白腐菌过氧化物酶对2，2’，4，4’-四溴联苯醚的去除研究
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摘要
多溴联苯醚(polybrominated diphenyl ethers, PBDEs)作为一类已在纺织、塑料、电器等行业被广泛使用的溴化阻燃剂,因其特殊的理化性质,易在生产、使用中释放到环境中,造成污染。PBDEs具有生物累积性及内分泌干扰活性,对生态系统构成了很大的威胁。2,2',4,4'-四溴联苯醚(BDE47)作为目前分布最广、生物样品中含量最高、对生物和人体毒性最强的同系物之一,受到越来越多的关注,而生物转化是其在环境中转化的一种重要途径。白腐菌作为一种非专一性高效降解木质素类结构化合物的广谱菌,被广泛应用于多种污染物的去除。因此,研究白腐菌及其过氧化物酶对BDE47的生物转化具有重要意义。
本研究首先优化了白腐菌分泌锰过氧化物酶的培养条件及其浓缩纯化方法；以BDE47作为研究对象,分别研究了白腐菌体系和浓缩纯化的LiP、MnP对BDE47的去除作用,并且考察了白腐菌体系下环糊精和吐温80的作用,以及浓缩酶体系下添加苯酚和NOM对BDE47去除率的影响。
研究结果主要概括如下：
(1)通过对比试验,得到了白腐菌产MnP的最佳培养条件,吐温80(0.05%,v/v),振荡培养(37℃,150r/min),在培养的第11天获得最大MnP活性2141.67U/L通过添加0.05%(v/v)吐温80并调整Mn2+的浓度至0.2mM,有效地实现了MnP与LiP的选择性产酶。
(2)考察了白腐菌体系以MnP为主导时对BDE47的去除作用,经过十五天的培养期,底物的平均去除率约为70%；在培养初期,菌丝体对BDE47有一定的吸附,但到培养后期吸附量在检测限以下；实验证实细胞色素P450在降解过程中不起作用。
(3)考察了添加吐温80和p-环糊精对BDE47去除率的影响,结果发现,添加不同浓度的吐温80或β-环糊精对去除BDE47均无明显效应,推测原因可能是BDE47特定的溶解度范围使其在较长的降解过程中能被充分利用,吐温80和p-环糊精的增溶作用对其整个降解过程作用不明显。
(4)以浓缩纯化后的LiP和MnP分别对BDE47进行酶促降解反应,分别考察了添加不同浓度的苯酚和NOM对BDE47去除率的影响,发现添加苯酚和NOM对LiP和MnP的作用趋势不同,实验浓度范围内的苯酚和NOM对LiP降解去除BDE47的作用都表现为促进趋势一直增大,不加苯酚和NOM时BDE47去除率为33.6%,加入2mM、10mM和50mM的苯酚BDE47去除率分别提高至41.3%、47.5%和56.1%,加入20mg/L和50mg/L TOC浓度的NOM BDE47的去除率则分别提高至51.3%和53.5%。苯酚和NOM对MnP降解去除BDE47的影响则表现为促进趋势先增大后减小。但是相比不加的条件下BDE47的去除率都得到了显著提高,不加苯酚和NOM时BDE47的去除率仅4.2%,加入2mM、10mM和50mM的苯酚BDE47去除率分别提高至18.1%、22.9%和8%。添加20mg/L TOC和50mg/L TOC的NOM时则分别提高至38.8%和24.6%。
Polybrominated diphenyl ethers (PBDEs) are flame retardants that have been widely integrated into textiles, plastics and electricity appliances for several decades. They are easily released into the environment from various commercial goods and caused pollution for their special physicochemical properties. Because of their bioaccumulative property and endocrine disrupting activity, the presence of PBDEs in the environment has potentially threatened the whole ecosystem and human health. 2,2',4,4'-tetraBDE(BDE47) which is currently the most widely distributed, with the highest concentrations in biological samples, and one of the most toxic polybrominated diphenyl ether congeners in living organisms and human beings attracts more and more attention. White rot fungi, which have high efficient and nonspecific degradation capacity to compounds with lignin structure, are widely applied on the removal of different kinds of recalcitrant pollutants. As biodegradation is an important pathway in the environment fate of PBDEs, it is of great significance to study the biotransformation of BDE47 with white rot fungi and their peroxidases.
