利用啤酒废弃物为原料进行Bt液态发酵的研究
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摘要
苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)是迄今最成功、最广泛使用的一种微生物杀虫剂。与使用化学农药导致严重的“3R”问题相比,该微生物杀虫剂具有易于生产、专一性、对脊椎动物无毒的特点。目前原料成本高是Bt难于推广的最重要原因之一,迫切需要研发一种便宜、易得的生产培养基及经济的发酵路线。啤酒厂废水及其废弃物来源广,废水属中、高浓度的有机废水,非常适合开发高附加值的生物制品,但目前国内外尚未见有关啤酒废水培养Bt的报道。本研究思路是以啤酒废弃物为培养基培养Bt,可缓解其他原料的缺陷,以期达到减少环境污染,降低Bt生产成本,促进Bt生产推广的目的。
(1)本研究跟踪观测了啤酒废水、城市污水COD的日/月变化,并以不同污水处理工段污水(+污泥)为原料培养苏云金杆菌BRC-WLY_1。研究表明:啤酒废水的年平均COD达1342.0 mg/L,是城市污水(COD 167.7 mg/L)的8倍,其平均COD月间变异系数分别为16.7%、57.3%,日变异系数分别在42-52.4%、184.4-391.3%范围;与城市污水相比,啤酒废水具有高且稳定的COD,可生化性强、卫生好等特点;以不同工段的啤酒废水+废弃物为原料发酵Bt,其活芽胞数均可达10~9 cfu/mL级,产晶体蛋白高、而发酵时间仅20 h左右;较好的组合是4/5(1/2酸化废水+1/2啤酒原废水)+1/5酵母液,适当地补充碳、氮有利于BRC-WLY_1的发酵。由于啤酒废弃物来源丰富且易得,完全适合做为Bt发酵的工业化生产原料。
(2)为了提高酵母泥的利用率,减轻后期发酵的灭菌工作,本研究率先采用环境工程预处理污水、污泥的处理方式,对啤酒废弃物进行预处理,研究发现A处理方式是较好的处理方法;并采用正交优化处理酵母泥,其最佳的预处理条件是新鲜酵母泥稀释6倍、A处理方式处理时间2 min、pH 5、高压蒸汽灭菌消毒时间45 min,其中稀释倍数对还原糖和氨基氮生成的影响最大。处理后啤酒废水、城市污泥、酵母泥氨基氮分别提高了73%、64.2%、198%,酵母泥按优化条件预处理后,氨基氮收得率为
4.37%(处理后称酵母液)。不但提高原料利用率,降低生产成本,又可达到无菌要求。(3)为了寻求适合污水为培养基的Bt菌株,本研究从不同的污水处理厂及其不同工段分离Bt菌株。40个样品中分离到芽胞菌112株,其中镜检有2株为Bt,占2.7%;对分离菌株的生物学特性、形态学、生理生化指标等进行测定;通过SDS-PAGE分析其蛋白质片段,并采用cry1-cry11、cyt、vip3A、aiiA和inhA 14对引物,通过PCR-RFLP鉴定体系对其cry基因型进行分析。结果表明:BRC-WLY_1、BRC-WLY2均含有65 kD蛋白片段,且都含有cry1(cry1Ag,cry1Ba,cry1Gb,cry1La)、cry2(cry2Ac)、vip3A和aiiA基因,BRC-WLY_1还含有inhA基因。与标准菌株8010和HD-1相比,分离的两株菌可缩短发酵时间6-8 h(缩短20%-30%),BRC-WLY_1发酵周期最短仅15 h,能缩短近50%的发酵时间,活菌数和晶体蛋白均较高,BRC-WLY_1发酵所得晶胞混合物为0.1312 g/25mL,对2-3龄小菜蛾具更高的毒力,48 h校正死亡率分别达到96.6%和100%,而8010和HD-1分别为89.7%,93.1%。
(4)为了寻找啤酒废水+酵母液培养BRC-WLY_1可能缺陷的营养因子,采用单因素实验确定葡萄糖、N物质、(NH4)2SO4、酵母液、KH_2PO_4、ZnSO_4、吐温80、NaCl为主要的营养限制因子,并采用PB(Plackett-Burman)、RSM(响应面优化)进一步优化培养基,获得的优化培养基组成(W/V)为:以1/2啤酒原废水+1/2酸化废水为溶液,添加葡萄糖0.2%、(NH_4)_2SO_4 0.1%、ZnSO_4 0.05%、吐温80 0.15%、NaCl 0.6%、N物质0.4%、酵母液30%(V/V)、KH_2PO_4 0.12%。优化后芽胞数可达10.86×10~8 cfu/mL,比优化前增加了5.1倍,OD_(595)由0.194增加至0.258。
优化的最佳发酵条件是:初始pH 7.5-8、装液量80 mL(500 mL三角瓶)、发酵温度30-34℃、接种量5%。最佳补料方式为发酵8 h,加入10%的酵母液,与未补料相比,芽胞数、晶体干重和生产强度和单位糖产量分别提高了8、1.78、0.98、3.07倍。
综上所述,啤酒废弃物适合作为Bt发酵的工业化生产原料,从“老”污水系统分离的高效Bt菌株,更适合于啤酒废弃物为培养基的发酵,可达到有效转化啤酒废弃物和生产高效低成本Bt杀虫剂的双盈利目的。
Bacillus thuringiensis(Bt) is the most widely and successfully used bio-insecticide. It has the characteristics of easily production , specificity and no toxicity to vertebrates , comparing to chemical pesticides which caused“3R”problems when used. However,production of Bt formulations using existing expensive raw material and fermentation technology incurs heavy expenditure. There is one of the primary causes why the use of these biopesticides has limitations. Hence,it is imperative to develop a cheaper media for culturing Bt and providing a feasible technical route,which would facilitate the production of biopesticides in a cost effective manner. The quantity of wastewater generated from brewery is large and is