CN113862186B - 一种微生物复合菌剂及其制备方法和应用 - Google Patents
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Abstract
本发明公开了一种微生物复合菌剂及其制备方法和应用,所述微生物复合菌剂由布氏乳杆菌、毕赤酵母菌、深红红螺菌、沼泽红假单胞菌、孢囊放线菌、少孢根霉、Halobacterium sp.和食苯假诺卡氏菌按CFU配比1~2:2~3:5~6:4~5:3~4:3~4:4~5:5~6混合得到。本发明的微生物复合菌剂中的布氏乳杆菌、毕赤酵母菌、深红红螺菌、沼泽红假单胞菌、孢囊放线菌、少孢根霉、Halobacteriumsp.及食苯假诺卡氏菌互相协调配合,形成一个完整的共生互营体系,能够高效处理污水中有机溶剂。
Description
技术领域
本发明涉及污水处理技术领域,具体地来说,涉及一种微生物复合菌剂及其制备方法和应用。
背景技术
当前,有机废水常见的处理方法有物理处理法、化学处理法以及生物处理法;其中,生物处理法为通过微生物复合菌剂来处理有机废水,即利用微生物的生长繁殖分解氧化有机物,还原水体的自净能力,其应用越来越广泛,但是由于有机废水中不仅含有好氧有机物,还含有重金属、植物营养素(氨氮等)、以及有毒有害有机物,处理难度极大,绝大多数的生物处理法的效果不是很理想。
针对上述问题,中国专利,公开号CN110240285A,公开一种降低重度污染废水中COD值的复合菌剂,文中提出“包括以下重量份数组分:恶臭假单胞菌1-8.5、圆红球菌2-8、深红红螺菌2-5、拜氏梭菌5-10、絮凝假丝酵母1-5、约氏不动杆菌2-5、巴氏醋杆菌5-12、混合芽孢杆菌13-34.5、混合乳杆菌22.5-48”,该现有技术虽然利用深红红螺菌等多种菌种组分来实现对重度污染废水中COD值的处理效果,但是仍然存在着对含甲苯、二氯甲烷的有机废水的处理效果一般、菌种易死亡的技术问题。
为此,需要一种新的技术方案以解决上述基本问题。
发明内容
本发明的目的在于提供一种微生物复合菌剂及其制备方法和应用,以解决上述背景技术中提出的现有微生物复合菌剂对含甲苯、二氯甲烷的有机废水的处理效果一般、菌种易死亡的技术问题。
为实现上述目的,本发明采取以下技术方案:
第一方面,本发明提供了一种微生物复合菌剂,所述微生物菌剂为液态菌剂,所述微生物菌剂中微生物的总浓度为3×108CFU/mL~5×108CFU/mL,由布氏乳杆菌、毕赤酵母菌、深红红螺菌、沼泽红假单胞菌、孢囊放线菌、少孢根霉、Halobacterium sp.和食苯假诺卡氏菌按CFU配比1~2:2~3:5~6:4~5:3~4:3~4:4~5:5~6混合得到;
进一步地,布氏乳杆菌、毕赤酵母菌、深红红螺菌、沼泽红假单胞菌、孢囊放线菌、少孢根霉、Halobacterium sp.和食苯假诺卡氏菌的CFU配比为1:2:5:4:3:3:4:5;
其中,所述布氏乳杆菌为lactobacillus buchneri,保藏编号为ATCC NO.11307;所述毕赤酵母菌为Pichia sp.,保藏编号为ATCC NO.28487;所述深红红螺菌为Rhodospirillum rubrum,属PNSB,保藏编号为ATCC NO.11170;所述沼泽红假单胞菌为Rhodopseudomonas palustris,保藏编号为CICC NO.23812;所述孢囊放线菌为Actinosporangium sp.,保藏编号为ATCCNO.31127;所述少孢根霉为Rhizopusoligosporus,保藏编号为ATCC NO.22959;所述Halobacterium sp.,保藏编号为ATCCNO.700922;所述食苯假诺卡氏菌为Pseudonocardia benzenivorans,保藏编号为DSMNO.44703。
