAF (no functional strain and degradation enzyme, functional strains, degradation enzymes) were97.21%,98.12%and98.68%(IOP),97.15%,96.53%and98.50%(IOM), respectively. Under the optimum treatment conditions of hydraulic load of0.08m3/m2-h, DO of7.5mg/L and carbon source of glucose for BZF, the removal rates in three BAF were90.04%,90.99%and93.08%for simulated micro-polluted water. The removal efficiencies of both conventional pollution indicators and PPCPs in natural water were lower than that in simulated micro-polluted water. However, once the additional carbon sources were added to the natural water, the target pollutants were biodegraded closed to the simulated water. In addition, backwashing parameters were optimized as below:as the simulated micro-polluted water was used as influent, the backwashing period was10d. The mode was air washing of1min with an intensity of9-12L/(m2-s) and the intermittent time was set at min for three cycles. If BAFs run over20days, the backwash mode had to be changed as air and water washing of2min with an air intensity of3-4L/(m2-s) and water intensity of7-8L/(mz·s). Furthermore, when natural water was used as influent, the backwashing period was5d, the mode was air combined with water washing of2min with an air intensity of4-5L/(m2·s) and water intensity of7-8L/(m2·s).
(1)探明了外加碳源与目标污染物的共同降解过程以及该过程中功能菌生长和代谢活性的变化趋势
结合已有的碳源检测方法,建立了葡萄糖、麦芽糖、淀粉和甘油在本实验过程中的优化浓度测定方法。分析了不同碳源条件下,功能菌I-24对IOP和IOM以及功能菌B-31对BZF的共代谢特性,发现淀粉和葡萄糖分别为功能菌I-24和B-31的最佳碳源。以淀粉为外加碳源时,功能菌I-24对IOP和IOM的最高降解率分别为92.70%和38.43%；以葡萄糖为外加碳源时,功能菌B-31对BZF的最高降解率为76.98%,然而功能菌I-24和B-31分别在葡萄糖和淀粉条件下生长情况最佳,故表观生长情况并非影响目标污染物降解率的决定性因素,但仍可作为降解率的指示因子之一。通过对不同碳源下功能菌电子传递系统活性(electron transport system activity, ETSA)的研究,得出麦芽糖对功能菌ETSA的促进效果最强,在IOP、IOM和BZF条件下,培养第一日分别为32.12μg/(g·h)、100.92μg/(g·h)和215.54μg/(g·h),显著高于无外加碳源的样品ETSA值,推测功能菌的表面需要先被外加碳源的电子活化后才会易于与目标污染物接触,因此ETSA可作为外加碳源参与共代谢过程中释放电子效率高低的表征数值。
(2)建立了便捷而有效的酶提取及活力表征方法
建立并优化功能菌I-24和B-31所分泌的降解酶活力表征方法,得出超声破碎运行时间20min,超声功率150W时,工作3s,休息2s和工作3s,休息1s分别可从功能菌B-31和I-24中提取最高浓度的降解酶。同时明确降解酶属于胞内酶,它们的测定条件为：反应时间2h,培养温度30℃,缓冲液pH=7,IOP降解酶和IOM降解酶浓度80～100mg/L,灭活温度80℃,BZF降解酶浓度90mg/L,灭活温度100℃。
(3)探讨了环境影响因子对酶促反应过程的作用
针对从I-24和B-31两种功能菌中所提取降解酶的性质进行了研究和测定,得出IOP降解酶在pH7-8、10-40℃,IOM降解酶在pH6-8、0-60℃,BZF降解酶在pH6-7、10～40℃时具有较好的pH和温度稳定性,IOP降解酶、IOM降解酶和BZF降解酶的米氏常数Km和最大反应速度Vm分别为136.70μmol/L、91.08μmol/L、41.85μmol/L和0.05μmol/(L·min)、0.04μmol/(L·min)、0.074gmol/(L·min).虽然IOP、IOM和BZF并非功能菌正常生长的必须因子,却是与PPCPs相关的降解酶的诱导因子之一。无外加碳源的条件下,降解酶通过内源呼吸保证菌体活性,诱导过程受到强烈抑制而导致其活力较低。不同外加碳源与PPCPs共同诱导的降解酶有活力差异,其中淀粉作为外加碳源时,IOP降解酶和IOM降解酶活力最高,分别为0.182mU和0.143mU,葡萄糖作为外加碳源时,BZF降解酶活力最高,达到0.188mU,但是培养3d后三种降解酶的活力都会逐渐减小,其原因可能包括代谢产物毒性累积和酶的自身老化。无机盐培养基中淀粉和葡萄糖的最佳投加浓度为1g/L和3g/L,淀粉浓度过高将与目标污染物产生竞争性抑制,减少降解酶的诱导量。虽然在1-3g/L的葡萄糖范围内其竞争性抑制尚未发生,但不代表更高浓度的葡萄糖不会抑制酶活力。对降解酶进行双底物条件的活力测定表明反应时间2h内IOP降解酶、IOM降解酶和BZF降解酶均无法测得酶活力,说明共代谢降解酶具有非专一特性,在反应体系中存在两种底物时,将优先降解结构简单的底物。
