餐厨垃圾半固态发酵产Bt生物农药及其稳定性
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摘要
为降低苏云金芽孢杆菌(Bacillus thuringiensis,Bt)生物农药的生产成本,该研究采用餐厨垃圾为原料半固态发酵生产Bt生物农药,并对其产物稳定性的影响因素进行了考察。结果表明,发酵48 h后半固态发酵样品中的伴孢晶体产率较相同体积的固态发酵样品和液态发酵样品分别提高了54%和162%。体系含盐量、p H值、温度及紫外线4种常见因素中,体系含盐量的变化对伴孢晶体稳定性的影响并不显著;温度对伴孢晶体稳定性的影响显著,但当体系温度在0~60℃间变化时,各组数据差异并不显著。p H值对伴孢晶体稳定性的影响显著,且各数据间存在显著性差异;紫外线对伴孢晶体稳定性影响极显著,试验表明,当伴孢晶体在紫外线(36 W,距离40 cm)下暴露3 h后,其毒力效价即下降50%。为进一步提高伴孢晶体的抗紫外能力,采用聚乳酸为载体包覆伴孢晶体制备缓释剂,结果表明该缓释农药可有效延缓晶体在紫外线照射下的失活速度,使其在紫外线照射72 h后仍然能保持85%的活性,较相同照射条件下的伴孢晶体原粉提高了10倍左右,并能在30 d的作用周期内稳定的释放伴孢晶体,该成果对Bt生物农药产品优化具有重要的应用价值。
Kitchen garbage is usually discharged from restaurants, public catering rooms and school canteens, and it is reported that 2000 t kitchen garbage would be produced per day in Beijing, China. In other big cities similar phenomena can be observed, and it is reported that half of the municipal waste is composed of kitchen garbage. Kitchen garbage is characterized by high moisture content, easily deteriorating and abundant in nutrition, but it should be treated as a kind of resource rather than simple waste because it is mainly composed of starch, protein, cellulose, fat, and trace element, such as potassium(K), sodium(Na), calcium(Ca), magnesium(Mg), and iron(Fe); kitchen garbage can be regarded as a suitable substrate for microbial growth. In this research, kitchen garbage was used as the raw material to produce Bacillus thuringiensis(Bt) bio-pesticide which is one of the most commonly used and extensively studied bio-pesticides because of its various field applications, such as in agriculture, forestry, and public health sectors. However, the costly raw materials, high equipment investment, and complicated operation procedures in traditional Bt production process have seriously constrained its use at the industrial production scale. In this work we conducted to determine the potential and superiority of using kitchen garbage for Bt bio-pesticide production through semi-solid fermentation to reduce the production cost, and the effects of several parameters on the stability of the product were also studied. In this work, the semi-solid state sample with 75% water content presented the highest δ-endotoxin efficiency of 833 μg/mL, which increased by 54% compared to that of the solid-state sample(water content of 50%) and 162% compared to that of the submerged sample(water content of 99%), and proved that using kitchen garbage as the raw material for Bt production through semi-solid state fermentation has obvious advantages compared with traditional techniques and some similar studies. Mainly because of its nutritious substrate and effective fermentation mode, this technology can not only solve the problem of civil solid waste pollution but also bring a cost-effective resource for Bt bio-pesticide production. Among the main affecting factors during Bt bio-pesticide manufacturing and application, salinity, p H value and temperature had the minimal impact on the stability of the parasporal crystal; when these 3 factors were controlled at 3%, 11 and 60 ℃, respectively, the entomotoxicity of parasporal crystal could still remain above 70% after the fermentation of less for 48 h, and therefore common fermentation process would not inactivate the goal product sharply. By contrast, ultraviolet irradiation had more significant impact on parasporal crystal's stability, and the entomotoxicity of parasporal crystal declined by 50% when exposed to the ultraviolet(36 W, 60 μW/cm2, 40 cm) for about 3 h. On account of this, polylactic acid was used as the carrier to produce Bt slow-release formulation to mitigate the effects of ultraviolet. Experiments showed that the activity of Bt slow-release formulation remained over 75% of its initial value after 72 h ultraviolet irradiation, which was 10 times the untreated parasporal crystal. And the release rates of parasporal crystal in formulation were generally metastable in a 30-day period, and the compact structure of polylactic acid was proven to effectively protect δ-endotoxin from ultraviolet damage.
