【作者】 吕剑；

【导师】 何义亮；

【作者基本信息】 上海交通大学 ， 环境工程， 2008， 博士

【摘要】 烷基酚聚氧乙烯醚(Alkylphenol polyethoxylates,APEOs)是全球第二大商用非离子表面活性剂,其中壬基酚聚氧乙烯醚(Nonylphenol polyeothoxylates,NPEOs)占该类产品总量的80%。近年来的研究表明,NPEOs的中间降解产物具有弱雌激素活性。因此,开展NPEOs生物降解的研究具有重要意义。在国家自然科学基金“烷基酚聚氧乙烯醚生物降解过程中的环境雌激素效应”(批准号:50478019)资助下,本文在对比NPEOs好氧与厌氧降解优缺点的基础上进一步开展了NPEOs在硫酸盐还原与Fe(III)还原等特殊厌氧环境及反硝化缺氧环境下的生物降解行为的研究,并进一步开展了几种因素对NPEOs在缺氧或厌氧条件下生物降解过程的影响的研究,最后又以前期研究结果为依据有针对性地对NPEOs高效降解菌株进行了大量的筛选工作。研究中既综合应用高效液相色谱(HPLC)、液相色谱-质谱(LC-MS)及气相色谱-质谱(GC-MS)等多种先进的化学分析检测技术对不同降解环境中NPEOs的降解效果及其雌激素中间产物浓度变化进行了检测,同时又应用生化与分子生物技术深入揭示了高效降解菌降解NPEOs类污染物的机理。所取得的主要研究成果如下:(1) NPEOs好氧与厌氧生物降解对比实验表明,NPEOs在好氧与厌氧环境下均可被降解。好氧处理对该类污染物具有更高的降解速率。厌氧处理最大降解速率高达28.37μM·d-1,而好氧处理最大降解速率则高达35.15μM·d-1。在好氧与厌氧条件下,长链NPEOs均通过一条不依赖于氧气的非末端氧化途径实现对乙氧基的脱除。但氧气对短链NPEOs的生物降解途径影响较大。短链NPEOs在通过非末端氧化途径脱除乙氧基的同时还可通过末端氧化途径生成短链的壬基酚聚氧乙烯基羧酸(Nonylphenol polyethoxylcarboxylates, NPECs)。NPEOs的好氧处理与厌氧处理均会产生雌激素活性中间产物。好氧处理可有效减弱壬基酚(Nonylphenol, NP)及短链NPEOs的积累,但是却会造成危害性更大的羧酸化产物NPECs的积累。NPEOs污染物的去除需要综合发挥好氧与厌氧处理各自的优势。(2) NPEOs在反硝化缺氧工艺中的降解实验表明,NPEOs在反硝化缺氧环境中亦可被快速降解。最大降解速率高达34.00μM·d-1,远高于普通厌氧处理,但略低于好氧处理。添加反硝化抑制剂钨酸盐可以抑制NPEOs在反硝化环境中的生物降解。这表明该体系中NPEOs的降解可能与硝酸盐还原过程相偶联。NPEOs通过非氧化的乙氧基脱除途径实现初级降解。该过程伴随乙醛的产生。总NPEOs快速降解导致NP及短链NPEOs积累。降解14天当约有85%的NPEOs被降解时,NP及短链NPEOs的浓度达到最大值。在随后的降解时间里,NP及短链NPEOs也逐渐被降解。NPEOs在反硝化活性污泥工艺中降解时,雌激素中间产物的积累量以及体系的雌激素活性均远低于普通厌氧活性污泥处理。由于NPEOs在反硝化缺氧环境中降解时既不会产生羧酸化产物NPECs,同时又可保持较高的降解速率,因此反硝化工艺处理NPEOs类污染物显示出一定的优越性。有关NPEOs在反硝化工艺中降解特性的研究国内外尚未见相关报道。(3) NPEOs在硫酸盐及Fe(III)还原条件下的厌氧生物降解实验表明,NPEOs在这些特殊厌氧条件下均可被生物降解。Fe(III)还原条件下,总NPEOs的最大降解速率则为34.95μM·d-1。硫酸盐还原条件下,总NPEOs的最大降解速率则为34.85μM·d-1。与NPEOs在普通厌氧条件下的降解途径相似,乙氧基链通过一条非末端氧化的途径被逐步脱除。在此过程中不会有羧酸化产物NPECs的生成。NPEOs的降解同样会引起NP及短链NPEOs的积累,进而导致雌激素活性的增加。Fe(III)还原处理的最大雌激素活性出现在第14天而硫酸盐还原处理的最大雌激素活性则推迟至21天。与普通厌氧处理相比,硫酸盐或Fe(III)还原条件下NPEOs雌激素中间产物的厌氧降解也得到了强化。国内外鲜见NPEOs在该两种特殊厌氧环境下生物降解行为的报道。(4)温度对NPEOs的缺氧与厌氧生物降解具有较大的影响。温度的降低会导致NPEOs降解效率的快速下降,低温可能是导致NPEOs生物降解效率低的一个主要原因。反硝化系统中NPEOs生物降解的温度系数为0.011℃-1。普通厌氧条件下的温度系数为0.01℃-1,与反硝化处理的温度系数较为接近。硫酸盐还原处理的NPEOs生物降解的温度系数最小,仅为0.008℃-1。这表明,NPEOs在硫酸盐还原条件下降解时对温度变化的敏感性稍弱于其它两种处理。温度对NPEOs好氧生物降解的影响已有大量报道,但鲜见温度对NPEOs厌氧及缺氧降解的影响的报道。(5)共存有机物与典型中间降解产物均会对NPEOs厌氧及缺氧生物降解产生抑制作用。共存有机物的存在会严重抑制NPEOs的厌氧与缺氧生物降解。并且,这种抑制作用会因共存有机物种类的不同而产生差异。反硝化工艺中添加甲醇对NPEOs生物降解的抑制作用最强。在500 mg·L-1的高初始浓度下NPEOs仍不会对本身的生物降解产生抑制作用。在典型厌氧、硫酸盐还原与反硝化缺氧处理中,NPEOs初始浓度每增加10 mM,最大降解速率分别增加1.24μM·d-1、1.3μM·d-1和2.51μM·d-1。有机物对NPEOs的抑制作用在废水处理中广泛发生,但NPEOs初始浓度与典型中间降解产物对NPEOs厌氧及缺氧生物降解产生的抑制作用在多数情况下可以忽略。(6)通过大量的筛选工作获得Serratia sp. LJ-1与Bacillus sp. LY两株NPEOs高效降解菌。两株细菌在好氧条件下均能高效降解NPEOs,同时避免危害性更大的羧酸化产物NPECs的生成。菌株LY及LJ-1的NPEOs降解准一级动力学常数分别高达0.325 d-1与0.599 d-1。与菌株LY相比,菌株LJ-1降解NPEOs类污染物的能力更强。尽管已有大量NPEOs降解菌株的报道,但鲜见Serratia菌与Bacillus菌对NPEOs生物降解的报道。由于菌株LY为异养脱氮菌,该菌株在降解NPEOs的同时会进行异养脱氮。两株细菌可能通过羟基转移途径脱除NPEOs的乙氧基链。菌株LY及LJ-1还可高效降解NP。降解NP的准一级降解动力学常数分别为0.435 d-1和0.852 d-1。通过对两菌株降解NP时相关酶活性的分析,初步推测它们对NP的降解涉及苯环的开裂,苯环可能通过邻位裂解途径开裂。更多还原

【Abstract】 Nonylphenol polyethoxylates (NPEOs) are used in large amounts in industrial and institutional applications as nonionic surfactants, encompassing more than 80% of the world market. Studies have found that some biodegradation intermediates of NPEOs such as nonylphenol (NP) are weakly estrogenic. Therefore, the biodegradation behaviors of NPEOs have raised public concern. The biodegradation behaviors of NPEOs under aerobic and different reducing conditions and effects of temperature, organic substrate, initial concentration, and typical intermediate were investigated which was financially supported by the National