苯酚降解嗜盐菌的筛选及分子生物学鉴定
摘要
高盐度苯酚废水污染环境且难以处理,是当前我国环境科学与环境工程领域的研究热点与难点,本项研究通过分离驯化苯酚降解嗜盐菌,通过对其进行分子生物学鉴定,明确其所属物种,在高盐度条件下对它们降解苯酚的性能分别进行了研究,然后对嗜盐组合菌群处理苯酚废水性能做了研究,最后对比了不同方法处理高盐度苯酚废水效果,所得到的嗜盐菌,试验技术,试验数据等可为工厂化处理高盐度苯酚废水提供微生物资源与技术支持。
本文在较系统地综述了国内外高盐度苯酚废水研究进展基础上,在下列四个方面进行了系统研究,并取得了一些相关成果:
1.盐田苯酚降解嗜盐菌HBCC-2和HBCC-3的筛选及分子生物学鉴定
从连云港台南盐田卤水样品中分离到两株极端嗜盐菌,编号分别为HBCC-2和HBCC-3。采用分子生物学方法对HBCC-2和HBCC-3的16S rRNA基因序列分析,并在此基础上初步确定HBCC-2属于Halorubrum, HBCC-3为Alkalibacillus salilacus。通过苯酚长期驯化嗜盐菌HBCC-2和HBCC-3后,在好氧条件下研究了HBCC-2和HBCC-3的苯酚降解效率,结果表明NaCl与KCl浓度分别为50g/L,100g/L,150g/L,200g/L与250g/L的条件下,HBCC-3的苯酚降解效率均高于HBCC-2。
2.焦化厂活性污泥苯酚降解嗜盐菌JHCFS1-JHCFS4的筛选及分子生物学鉴定
从正在运行的焦化厂活性污泥中分离筛选到四株苯酚降解中度嗜盐菌,编号分别为JHCFS1,JHCFS2,JHCFS3与JHCFS4。采用分子生物学方法对它们的16S rRNA基因序列进行了分析,初步鉴定四株菌分别为Bacillus simplex,Bacillus cereus,Bacillus pumilus与Bacillus cibi。四株菌在好氧条件下以苯酚为唯一碳源生长时,最大耐受NaCl浓度为30g/L,此时JHCFS2的降解效率最高,达到了83%,JHCFS4的降解效率最低,仅为47.20%;它们对KCl的最大耐受浓度为30g/L,此时JHCFS2的降解效率最高,达到了99%,JHCFS4的降解效率最低,仅为48%。
3.焦化废水污染土壤苯酚降解嗜盐菌CC2的筛选及分子生物学鉴定
从废弃焦化厂焦化废水污染土壤中分离筛选到一株苯酚降解中度嗜盐菌,编号为CC2。采用分子生物学方法对它的16S rRNA基因序列分析进行了分析,初步鉴定该菌株为Alcaligenes faecalis。采用引物Ph1和Ph2对其苯酚羟化酶基因进行了PCR扩增,经克隆后测出其序列长度为642bp(GenBank收录号:FJ226422),经BlastP在线比较,推断其苯酚羟化酶与固氮粪产碱菌(Alcaligenes faecalis) (ABG91754)高度同源,相似性为99%。在好氧条件下CC2能以苯酚为唯一碳源生长,最大耐受NaCl,KCl的浓度均为30g/L,苯酚降解率分别为75%与92.4%。
4.嗜盐组合菌群处理高盐度苯酚废水研究
在NaCl浓度为100g/L,苯酚废水CODcr为1255.26mg/L的条件下,采用正交试验方法,比较了7株嗜盐菌的不同配比组合处理效果,试验结果表明HBCC-2∶HBCC-3∶CC2∶JHCFS1∶JHCFS2∶JHCFS3∶JHCFS4的最佳组合配比为1∶2∶2∶2∶2∶2∶2。实验室条件下模拟SBR反应器处理装置,采用海藻酸钠+活性炭包埋最佳嗜盐组合菌群固定后处理高盐度苯酚废水效果最好,在NaCl,KCl浓度分别为100g/L,苯酚废水处理24h后CODcr由1255.26mg/L分别降为80.42mg/L,69.8 mg/L。
上述工作为高盐度苯酚废水的生物处理提供了理论基础与技术参考。该论文有图70幅,表10个,参考文献162篇。
The study on the treatment problem of phenol wastewater under high salinity condition is the present focus on environmental science and engineering fields in China.Isolation and screening of halophilic microbial strains , determination of their taxonomic status using molecular biological techniques,phenol-degrading efficiency of these microbial strains, combined microbial consortium and compared of the treatment performance using different treatment methods were made.They offered the microbial strains,data support ,experiment techniques and scientific guidance for the treatment of high salinity phenol wastewater in industry.