第二方面,本发明提供了上述微生物复合菌剂的制备方法:
S1、分别将所述布氏乳杆菌、毕赤酵母菌、孢囊放线菌、少孢根霉、Halobacteriumsp.和食苯假诺卡氏菌进行低温胁迫培养,所述深红红螺菌、沼泽红假单胞菌另行在无光的条件下进行低温胁迫培养;
具体而言,低温胁迫培养包括:
第一培养阶段,将所述布氏乳杆菌、毕赤酵母菌、孢囊放线菌、少孢根霉、Halobacterium sp.和食苯假诺卡氏菌分别接种于第一低温胁迫培养基中,80rpm/min,28℃,培养72h,所述第一低温胁迫培养基的配方为:酵母抽提物0.5g/L,酪蛋白水解物0.5g/L,马铃薯淀粉0.5g/L,胰蛋白胨0.25g/L,葡萄糖0.5g/L,丙酮酸钠0.3g/L,KH2PO4 0.5g/L,谷氨酸钠0.2g/L,NH4Cl 0.8g/L,MgSO4·7H2O 0.25g/L,CaCl2·2H2O 0.05g/L;微量元素溶液3ml,pH为6.5~7.5;
将所述深红红螺菌、沼泽红假单胞菌分别另行接种于第一低温胁迫无光培养基,170rpm/min,28℃,培养5D,无光;5天后转为塑料透明桶静置培养,设光源2*40瓦,照度750Lx,28℃,培养2D;所述第一低温胁迫无光培养基的配方为:NH4Cl 0.1g/L,NaHCO30.1 g/L,K2HPO40.02 g/L,葡萄糖0.5g/L,CH3COONa 0.3~0.5g/L,MgSO4·7H2O 0.02g/L,Na2S0.01g/L,蛋白胨1.0g/L,酵母抽提物0.5g/L;生长因子1ml,微量元素溶液1ml,pH为7.0~8.0;
第二培养阶段,将经过第一培养阶段培养的布氏乳杆菌、毕赤酵母菌、孢囊放线菌、少孢根霉、Halobacterium sp.和食苯假诺卡氏菌,按照15%的接种量分别接种于第二低温胁迫培养基中,40rpm/min,20℃,培养60h,所述第二低温胁迫培养基的配方为:酵母抽提物0.3g/L,酪蛋白水解物0.1g/L,马铃薯淀粉0.5g/L,胰蛋白胨0.5g/L,葡萄糖0.3g/L,丙酮酸钠0.5g/L,KH2PO4 0.5g/L,谷氨酸钠0.2g/L,NH4Cl 0.8g/L,MgSO4·7H2O 0.25g/L,CaCl2·2H2O 0.05g/L,微量元素溶液5ml,pH为6.5~7.5;
将经过第一培养阶段培养的深红红螺菌、沼泽红假单胞菌分别另行接种于第二低温胁迫无光培养基,100rpm/min,20℃,培养5D,无光;5天后转为塑料透明桶静置培养,设光源2*40瓦,照度750Lx,20℃,培养2D;所述第二低温胁迫无光培养基的配方为:NH4Cl 0.1g/L,NaHCO30.1g/L,K2HPO40.02g/L,葡萄糖0.5g/L,CH3COONa 0.3~0.5g/L,MgSO4·7H2O0.02g/L,Na2S 0.01g/L,蛋白胨1.0g/L,酵母抽提物0.5g/L;生长因子1ml,微量元素溶液1ml,pH为7.0~8.0;
S2、胁迫培养后的微生物按比例混合均匀,得到所述微生物复合菌剂;
具体而言,将经过第二培养阶段培养的微生物,按照比例混合并悬浮于浓度6%的糖蜜溶液,得到微生物复合菌剂,并常温保存;
