解淀粉芽孢杆菌M1摇瓶发酵条件优化及脱氮性能评价
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摘要
旨在提高解淀粉芽孢杆菌M1液体发酵产芽孢量,并考察其对实际废水的反硝化脱氮效果。首先采用单因子实验研究了碳源、氮源、初始pH值、温度、转速和装液量等因子对M1液体发酵产芽孢量的影响。然后对其中显著性因子:氮源浓度、接种量、装液量、温度4个因素进行正交试验,进一步优化发酵条件。结果显示,优化后的培养基成分为:5 g/L碳源(可溶性淀粉)、10 g/L氮源(酵母粉∶蛋白胨=2∶1)、1 g/L NaCl;最佳培养条件为:初始pH6.5、发酵温度34℃、摇床转速180 r/min、培养瓶装液量30%、接种量7%。在此条件下,芽孢杆菌M1芽孢数可达到6.8×108 CFU/mL,高于优化前的2.08×108 CFU/mL。将芽孢杆菌M1投加于反应器中,与不加菌种相比,反硝化脱氮效果提升15%-34%。
This work aims to increase the total spores of Bacillus amyloliquefaciens M1 in fermentation,and investigate the anaerobic denitrification effect in wastewater. First,single factor experiments were used to investigate the effects of species and concentrations of various carbon and nitrogen sources,initial pH,temperature,shaking speed,medium volume etc. on the spore yield of M1.Then orthogonal experiments were carried out to optimize 4 significant factors of nitrogen source concentration,inoculation amount,liquid loading amount and temperature. Results showed that the optimal composition of the medium were as such :5 g/L carbon source(soluble starch),10 g/L nitrogen source[yeast powder and tryptone mix(2∶1,V/V)],1 g/L NaCl. The optimal cultural condition was as :initial pH6.5,temperature 34℃,shaking speed 180 r/min,medium capacity 30%(75 mL/250 mL),and inoculation rate 7%. Under the optimal fermentation condition,the total spores yield of B. amyloliquefaciens M1 was up to 6.8×108 CFU/mL,2.3 times higher than that cultured in LB medium(2.08×108 CFU/mL). Compared with the control test,test with M1 addition showed 15%-34% higher denitrification.
This work aims to increase the total spores of Bacillus amyloliquefaciens M1 in fermentation,and investigate the anaerobic denitrification effect in wastewater. First,single factor experiments were used to investigate the effects of species and concentrations of various carbon and nitrogen sources,initial pH,temperature,shaking speed,medium volume etc. on the spore yield of M1.Then orthogonal experiments were carried out to optimize 4 significant factors of nitrogen source concentration,inoculation amount,liquid loading amount and temperature. Results showed that the optimal composition of the medium were as such :5 g/L carbon source(soluble starch),10 g/L nitrogen source[yeast powder and tryptone mix(2∶1,V/V)],1 g/L NaCl. The optimal cultural condition was as :initial pH6.5,temperature 34℃,shaking speed 180 r/min,medium capacity 30%(75 mL/250 mL),and inoculation rate 7%. Under the optimal fermentation condition,the total spores yield of B. amyloliquefaciens M1 was up to 6.8×108 CFU/mL,2.3 times higher than that cultured in LB medium(2.08×108 CFU/mL). Compared with the control test,test with M1 addition showed 15%-34% higher denitrification.
引文
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[15]张子豪,康建依,谢远红,等.高效抑制大肠埃希氏菌的芽孢杆菌的发酵培养基及发酵条件优化[J].食品工业科技,2018, 39(19):114-118.
[16]汪晶晶,曹雪梅,李欢,等.抗菌海洋解淀粉芽孢杆菌GM-1-2菌株摇瓶发酵培养基和发酵条件优化[J].江苏农业科学,2018, 46(8):291-295.
[17]李鑫,郝林,马玲.解淀粉芽孢杆菌HRH317抑菌物质发酵条件的优化[J].中国酿造, 2017, 36(2):25-29.
[18]曹雪梅,汪晶晶,贾杰,等.海洋细菌GM-1-1菌株摇瓶发酵培养基和发酵条件优化[J].食品科技, 2017, 42(6):23-29.
[19]He X, Zhang W, Ren F, et al. Screening fermentation parameters of the milk-clotting enzyme produced by newly isolated Bacillus amyloliquefaciens D4 from the Tibetan Plateau in China[J].Annals of Microbiology, 2012, 62(1):357-365.
[20]王卉,尚庆茂,张志刚,等.解淀粉芽孢杆菌L-H15产促生物质分析及发酵工艺优化[J].食品科学, 2017, 38(10):74-81.
[21]王涛,刘健,左建英,等.解淀粉芽孢杆菌高密度发酵条件的优化[J].中国测试, 2017, 43(5):49-52.
[22]Ren H, Su YT, Guo XH. Rapid optimization of spore production from Bacillus amyloliquefaciens in submerged cultures based on dipicolinic acid fluorimetry assay[J]. AMB Express, 2018, 8(1):21.