Systematically summarized the development of research at home and abroad on high-salinity wastewater containing phenol,this paper focused on the following four research works:
1. Screening and molecular biological identification of phenol-degrading halophilies from salt ponds
Halophilies were isolated from salt ponds of Lianyungang Tai Nai District,and two halophilic microbial strains designated HBCC-2 and HBCC-3 were screened respectively. HBCC-2 and HBCC-3 were identified according to the biochemical and physical characteristics ,16S rRNA gene of the two microbial strains were sequenced and analyzed. They could use phenol as sole carbon source afer being acclimated for about three years by phenol ,and the efficiency of phenol treatment of HBCC-3 was better than HBCC-2.
2. Screening and molecular biological identification of phenol-degrading halophilies from activated sludge
Four middle halophilic microbial strains were isolated from activated sludge of a operating coking plant, respectively.They were designated as JHCFS1, JHCFS2, JHCFS3 and JHCFS4 respectively. JHCFS1, JHCFS2, JHCFS3 and JHCFS4 were identified according to the biochemical and physical characteristics and 16S rRNA gene of the four microbial strains were sequenced and analyzed. The efficiency of phenol treatment were tested by them when NaCl and KCl concentration were 30g/L respectively. The result illustrated the best treatment performance of wastewater was used of JHCFS2 and the worse treatment performance of wastewater was used of JHCFS4.
3. Screening and molecular biological identification of phenol-degrading halophilies from soil polluted by coking wastewater
One halophilic and phenol-degrading microbial strain designated as CC2 was isolated from polluted soil of a abandoned coking plant.16S rRNA gene of CC2 was sequenced and analyzed, it was identified according to its biochemical and physical characteristics.The phenol hydroxylase gene of CC2 was detected directly using the pair of primers Ph1 and Ph2.The phenol degradation characteristics of CC2 was tested,the resulted showed CC2 could use phenol as a sole carbon source when NaCl and KCl concentration were 30g/L for its growth, respectively,
4. Research on high salinity phenol wastewater treatment performance by halophilic consortium
The treatment performance of high-salinity phenol wastewater was investigated and compared by halophilic microbial consortium consisting of different mixture ratio of seven phenol-degrading halophilies at a orbital shaker. Based on the best treatment efficiency of phenol by combined microbial consortium , the treatment performance by four different methods, including suspension of microorganisms,absorption of activated carbon,embedding immobilization by sodium alginate, embedding immobilization by sodium alginate and active carbon simulating SBR reactor equipment were investigated repectively,the result showed embedding immobilization by sodium alginate and active carbon was best one among the four methods.
本文在较系统地综述了国内外高盐度苯酚废水研究进展基础上,在下列四个方面进行了系统研究,并取得了一些相关成果:
1.盐田苯酚降解嗜盐菌HBCC-2和HBCC-3的筛选及分子生物学鉴定
从连云港台南盐田卤水样品中分离到两株极端嗜盐菌,编号分别为HBCC-2和HBCC-3。采用分子生物学方法对HBCC-2和HBCC-3的16S rRNA基因序列分析,并在此基础上初步确定HBCC-2属于Halorubrum, HBCC-3为Alkalibacillus salilacus。通过苯酚长期驯化嗜盐菌HBCC-2和HBCC-3后,在好氧条件下研究了HBCC-2和HBCC-3的苯酚降解效率,结果表明NaCl与KCl浓度分别为50g/L,100g/L,150g/L,200g/L与250g/L的条件下,HBCC-3的苯酚降解效率均高于HBCC-2。
2.焦化厂活性污泥苯酚降解嗜盐菌JHCFS1-JHCFS4的筛选及分子生物学鉴定
从正在运行的焦化厂活性污泥中分离筛选到四株苯酚降解中度嗜盐菌,编号分别为JHCFS1,JHCFS2,JHCFS3与JHCFS4。采用分子生物学方法对它们的16S rRNA基因序列进行了分析,初步鉴定四株菌分别为Bacillus simplex,Bacillus cereus,Bacillus pumilus与Bacillus cibi。四株菌在好氧条件下以苯酚为唯一碳源生长时,最大耐受NaCl浓度为30g/L,此时JHCFS2的降解效率最高,达到了83%,JHCFS4的降解效率最低,仅为47.20%;它们对KCl的最大耐受浓度为30g/L,此时JHCFS2的降解效率最高,达到了99%,JHCFS4的降解效率最低,仅为48%。
3.焦化废水污染土壤苯酚降解嗜盐菌CC2的筛选及分子生物学鉴定
从废弃焦化厂焦化废水污染土壤中分离筛选到一株苯酚降解中度嗜盐菌,编号为CC2。采用分子生物学方法对它的16S rRNA基因序列分析进行了分析,初步鉴定该菌株为Alcaligenes faecalis。采用引物Ph1和Ph2对其苯酚羟化酶基因进行了PCR扩增,经克隆后测出其序列长度为642bp(GenBank收录号:FJ226422),经BlastP在线比较,推断其苯酚羟化酶与固氮粪产碱菌(Alcaligenes faecalis) (ABG91754)高度同源,相似性为99%。在好氧条件下CC2能以苯酚为唯一碳源生长,最大耐受NaCl,KCl的浓度均为30g/L,苯酚降解率分别为75%与92.4%。