其中,所述生长因子的制备方法如下:取维生素B1 0.1mg、乙尼克丁酸0.01mg、对氨基苯甲酸0.01mg、生物素0.0001mg,溶于蒸馏水中,定容至10ml,然后过滤除菌;所述微量元素溶液的制备方法如下:取FeCl3·6H2O 5.0mg、CuS04·5H2O 0.05mg、H3BO4 l.0mg、MnCl2·4H2O 0.05mg、ZnSO4·7H2O 1.0mg、Co(NO3)2·6H2O 0.5mg,溶于蒸馏水中,定容至1000ml。
第三方面,本发明还提供了上述微生物复合菌剂在有机废水处理中的应用:
所述应用为向每升待处理有机废水中加入总量为3×109CFU~5×109CFU的微生物复合菌剂,其中,所述有机废水中含有甲苯、二氯甲烷。
与现有技术相比,本发明的有益效果是:
1、本发明的布氏乳杆菌、毕赤酵母菌、深红红螺菌、沼泽红假单胞菌、孢囊放线菌、少孢根霉、Halobacterium sp.和食苯假诺卡氏菌互相协调配合,能够快速高效地处理污水中的有机溶剂,对有机废水中的COD、NH3-N、TN、TP都有着极高的去除率,其中,COD最高去除率可达81.1%、NH3-N可达91.7%、TN可达85.3%、TP可达79%,使含甲苯、二氯甲烷的有机废水处理效果和效率明显提高;
2、本发明的制备方法中的各阶段培养基有效地模拟了有机废水的富营养、低温、无光的复杂环境,其制备的布氏乳杆菌、毕赤酵母菌、深红红螺菌、沼泽红假单胞菌、孢囊放线菌、少孢根霉、Halobacterium sp.和食苯假诺卡氏菌在混合均匀后,形成一个完整的共生互营体系,能够适应有机废水的复杂水质,菌种不易死亡,活性高,进一步提升了有机废水的处理效果。
具体实施方式
下面将结合实施例对本发明的技术方案进行清楚、完整地描述。
实施例1
一种微生物复合菌剂,包括保藏编号为ATCC NO.11307的布氏乳杆菌(lactobacillus buchneri)、保藏编号为ATCC NO.28487的毕赤酵母菌(Pichia sp.)、属PNSB且保藏编号为ATCC NO.11170的深红红螺菌(Rhodospirillum rubrum)、保藏编号为CICC NO.23812的沼泽红假单胞菌(Rhodopseudomonas palustris)、保藏编号为ATCCNO.31127的孢囊放线菌(Actinosporangium sp.)、保藏编号为ATCC NO.22959的少孢根霉(Rhizopus oligosporus)、保藏编号为ATCC NO.700922的Halobacterium sp.和保藏编号为DSM NO.44703的食苯假诺卡氏菌(Pseudonocardia benzenivorans),其CFU配比为1:2:5:4:3:3:4:5。
上述微生物复合菌剂的制备:
S1、将布氏乳杆菌、毕赤酵母菌、孢囊放线菌、少孢根霉、Halobacterium sp.和食苯假诺卡氏菌分别接种于第一低温胁迫培养基中,80rpm/min,28℃,培养72h;将深红红螺菌、沼泽红假单胞菌另行接种于第一低温胁迫无光培养基,170rpm/min,28℃,培养5D,无光;5天后转为塑料透明桶静置培养,设光源2*40瓦,照度750Lx,28℃,培养2D;第一低温胁迫培养基的配方为:酵母抽提物0.5g/L,酪蛋白水解物0.5g/L,马铃薯淀粉0.5g/L,胰蛋白胨0.25g/L,葡萄糖0.5g/L,丙酮酸钠0.3g/L,KH2PO4 