微生物絮凝菌的固定化及混合菌种培养
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摘要
微生物絮凝剂(Microbial Flocculant,MBF)是微生物生长过程中产生的一类次生代谢产物,可絮凝,沉淀水体中的悬浮颗粒、菌体细胞及胶体粒子。由于其具有易生物降解、对环境和人类无害等特点,微生物絮凝剂已成为絮凝剂研究的一个新热点。微生物固定化技术具有细胞密度高、反应速度快、稳定性强、耐毒害能力强、微生物流失少、产物分离容易和剩余污泥少等优点,因此在废水处理中受到重视并得到广泛的应用。为了使微生物絮凝剂在实际中能更好的应用,本论文将絮凝菌与微生物固定化技术结合运用,并对混合絮凝菌株产生的微生物絮凝剂进行了研究。
本文在实验室经过多重筛选和富集,分离出3株高效絮凝剂产生菌株,分别命名为X-1,X-2,X-3,其中X-1的絮凝效率最佳。在最优絮凝条件,即5ml的菌液投加到100ml pH为8.0的5%高岭土中,添加4ml的1%CaCl_2,沉降15min时,其絮凝率可达到83.45%。
选取X-1作为研究菌株,海藻酸钠、聚乙烯醇(PVA)、海藻酸钠与聚丙烯酰胺的复合物作为固定化载体,以固定化载体的浓度、微生物与载体的重量比、交联时间、交联剂的浓度为影响因素,进行了L_9(3~4)正交实验,以悬浮菌种作为对照试验,寻找出影响因素的重要性顺序及最佳的固定化条件。
为了进一步提高固定化的菌种颗粒的絮凝性,首先对絮凝菌液用硅藻土或活性碳进行物理吸附,再用固定化载体包埋,然后对成形的固定化颗粒进行化学处理,以增强其机械性能。
试验还对固定化细胞的絮凝条件进行了研究,找出最佳的絮凝条件;并用电镜观察固定化菌种颗粒的内部结构,以进一步了解不同固定化载体的结构对微生物絮凝剂的影响。
经过实验证明:海藻酸钠和聚丙烯酰胺(PAM)组成的复合载体固定菌株能取得最佳絮凝性。最佳固定化条件为:2%海藻酸钠和3%PAM,包埋菌种与载体体积比为1∶1,质量分数为4%的CaCl_2作为交联剂,交联剂的pH为6,交联温度为25°C。加入吸附剂活性炭时,其絮凝性提高,当活性碳的量为0.4g/(10ml湿菌种)时,达到最高絮凝率94.23%,比悬浮菌液,海藻酸钠固定菌种,PVA固定菌种的絮凝率分别提高了10.7%,2.46%,6.44%。且最佳絮凝条件为:高岭土的最初pH值为9,沉降时间为15min,最佳助凝剂投加量为4ml/(100ml5%高岭土悬浮液),絮凝菌菌液投加量为1ml/(100ml5%高岭土悬浮液)。
将筛选出的三种菌种,进行不同方式的搭配混合培养,测混合菌种的絮凝效果。发现X-1+X-2(后称J-1)的絮凝性最佳,要高于各菌种单独发酵所得的发酵液的絮凝效果,絮凝率可达到87.56%。
Microbial flocculants are the product sresulted from the synthesis and secretion of extracellular biopolymeric flocculants(EBFs)by microorganisms. With the properties of high flocculating efficiency,without toxicity and second-pollution,self-degradability and high-produced rate,the research on development and application of bioflocculant has been a focus of the area of biological preparation in recent years.And moreover,the immobilization of microorganisms has been widely used in the applications as a new kind of technology in the wastewater treatment because of the characteristics of high removal efficiency,stability of operation,and less yield of sludge.In order to make microbial flocculants have more efficiency in practice,the immobilization of flocculating bacterium and the flocculant produced by effective microorganisms(EM)were discussed in the paper.
