摘要
多环芳烃是环境中常见的有毒难降解有机污染物,广泛分布于水体、空气以及土壤中,并可通过生物链累积,最终对人类健康造成危害。从目前国内外研究发展趋势看,多环芳烃的生物修复是一个涉及许多环节的、复杂的系统工程。对多环芳烃生物降解诸环节进行深入研究,从理论和实际上均具有重要的意义。
本论文首先成功分离出多环芳烃优势降解菌,利用所筛选的菌株考察混合多环芳烃分子间的竞争代谢能力、混合菌体间的相互作用关系、解析多环芳烃的传质及其降解动力学过程,获得以下研究结果：
(1)以焦化废水底泥为菌源,经驯化培养分离出10株降解多环芳烃的菌株,其形态特征互有差异,细胞表面疏水性差异显著,具有一定的多样性。经分子生物学和生理生化特征鉴定,其中CY4为杀鲑气单胞菌(Aeromonas salmonicida),目前从多环芳烃的降解文献中未见该株菌的报道；HY7为施氏假单胞菌(Pseudomonas stutzeri)。且CY4菌和HY7菌能利用的底物较为广泛,对芘的5d去除率达到35%以上。
(2)优势菌对混合多环芳烃降解时,分子结构越简单、溶解度越大,其竞争代谢能力越强,表明优势菌降解混合多环芳烃时,不同结构的多环芳烃分子间存在明显的竞争代谢；同时,在混合多环芳烃降解时,会使其中某个多环芳烃的代谢速率比单一降解时加快,说明多环芳烃分子间存在协同代谢。实验结果表明,按照顺序芴、菲、蒽、荧蒽、芘,竞争代谢能力逐渐减小,HY7菌对混合蒽菲芘中的蒽、芘降解率分别比单一碳源时的降解率提高了12.7%、5.5%。
(3)混合菌种降解多环芳烃时,菌种间也存在着相互影响。受试的三种菌株HY3菌、HY6菌和HY7菌均有降解多环芳烃的能力,混合时相互可产生抑制、协同等作用,其中HY7菌在降解体系中是一个强势的菌株,HY3菌和HY6菌的存在能提高HY7菌的降解作用。
(4) GC-MS结果表明,多环芳烃转化产物一般是含有多酚羟基和羰基或羧基的化合物。降解前期酚类化合物含量较多,后期羧酸性化合物含量较多。转化产物对母体环进一步降解一般表现出促进作用。外加蒽的转化产物混合物,156h蒽的累积降解率可提高3.07-6.10%,外加菲的转化产物混合物使菲60h内累积降解率可提高13.14-30.35%。
(5)多环芳烃降解过程涉及到多个环节：多环芳烃的溶解、吸附、“溶入”细胞壁、跨过细胞膜进入细胞内部、胞内酶氧化等。实验结果表明,传质过程各环节对整个降解速率的影响很大。菌体对多环芳烃的吸附速率远远大于其降解速率,造成降解时多环芳烃在菌体表面和内部的富集。多环芳烃的表观降解速率远大于其溶解速率,因此,多环芳烃降解过程中,溶解过程是降解过程中的限速步。
(6)论文考察了降解过程中,菌体对多环芳烃分子的捕获富集特征。整个降解过程中,细胞表面、细胞壁中、细胞内部对芘均有富集迹象,从芘的分布特点看,细胞表面富集的芘最多,占降解体系中芘总量的1.1～2.9%。细胞内部和细胞壁中的芘富集量相差不大,占降解体系中芘总量的0.3～0.6%。并且,芘不会在细胞表面持续累积,而是不断的传到细胞内部被降解。
(7)多环芳烃溶解过程可用方程Ct=Cs+Ce-kAt描述,通过实验得到了几种多环芳烃溶解动力学参数Cs和kA,计算Vmax=kACs得到了最大溶解速率。加入微生物表面活性剂,蒽的溶解度可增大1.23倍,最大溶解速率可增大3.15倍；菲的溶解度可增大1.47倍,最大溶解速率增大了1.62倍；芘的溶解度增大4.50倍,其最大溶解速率增大了7.51倍。
(8)菌体表面对多环芳烃的吸附动力学实验结果表明,菌体对菲、芘的吸附可以快速达到饱和。其吸附过程可以用拟二级动力学方程描述,通过实验得到了相应参数k2和qe值。
上述研究结果,对于深入理解多环芳烃的生物降解过程,实现多环芳烃污染环境的生物修复过程有一定的指导意义。
Polycyclic aromatic hydrocarbons (PAHs) are a class of ubiquitous toxic recalcitrant organic pollutants. They are widely distributed in various environmental media including atmosphere, water, sediment, soil and organism. And they have done harm to human health by biological-chains accumulation. Based on the review of related study in the worldwide, microbial degradation of PAHs is considered to be the major decomposition process for these contaminants in nature, and which is a complicated systematic project concerning many aspects. Thus, the further study on bioremediation aspects of PAHs is of theoretical and realistic significance.