0.5g/L,谷氨酸钠0.2g/L,NH4Cl 0.8g/L,MgSO4·7H2O 0.25g/L,CaCl2·2H2O 0.05g/L,微量元素溶液3ml,pH为6.5~7.5;第一低温胁迫无光培养基的配方为:NH4Cl 0.1g/L,NaHCO30.1g/L,K2HPO40.02g/L,葡萄糖0.5g/L,CH3COONa 0.3~0.5g/L,MgSO4·7H2O 0.02g/L,Na2S 0.01g/L,蛋白胨1.0g/L,酵母抽提物0.5g/L;生长因子1ml,微量元素溶液1ml,pH为7.0~8.0;
S2、第二培养阶段,将经过第一培养阶段培养的布氏乳杆菌、毕赤酵母菌、孢囊放线菌、少孢根霉、Halobacterium sp.和食苯假诺卡氏菌,按照15%的接种量分别接种于第二低温胁迫培养基中,40rpm/min,20℃,培养60h;将经过第一培养阶段培养的深红红螺菌、沼泽红假单胞菌另行接种于第二低温胁迫无光培养基,100rpm/min,20℃,培养5D,无光;5天后转为塑料透明桶静置培养,设光源2*40瓦,照度750Lx,20℃,培养2D;
第二低温胁迫培养基的配方为:酵母抽提物0.3g/L,酪蛋白水解物0.1g/L,马铃薯淀粉0.5g/L,胰蛋白胨0.5g/L,葡萄糖0.3g/L,丙酮酸钠0.5g/L,KH2PO4 0.5g/L,谷氨酸钠0.2g/L,NH4Cl 0.8g/L,MgSO4·7H2O 0.25g/L,CaCl2·2H2O 0.05g/L,微量元素溶液5ml,pH为6.5~7.5;第二低温胁迫无光培养基的配方为:NH4Cl 0.1g/L,NaHCO30.1 g/L,K2HPO40.02g/L,葡萄糖0.5g/L,CH3COONa 0.3~0.5g/L,MgSO4·7H2O 0.02g/L,Na2S 0.01g/L,蛋白胨1.0g/L,酵母抽提物0.5g/L;生长因子1ml,微量元素溶液1ml,pH为7.0~8.0;
其中,生长因子的制备方法如下:取维生素B1 0.1mg、乙尼克丁酸0.01mg、对氨基苯甲酸0.01mg、生物素0.0001mg,溶于蒸馏水中,定容至10ml,然后过滤除菌;微量元素溶液的制备方法如下:取FeCl3·6H2O 5.0mg、CuS04·5H2O 0.05mg、H3BO4 l.0mg、MnCl2·4H2O0.05mg、ZnSO4·7H2O 1.0mg、Co(NO3)2·6H2O 0.5mg,溶于蒸馏水中,定容至1000ml;S3、将经过第二培养阶段培养的布氏乳杆菌、毕赤酵母菌、深红红螺菌、沼泽红假单胞菌、孢囊放线菌、少孢根霉、Halobacterium sp.和食苯假诺卡氏菌,按照CFU配比1:2:5:4:3:3:4:5混合并悬浮于浓度6%的糖蜜溶液,得到总CFU浓度为5×108CFU/mL的液态微生物复合菌剂。
上述微生物复合菌剂在有机废水处理中的应用:向每升待处理的含有甲苯、二氯甲烷的有机废水中加入总量为5×109CFU的微生物复合菌剂。
实施例2
本实施例的微生物复合菌剂,包括布氏乳杆菌、毕赤酵母菌、深红红螺菌、沼泽红假单胞菌、孢囊放线菌、少孢根霉、Halobacterium sp.和食苯假诺卡氏菌,其CFU配比为2:3:6:5:4:4:5:6,总CFU浓度为5×108CFU/mL。
其余制备方法及应用同实施例1。
实施例3
本实施例中的微生物复合菌剂,包括布氏乳杆菌、毕赤酵母菌、深红红螺菌、沼泽红假单胞菌、孢囊放线菌、少孢根霉、Halobacterium sp.和食苯假诺卡氏菌,其CFU配比为1:2.5:5:4.5:3:3.5:4:5.5,总CFU浓度为5×108CFU/mL。