characterized by a high pollution load,and its sustainable management via value-addition is an alternative efficacious route to other disposal options,namely,biological wastewater treatment processes. So far, there is no report about that Bt-based biopesticide production from brewery wastewater and its castoff. In this study, brewery wastewater and its castoff were used as a novel medium to cultivate Bt based on clean-up option as well as augmentation path for commercialization of economical biopesticides.
The wastewater formed flow and COD variations days/months of sewage from urban sewage-digestive plant and brewery wastewater-digestive plant were extensively investigated. In the year,the average COD of sewage from brewery wastewater-digestive plant is 1342.0 mg/L which is 8 folds higher than that of sewage from urban sewage-digestive plant. The variability coefficient of months for urban sewage-digestive plant and brewery wastewater is 16.7% and 57.3% respectively, and 42-52.4% and 184.4-391.3% for variation days. Wastewater from brewery wastewater-digestive plant possesed relatively invariable COD and with abundance nutrition and sanitation.
Brewery raw wastewater/acidified waste water+yeast fluid (brewery-waste-medium) from different working procedure were found to be suitable for culturing Bacillus thuringiensis strain BRC-WLY1,which could produce spores up to 10~9cfu/mL and high crystal compound as well as short fermentation cycle (around 20h). The optium combination is 4/5 (1/2 acidified wastewater + 1/2 brewery raw wastewater)+ 1/5 pretreatmented yeast fluid. Supply carbon and nitrogen is favor to BRC-WLY_1 production. Due to brewery wastewater/sludge/ waste yeast slurry is a cheaper raw material and very easy to obtain,it would be a good material for the industrialisation production of Bt-based bioinsecticide.
In order to improve the utilization ratio of brewery wastes and to ease the sterilization with high pressure steam,the methods to pretreat sludge/sewage used in environment engineering were selected to pretreat brewery sludge /sewage. It showed that method A is the top choice. An orthogonal design was furthur used to optimize the condition, including dilution ratio, pH, pretreatment time, and/or the time of sterilization with high pressure steam, for the pretreament of brewery wastewater and sludge, and waste yeast slurry. The optimal condition comprised 6 times diluent of the waste yeast slurry ,pH at 5.0,pretreatment time for 2 min and/or the time of sterilization with high pressure steam for 45min was obtained. By the optimal pretreatment condition, the produce ofα-amino nitrogen after pretreatment improved up to 73%, 64.2% and 198% respectively for brewery wastewater, sludge and waste yeast slurry, and the harvest rate of theα-amino nitrogen for waste yeast slurry is 4.37%.