In this paper, we first explored the optimized cultivating conditions for white rot fungi to selectively excret manganese peroxidase (MnP) and its purification method, and then studied the removal efficiency of BDE47 with the white rot fungi cell culture, concentrated LiP, and MnP separately. The effects ofβ-CD and Tween80 on the removal of BDE47 by white rot fungi cell culture and the effects of phenol and NOM on the removal of BDE47 with the concentrated LiP and MnP were further investigated.
The main results obtained from the studies are as follows:
(1) Through comparative tests, we got the optimal cultivating condition of white rot fungi to selectively excret MnP:Tween80(0.05%, v/v), agitated culture(37℃,150r/min), and the MnP activity reached the maximum of 2141.67U/L at the 11th day after incubation. Effective separation of MnP and LiP was achieved by adding Tween80 and increasing the amount of Mn2+.
(2) The average removal efficiency of BDE47 in the white rot fungi cell culture system was about 70% after incubation for 15 days; In early days of the incubation, some of the BDE47 were adsorbed by the mycelium, but the adsorption was under the detection limits in later period. The contribution of P450 enzyme on the degradation of BDE47 were also examed and no effect of P450 on the biodegradation of BDE47 were observed.
(3) Addition of Tween80 andβ-CD in the cell culture system were studied, and no significant effects were observed on the removal efficiency of BDE47 no matter by adding Tween80 orβ-CD, which might be mainly because of the relatively high bioavailability of BDE47 which made the solubilization effects of Tween 80 andβ-CD on the apparent solubility of BDE47 not significant.
(4) The role of purified LiP and MnP on the removal of BDE47 was also investigated. The effect of phenol and natural organic matter (NOM) on enzymatic reaction of BDE47 revealed that phenol and NOM have different effects on the LiP and MnP reaction. In the experiment concentration, phenol and NOM could increase the removal efficiency of BDE47 and removal efficiency is parallel with the concentration of phenol and NOM for LiP. The removal efficiency of BDE47 is achieved:33.6% without phenol and NOM,41.3% with 2mM phenol,47.5% with lOmM phenol,56.1% with 50mM phenol,51.3% with 20mg TOC/L and 53.5% with 50mg TOC/L. However, for MnP, the enhancement effects were increased at low concentration and decreased at high concentration both with phenol and NOM, but the removal efficiency of BDE47 was still higher than the system without phenol and NOM, it is achieved: 4.2% without phenol and NOM,18.1% with 2mM phenol,22.9% with lOmM phenol,8% with 50mM phenol,38.8% with 20mg TOC/L and 24.6% with 50mg TOC/L.
本研究首先优化了白腐菌分泌锰过氧化物酶的培养条件及其浓缩纯化方法；以BDE47作为研究对象,分别研究了白腐菌体系和浓缩纯化的LiP、MnP对BDE47的去除作用,并且考察了白腐菌体系下环糊精和吐温80的作用,以及浓缩酶体系下添加苯酚和NOM对BDE47去除率的影响。
研究结果主要概括如下：
(1)通过对比试验,得到了白腐菌产MnP的最佳培养条件,吐温80(0.05%,v/v),振荡培养(37℃,150r/min),在培养的第11天获得最大MnP活性2141.67U/L通过添加0.05%(v/v)吐温80并调整Mn2+的浓度至0.2mM,有效地实现了MnP与LiP的选择性产酶。
(2)考察了白腐菌体系以MnP为主导时对BDE47的去除作用,经过十五天的培养期,底物的平均去除率约为70%；在培养初期,菌丝体对BDE47有一定的吸附,但到培养后期吸附量在检测限以下；实验证实细胞色素P450在降解过程中不起作用。
(3)考察了添加吐温80和p-环糊精对BDE47去除率的影响,结果发现,添加不同浓度的吐温80或β-环糊精对去除BDE47均无明显效应,推测原因可能是BDE47特定的溶解度范围使其在较长的降解过程中能被充分利用,吐温80和p-环糊精的增溶作用对其整个降解过程作用不明显。
(4)以浓缩纯化后的LiP和MnP分别对BDE47进行酶促降解反应,分别考察了添加不同浓度的苯酚和NOM对BDE47去除率的影响,发现添加苯酚和NOM对LiP和MnP的作用趋势不同,实验浓度范围内的苯酚和NOM对LiP降解去除BDE47的作用都表现为促进趋势一直增大,不加苯酚和NOM时BDE47去除率为33.6%,加入2mM、10mM和50mM的苯酚BDE47去除率分别提高至41.3%、47.5%和56.1%,加入20mg/L和50mg/L TOC浓度的NOM BDE47的去除率则分别提高至51.3%和53.5%。苯酚和NOM对MnP降解去除BDE47的影响则表现为促进趋势先增大后减小。但是相比不加的条件下BDE47的去除率都得到了显著提高,不加苯酚和NOM时BDE47的去除率仅4.2%,加入2mM、10mM和50mM的苯酚BDE47去除率分别提高至18.1%、22.9%和8%。添加20mg/L TOC和50mg/L TOC的NOM时则分别提高至38.8%和24.6%。