其余制备方法及应用同实施例1。
实施例4
本实施例中的微生物复合菌剂,包括布氏乳杆菌、毕赤酵母菌、深红红螺菌、沼泽红假单胞菌、孢囊放线菌、少孢根霉、Halobacterium sp.和食苯假诺卡氏菌,其CFU配比为1.5:3:5.5:5:3.5:4:4.5:6,总CFU浓度为5×108CFU/mL。
其余制备方法及应用同实施例1。
实施例5
本实施例中的微生物复合菌剂,包括布氏乳杆菌、毕赤酵母菌、深红红螺菌、沼泽红假单胞菌、孢囊放线菌、少孢根霉、Halobacterium sp.和食苯假诺卡氏菌,其CFU配比为1:3:5:5:4:4:4:5,总CFU浓度为5×108CFU/mL。
其余制备方法及应用同实施例1。
将实施例1~5制成的微生物复合菌剂分别以5×109CFU的量投入于1L有机废水水体中作为实验组;不做任何处理的1L废水水体作为对照组,所取水样与实验组相同。36h后检测实验组与对照组相关指标的实验结果如下表所示:
综上,本发明制备的微生物复合菌剂对有机废水中的COD、NH3-N、TN、TP等都有着极高的去除率,其中,COD的去除率最高可达81.1%、NH3-N可达91.7%、TN可达85.3%、TP可达79%。
Claims (4)
1.一种微生物复合菌剂,其特征在于,由布氏乳杆菌、毕赤酵母菌、深红红螺菌、沼泽红假单胞菌、孢囊放线菌、少孢根霉、Halobacterium sp.和食苯假诺卡氏菌按CFU配比1~2:2~3:5~6:4~5:3~4:3~4:4~5:5~6混合得到;
其中,所述布氏乳杆菌为lactobacillus buchneri,保藏编号为ATCC NO.11307;所述毕赤酵母菌为Pichia sp.,保藏编号为ATCC NO.28487;所述深红红螺菌为Rhodospirillum rubrum,属PNSB,保藏编号为ATCC NO.11170;所述沼泽红假单胞菌为Rhodopseudomonas palustris,保藏编号为CICC NO.23812;所述孢囊放线菌为Actinosporangium sp.,保藏编号为ATCC NO.31127;所述少孢根霉为Rhizopus oligosporus,保藏编号为ATCC NO.22959;所述Halobacterium sp.,保藏编号为ATCC NO.700922;所述食苯假诺卡氏菌为Pseudonocardia benzenivorans,保藏编号为DSM NO.44703;
所述微生物复合菌剂采用如下制备方法:
S1、分别将所述布氏乳杆菌、毕赤酵母菌、孢囊放线菌、少孢根霉、Halobacterium sp.和食苯假诺卡氏菌进行低温胁迫培养,所述深红红螺菌、沼泽红假单胞菌另行在无光的条件下进行低温胁迫培养;
S2、胁迫培养后的微生物按比例混合均匀,得到所述微生物复合菌剂;
其中,所述步骤S1中,低温胁迫培养包括:
第一培养阶段,将所述布氏乳杆菌、毕赤酵母菌、孢囊放线菌、少孢根霉、Halobacterium sp.和食苯假诺卡氏菌分别接种于第一低温胁迫培养基中,80rpm/min,28℃,培养72h,所述第一低温胁迫培养基的配方为:酵母抽提物0.5g/L,酪蛋白水解物0.5g/L,马铃薯淀粉0.5g/L,胰蛋白胨0.25g/L,葡萄糖0.5g/L,丙酮酸钠0.3g/L,KH2PO4 0.5g/L,谷氨酸钠0.2g/L,NH4Cl 0.8g/L,MgSO4·7H2O 0.25g/L,CaCl2·2H2O 0.05g/L;微量元素溶液3ml,pH为6.5~7.5;
