絮凝益生菌的筛选及絮凝效果研究
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摘要
微生物絮凝剂(Microbial flocculant)是一类由微生物产生的,具有絮凝活性的代谢产物,它是利用微生物技术,通过细菌、真菌、藻类等微生物发酵、提取、精制而得的,主要成分有糖蛋白、多糖、蛋白质、脂类、纤维素和DNA等。它具有易被微生物降解、无毒无害、安全性高、适用范围广、无二次污染等特点,以其作为污水处理剂,应用于工业或者生活污水处理,可最终实现无污染排放,因其特点,近年来得到了国内外学者的广泛关注并成为新型絮凝剂研究开发的热点。
本研究通过稀释涂板法和划线法,从河道污泥、花园土壤、鲈鱼肠道内分离筛选到18株絮凝剂产生菌,经过复筛得到5株高效稳定的絮凝剂产生菌,最终筛选出一株絮凝益生菌DK1,并对该菌生理生化特征及16S rRNA基因进行了研究,初步确定该菌为地衣芽孢杆菌(Bacillus licheniformis)。
该菌的培养条件优化实验研究确定该菌最佳培养基及最佳产絮凝剂培养条件为:乳糖20 g/L,尿素0.5 g/L,酵母膏0.5g/L,K_2HPO_4 5g/L,KH_2PO_42 g/L,NaCl 0.1g/L,(NH_4)_2SO_40.2g/L,尿素0.5g/L,琼脂15～20g,双蒸水1000mL,pH为7.5～9.5,装液量30/250mL～60/250mL,摇床转速160r/min,37℃培养48～72h。其发酵液对高岭土悬液的絮凝率高达80%以上。该菌在3%～5%接种量条件下可获得最短2d的高效产絮凝剂的培养周期。
地衣芽孢杆菌(Bacillus licheniformis)DK1在液体培养情况下产絮凝剂主要分布于发酵液中,在固体培养的条件下,絮凝剂分泌到细胞外,在培养基的表面形成液滴,初步确定为其絮凝成分为地衣芽孢杆菌的胞外代谢产物,通过因子破坏法和化学分析法,确定其分泌物中的多糖和蛋白质类成分有絮凝活性。
对地衣芽孢杆菌(Bacillus licheniformis)处理鲤鱼养殖水体的研究表明,该微生物絮凝剂对养殖水体的BOD_5,COD_((Mn)),pH值,亚硝酸盐态氮的含量,以及浊度都有很大的改善,又由于地衣芽孢杆菌(Bacillus licheniformis)是传统的益生菌,对养殖水产几乎不存在的有害性,因此,地衣芽孢杆菌(Bacillus licheniformis)在水产养殖水体水处理中显现出良好的应用前景。
Microbial flocculant is a sort of flocculatant metabolite produced by microorganisms,Microbial flocculant can be obtained through fermenation, concentration and refining of microorganisms such as bateria and epiphyte.It is the application of microbial technique.It mainly contains glucoprotein, polysaccharides,protein,lipid,Fibrin,DNA,etc.It has many strongpoints such as high safety,wide application,easy decomposition by micro organisms,and harmless to the environment.If microbial flocculant was used to dispose sewage, there would be no pollution in the drains.So it is paid so much attention to by scholars of all countries and has become the focus of research and development.
This paper summarized a series of research and development on preparation of microbial flocculants systematically.The isolation of flocculant-producing bacterium,optimization of culture condition for flocculants production,and identification of the bioflocculant-producing bacterium.The composition and characterisitics of microbial flocculants were studied and discussed.The main contents of the research and the results are as follows:
In this paper,5 stubs of strains have been sorted and dissolved from the sludge,intestines of the weever and the garden soil.A strain,DK1,with high flocculating activity was screened form sludge.It was identified as Bacillus licheniformis by it is characters and molecular biology(16S rRNA)to find a beneficial bacterium.
The culture conditions were optimized for Bacillus licheniformis.The strain could grow in various situations and produce high-effect flocculants.The sudies showed that the optimum carbon sources were lactose and nitrogen sourcesm in the mixture of yeast extract(0.5g/L),ammonium sulfate(0.2g/L),and carbamide(0.5g/L).Under the cultivating conditions with primary pH value7.5～9.5,the liquid pack of 30～60 mL/250mL inoculum size being 3%～6%, at 37℃,on a shaking table at 160r/min.Strain DK1 cultivated for 48～72h can produce a highly efficient flocculant,with a flocculation ratio of 80%being against kaolin clay suspension.The periods of the bioflocculants producing was two days'time.
The microbial flocculants produced by Bacillus licheniformis are excreted out of the cell,and the flocculants are mostly distributed in zymolytic medium when cultured in liquid culture mediums,and excreted on the surfac,e of the solid culture medium.Polysaccharides and protein in the secretion were made sure to have a property of flocculant through the method of factor destruction and chemistry analysis.