Polybrominated diphenyl ethers (PBDEs) are flame retardants that have been widely integrated into textiles, plastics and electricity appliances for several decades. They are easily released into the environment from various commercial goods and caused pollution for their special physicochemical properties. Because of their bioaccumulative property and endocrine disrupting activity, the presence of PBDEs in the environment has potentially threatened the whole ecosystem and human health. 2,2',4,4'-tetraBDE(BDE47) which is currently the most widely distributed, with the highest concentrations in biological samples, and one of the most toxic polybrominated diphenyl ether congeners in living organisms and human beings attracts more and more attention. White rot fungi, which have high efficient and nonspecific degradation capacity to compounds with lignin structure, are widely applied on the removal of different kinds of recalcitrant pollutants. As biodegradation is an important pathway in the environment fate of PBDEs, it is of great significance to study the biotransformation of BDE47 with white rot fungi and their peroxidases.
In this paper, we first explored the optimized cultivating conditions for white rot fungi to selectively excret manganese peroxidase (MnP) and its purification method, and then studied the removal efficiency of BDE47 with the white rot fungi cell culture, concentrated LiP, and MnP separately. The effects ofβ-CD and Tween80 on the removal of BDE47 by white rot fungi cell culture and the effects of phenol and NOM on the removal of BDE47 with the concentrated LiP and MnP were further investigated.
The main results obtained from the studies are as follows:
(1) Through comparative tests, we got the optimal cultivating condition of white rot fungi to selectively excret MnP:Tween80(0.05%, v/v), agitated culture(37℃,150r/min), and the MnP activity reached the maximum of 2141.67U/L at the 11th day after incubation. Effective separation of MnP and LiP was achieved by adding Tween80 and increasing the amount of Mn2+.
(2) The average removal efficiency of BDE47 in the white rot fungi cell culture system was about 70% after incubation for 15 days; In early days of the incubation, some of the BDE47 were adsorbed by the mycelium, but the adsorption was under the detection limits in later period. The contribution of P450 enzyme on the degradation of BDE47 were also examed and no effect of P450 on the biodegradation of BDE47 were observed.
(3) Addition of Tween80 andβ-CD in the cell culture system were studied, and no significant effects were observed on the removal efficiency of BDE47 no matter by adding Tween80 orβ-CD, which might be mainly because of the relatively high bioavailability of BDE47 which made the solubilization effects of Tween 80 andβ-CD on the apparent solubility of BDE47 not significant.
(4) The role of purified LiP and MnP on the removal of BDE47 was also investigated. The effect of phenol and natural organic matter (NOM) on enzymatic reaction of BDE47 revealed that phenol and NOM have different effects on the LiP and MnP reaction. In the experiment concentration, phenol and NOM could increase the removal efficiency of BDE47 and removal efficiency is parallel with the concentration of phenol and NOM for LiP. The removal efficiency of BDE47 is achieved:33.6% without phenol and NOM,41.3% with 2mM phenol,47.5% with lOmM phenol,56.1% with 50mM phenol,51.3% with 20mg TOC/L and 53.5% with 50mg TOC/L. However, for MnP, the enhancement effects were increased at low concentration and decreased at high concentration both with phenol and NOM, but the removal efficiency of BDE47 was still higher than the system without phenol and NOM, it is achieved: 4.2% without phenol and NOM,18.1% with 2mM phenol,22.9% with lOmM phenol,8% with 50mM phenol,38.8% with 20mg TOC/L and 24.6% with 50mg TOC/L.
引文
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