(4)分析了功能菌I-24蛋白质受碳源影响所产生的差异表达
碳源对功能菌及降解酶共代谢过程和降解特性的表观影响已有所研究,但其对功能菌蛋白质的影响尚未明确,因此本研究进一步着眼于功能菌I-24蛋白质受碳源影响所产生的差异表达,通过对碳源条件分别为IOP、IOP+淀粉和IOP+葡萄糖这三组不同培养基条件下的功能菌蛋白质进行双向电泳测试,发现在只有IOP的样品中蛋白质等电点在4.5-6.0之间,其他两种蛋白质为4.5-8.5,三组蛋白质分子量均位于25kDa和45kDa之间。对淀粉和葡萄糖条件下的蛋白质进行差异点分析,得出28个差异点,相对于葡萄糖条件下的蛋白质,淀粉条件下的蛋白质共有23个点位上调表达,5个点位下调表达。MALDI-TOF MS成功鉴定了其中24个点位,经过分析得出淀粉通过ATP合成、蛋白质转录和运输等重要生理环节提高了降解酶的诱导水平,它对于谷氨酸盐利用效率高于葡萄糖,由此获得更为显著的ATP合成作用,与此同时,在细胞蛋白代谢过程和超氧化物代谢水平上淀粉都有所提升。
(5)检测了功能菌和降解酶在曝气生物滤池反应器中的处理效果
本研究采用实验室设计的小型BAF装置,选择两种微污染水进行小试研究,将两种功能菌及其提取的降解酶投加到两组滤池中,与一组只接种活性污泥的滤池对照,分析比较常规工况和碳源种类对三组滤池(空白,投加功能菌,投加降解酶)中常规污染指标和三种PPCPs的处理情况,分析功能菌和降解酶的实际应用效能。结果显示功能菌和降解酶对CODMn、氨氮、UV254等常规污染指标的去除无明显提升作用,但是对于IOP、IOM和BZF则体现出了较好的去除效果和抗负荷性能。降解酶由于存在酶的流失现象,因此其处理效率会产生较大的波动。相对于复杂的自然水体,模拟污水条件下,滤池对各种污染物指标的去除更加有效,水力负荷越低,PPCPs与生物膜及滤料的接触机会越多；溶解氧在一定范围内逐渐增高可促进PPCPs去除,但若持续增高,将可能破坏生物膜,影响去除效果；碳源种类同样影响滤池运行状况,且最佳碳源与静态实验一致,表明在复杂的环境中,功能菌和降解酶同样。IOP和IOM的最佳处理条件为：水力负荷0.08m3/m2-h,DO9.5mg/L,碳源淀粉,BZF最佳处理条件为：水力负荷0.08m3/m2-h,DO7.5mg/L,碳源葡萄糖,三组滤池的PPCPs平均去除率分别为IOP：97.21%、98.12%和98.68%,IOM：97.15%、96.53%和98.50%,BZF：90.04%、90.26%和93.08%。自然水体下,三组滤池的常规污染指标和PPCPs的去除率均低于模拟污水,但是向其投加碳源后,各项PPCPs去除率均得到提升,接近模拟污水的去除水平。由于曝气生物滤池反冲洗会影响装置运行效果,因此对反冲洗方式进行了选择,其最适运行方式如下：以模拟污水作为来源时,反冲洗周期为10d,运行方式为气冲,时间1min,强度9-12L/(m2·s),间歇时间1min,频率3次。20d后改为气水联合反冲,时间2min,气体强度3-4L/(m2.s),水力强度7-8L/(m2·s)。以自然水体作为来源时,反冲洗周期为5d,运行方式为气水联合反冲,时间2min,气体强度4-5L/(m2·s),水力强度7-8L/(m2.s)。
Pharmaceuticals and personal care products (PPCPs) are one kind of emerging micropollutant, which are used frequently in daily life and discharged into the environment via sewage and surface runoff. In recent years, many PPCPs were detected in soil, ground water, surface water, etc. In fact, PPCPs can be concentrated through food chains, potentially threatening human health. As a result, various methods have been used to remove PPCPs from different environmental media. The existing studies reported that co-metabolism was one of the most important removal pathways for the persistent organic pollutants and PPCPs as well. However, co-metabolism relied on the additional carbon sources for providing energy to microorganisms as growth substrate to degrade the organic pollutants. Therefore, this research aimed at both the macroscopic and microcosmic influences of different carbon sources on the typical PPCPs co-metabolic processes. Iopromide (IOP), iomeprol (IOM) and bezafibrate (BZF) were chosen as the target PPCPs and two pre-isolated functional strains named Pseudomonas SP.1-24(1-24) and Pseudomonas putida B-31(B-31), were investigated. The effects of different carbon sources on the co-metabolic processes were studied. Also, the enzymology regulatory mechanism was investigated and the enzymatic activity detection methods were established. The optimal reactive