Kitchen garbage is usually discharged from restaurants, public catering rooms and school canteens, and it is reported that 2000 t kitchen garbage would be produced per day in Beijing, China. In other big cities similar phenomena can be observed, and it is reported that half of the municipal waste is composed of kitchen garbage. Kitchen garbage is characterized by high moisture content, easily deteriorating and abundant in nutrition, but it should be treated as a kind of resource rather than simple waste because it is mainly composed of starch, protein, cellulose, fat, and trace element, such as potassium(K), sodium(Na), calcium(Ca), magnesium(Mg), and iron(Fe); kitchen garbage can be regarded as a suitable substrate for microbial growth. In this research, kitchen garbage was used as the raw material to produce Bacillus thuringiensis(Bt) bio-pesticide which is one of the most commonly used and extensively studied bio-pesticides because of its various field applications, such as in agriculture, forestry, and public health sectors. However, the costly raw materials, high equipment investment, and complicated operation procedures in traditional Bt production process have seriously constrained its use at the industrial production scale. In this work we conducted to determine the potential and superiority of using kitchen garbage for Bt bio-pesticide production through semi-solid fermentation to reduce the production cost, and the effects of several parameters on the stability of the product were also studied. In this work, the semi-solid state sample with 75% water content presented the highest δ-endotoxin efficiency of 833 μg/mL, which increased by 54% compared to that of the solid-state sample(water content of 50%) and 162% compared to that of the submerged sample(water content of 99%), and proved that using kitchen garbage as the raw material for Bt production through semi-solid state fermentation has obvious advantages compared with traditional techniques and some similar studies. Mainly because of its nutritious substrate and effective fermentation mode, this technology can not only solve the problem of civil solid waste pollution but also bring a cost-effective resource for Bt bio-pesticide production. Among the main affecting factors during Bt bio-pesticide manufacturing and application, salinity, p H value and temperature had the minimal impact on the stability of the parasporal crystal; when these 3 factors were controlled at 3%, 11 and 60 ℃, respectively, the entomotoxicity of parasporal crystal could still remain above 70% after the fermentation of less for 48 h, and therefore common fermentation process would not inactivate the goal product sharply. By contrast, ultraviolet irradiation had more significant impact on parasporal crystal's stability, and the entomotoxicity of parasporal crystal declined by 50% when exposed to the ultraviolet(36 W, 60 μW/cm2, 40 cm) for about 3 h. On account of this, polylactic acid was used as the carrier to produce Bt slow-release formulation to mitigate the effects of ultraviolet. Experiments showed that the activity of Bt slow-release formulation remained over 75% of its initial value after 72 h ultraviolet irradiation, which was 10 times the untreated parasporal crystal. And the release rates of parasporal crystal in formulation were generally metastable in a 30-day period, and the compact structure of polylactic acid was proven to effectively protect δ-endotoxin from ultraviolet damage.
