摘要
以福建龙海浮宫九龙江口红树林为研究区域,设置四个断面16个采样站位,于冬季和夏季采集红树林表层沉积物样品。对16个站位的理化因子、胞外酶活性、微生物数量分布特征以及PAHs污染胁迫下红树林沉积物微生物群落响应等方面进行了研究。同时,从红树林沉积物中分离得到了多株PAHs降解菌,采用PCR-DGGE技术分析了PAHs降解单菌的不同组合对单一和混合PAHs的降解规律。主要的研究结果如下:
1、分别测定了冬季和夏季16个站位红树林表层沉积物的pH值、盐度、温度、有机碳(TOC)、有机质(TOM)、油类含量等理化参数,同时,应用荧光模拟底物的方法和GC/MS的方法对红树林沉积物中的胞外酶活性和PAHs含量也进行了测定,并分析了这些环境参数的相关关系。结果表明,冬季与夏季各环境参数之间有显著差异;各站位α-葡萄糖苷酶活性(α-GlcA)的变化范围分别为10.63μmol/gh-100.86μmol/gh和43.80μmol/gh-197.78μmol/gh;β-葡萄糖苷酶活性(β-GlcA)的变化范围分别为39.60μmol/gh-222.75μmol/gh和169.88μmol/gh-676.93μmol/gh,各站位α-GlcA与β-GlcA呈显著相关性(R~2=0.8215和R~2=0.7582),且β-GlcA显著高于α-GlcA (p<0.05):α-GlcA和β-GlcA与各站位TOC、TOM呈显著相关性,与其它理、化参数无相关关系:不同站位的PAHs含量范围为279.98 ng/g-1074.50 ng/g,各站位均表现为高分子量的PAHs组分占优势,该区域PAHs主要来源于矿物如柴油、汽油和煤的不完全燃烧。
2、对两个季节红树林沉积物各站位异养细菌、菲(Phe)、芘(Pyr)、荧葸(Flu)、苯并芘(Bap)和混合PAHs(M-PAHs)降解菌进行了计数,分析了它们与各环境参数的相关关系。结果表明,夏季的异养细菌、Phe降解菌、Pyr降解菌、Flu降解菌、Bap降解菌及M-PAHs降解菌数量明显高于冬季;不同季节异养细菌数与各环境因子有不同程度的相关性,同一季节异养细菌数与α-GlcA和β-GlcA均呈显著正相关;各站位不同PAHs降解菌的数量与相应PAHs的含量无显著相关性。
应用PCR-DGGE技术对红树林沉积物各站位的微生物群落结构进行了分析。结果表明,每个站位都存在着在丰富的微生物多样性,夏季各站位的细菌种类数多于冬季,红树林区细菌多样性高于非红树林区细菌多样性;各站位细菌多样性指数、丰度和均匀度均有不同,不同站位的相似性系数变化较大;聚类分析表明同一断面相邻站位相似性系数较高分类位置较近。
应用16S rDNA文库技术对红树林沉积物各站位的微生物多样性也进行了分析。结果表明,绝大多数序列对应的细菌均为未培养微生物,这些微生物中变形菌门(Proteobacteria)占优势(70.0%),其次还有拟杆菌门(Bacteroidetes)占8.0%,浮霉菌门(Planctomycetacia)、放线菌门(Actinobacteria)和疣微菌门(Verrucomicrobia)分别占2.0%,除此之外,还检测到未可培养,性质以及分类尚不清楚的细菌占16.0%。无论是PCR-DGGE还是16S rDNA文库的方法,结果均表明红树林沉积物存在着丰富的微生物多样性。
3、通过PCR-DGGE技术研究红树林沉积物微生物在PAHs污染胁迫下的群落结构变化。结果表明,当加入不同浓度的Phe、Pyr、Bap、M-PAHs后,在不同的时间,微生物群落结构发生了不同的变化。不同浓度的PAHs对红树林沉积物中的微生物群落结构的影响不同,较高浓度的PAHs对微生物群落结构的影响也较大,同时在高浓度的PAHs胁迫下,微生物群落结构发生改变的响应时间也较短。发生明显变化的DGGE条带序列所代表的微生物大部分为未培养微生物。
4、从红树林冬季沉积物样品中分离得到14株PAHs降解菌,它们都属于变形菌门(Protcobactcria),其中7个属于α-proteobacteria,占50%,7个属于γ-proteobacteria,占50%。对夏季样品以选用不同培养基为筛选策略,经细菌形态分类和RFLP分型后,分离得到67株PAHs降解菌。两个季节共得到不同的PAHs降解菌56株,变形菌门(Proteobacteria)38株,占67.8%,包括α-proteobacteria为14株,β-proteobacteria为1株,γ-proteobacteria为23株;拟杆菌门(Bacteroidetes)5株,占8.9%,包括鞘脂杆菌纲(Sphingobactcria)2株,黄杆菌纲(Flavobacteria)3株;放线菌门(Actinobacteria)5株,占8.9%;厚壁菌门(Firmicutes)的芽孢杆菌纲(Bacilli)8株,占14.3%。
5、利用平板升华法观察降解菌对PAHs利用能力,结果发现Phe降解菌F2在MM2平板和2216E平板均形成明显的透明圈,同时发现不同降解菌利用PAHs做为碳源进行生长的情况不同。另外,以不同PAHs降解菌驯化过程中DGGE条带的变化信息为依据,将优势条带对应的降解单菌进行组合,研究混合菌系对不同PAHs的降解效果,结果表明,Phe降解菌系和M-PAHs降解菌系对Phe均有较高的降解率,这些降解菌系对三环PAHs的降解率要高于四环和五环PAHs的降解率。
In this research, mangrove swamp has been selected as a research region in Fugong, Longhai, Fujian Province of China to study the potential role in bioremediation of polluted environment. The four transects including sixteen stations were established, and the surficial sediments were collected in Winter and Summer, respectively. Physico-chemical and microbiological parameters were determined in 16 stations from mangrove swamps. The activities of extracellular enzymes were analyzed, and the changes of microbial community constructure were also evaluated when added different PAHs to the samples from mangrove sediments. In addition, according to PCR-DGGE profiles, the research of degradation characteristics of individual and mixed PAHs have been performed by different mixture of PAH-degrading bacteria isolated from mangrove swamp.