conditions and characteristics were discussed. The differential expression of proteome studies revealed inducement of various proteins by different carbon sources. In order to verify the practical applicability of the functional strains and degradation enzymes, biological aerated filters (BAF) were used to compare their performance for the removal of PPCPs and conventional pollution indicators. The results are shown as follows:
(1) The investigation of degradation processes of additional carbon sources and target pollutants, as well as the tendency of growth and metabolic activity of functional strains.
Methods for the detection of glucose, malt sugar, starch and glycerol were optimized according to the established pathways. The tests of co-metabolism process using different carbon sources indicated that starch and glucose were the most suitable carbon sources for I-24and B-31, respectively. Removal efficiencies of92.70%and38.43%for IOP and IOM by I-24, respectively, were obtained by using starch. While using glucose, B-31degraded BZF by76.98%. However, I-24grew best under glucose condition, indicating that growth condition did not determine degradation efficiency, but it could still exist as one of indicative factors for degradation efficiency. The observation of electron transport system activity (ETSA) of functional strains suggested that malt sugar promoted ETSA mostly. ETSA values of I-24in IOP and IOM, and B-31in BZF were32.12μg/(g·h),100.92μg/(g·h) and215.54μg/(g·h) in the first cultivation day, respectively. From the comparison between non-growth substrate and growth substrate, we guess that the functional strains can hardly get in touch with target pollutants until their surface are activated by carbon electron. As a result, ETSA was useful for evaluating electron releasing efficiency of additional carbon sources in co-metabolism conditon.
(2) Establishment of effective and easy detection methods for enzymatic activity
The degradation enzyme excreted by I-24and B-31were defined as IOP enzyme, IOM enzyme and BZF enzyme according to their substrate. The optimum conditions for IOP enzyme and IOM enzyme extraction were:ultrasonic power of150W, running time of20min, working time of3s and resting time of1s. The optimum conditions for BZF enzyme extraction were: ultrasonic power of150W, running time of20min, working time of3s and resting time of2s. The detection methods for enzymatic activity were established and optimized as pH7, reaction temperature of30℃, reaction time of2h and enzyme concentration of80～100mg/L for both IOP and IOM enzymes, and90mg/L for BZF enzyme. The inactivation temperature for IOP and IOM enzymes was80℃, while for BZF enzyme was100℃. Based on the activity tests, the degradation enzymes were found to be intracellular enzyme.