引文
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[8]张玮玮.以沼渣为原料固态发酵生产Bt生物农药[J].农业工程学报,2013,29(8):212-217.Zhang Weiwei,Gong Aijun,Qiu Lina,et al.Solid state fermentation of biogas residues for production of Bacillus Thuringiensis based bio-pesticide[J].Transactions of the Chinese Society of Agricultural Engineering(Transactions of the CSAE),2013,29(8):212-217.(in Chinese with English abstract)
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[12]马鸿志,宫利娟,汪群慧,等.Plackett-Burman试验设计优化餐厨垃圾发酵产燃料酒精的研究[J].环境科学,2008,29(5):1452-1456.Ma Hongzhi,Gong Lijuan,Wang Qunhui,et al.Optimization of fuel ethanol production from kitchen waste by plackett-burman design[J].Environmental Science,2008,29(5):1452-1456.(in Chinese with English abstract)
[13]周建强,方海兰,朱丽,等.3种测定餐厨垃圾中氯离子含量的方法比较[J].上海农业学报,2014,30(3):45-48.Zhou Jianqiang,Fang Hailan,Zhu Li,et al.Comparison of three methods for determining the content of chloride ion in food waste[J].Acta Agriculturae Shanghai,2014,30(3):45-48.(in Chinese with English abstract)
[14]崔云龙,田明,邵宗泽.紫外线使苏云金芽孢杆菌伴孢晶体失活机理的研究[J].微生物学通报,2003,28(4):193-197.
[15]游红,镇达,徐家文,等.苏云金芽胞杆菌微胶囊剂的制备及其抗紫外线降解活性研究[J].湖北农业科学,2008,47(11):1288-1291.
[16]Navon A,Keren S,Levski S,et al.Granular feeding baits based on Bacillus thuringiensis products for the control of lepidopterous pests[J].Phytoparasitica,1997,25:S101-S110.
[17]王子佳,李红梅,弓爱君,等.苏云金芽孢杆菌伴孢晶体的制备[J].化学与生物工程,2009,26(9):56-58.Wang Zijia,Li Hong Mei,Gong Aijun,et al.Preparation of Crystal Protein from Bacillus Thuringiensis[J].Chemistry&Bioengineering,2009,26(9):56-58.(in Chinese with English abstract)
[18]陈洪章.现代固态发酵技术-理论与实践[M].北京:化学工业出版社,2013.
[19]Zhang W W,Qiu L N,Gong A J,et al.Solid-state fermentation of food waste for production of Bacillus thuringiensis-based bio-pesticide[J].Bioresource,2013(8):1124-1135.
[20]Naveena B J,Altaf M,Bhadrayya K,et al.Production of L(+)-lactic acid by Lactobacillus amylophilus GV6 in semi-solid state fermentation using wheat bran[J].Food Technol Biotechnol,2004,42:147-152.
[21]韩北忠.发酵工程.中国轻工业出版社,2013.
[22]Economou C N,Makri A,Aggelis G,et al.Semi-solid state fermentation of sweet sorghum for the biotechnological production of single cell oil[J].Bioresour Technol,2010,101:1385-1388.
[23]史双枝,陈复生,赵俊廷.温度和盐离子强度对蛋白质和AOT分离及相互作用的影响[J].河南工业大学学报:自然科学版,2006,25(5):9-12.Shi Shuangzhi,Chen Fusheng,Zhao Junting,et al.The influence of temperature and Nacl ionic intension on the separation and combination of protein and aot[J].Journal of Henan University of Technology:Natural Science Edition,2006,25(5):9-12.(in Chinese with English abstract)
[24]许长明.温度对航空煤油中微生物生长繁殖的影响[J].石油化工腐蚀与护,2011,28(1):23-25.
[25]Marcel J I.The influence of salt and temperature on the natural fermentation of African locust bean[J].International Journal of Food Microbiology,1998,8:133-139.
[26]李淑艳,王建中.萌发p H值对大豆种子蛋白质代谢的影响研究[J].种子,2009,28(3):1-8.Li Shuyan,Wang Jianzhong.Study the effect of p H on protein metabolism during germination of soybean seed[J].Seed,2009,28(3):1-8.(in Chinese with English abstract)
[27]Ignoffo C M,Hostetter D L,Sikorowski P P,et al.Inactivation of representative species of entomo-pathogenic viruses,a bacterium,fungs and protozoan by an ultraviolet light source[J].Environ Entomli,2007,6(3):411-415.