The sewages collected from the different process stages and sewage-digestive plants were used to isolated Bt strains, and 2 strains (named BRC-WLY_1 and BRC-WLY2) were obtained. Besides the investigation of the morphology, biochemical characteristics and fermentation performance, the gene and protein types of the above two strains were analysis by PCR-RFLP with 14 couples specific primers and by SDS-PAGE respectively. The results indicated that both BRC-WLY_1 and BRC-WLY2 harbored the genes of cry1(cry1Ag,cry1Ba,cry1Gb,cry1La) , cry2(cry2Ac) , vip3A and aiiA , and the protein with 65KD. Compared to the two control strains HD-1 and 8010, BRC-WLY_1 and BRC-WLY2 possess shorter fermentation cycle(nearly cut 20%-30%). The minimum fermentation cycle of BRC-WLY_1 is 15h and its hybrid of crystal and sporangium in the ferment liquid is 0.1312g/25mL. Bioassay results showed that the correction mortalitie at 48h of BRC-WLY_1, BRC-WLY2, 8010 and HD-1 was 96.6%, 100%, 89.7% and 93.1% respectively for 2-3rd instar larvae of Plutella xylostella.
To seek the nutritional deficiency factors in brewery-waste-medium for BRC-WLY1,8 nutritional factors were optimized by single factor experiments. Based on the above result, the Plackett-Burman (PB)Methods and the response surface methodology(RSM) were used to further optimize the fermentation medium.The optimal brewery-waste-medium comprised 0.2% of glucose,0.1% of (NH4)2SO_4,0.05% of ZnSO_4,0.6% of NaCl,0.15% of Tuween80,0.4% of material A,30% of pretreatmented yeast fluid , 0.12% of KH_2PO_4 and 1/2brewery waste water+1/2 acidified wastewater was obtained, which could produce spores up to 10.86×10~8 cfu/mL resulted in a 5.1 times increase of sporangium yield in comparison with the original medium. OD595 increased to 0.258 from 0.194 that of fermentation without optimized.
The single factor experiment were used to optimize fermentation conditions and the optimal conditions comprised initial pH at 7.5-8,80 mL of liquid volume in the 500mL flask,fermentation temperature at 30-34℃and 5% of inoculation volume was obtained . The optimal fed batch mode is fedding 10%(V/V) of yeast fluid after culturing for 8 h,which resulted in spores yield, crystal weightiness, production intensity and yield of units sugar increased 8, 1.78, 0.98 and 3.07 times than those of fermentation without fed batched.
In conclusion,BRC-WLY1 and BRC-WLY2 isolated from sewage/sludge are suitable to grow in brewery-wastewater-medium. It suggested that brewery wastewater could be used for the industrialisation production of Bt based bioinsecticide,which would effectively utilize the bio-organic waste materials from the environment,on the one hand, and reduce the cost the Bt formulation on the other.
(1)本研究跟踪观测了啤酒废水、城市污水COD的日/月变化,并以不同污水处理工段污水(+污泥)为原料培养苏云金杆菌BRC-WLY_1。研究表明:啤酒废水的年平均COD达1342.0 mg/L,是城市污水(COD 167.7 mg/L)的8倍,其平均COD月间变异系数分别为16.7%、57.3%,日变异系数分别在42-52.4%、184.4-391.3%范围;与城市污水相比,啤酒废水具有高且稳定的COD,可生化性强、卫生好等特点;以不同工段的啤酒废水+废弃物为原料发酵Bt,其活芽胞数均可达10~9 cfu/mL级,产晶体蛋白高、而发酵时间仅20 h左右;较好的组合是4/5(1/2酸化废水+1/2啤酒原废水)+1/5酵母液,适当地补充碳、氮有利于BRC-WLY_1的发酵。由于啤酒废弃物来源丰富且易得,完全适合做为Bt发酵的工业化生产原料。