The results obtained are summarized as follows:
1. The physico-chemical parameters were determined in mangrove surficialsediments from 16 stations, such as pH, salinity, temperature, TOC, TOM, and contents of oil. The glucosidase activities and PAHs contents were also determined in different stations by the method of fluorogenic model substrate (FMS) and GC/MS, respectively. The activities of a-glucosidase (α-GlcA) ranged from 10.63μmol/gh to 100.86μmol/gh and from 43.80μmol/gh to 197.78μmol/gh in Winter and Summer, respectively, and the activities ofβ-glucosidase (β-GlcA) varied from 39.60μmol/gh to 222.75μmol/gh in Winter and from 169.88μmol/gh to 676.93μmol/gh in Summer. There was significant positive relationship betweenα-GlcA andβ-GlcA (R~2=0.8215 and R~2=0.7582), andβ-GlcA was higher thanα-GlcA in different seasons. The significant positive relationship was found between glucosidase activities and TOC, also TOM. Except, there was no significant relationship between glucosidase activities and other environmental parameters. The contents of PAHs ranged from 279.98 ng/g to 1074.50 ng/g,and HMW PAHs were dominant in all of the stations. Based on our data, it suggested the source of PAHs maybe came from fuel combustion.
2. The numbers of several different bacterial groups, including culturable heterotrophic bacteria, Phe-degrading bacteria, Pyr-degrading bacteria, Flu-degrading bacteria, Bap-degrading bacteria and mixture of PAH-degrading bacteria were counted. Correlated relationship was analyzed between bacterial numbers and environmental parameters. The results showed the numbers of these bacteria were higher in Summer than those in Winter. As to the numbers of culturable heterotrophic bacteria, there was different relationship between them and the environmental parameters in different seasons, and significant positive relationship between them and glucosidase activities. While, there were no differences in correlations between the numbers of PAH-degrading bacteria and contents of PAHs.
The results of analysis of microbial community structure by the method of PCR-DGGE indicated that the diversity of microbial population was higher in Summer than that in Winter by DGGE profiles. The microbial diversity was also higher in mangrove swamp than that in non-mangrove swamp. There were various Shannon-wiener Index (H), Richness (S), Evenness (EH) and community similar coefficient in mangrove sediments at different stations.
The results of evaluation to microbial diversity by the method of constructed 16S rDNA library showed that the most colonies were uncultured microbes in 16S rDNA library. The dominant microbes belonged to Proteobacteria (70.0%), the other microbes were Bacteroidetes (8.0%), Planctomycetacia (2.0%), Actinbacteria (2.0%) and Verrucomicrobia (2.0%), respectively. Additional, uncultured and unclassified microbes (16.0%) were also detected. Finally, it demonstrated that there were abundant microbial diversity in mangrove sediments by using the methods of either PCR-DGGE or 16S rDNA library.
3. The results of research on community structure through the response of microbes in mangrove swamp under PAHs stress revealed that different changes of microbial community structure had occurred in different concentrations of Phe, Pyr, Flu, Bap and M-PAHs. The more obvious influence would happen to microbial community structure in higher concentrations of PAHs, and responsive time would also be short. The most bands of distinct difference were as uncultured microorganisms that were associated with biodegradation of pollutants.
4.14 strains of PAH-degrading bacteria were isolated from mangrove sediments in Winter. All of these strains belonged to Proteobacteria, includingα-proteobacteria (7 strains, 50%) andγ-proteobacteria (7 strains, 50%). Based on microbial morphology and results of RFLP analysis, 67 strains PAH-degrading bacteria were isolated from mangrove swamp in Summer by some screening strategies with different medium. All of them were chosen to carry out 16S rDNA identification. The results indicated that these strains were grouped into four phylum from samples in Winter and Summer, including 38 strains of proteobacteria (α-proteobacteria, 14 strains;β-proteobacteria,1 strains;γ-proteobacteria,23 strains), 5 strains of Bacteroidetes (Sphingobacteria,2 strains; Flavobacteria 3 strains), 5 strains of Actinobacteria and 8 strains of Firmicutes (Bacilli, 8 strains).
5. Phe- degrading bacterium F2 formed the clear zones in MM2 and 2216E plates by the sublimate-plate method, and no clear zones were observed in other PAH-degrading bacteria. The different PAH-degrading bacteria had various growth ways to utilize PAHs as sole carbon source. In addition, bacterial degradation effects to PAHs were also studied by mixture of several PAH-degrading bacteria according to information from DGGE profiles. The results of degrading rates by HPLC showed that either Phe-degrading bacteria or mixture of PAH-degrading bacteria had the stronger degradation ability to phenanthrene. The degradation rates to the three rings of PAHs were higher than those of the four or five rings of PAHs.
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