一株微生物絮凝剂产生菌的分离鉴定及其特性研究
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摘要
生物絮凝剂主要是由微生物在特定条件下培养到一定阶段分泌产生的具有絮凝活性的代谢产物,主要为多糖、蛋白、核酸等高分子化合物。它们通过电荷中和、吸附、桥联、网捕等作用,使胶体脱稳、絮凝沉淀达到固液分离的目的,具有安全、高效、易降解等优点,可广泛应用于废水处理、医药、食品生产等领域,是一种具有发展前途的水处理剂。
本课题从污泥样品中分离得到245株单菌落,通过进一步筛选得到一株絮凝活性稳定的菌株JX18,絮凝率达到84%。通过菌落形态、生理生化和分子生物学鉴定,初步表明该菌为荧光假单胞菌。
研究了P. fluorescens JX18的菌体生长曲线,该菌在12h时进入对数生长期,42h进入稳定期,培养48h时絮凝活性达到最大。通过单因素实验得出菌株P. fluorescens JX18在30℃,培养基初始pH 7.0,摇床转速160r/min的条件下发酵液絮凝活性最好。通过单因素和正交试验,确定了P. fluorescens JX18的最适发酵培养基为:葡萄糖7g,蛋白胨1.3g,KH_2PO_4 2g,K2HPO_4 5g,MgSO_4·7H_2O 0.1g,NaCl 0.3g,FeSO_4·7H_2O 0.02g,水1000mL,在此条件下絮凝率达到91.19%。
采用5L发酵罐进行间歇发酵实验,定时测定生物量、葡萄糖含量、絮凝物质产量,研究了P. fluorescens JX18的培养动力学方程。以Logistic方程和Luedeking-Piret方程为基础,建立了该菌株在发酵过程中菌体生长、产物形成和底物消耗的动力学模型,其模型能较好地反应P. fluorescens JX18产絮凝剂的发酵规律。
进一步研究发现,絮凝物质主要分布在发酵液中。通过对絮凝物质进行提取、纯化,化学、物理方法分析表明该絮凝物质主要为中性多糖,占粗产物的68%。在扫描电子显微镜下观察,粗产物为长纤维丝状。通过薄层层析(TLC)、高效液相色谱(HPLC)分析,该多糖主要由葡萄糖、半乳糖、甘露糖组成。
以高岭土悬浮液模拟实际废水,研究了P. fluorescens JX18所产絮凝物质的絮凝特性:该絮凝剂具有较宽的温度和pH值使用范围。温度在20~100℃范围内,絮凝活性在90%以上,当温度在100℃时,絮凝活性基本无损失,表现出良好的热稳定性;pH值在3~10范围内,絮凝率稳定在85%以上。对于4g/L的高岭土悬浮液体系,絮凝物质的最适用量为2.5 mg/L,最适助凝剂为CaCl_2,其最适用量为0.5 g/L。
通过电子显微镜观察和红外光谱分析,结合絮凝特性,初步描述絮凝过程为:高岭土本身为小颗粒物质,在溶液中分散比较均匀。加入助凝剂CaCl_2后,CaCl_2可中和溶液中部分负电荷,使胶体颗粒和絮凝物质之间的静电斥力减小。线性絮凝物质的加入,伸展的絮凝剂分子通过不同的基团与不同的颗粒发生吸附,同时长的线性分子之间容易交互连接,形成大的颗粒快速沉降。
Microbial flocculants were mainly produced by microorganisms under certain conditions of cultivation, including polysaccharides, proteins and nucleic acid polymers with flocculation activity. By charge neutralization, adsorption, bridging effect and immesh, the flocculants cause the colloid aggregation. Since microbial flocculants are biodegradable, nontoxic, and no secondary pollution, they were deemed as highly promising water treatment chemicals and could be widely used in wastewater treatment, pharmaceutical, food-production and other fields.
245 strains were isolated from sludge samples and a new kind of high flocculants-producing bacteria JX18 with flocculation rate of 84% was obtained. It was identified as a Pseudomonas fluorescens based on its morphological, physiological characteristics and the partial sequences of its 16S rRNA.