In this paper, firstly, some predominant strains for biodegradation of PAHs were successful isolated. Then, the competitive metabolism of mixed PAHs and the interaction relationship between mixed cultures were investigated respectively. Subsequently, the mass transferring process and degradation kinetics of PAHs were studied. The main results were as follows:
(1) Ten strains isolated were of different morphological character and of different cell surface hydrophilicity, which showed a certain microbial diversity. Based on modern molecular identification results, CY4 were identified as Aeromonas salmonicida, which was a new strain in PAHs biodegradation reports to our best known; HY7 was identified as Pseudomonas stutzeri. The two strains could utilized extensive carbon source and had a higher pyrene degradation rate over 35% after inoculated 5days.
(2) PAHs biodegradation under the condition of mixed substrate by single strain showed that there were obviously competitive metabolism phenomenon between different kinds of PAHs, and the more simple molecular structure it was or the higher solubility it had, the stronger competitive ability it displayed. Moreover, the metabolic rate of some PAH could be enhanced compared with biodegradation under the condition of single substrate. The results showed a gradually decreased order of competitive ability according to fluorine, phenanthrene, anthracene, fluoranthene and pyrene. For HY7 strain, the anthracene biodegradation rate could be enhanced by 12.7% and the pyrene biodegradation rate could be enhanced by 5.5%.
PAHs biodegradation by mixed strains showed that there were interactional relationship among them, and it could be synergism or inhibition. Among it, HY7 strain was a most predominant bacteria, its biodegradation could be enhanced under the existing of HY3 strain and HY6 strain.
(3) Analysed intermediates from poly cyclic aromatic hydrocarbon biodegradation using GC/MS, it showed that the mixed intermediates including chemical species with. Dihydroxyl, carbonyl or carboxyl. It had higher content of phenolic compounds in the previous period and of carboxylic acid in the later period, it displayed promoting effect of intermediates on further biodegradation of PAHs. The removing rates of anthracene were increased 3.07-6.10% within 156h, and those of phenanthrene were 13.14-30.35% within 60h.
(4) There were at least five steps involved in PAHs biodegradations:the dissolution of PAHs, the sorption of PAHs, dissolved into cell wall, across cell membrane into cells, oxidized by endoenzyme. The experimental results show that the steps in mass transformation had large effects on degradation rate. The adsorption rate of PAHs by strain was much larger than the degradation rate, as a result, that PAHs could exist on the cell surface, within cell wall or inside cells for some time. The apparent degradation rate of PAHs was much larger than the dissolution rate, thus, the dissolution process was the ratelimiting step of the degradation process.
(5) In the biodegradation process, the PAHs captured by cells was investigated. Take pyrene for instance, pyrene could exist on the cell surface, within cell wall or inside cells during the biodegradation process. And it always had the most amount of pyrene on the surface of cells, which accounted forl.1～2.9%of the pyrene total amount of biodegradation system. There were little difference between the cell wall and the inside cells of pyrene content, was about 0.3～0.6% of the pyrene total amount of biodegradation system.That pyrene would't continuously accumulation on cell surface but continuously transferred into the cells and was biodegradated there.
(6) The dissolution process could be described by a first-order model Ct=Cs+Ce-kAC, and the kinetic parameters Cs and kA could be determinated by experiment data, the maximum dissolution rate could be calculated by vmax=kACs.by adding biosurfactant, the solubility and the maximum dissolution rate of anthracene could be enhanced by 1.23 tines and by 3.15 times respectively, that of phenanthrene could be enhanced by 1.47 tines and by 1.62 times respectively, and that of pyrene could be enhanced by 4.50 tines and by 7.51 times respectively.
(7) The kinetics experiments of PAHs adsorption on cell surface showed that, the adsorption process of phenanthrene or pyrene could rapidly up to the adsorption equilibrium, and the adsorption process could be described by a quasi-second order kinetic equation and the kinetic parameters k2 and qe could be determinated by experiment data.
The above experimental results, providing a further understanding on the PAHs biodegradarion process, and was meanful to realizing the bioremediation of PAHs-polluted environment.
引文
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[1]唐玉斌,毛莉,吕锡武,等.一株蒽降解菌的分离鉴定及其降解特性研究[J].环境与技术,2007,30(9)：11-13.
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