[28]Zhao J,Wilk R M.Low molecular weight polylactic acid as amatrix for the delayed release of pesticides[J].Journal of Agricultural and Food Chemistry,2005,53:4076-4082.
[29]郭瑞峰,黄彬彬,杨晓伟,等.毒死蜱-聚乳酸微球的制备及其性能评价[J].农药学学报,2011,13(4):409-414.Guo Ruifeng,Huang Binbin,Yang Xiaowei,et al.Preparation and characteristics analysis of microspheres of chlorpyrifos and polylactic acid[J].Chinese Journal of Pesticide Science,2005,53:4076-4082.(in Chinese with English abstract)
[2]Benoit T G,Wilson G R,Baugh C L.Fermentation during growth and sporulation of Bacillus thuringiensis HD-1[J].Appl Microbiol,1990,l0:1518.
[3]Brar S K,Verma M,Tyagi R D,et al.Bacillus thuringiensis fermentation of hydrolyzed sludge-Rheology and formulation studies[J].Chemosphere,2007,67:674-683.
[4]Adams T T,Eiteman M A,Hanel B M.Solid state fermentation of broiler litter for production of biocontrol agents[J].Bioresource Technology,2002,82(1):33-41.
[5]杜雷,赵高岭,席宇,等.烟梗废料固态发酵生产苏云金芽孢杆菌的适宜条件筛选[J].烟草科技,2011(12):69-72.Du Lei,Zhao Gaoling,Xi Yu,et al.Screening of solid state fermentation conditions of waste stem for producing Bacillus Thuringiensis[J].Tobacco Science&Technology,2011,12:69-72.(in Chinese with English abstract)
[6]Zhuang Li,Zhou Shungui,Wang Yueqiang,et al.Cost-effective production of Bacillus thuringiensis biopesticides by solid-state fermentation using wastewater sludge:Effects of heavy metals[J].Bioresource Technology,2011,102(7):4820-4826.
[7]Wang X Q,Wang Q H,Ma H Z,et al.Lactic acid fermentation of food waste using integrated glucoamylase production[J].J Chem Technol.Biotechnol,2009,84:139-143.
[8]张玮玮.以沼渣为原料固态发酵生产Bt生物农药[J].农业工程学报,2013,29(8):212-217.Zhang Weiwei,Gong Aijun,Qiu Lina,et al.Solid state fermentation of biogas residues for production of Bacillus Thuringiensis based bio-pesticide[J].Transactions of the Chinese Society of Agricultural Engineering(Transactions of the CSAE),2013,29(8):212-217.(in Chinese with English abstract)
[9]Poopathi S,Abidha S.Use of feather-based culture media for the production of mosquitocidal bacteria[J].Biocontrol,2007,43:49-55.
[10]El-Bendary M A.Production of mosquitocidal Bacillus sphaericus by solid state fermentation using agricultural wastes[J].World J Microbiol.Biotechnol,2010,26:153-159.
[11]Holmberg A,Sievanen R.Fermentation of Bacillus thuringiensis for exotoxin Production:Process analysis study[J].Biotechnol Bioeng,1980,22:1707-1724.
[12]马鸿志,宫利娟,汪群慧,等.Plackett-Burman试验设计优化餐厨垃圾发酵产燃料酒精的研究[J].环境科学,2008,29(5):1452-1456.Ma Hongzhi,Gong Lijuan,Wang Qunhui,et al.Optimization of fuel ethanol production from kitchen waste by plackett-burman design[J].Environmental Science,2008,29(5):1452-1456.(in Chinese with English abstract)
[13]周建强,方海兰,朱丽,等.3种测定餐厨垃圾中氯离子含量的方法比较[J].上海农业学报,2014,30(3):45-48.Zhou Jianqiang,Fang Hailan,Zhu Li,et al.Comparison of three methods for determining the content of chloride ion in food waste[J].Acta Agriculturae Shanghai,2014,30(3):45-48.(in Chinese with English abstract)
[14]崔云龙,田明,邵宗泽.紫外线使苏云金芽孢杆菌伴孢晶体失活机理的研究[J].微生物学通报,2003,28(4):193-197.