The bacteria entered the logarithmic phase after culturing12 h, reached stationary phase after culturing 40 h, and had a maximum ?occulating activity after culturing 48 h. The further investigation indicated that the optimum flocculants-producing conditions obtained were initial pH 7.0, temperature 30℃, rotation speed 160 r/min. The optimum compent proportion of medium of P. fluorescens JX18 were glucose 7g, peptone 1.3g, KH_2PO_4 2g, K2HPO_4 5g, NaCl 0.3g, MgSO_4?7H_2O 0.1g, FeSO_4?7H_2O 0.02g, water 1L according to the single factor experiments and the orthogonal experiment. Optimum cultivation conditions, the flocculation efficiency toward Kaolin clay suspension reached 91.19%.
Biomasses, content of flocculant, concentration of glucose in fermentation broth during different time were detected in 5L fermentator. Based on the Logistic equation and Luedeking-Piret equation, the fermentation dynamics models on growth of cell, consumpution of glucose and the content of flocculant were founded. The model could well reflect the changing pattern in growth of cell, the consumpution of glucose and flocculants yield.
The further study showed that the materials with flocculation activity were mainly distributed in the fermentation broth. Measurements of chemical and physical analysis revealed that the flocculant is a kind of polysaccharidesthe, accounting for 68% of the crude product. Scanning electron microscopy (SEM) image of the solid-state crude product showed that the flocculant has a linear morphology. It was consisted of glucose, galactose and mannose with the thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analysis.
As Kaolin suspension modeling waste water, the results of the flocculate properties showed that the flocculant has high heated stability and wide pH serviceable range. At 20~100℃, the flocculating activity was almost not descended, and the flocculating activity was above 85% at pH 3.0~10.0. Toward 4g/L Kaolin suspension, the optimum concentration of bioflocculant was 2.5 mg/L, and CaCl_2 was proved to be the best coagulant aid in this flocculating system, in which the optimum concentration was 0.5 g/L.
By flocculate property, electron microscopy images and infrared spectra analysis, the initial description of flocculation process are that: kaolin itself is small particulate matter, more evenly dispersed in solution; CaCl_2 is a coagulant aid, the major role of Ca2+ in flocculation is neutralizing negative charge and reducing repulsion of electric charge. Adding the flocculant, bridge and adsorption effect occured. The bioflocculant has a long-linear morphology, and connected the molecule of Kaolin clay together.
本课题从污泥样品中分离得到245株单菌落,通过进一步筛选得到一株絮凝活性稳定的菌株JX18,絮凝率达到84%。通过菌落形态、生理生化和分子生物学鉴定,初步表明该菌为荧光假单胞菌。
研究了P. fluorescens JX18的菌体生长曲线,该菌在12h时进入对数生长期,42h进入稳定期,培养48h时絮凝活性达到最大。通过单因素实验得出菌株P. fluorescens JX18在30℃,培养基初始pH 7.0,摇床转速160r/min的条件下发酵液絮凝活性最好。通过单因素和正交试验,确定了P. fluorescens JX18的最适发酵培养基为:葡萄糖7g,蛋白胨1.3g,KH_2PO_4 2g,K2HPO_4 5g,MgSO_4·7H_2O 0.1g,NaCl 0.3g,FeSO_4·7H_2O 0.02g,水1000mL,在此条件下絮凝率达到91.19%。
采用5L发酵罐进行间歇发酵实验,定时测定生物量、葡萄糖含量、絮凝物质产量,研究了P. fluorescens JX18的培养动力学方程。以Logistic方程和Luedeking-Piret方程为基础,建立了该菌株在发酵过程中菌体生长、产物形成和底物消耗的动力学模型,其模型能较好地反应P. fluorescens JX18产絮凝剂的发酵规律。
进一步研究发现,絮凝物质主要分布在发酵液中。通过对絮凝物质进行提取、纯化,化学、物理方法分析表明该絮凝物质主要为中性多糖,占粗产物的68%。在扫描电子显微镜下观察,粗产物为长纤维丝状。通过薄层层析(TLC)、高效液相色谱(HPLC)分析,该多糖主要由葡萄糖、半乳糖、甘露糖组成。
以高岭土悬浮液模拟实际废水,研究了P. fluorescens JX18所产絮凝物质的絮凝特性:该絮凝剂具有较宽的温度和pH值使用范围。温度在20~100℃范围内,絮凝活性在90%以上,当温度在100℃时,絮凝活性基本无损失,表现出良好的热稳定性;pH值在3~10范围内,絮凝率稳定在85%以上。对于4g/L的高岭土悬浮液体系,絮凝物质的最适用量为2.5 mg/L,最适助凝剂为CaCl_2,其最适用量为0.5 g/L。