4.嗜盐组合菌群处理高盐度苯酚废水研究
在NaCl浓度为100g/L,苯酚废水CODcr为1255.26mg/L的条件下,采用正交试验方法,比较了7株嗜盐菌的不同配比组合处理效果,试验结果表明HBCC-2∶HBCC-3∶CC2∶JHCFS1∶JHCFS2∶JHCFS3∶JHCFS4的最佳组合配比为1∶2∶2∶2∶2∶2∶2。实验室条件下模拟SBR反应器处理装置,采用海藻酸钠+活性炭包埋最佳嗜盐组合菌群固定后处理高盐度苯酚废水效果最好,在NaCl,KCl浓度分别为100g/L,苯酚废水处理24h后CODcr由1255.26mg/L分别降为80.42mg/L,69.8 mg/L。
上述工作为高盐度苯酚废水的生物处理提供了理论基础与技术参考。该论文有图70幅,表10个,参考文献162篇。
The study on the treatment problem of phenol wastewater under high salinity condition is the present focus on environmental science and engineering fields in China.Isolation and screening of halophilic microbial strains , determination of their taxonomic status using molecular biological techniques,phenol-degrading efficiency of these microbial strains, combined microbial consortium and compared of the treatment performance using different treatment methods were made.They offered the microbial strains,data support ,experiment techniques and scientific guidance for the treatment of high salinity phenol wastewater in industry.
Systematically summarized the development of research at home and abroad on high-salinity wastewater containing phenol,this paper focused on the following four research works:
1. Screening and molecular biological identification of phenol-degrading halophilies from salt ponds
Halophilies were isolated from salt ponds of Lianyungang Tai Nai District,and two halophilic microbial strains designated HBCC-2 and HBCC-3 were screened respectively. HBCC-2 and HBCC-3 were identified according to the biochemical and physical characteristics ,16S rRNA gene of the two microbial strains were sequenced and analyzed. They could use phenol as sole carbon source afer being acclimated for about three years by phenol ,and the efficiency of phenol treatment of HBCC-3 was better than HBCC-2.
2. Screening and molecular biological identification of phenol-degrading halophilies from activated sludge
Four middle halophilic microbial strains were isolated from activated sludge of a operating coking plant, respectively.They were designated as JHCFS1, JHCFS2, JHCFS3 and JHCFS4 respectively. JHCFS1, JHCFS2, JHCFS3 and JHCFS4 were identified according to the biochemical and physical characteristics and 16S rRNA gene of the four microbial strains were sequenced and analyzed. The efficiency of phenol treatment were tested by them when NaCl and KCl concentration were 30g/L respectively. The result illustrated the best treatment performance of wastewater was used of JHCFS2 and the worse treatment performance of wastewater was used of JHCFS4.
3. Screening and molecular biological identification of phenol-degrading halophilies from soil polluted by coking wastewater
One halophilic and phenol-degrading microbial strain designated as CC2 was isolated from polluted soil of a abandoned coking plant.16S rRNA gene of CC2 was sequenced and analyzed, it was identified according to its biochemical and physical characteristics.The phenol hydroxylase gene of CC2 was detected directly using the pair of primers Ph1 and Ph2.The phenol degradation characteristics of CC2 was tested,the resulted showed CC2 could use phenol as a sole carbon source when NaCl and KCl concentration were 30g/L for its growth, respectively,
4. Research on high salinity phenol wastewater treatment performance by halophilic consortium
The treatment performance of high-salinity phenol wastewater was investigated and compared by halophilic microbial consortium consisting of different mixture ratio of seven phenol-degrading halophilies at a orbital shaker. Based on the best treatment efficiency of phenol by combined microbial consortium , the treatment performance by four different methods, including suspension of microorganisms,absorption of activated carbon,embedding immobilization by sodium alginate, embedding immobilization by sodium alginate and active carbon simulating SBR reactor equipment were investigated repectively,the result showed embedding immobilization by sodium alginate and active carbon was best one among the four methods.
引文
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