The results of the flocculating experiment on different types of aquaculture with the bioflocculant produced by Bacillus licheniformis indicated that biofiocculant had excellent effect on the water-clarity of aquaculture and could remove indiscerptible COD_((Mn))and BOD_5,and showed excellent effects on the treatment of aquaiculture water.
本研究通过稀释涂板法和划线法,从河道污泥、花园土壤、鲈鱼肠道内分离筛选到18株絮凝剂产生菌,经过复筛得到5株高效稳定的絮凝剂产生菌,最终筛选出一株絮凝益生菌DK1,并对该菌生理生化特征及16S rRNA基因进行了研究,初步确定该菌为地衣芽孢杆菌(Bacillus licheniformis)。
该菌的培养条件优化实验研究确定该菌最佳培养基及最佳产絮凝剂培养条件为:乳糖20 g/L,尿素0.5 g/L,酵母膏0.5g/L,K_2HPO_4 5g/L,KH_2PO_42 g/L,NaCl 0.1g/L,(NH_4)_2SO_40.2g/L,尿素0.5g/L,琼脂15～20g,双蒸水1000mL,pH为7.5～9.5,装液量30/250mL～60/250mL,摇床转速160r/min,37℃培养48～72h。其发酵液对高岭土悬液的絮凝率高达80%以上。该菌在3%～5%接种量条件下可获得最短2d的高效产絮凝剂的培养周期。
地衣芽孢杆菌(Bacillus licheniformis)DK1在液体培养情况下产絮凝剂主要分布于发酵液中,在固体培养的条件下,絮凝剂分泌到细胞外,在培养基的表面形成液滴,初步确定为其絮凝成分为地衣芽孢杆菌的胞外代谢产物,通过因子破坏法和化学分析法,确定其分泌物中的多糖和蛋白质类成分有絮凝活性。
对地衣芽孢杆菌(Bacillus licheniformis)处理鲤鱼养殖水体的研究表明,该微生物絮凝剂对养殖水体的BOD_5,COD_((Mn)),pH值,亚硝酸盐态氮的含量,以及浊度都有很大的改善,又由于地衣芽孢杆菌(Bacillus licheniformis)是传统的益生菌,对养殖水产几乎不存在的有害性,因此,地衣芽孢杆菌(Bacillus licheniformis)在水产养殖水体水处理中显现出良好的应用前景。
Microbial flocculant is a sort of flocculatant metabolite produced by microorganisms,Microbial flocculant can be obtained through fermenation, concentration and refining of microorganisms such as bateria and epiphyte.It is the application of microbial technique.It mainly contains glucoprotein, polysaccharides,protein,lipid,Fibrin,DNA,etc.It has many strongpoints such as high safety,wide application,easy decomposition by micro organisms,and harmless to the environment.If microbial flocculant was used to dispose sewage, there would be no pollution in the drains.So it is paid so much attention to by scholars of all countries and has become the focus of research and development.
This paper summarized a series of research and development on preparation of microbial flocculants systematically.The isolation of flocculant-producing bacterium,optimization of culture condition for flocculants production,and identification of the bioflocculant-producing bacterium.The composition and characterisitics of microbial flocculants were studied and discussed.The main contents of the research and the results are as follows:
In this paper,5 stubs of strains have been sorted and dissolved from the sludge,intestines of the weever and the garden soil.A strain,DK1,with high flocculating activity was screened form sludge.It was identified as Bacillus licheniformis by it is characters and molecular biology(16S rRNA)to find a beneficial bacterium.
The culture conditions were optimized for Bacillus licheniformis.The strain could grow in various situations and produce high-effect flocculants.The sudies showed that the optimum carbon sources were lactose and nitrogen sourcesm in the mixture of yeast extract(0.5g/L),ammonium sulfate(0.2g/L),and carbamide(0.5g/L).Under the cultivating conditions with primary pH value7.5～9.5,the liquid pack of 30～60 mL/250mL inoculum size being 3%～6%, at 37℃,on a shaking table at 160r/min.Strain DK1 cultivated for 48～72h can produce a highly efficient flocculant,with a flocculation ratio of 80%being against kaolin clay suspension.The periods of the bioflocculants producing was two days'time.
The microbial flocculants produced by Bacillus licheniformis are excreted out of the cell,and the flocculants are mostly distributed in zymolytic medium when cultured in liquid culture mediums,and excreted on the surfac,e of the solid culture medium.Polysaccharides and protein in the secretion were made sure to have a property of flocculant through the method of factor destruction and chemistry analysis.
The results of the flocculating experiment on different types of aquaculture with the bioflocculant produced by Bacillus licheniformis indicated that biofiocculant had excellent effect on the water-clarity of aquaculture and could remove indiscerptible COD_((Mn))and BOD_5,and showed excellent effects on the treatment of aquaiculture water.
引文
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[2]蔡泽文,渔用微生物絮凝剂产生菌筛选及特性研究(D).2006.
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