[15]游红,镇达,徐家文,等.苏云金芽胞杆菌微胶囊剂的制备及其抗紫外线降解活性研究[J].湖北农业科学,2008,47(11):1288-1291.
[16]Navon A,Keren S,Levski S,et al.Granular feeding baits based on Bacillus thuringiensis products for the control of lepidopterous pests[J].Phytoparasitica,1997,25:S101-S110.
[17]王子佳,李红梅,弓爱君,等.苏云金芽孢杆菌伴孢晶体的制备[J].化学与生物工程,2009,26(9):56-58.Wang Zijia,Li Hong Mei,Gong Aijun,et al.Preparation of Crystal Protein from Bacillus Thuringiensis[J].Chemistry&Bioengineering,2009,26(9):56-58.(in Chinese with English abstract)
[18]陈洪章.现代固态发酵技术-理论与实践[M].北京:化学工业出版社,2013.
[19]Zhang W W,Qiu L N,Gong A J,et al.Solid-state fermentation of food waste for production of Bacillus thuringiensis-based bio-pesticide[J].Bioresource,2013(8):1124-1135.
[20]Naveena B J,Altaf M,Bhadrayya K,et al.Production of L(+)-lactic acid by Lactobacillus amylophilus GV6 in semi-solid state fermentation using wheat bran[J].Food Technol Biotechnol,2004,42:147-152.
[21]韩北忠.发酵工程.中国轻工业出版社,2013.
[22]Economou C N,Makri A,Aggelis G,et al.Semi-solid state fermentation of sweet sorghum for the biotechnological production of single cell oil[J].Bioresour Technol,2010,101:1385-1388.
[23]史双枝,陈复生,赵俊廷.温度和盐离子强度对蛋白质和AOT分离及相互作用的影响[J].河南工业大学学报:自然科学版,2006,25(5):9-12.Shi Shuangzhi,Chen Fusheng,Zhao Junting,et al.The influence of temperature and Nacl ionic intension on the separation and combination of protein and aot[J].Journal of Henan University of Technology:Natural Science Edition,2006,25(5):9-12.(in Chinese with English abstract)
[24]许长明.温度对航空煤油中微生物生长繁殖的影响[J].石油化工腐蚀与护,2011,28(1):23-25.
[25]Marcel J I.The influence of salt and temperature on the natural fermentation of African locust bean[J].International Journal of Food Microbiology,1998,8:133-139.
[26]李淑艳,王建中.萌发p H值对大豆种子蛋白质代谢的影响研究[J].种子,2009,28(3):1-8.Li Shuyan,Wang Jianzhong.Study the effect of p H on protein metabolism during germination of soybean seed[J].Seed,2009,28(3):1-8.(in Chinese with English abstract)
[27]Ignoffo C M,Hostetter D L,Sikorowski P P,et al.Inactivation of representative species of entomo-pathogenic viruses,a bacterium,fungs and protozoan by an ultraviolet light source[J].Environ Entomli,2007,6(3):411-415.
[28]Zhao J,Wilk R M.Low molecular weight polylactic acid as amatrix for the delayed release of pesticides[J].Journal of Agricultural and Food Chemistry,2005,53:4076-4082.
[29]郭瑞峰,黄彬彬,杨晓伟,等.毒死蜱-聚乳酸微球的制备及其性能评价[J].农药学学报,2011,13(4):409-414.Guo Ruifeng,Huang Binbin,Yang Xiaowei,et al.Preparation and characteristics analysis of microspheres of chlorpyrifos and polylactic acid[J].Chinese Journal of Pesticide Science,2005,53:4076-4082.(in Chinese with English abstract)
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