First of all,three kinds of flocculating microbes with good flocculating ability,namely X-1,X-2 and X-3,were obtained by repetitious enrichment and reselection from three kinds of activated sludge.At the optimum flocculation condition(8.0 for initial pH,4ml/100ml for the dosage of CaCl_2, 5ml/100ml for the dosage of strain,15min for the sedimentation time),X-1 had the best flocculating ability,the efficiency of which could reach 83.45%.
X-1 was separately imbedded in the imbedding beads including Sodium Alginate(SA),PVA and SA and polyacrylamide(PAM)composite gel to research the prime immobilizing conditions and the optimal flocculating conditions,using the free bacterium as comparison.
Aiming to find out the optimal immobilization condition,L9(3~4) orthogonal experiments were carried out for each imbedding material to research the influence of the concentration of imbedding material,the volume rate between microbe and imbedding material,the crossing time and the concentration of additives.
To enhance the flocculating rate,free bacteria was absorbed by kieselgur or activated carbon before imbedded in the immobilized beads.Then the imbedded beads were treated by chemical method to improve their mechanical characteristics.
For the purpose of obtaining the highest flocculating rate of immobilized bacteria,some experiments were carried out to find the optimal flocculating condition.Then,the structure of imbedding bead and distribution of immobilized microbial were observed and analyzed by electron microscope.
The highest flocculation was obtained when X-1 was imbedded in SA and polyacrylamide(PAM)composite gel beads.An optimal immobilization condition was achieved when the imbedded beads were composed of 2%SA and 3%PAM,the strains and imbedded material mixed as the volume ratio of 1:1,4%CaCl_2 as additives,pH 6 of additives,the reaction time of 24h,25℃in the temperature of additives.And the highest of the flocculating rate was gotten when 10ml flocculating bacteria were absorbed by 0.4g activated carbon with the efficiency(up to 94.23%),which was enhanced 10.7%, 2.46%and 6.44%compared to free bacteria,immobilized bacteria in SA beads and PVA beads respectively.The optimum flocculation condition were 9.0 for initial pH,4ml/100ml for the dosage of CaCl_2,1ml/100ml for the dosage of strain,15min for the sedimentation time.
The complex microbial flocculant was developed by inexpensive substrate through fermentation.When the flocculating efficiency was determined by mixed fermentation of the three chosen flocculating strains (X-1,X-2 and X-3)in different combinatorial ways,the best efficiency(up to 87.56%)was found out when X-1 and X-2 were combined.The mixed fermentation produce of X-1 and X-2 had more efficiency than the one of single strain.
本文在实验室经过多重筛选和富集,分离出3株高效絮凝剂产生菌株,分别命名为X-1,X-2,X-3,其中X-1的絮凝效率最佳。在最优絮凝条件,即5ml的菌液投加到100ml pH为8.0的5%高岭土中,添加4ml的1%CaCl_2,沉降15min时,其絮凝率可达到83.45%。
选取X-1作为研究菌株,海藻酸钠、聚乙烯醇(PVA)、海藻酸钠与聚丙烯酰胺的复合物作为固定化载体,以固定化载体的浓度、微生物与载体的重量比、交联时间、交联剂的浓度为影响因素,进行了L_9(3~4)正交实验,以悬浮菌种作为对照试验,寻找出影响因素的重要性顺序及最佳的固定化条件。
为了进一步提高固定化的菌种颗粒的絮凝性,首先对絮凝菌液用硅藻土或活性碳进行物理吸附,再用固定化载体包埋,然后对成形的固定化颗粒进行化学处理,以增强其机械性能。
试验还对固定化细胞的絮凝条件进行了研究,找出最佳的絮凝条件;并用电镜观察固定化菌种颗粒的内部结构,以进一步了解不同固定化载体的结构对微生物絮凝剂的影响。
经过实验证明:海藻酸钠和聚丙烯酰胺(PAM)组成的复合载体固定菌株能取得最佳絮凝性。最佳固定化条件为:2%海藻酸钠和3%PAM,包埋菌种与载体体积比为1∶1,质量分数为4%的CaCl_2作为交联剂,交联剂的pH为6,交联温度为25°C。加入吸附剂活性炭时,其絮凝性提高,当活性碳的量为0.4g/(10ml湿菌种)时,达到最高絮凝率94.23%,比悬浮菌液,海藻酸钠固定菌种,PVA固定菌种的絮凝率分别提高了10.7%,2.46%,6.44%。且最佳絮凝条件为:高岭土的最初pH值为9,沉降时间为15min,最佳助凝剂投加量为4ml/(100ml5%高岭土悬浮液),絮凝菌菌液投加量为1ml/(100ml5%高岭土悬浮液)。
将筛选出的三种菌种,进行不同方式的搭配混合培养,测混合菌种的絮凝效果。发现X-1+X-2(后称J-1)的絮凝性最佳,要高于各菌种单独发酵所得的发酵液的絮凝效果,絮凝率可达到87.56%。