[23]Bricker SB, Ferreira JG, Zhu C, et al. Role of shellfish aquaculture in the reduction of eutrophication in an urban estuary[J].Environmental Science&Technology, 2018, 52(1):173.
[24]Prosser JI. Autotrophic nitrification in bacteria[J]. Advances in Microbial Physiology, 1990, 30:125-181.
[25]郭丽芸,时飞,杨柳燕,等.好氧反硝化菌处理低浓度含氮污水[J].江苏农业科学, 2016, 44(8):523-526, 575.
[2]李文超,王文浩,何岩,等.黑臭河道沉积物中硫铁行为与氮磷循环的耦合机制[J].华东师范大学学报:自然科学版, 2015(2):1-8, 29.
[3]吕佳佳,杨娇艳,廖卫芳,等.黑臭水形成的水质和环境条件研究[J].华中师范大学学报:自然科学版, 2014, 48(5):711-716.
[4]Stutter MI, Graeber D, Evans CD, et al. Balancing macronutrient stoichiometry to alleviate eutrophication[J]. Science of the Total Environment, 2018, 634:439-447.
[5]闫兴成,王明玥,许晓光,等.富营养化湖泊沉积物有机质矿化过程中碳、氮、磷的迁移特征[J].湖泊科学, 2018, 30(2):306-313.
[6]桓清柳,庞仁松,周秋伶,冷科明.深圳近岸海域氮、磷营养盐变化趋势及其与赤潮发生的关系[J].海洋环境科学, 2016,35(6):908-914.
[7]Ménesguen A, Desmit X, Dulière V, et al. How to avoid eutrophication in coastal seas? A new approach to derive river-specific combined nitrate and phosphate maximum concentrations[J]. Science of the Total Environment, 2018, 628-629:400-414.
[8]孙锦宜.含氮废水处理技术与应用[M].北京:化学工业出版社, 2003.
[9]沈萍,陈向东.微生物学实验[M].北京:高等教育出版社,2007:26-30.
[10]国家环境保护总局.水和废水监测分析方法[M].第4版.北京:中国环境科学出版社, 2002:53-57.
[11]李娟,夏凯丽,王远宏,等.解淀粉芽孢杆菌LJ1摇瓶发酵条件优化[J].生物技术通报, 2015, 31(12):214-220.
[12]Fang CJ, Lu ZX, Sun LJ, et al. Optimization of fermentation technologyforlipopeptidesproducingbacteriaBacillus amyloliquefaciens ES-2-4[J]. Scientia Agricultura Sinica, 2008,41(2):533-539.
[13]梁昌聪,郭立佳,刘磊,等.响应面法优化解淀粉芽孢杆菌C101发酵培养基[J].生物技术通报, 2014(8):169-174.
[14]刘京兰,薛雅蓉,刘常宏.内生解淀粉芽孢杆菌CC09产Iturin A摇瓶发酵条件优化[J].微生物学通报, 2014, 41(1):75-82.
[15]张子豪,康建依,谢远红,等.高效抑制大肠埃希氏菌的芽孢杆菌的发酵培养基及发酵条件优化[J].食品工业科技,2018, 39(19):114-118.
[16]汪晶晶,曹雪梅,李欢,等.抗菌海洋解淀粉芽孢杆菌GM-1-2菌株摇瓶发酵培养基和发酵条件优化[J].江苏农业科学,2018, 46(8):291-295.
[17]李鑫,郝林,马玲.解淀粉芽孢杆菌HRH317抑菌物质发酵条件的优化[J].中国酿造, 2017, 36(2):25-29.
[18]曹雪梅,汪晶晶,贾杰,等.海洋细菌GM-1-1菌株摇瓶发酵培养基和发酵条件优化[J].食品科技, 2017, 42(6):23-29.
[19]He X, Zhang W, Ren F, et al. Screening fermentation parameters of the milk-clotting enzyme produced by newly isolated Bacillus amyloliquefaciens D4 from the Tibetan Plateau in China[J].Annals of Microbiology, 2012, 62(1):357-365.
[20]王卉,尚庆茂,张志刚,等.解淀粉芽孢杆菌L-H15产促生物质分析及发酵工艺优化[J].食品科学, 2017, 38(10):74-81.
[21]王涛,刘健,左建英,等.解淀粉芽孢杆菌高密度发酵条件的优化[J].中国测试, 2017, 43(5):49-52.
[22]Ren H, Su YT, Guo XH. Rapid optimization of spore production from Bacillus amyloliquefaciens in submerged cultures based on dipicolinic acid fluorimetry assay[J]. AMB Express, 2018, 8(1):21.
[23]Bricker SB, Ferreira JG, Zhu C, et al. Role of shellfish aquaculture in the reduction of eutrophication in an urban estuary[J].Environmental Science&Technology, 2018, 52(1):173.
[24]Prosser JI. Autotrophic nitrification in bacteria[J]. Advances in Microbial Physiology, 1990, 30:125-181.
[25]郭丽芸,时飞,杨柳燕,等.好氧反硝化菌处理低浓度含氮污水[J].江苏农业科学, 2016, 44(8):523-526, 575.