将所述深红红螺菌、沼泽红假单胞菌分别另行接种于第一低温胁迫无光培养基,170rpm/min,28℃,培养5D,无光;5天后转为塑料透明桶静置培养,设光源2*40瓦,照度750Lx,28℃,培养2D;所述第一低温胁迫无光培养基的配方为:NH4Cl 0.1g/L,NaHCO30.1 g/L,K2HPO40.02 g/L,葡萄糖0.5g/L,CH3COONa 0.3~0.5g/L,MgSO4·7H2O 0.02g/L,Na2S0.01g/L,蛋白胨1.0g/L,酵母抽提物0.5g/L;生长因子1ml,微量元素溶液1ml,pH为7.0~8.0;
第二培养阶段,将经过第一培养阶段培养的布氏乳杆菌、毕赤酵母菌、孢囊放线菌、少孢根霉、Halobacterium sp.和食苯假诺卡氏菌,按照15%的接种量分别接种于第二低温胁迫培养基中,40rpm/min,20℃,培养60h,所述第二低温胁迫培养基的配方为:酵母抽提物0.3g/L,酪蛋白水解物0.1g/L,马铃薯淀粉0.5g/L,胰蛋白胨0.5g/L,葡萄糖0.3g/L,丙酮酸钠0.5g/L,KH2PO4 0.5g/L,谷氨酸钠0.2g/L,NH4Cl 0.8g/L,MgSO4·7H2O 0.25g/L,CaCl2·2H2O 0.05g/L;微量元素溶液5ml,pH为6.5~7.5;
将经过第一培养阶段培养的深红红螺菌、沼泽红假单胞菌分别另行接种于第二低温胁迫无光培养基,100rpm/min,20℃,培养5D,无光;5天后转为塑料透明桶静置培养,设光源2*40瓦,照度750Lx,20℃,培养2D;所述第二低温胁迫无光培养基的配方为:NH4Cl0.1g/L,NaHCO30.1 g/L,K2HPO40.02 g/L,葡萄糖0.5g/L,CH3COONa 0.3~0.5g/L,MgSO4·7H2O0.02g/L,Na2S 0.01g/L,蛋白胨1.0g/L,酵母抽提物0.5g/L;生长因子1ml,微量元素溶液1ml,pH为7.0~8.0;
所述生长因子的制备方法如下:取维生素B1 0.1mg、乙尼克丁酸0.01mg、对氨基苯甲酸0.01mg、生物素0.0001mg,溶于蒸馏水中,定容至10ml,然后过滤除菌;所述微量元素溶液的制备方法如下:取FeCl3·6H2O 5.0mg、CuS04·5H2O 0.05mg、H3BO4 l.0mg、MnCl2·4H2O0.05mg、ZnSO4·7H2O 1.0mg、Co(NO3)2·6H2O 0.5mg,溶于蒸馏水中,定容至1000ml。
2.根据权利要求1所述的一种微生物复合菌剂,其特征在于,由布氏乳杆菌、毕赤酵母菌、深红红螺菌、沼泽红假单胞菌、孢囊放线菌、少孢根霉、Halobacterium sp.和食苯假诺卡氏菌按CFU配比1:2:5:4:3:3:4:5混合得到。
3.根据权利要求1所述的一种微生物复合菌剂,其特征在于,所述微生物菌剂为液态菌剂,所述微生物菌剂中微生物的总浓度为3×108CFU/mL~5×108CFU/mL。
4.根据权利要求1所述的一种微生物复合菌剂,其特征在于,所述步骤S2的具体操作如下:将经过第二培养阶段培养的微生物,按照比例混合并悬浮于浓度6%的糖蜜溶液,得到微生物复合菌剂,并常温保存。
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