通过电子显微镜观察和红外光谱分析,结合絮凝特性,初步描述絮凝过程为:高岭土本身为小颗粒物质,在溶液中分散比较均匀。加入助凝剂CaCl_2后,CaCl_2可中和溶液中部分负电荷,使胶体颗粒和絮凝物质之间的静电斥力减小。线性絮凝物质的加入,伸展的絮凝剂分子通过不同的基团与不同的颗粒发生吸附,同时长的线性分子之间容易交互连接,形成大的颗粒快速沉降。
Microbial flocculants were mainly produced by microorganisms under certain conditions of cultivation, including polysaccharides, proteins and nucleic acid polymers with flocculation activity. By charge neutralization, adsorption, bridging effect and immesh, the flocculants cause the colloid aggregation. Since microbial flocculants are biodegradable, nontoxic, and no secondary pollution, they were deemed as highly promising water treatment chemicals and could be widely used in wastewater treatment, pharmaceutical, food-production and other fields.
245 strains were isolated from sludge samples and a new kind of high flocculants-producing bacteria JX18 with flocculation rate of 84% was obtained. It was identified as a Pseudomonas fluorescens based on its morphological, physiological characteristics and the partial sequences of its 16S rRNA.
The bacteria entered the logarithmic phase after culturing12 h, reached stationary phase after culturing 40 h, and had a maximum ?occulating activity after culturing 48 h. The further investigation indicated that the optimum flocculants-producing conditions obtained were initial pH 7.0, temperature 30℃, rotation speed 160 r/min. The optimum compent proportion of medium of P. fluorescens JX18 were glucose 7g, peptone 1.3g, KH_2PO_4 2g, K2HPO_4 5g, NaCl 0.3g, MgSO_4?7H_2O 0.1g, FeSO_4?7H_2O 0.02g, water 1L according to the single factor experiments and the orthogonal experiment. Optimum cultivation conditions, the flocculation efficiency toward Kaolin clay suspension reached 91.19%.
Biomasses, content of flocculant, concentration of glucose in fermentation broth during different time were detected in 5L fermentator. Based on the Logistic equation and Luedeking-Piret equation, the fermentation dynamics models on growth of cell, consumpution of glucose and the content of flocculant were founded. The model could well reflect the changing pattern in growth of cell, the consumpution of glucose and flocculants yield.
The further study showed that the materials with flocculation activity were mainly distributed in the fermentation broth. Measurements of chemical and physical analysis revealed that the flocculant is a kind of polysaccharidesthe, accounting for 68% of the crude product. Scanning electron microscopy (SEM) image of the solid-state crude product showed that the flocculant has a linear morphology. It was consisted of glucose, galactose and mannose with the thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analysis.
As Kaolin suspension modeling waste water, the results of the flocculate properties showed that the flocculant has high heated stability and wide pH serviceable range. At 20~100℃, the flocculating activity was almost not descended, and the flocculating activity was above 85% at pH 3.0~10.0. Toward 4g/L Kaolin suspension, the optimum concentration of bioflocculant was 2.5 mg/L, and CaCl_2 was proved to be the best coagulant aid in this flocculating system, in which the optimum concentration was 0.5 g/L.
By flocculate property, electron microscopy images and infrared spectra analysis, the initial description of flocculation process are that: kaolin itself is small particulate matter, more evenly dispersed in solution; CaCl_2 is a coagulant aid, the major role of Ca2+ in flocculation is neutralizing negative charge and reducing repulsion of electric charge. Adding the flocculant, bridge and adsorption effect occured. The bioflocculant has a long-linear morphology, and connected the molecule of Kaolin clay together.
引文
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