Foundation of Science of China (Grant No. 50478019). High-performance liquid chromatography (HPLC) analysis, gas chromatography-mass spectrometry (GC-MS) analysis, and liquid chromatography-mass spectrometry (LC-MS) analysis were performed to monitor the biodegradation behaviors of NPEOs under aerobic and different reducing conditions. Biochemistry and molecular biology analysis methods were also used to elucidate the mechanism for NPEO biodegradation by isolated bacteria. The results were shown as follows: (1) NPEOs were readily degraded under both aerobic and anaerobic conditions. The removal efficiency of the aerobic treatment was much higher than that of the anaerobic treatment. The maximum biodegradation rates in aerobic and anaerobic bioprocesses were 35.15μM·d-1 and 28.37 M·d-1, respectively. NPEOs were biodegraded through an oxygen-independent nonoxidative pathway, through which NPEOs were degraded via sequential removal of ethoxyl units to the nonyphenol (NP), under both aerobic and anaerobic conditions. Oxygen played an important role in the biodegradation pathway of short-chain NPEOs. Nonylphenol polyethoxycarboxylates (NPECs) were formed under aerobic conditions. Typical estrogenic intermediates of NPEOs accumulated under both aerobic and anaerobic conditions. Although the accumulation of these estrogenic intermediates was alleviated under aerobic conditions, significant accumulation of short-chain NPECs, which were more harmful than their parent compounds, occurred.(2) The results showed that NPEOs were readily degraded in the denitrifying activated sludge process. NPEO maximum biodegradation rate in nitrate-reducing treatment was 34.00μM·d-1, which was much higher than that of anaerobic treatment and a little lower than that of aerobic treatment. The addition of tungstate resulted in immediate inhibition of NPEOs biodegradation, suggesting that nitrate reduction was necessary for NPEOs biodegradation. NPEOs were biodegraded through a non-oxidative pathway, through which NPEOs were degraded via sequential removal of ethoxyl units (as acetaldehyde) to NP. The accumulation of NP, nonylphenol monoethoxylate (NP1EO), and nonylphenol diethoxylate (NP2EO) coincided with the rapid removal of total NPEOs. Concentrations of NP, NP1EO, and NP2EO reached their tops on day 14 when about 85 percent of the total NPEOs were removed. The subsequent decrease in the concentrations of NP, NP1EO and NP2EO suggested that these persistent intermediates could also be biodegraded in denitrifying activated sludge process. Concentrations of the estrogenic intermediates and calculated estrogen equivalent in denitrifying treatment were much lower than those in anaerobic treatment during NPEO biodegradation. Since NPEO contaminants could be rapidly biodegraded under denitrifying conditions without forming NPECs, it seemed that denitrifying bioprocess may have advantage in NPEO removal. To our knowledge, it is the first report on the biodegradation of NPEOs in denitrifying activated sludge process.