(2)为了提高酵母泥的利用率,减轻后期发酵的灭菌工作,本研究率先采用环境工程预处理污水、污泥的处理方式,对啤酒废弃物进行预处理,研究发现A处理方式是较好的处理方法;并采用正交优化处理酵母泥,其最佳的预处理条件是新鲜酵母泥稀释6倍、A处理方式处理时间2 min、pH 5、高压蒸汽灭菌消毒时间45 min,其中稀释倍数对还原糖和氨基氮生成的影响最大。处理后啤酒废水、城市污泥、酵母泥氨基氮分别提高了73%、64.2%、198%,酵母泥按优化条件预处理后,氨基氮收得率为
4.37%(处理后称酵母液)。不但提高原料利用率,降低生产成本,又可达到无菌要求。(3)为了寻求适合污水为培养基的Bt菌株,本研究从不同的污水处理厂及其不同工段分离Bt菌株。40个样品中分离到芽胞菌112株,其中镜检有2株为Bt,占2.7%;对分离菌株的生物学特性、形态学、生理生化指标等进行测定;通过SDS-PAGE分析其蛋白质片段,并采用cry1-cry11、cyt、vip3A、aiiA和inhA 14对引物,通过PCR-RFLP鉴定体系对其cry基因型进行分析。结果表明:BRC-WLY_1、BRC-WLY2均含有65 kD蛋白片段,且都含有cry1(cry1Ag,cry1Ba,cry1Gb,cry1La)、cry2(cry2Ac)、vip3A和aiiA基因,BRC-WLY_1还含有inhA基因。与标准菌株8010和HD-1相比,分离的两株菌可缩短发酵时间6-8 h(缩短20%-30%),BRC-WLY_1发酵周期最短仅15 h,能缩短近50%的发酵时间,活菌数和晶体蛋白均较高,BRC-WLY_1发酵所得晶胞混合物为0.1312 g/25mL,对2-3龄小菜蛾具更高的毒力,48 h校正死亡率分别达到96.6%和100%,而8010和HD-1分别为89.7%,93.1%。
(4)为了寻找啤酒废水+酵母液培养BRC-WLY_1可能缺陷的营养因子,采用单因素实验确定葡萄糖、N物质、(NH4)2SO4、酵母液、KH_2PO_4、ZnSO_4、吐温80、NaCl为主要的营养限制因子,并采用PB(Plackett-Burman)、RSM(响应面优化)进一步优化培养基,获得的优化培养基组成(W/V)为:以1/2啤酒原废水+1/2酸化废水为溶液,添加葡萄糖0.2%、(NH_4)_2SO_4 0.1%、ZnSO_4 0.05%、吐温80 0.15%、NaCl 0.6%、N物质0.4%、酵母液30%(V/V)、KH_2PO_4 0.12%。优化后芽胞数可达10.86×10~8 cfu/mL,比优化前增加了5.1倍,OD_(595)由0.194增加至0.258。
优化的最佳发酵条件是:初始pH 7.5-8、装液量80 mL(500 mL三角瓶)、发酵温度30-34℃、接种量5%。最佳补料方式为发酵8 h,加入10%的酵母液,与未补料相比,芽胞数、晶体干重和生产强度和单位糖产量分别提高了8、1.78、0.98、3.07倍。
综上所述,啤酒废弃物适合作为Bt发酵的工业化生产原料,从“老”污水系统分离的高效Bt菌株,更适合于啤酒废弃物为培养基的发酵,可达到有效转化啤酒废弃物和生产高效低成本Bt杀虫剂的双盈利目的。
Bacillus thuringiensis(Bt) is the most widely and successfully used bio-insecticide. It has the characteristics of easily production , specificity and no toxicity to vertebrates , comparing to chemical pesticides which caused“3R”problems when used. However,production of Bt formulations using existing expensive raw material and fermentation technology incurs heavy expenditure. There is one of the primary causes why the use of these biopesticides has limitations. Hence,it is imperative to develop a cheaper media for culturing Bt and providing a feasible technical route,which would facilitate the production of biopesticides in a cost effective manner. The quantity of wastewater generated from brewery is large and is characterized by a high pollution load,and its sustainable management via value-addition is an alternative efficacious route to other disposal options,namely,biological wastewater treatment processes. So far, there is no report about that Bt-based biopesticide production from brewery wastewater and its castoff. In this study, brewery wastewater and its castoff were used as a novel medium to cultivate Bt based on clean-up option as well as augmentation path for commercialization of economical biopesticides.
The wastewater formed flow and COD variations days/months of sewage from urban sewage-digestive plant and brewery wastewater-digestive plant were extensively investigated. In the year,the average COD of sewage from brewery wastewater-digestive plant is 1342.0 mg/L which is 8 folds higher than that of sewage from urban sewage-digestive plant. The variability coefficient of months for urban sewage-digestive plant and brewery wastewater is 16.7% and 57.3% respectively, and 42-52.4% and 184.4-391.3% for variation days. Wastewater from brewery wastewater-digestive plant possesed relatively invariable COD and with abundance nutrition and sanitation.