(3) Influence of environmental factors on enzyme reactive processes
The characteristics of IOP enzyme, IOM enzyme and BZF enzyme were studied as follows: the pH stability ranges were7～8for IOP enzyme,6～8for IOM enzyme and6～7for BZF enzyme, the temperature stability ranges were10～40℃for IOP enzyme,0～60℃for IOM enzyme and10～40℃for BZF enzyme. Michaelis constant tests indicated that Km of IOP enzyme, IOM enzyme and BZF enzyme were136.70μmol/L,91.08μmol/L and41.85μmol/L, respectively, while the Vm were0.05μmol/(L·min),0.04μmol/(L·min) and0.074μmol/(L·min), respectively. Though IOP, IOM and BZF were not the limiting factors of strain growth, they were still found to be one of inductors of the degradation enzymes. Once there was other carbon source, both of them would induce degradation enzyme in union. However, enzyme inducement was supposed to be strongly restrained in poor energy environment. Enzymatic activities induced by different additional carbon sources verified that starch accelerated the activities of IOP enzyme and IOM enzyme to0.182mU and0.143mU, while glucose accelerated BZF enzyme activity to0.188mU. Due to the accumulation of intermediate products and enzyme aging, their activities decreased. The suitable dosage of starch and glucose were determined as1g/L and3g/L. Excessive starch might result in competitive inhibition with target pollutants and reduced enzyme inducement. Though there was no circumstance suggesting glucose in the concentration between1and3g/L induced inhibition, it did not represent that no inhibition would occur by high concentration of glucose. Double-substrate enzyme reaction method was also established, which showed no IOP, IOM and BZF enzyme activity during2h reaction time, demonstrating the non-specific characteristic of co-metabolic enzymes. Once there was two substrates, the substrate with simple structure would be superior to be degraded.
(4) The differential expression of functional strain1-24influenced by verified carbon sources
In order to see the influence of verified carbon sources (IOP, IOP+starch and IOP+glucose) on strain, the differential expression of1-24was studied. By means of two-dimensional gel electrophoresis tests, the isoelectric point (pI) of protein in IOP condition was suggested to be between4.5and6.0, while the pIs of the other two proteins were both between4.5and8.5. Nevertheless, the molecular mass ranges were all between25kDa and45kDa. The analysis of protein pages between IOP+starch and IOP+glucose samples pointed out28different spots, of which23spots up regulated and5spots down regulated in IOP+starch sample according to IOP+glucose sample. MALDI-TOF MS analysis of these spots showed that ATP synthesis, protein transcription and transportation were involved in carbon influence. Starch utilized glutamate more efficiency than glucose, therefore, ATP synthesis process was much more outstanding. In the mean time, starch was superior in cell protein metabolism and superoxide metabolism processes.
(5) The application of functional strains and degradation enzyme in BAFs
The functional strains and degradation enzymes were applied to two BAF separately to compare the removal efficiencies of conventional pollution indicators and PPCPs under different operating conditions and carbon sources. The results showed that the functional strains and degradation enzymes did not exhibit any acceleration on CODMn, NH3-N and UV254removal, but it showed favorable removal efficiency and load resistance of IOP, IOM and BZF. The BAF exhibited fluctuant removal efficiencies because degradation enzymes could be washed away easily. Compared to complex natural water, simulated micro-polluted water was easier to treat. Carbon sources affected PPCPs removal in accordance with static tests. The lower the hydraulic load was, the contact opportunity the PPCPs would get with biological membrane and filter materials. However, if the DO was too high, the biological membrane might be destroyed and the removal efficiency would be restrained as well. Under the optimum treatment conditions of hydraulic load of0.08m3/m2-h, DO of9.5mg/L and carbon source of starch for IOP and IOM, in simulated micro-polluted water, the IOP and IOM removal rates of three BAF (no functional strain and degradation enzyme, functional strains, degradation enzymes) were97.21%,98.12%and98.68%(IOP),97.15%,96.53%and98.50%(IOM), respectively. Under the optimum treatment conditions of hydraulic load of0.08m3/m2-h, DO of7.5mg/L and carbon source of glucose for BZF, the removal rates in three BAF were90.04%,90.99%and93.08%for simulated micro-polluted water. The removal efficiencies of both conventional pollution indicators and PPCPs in natural water were lower than that in simulated micro-polluted water. However, once the additional carbon sources were added to the natural water, the target pollutants were biodegraded closed to the simulated water. In addition, backwashing parameters were optimized as below:as the simulated micro-polluted water was used as influent, the backwashing period was10d. The mode was air washing of1min with an intensity of9-12L/(m2-s) and the intermittent time was set at min for three cycles. If BAFs run over20days, the backwash mode had to be changed as air and water washing of2min with an air intensity of3-4L/(m2-s) and water intensity of7-8L/(mz·s). Furthermore, when natural water was used as influent, the backwashing period was5d, the mode was air combined with water washing of2min with an air intensity of4-5L/(m2·s) and water intensity of7-8L/(m2·s).
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