(3) The study demonstrated that NPEOs could also be rapidly biodegraded under both Fe(III)-reducing and sulfate-reducing conditions. The maximum biodegradation rate was 34.95μM·d-1 under Fe(III)-reducing conditions. The maximum biodegradation rate was 34.85μM·d-1 under sulfate-reducing conditions. NPEOs were degraded via sequential removal of ethoxyl units under both Fe(III)-reducing and sulfate-reducing conditions. No NPECs were formed in this process. NP, NP1EO, and NP2EO slightly accumulated in the anaerobic biodegradation process. The accumulation of these estrogenic metabolites led to a significant increase in the estrogenic activity during the biodegradation period. The calculated estrogenic activity reached its top on day 14 when the total concentration of these estrogenic metabolites was maximal under Fe(III)-reducing conditions. The highest estrogenic activity appeared on day 21 when the total concentration of these metabolites reached its top (18.03μM) under sulfate-reducing conditions. Concentrations of estrogenic intermediates in sulfate-reducing and Fe(III)-reducing treatments were also lower than those of normal anaerobic treatment, suggesting that the anaerobic biodegradation of these contaminants was enhanced under sulfate-reducing and Fe(III)-reducing conditions. This is the first report of the primary biodegradation behavior of NPEOs under Fe(III)-reducing and sulfate-reducing conditions.(4) Temperature had great influence on the biodegradation of NPEOs. The decrease in temperature caused a sharp decrease in the removal efficiency of NPEOs. The temperature coefficient (Ф) for the biodegradation of NPEOs in the denitrifying activated sludge process was 0.011℃-1. The decrease in temperature caused a sharp decrease in the removal efficiency of NPEOs. The temperature coefficient (Ф) for typical anaerobic biodegradation of NPEOs was 0.01℃-1. Compared to the biodegradation of NPEOs under sulfate reducing-conditions whoseФwas 0.008℃-1, the NPEO biodegradation in denitrifying or normal anaerobic activated sludge process was more sensitive to temperature.(5) Organic substance and biodegradation intermediates such as NP had inhibition effect on anaerobic and anoxic biodegradation of NPEOs. The biodegradation of NPEOs was severely inhibited in the presence of organic substance. Different organic substances had different inhibition ability on the biodegradation of NPEOs. Anaerobic and anoxic biodegradation of NPEOs was not inhibited even at very high initial concentrations of NPEOs. The maximum biodegradation rate increased 1.24μM·d-1, 1.3μM·d-1, and 2.51μM·d-1 for each ten micromoles increase in initial concentration under typical anaerobic, sulfate-reducing, and nitrate-reducing conditions, respectively. NP, the typical intermediate of NPEOs, could inhibit anaerobic and anoxic biodegradation of NPEOs only at high concentration.(6) Two strains capable of NPEO removal were isolated. NPEOs were readily degraded by strain Serratia sp. LY and Bacillus sp. LJ-1 without forming more harmful NPECs. More than 80 percent of the total NPEOs were removed within seven days. NPEO biodegradation rate constants for strain LY and strain LJ-1 were 0.325 d-1 and 0.599 d-1, respectively. To our knowledge, this is the first report of NPEO biodegradation by Serratia and Bacillus strains. Heterotrophic nitrogen removal simultaneously occurred during NPEO biodegradation by strain LY. Moreover, NPEOs could be degraded by strain LY in the presence of different nitrogen contaminants. NPEOs were biodegraded through a nonoxidative pathway, through which NPEOs were degraded via sequential removal of ethoxyl units to NP. NP could also be removed by strain LY and LJ-1. The biodegradation rate constants for NP were 0.435 d-1 and 0.852 d-1, respectively. Enzyme activity analysis showed that NP phenyl was biodegraded through the ortho cleave pathway.更多还原