Brewery raw wastewater/acidified waste water+yeast fluid (brewery-waste-medium) from different working procedure were found to be suitable for culturing Bacillus thuringiensis strain BRC-WLY1,which could produce spores up to 10~9cfu/mL and high crystal compound as well as short fermentation cycle (around 20h). The optium combination is 4/5 (1/2 acidified wastewater + 1/2 brewery raw wastewater)+ 1/5 pretreatmented yeast fluid. Supply carbon and nitrogen is favor to BRC-WLY_1 production. Due to brewery wastewater/sludge/ waste yeast slurry is a cheaper raw material and very easy to obtain,it would be a good material for the industrialisation production of Bt-based bioinsecticide.
In order to improve the utilization ratio of brewery wastes and to ease the sterilization with high pressure steam,the methods to pretreat sludge/sewage used in environment engineering were selected to pretreat brewery sludge /sewage. It showed that method A is the top choice. An orthogonal design was furthur used to optimize the condition, including dilution ratio, pH, pretreatment time, and/or the time of sterilization with high pressure steam, for the pretreament of brewery wastewater and sludge, and waste yeast slurry. The optimal condition comprised 6 times diluent of the waste yeast slurry ,pH at 5.0,pretreatment time for 2 min and/or the time of sterilization with high pressure steam for 45min was obtained. By the optimal pretreatment condition, the produce ofα-amino nitrogen after pretreatment improved up to 73%, 64.2% and 198% respectively for brewery wastewater, sludge and waste yeast slurry, and the harvest rate of theα-amino nitrogen for waste yeast slurry is 4.37%.
The sewages collected from the different process stages and sewage-digestive plants were used to isolated Bt strains, and 2 strains (named BRC-WLY_1 and BRC-WLY2) were obtained. Besides the investigation of the morphology, biochemical characteristics and fermentation performance, the gene and protein types of the above two strains were analysis by PCR-RFLP with 14 couples specific primers and by SDS-PAGE respectively. The results indicated that both BRC-WLY_1 and BRC-WLY2 harbored the genes of cry1(cry1Ag,cry1Ba,cry1Gb,cry1La) , cry2(cry2Ac) , vip3A and aiiA , and the protein with 65KD. Compared to the two control strains HD-1 and 8010, BRC-WLY_1 and BRC-WLY2 possess shorter fermentation cycle(nearly cut 20%-30%). The minimum fermentation cycle of BRC-WLY_1 is 15h and its hybrid of crystal and sporangium in the ferment liquid is 0.1312g/25mL. Bioassay results showed that the correction mortalitie at 48h of BRC-WLY_1, BRC-WLY2, 8010 and HD-1 was 96.6%, 100%, 89.7% and 93.1% respectively for 2-3rd instar larvae of Plutella xylostella.
To seek the nutritional deficiency factors in brewery-waste-medium for BRC-WLY1,8 nutritional factors were optimized by single factor experiments. Based on the above result, the Plackett-Burman (PB)Methods and the response surface methodology(RSM) were used to further optimize the fermentation medium.The optimal brewery-waste-medium comprised 0.2% of glucose,0.1% of (NH4)2SO_4,0.05% of ZnSO_4,0.6% of NaCl,0.15% of Tuween80,0.4% of material A,30% of pretreatmented yeast fluid , 0.12% of KH_2PO_4 and 1/2brewery waste water+1/2 acidified wastewater was obtained, which could produce spores up to 10.86×10~8 cfu/mL resulted in a 5.1 times increase of sporangium yield in comparison with the original medium. OD595 increased to 0.258 from 0.194 that of fermentation without optimized.
The single factor experiment were used to optimize fermentation conditions and the optimal conditions comprised initial pH at 7.5-8,80 mL of liquid volume in the 500mL flask,fermentation temperature at 30-34℃and 5% of inoculation volume was obtained . The optimal fed batch mode is fedding 10%(V/V) of yeast fluid after culturing for 8 h,which resulted in spores yield, crystal weightiness, production intensity and yield of units sugar increased 8, 1.78, 0.98 and 3.07 times than those of fermentation without fed batched.
In conclusion,BRC-WLY1 and BRC-WLY2 isolated from sewage/sludge are suitable to grow in brewery-wastewater-medium. It suggested that brewery wastewater could be used for the industrialisation production of Bt based bioinsecticide,which would effectively utilize the bio-organic waste materials from the environment,on the one hand, and reduce the cost the Bt formulation on the other.
引文
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