Microbial flocculants are the product sresulted from the synthesis and secretion of extracellular biopolymeric flocculants(EBFs)by microorganisms. With the properties of high flocculating efficiency,without toxicity and second-pollution,self-degradability and high-produced rate,the research on development and application of bioflocculant has been a focus of the area of biological preparation in recent years.And moreover,the immobilization of microorganisms has been widely used in the applications as a new kind of technology in the wastewater treatment because of the characteristics of high removal efficiency,stability of operation,and less yield of sludge.In order to make microbial flocculants have more efficiency in practice,the immobilization of flocculating bacterium and the flocculant produced by effective microorganisms(EM)were discussed in the paper.
First of all,three kinds of flocculating microbes with good flocculating ability,namely X-1,X-2 and X-3,were obtained by repetitious enrichment and reselection from three kinds of activated sludge.At the optimum flocculation condition(8.0 for initial pH,4ml/100ml for the dosage of CaCl_2, 5ml/100ml for the dosage of strain,15min for the sedimentation time),X-1 had the best flocculating ability,the efficiency of which could reach 83.45%.
X-1 was separately imbedded in the imbedding beads including Sodium Alginate(SA),PVA and SA and polyacrylamide(PAM)composite gel to research the prime immobilizing conditions and the optimal flocculating conditions,using the free bacterium as comparison.
Aiming to find out the optimal immobilization condition,L9(3~4) orthogonal experiments were carried out for each imbedding material to research the influence of the concentration of imbedding material,the volume rate between microbe and imbedding material,the crossing time and the concentration of additives.
To enhance the flocculating rate,free bacteria was absorbed by kieselgur or activated carbon before imbedded in the immobilized beads.Then the imbedded beads were treated by chemical method to improve their mechanical characteristics.
For the purpose of obtaining the highest flocculating rate of immobilized bacteria,some experiments were carried out to find the optimal flocculating condition.Then,the structure of imbedding bead and distribution of immobilized microbial were observed and analyzed by electron microscope.
The highest flocculation was obtained when X-1 was imbedded in SA and polyacrylamide(PAM)composite gel beads.An optimal immobilization condition was achieved when the imbedded beads were composed of 2%SA and 3%PAM,the strains and imbedded material mixed as the volume ratio of 1:1,4%CaCl_2 as additives,pH 6 of additives,the reaction time of 24h,25℃in the temperature of additives.And the highest of the flocculating rate was gotten when 10ml flocculating bacteria were absorbed by 0.4g activated carbon with the efficiency(up to 94.23%),which was enhanced 10.7%, 2.46%and 6.44%compared to free bacteria,immobilized bacteria in SA beads and PVA beads respectively.The optimum flocculation condition were 9.0 for initial pH,4ml/100ml for the dosage of CaCl_2,1ml/100ml for the dosage of strain,15min for the sedimentation time.
The complex microbial flocculant was developed by inexpensive substrate through fermentation.When the flocculating efficiency was determined by mixed fermentation of the three chosen flocculating strains (X-1,X-2 and X-3)in different combinatorial ways,the best efficiency(up to 87.56%)was found out when X-1 and X-2 were combined.The mixed fermentation produce of X-1 and X-2 had more efficiency than the one of single strain.
引文
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