餐饮废水高效降解蛋白菌株的分离筛选及诱变选育研究

Reserch of Isolation and Mutagenesis of Highly Effective Protein-degrading Strain

作者: 专业:生物物理 导师:秦广雍 年度:2010 学位:硕士  院校: 郑州大学

Keywords

restaurant wastewater, rate of protein degradation, isolation and screening, Bacillus megaterium, UV mutagenesis, ion beam implantion

        餐饮废水排放量大,污染严重,其处理逐渐成为城市生活污水处理的新重点。国内外,通过微生物发酵处理餐饮废水的技术逐渐兴起。本实验从采自屠宰场、豆制品加工厂、餐馆及学校餐厅下水道口的4个样品中分离筛选得到高效降解餐饮废水蛋白的菌株D-4,对该菌株的菌落形态、生理生化指标等方面进行了研究,初步鉴定为巨大芽孢杆菌(Bacillus megaterium)。对D-4菌株的生长曲线研究显示:0~4h该菌生长处于迟缓期,4~28h为对数增长期,28h后进入稳定期;蛋白降解曲线研究发现:降解36h时,蛋白降解率已达到81.67%。以从自然界分离筛选出的B. megaterium D-4为出发原始菌株,利用紫外照射和离子束注入诱变进行复合诱变育种,在紫外诱变时,选择照射时间为4min(存活率为17.10%);低能离子注入时,选择能量30 keV,剂量6×1015·ion/cm2,存活率为13.40%。在该条件下对出发菌株进行复合诱变,通过初筛和复筛,选育出一株突变株D-4-29-3,其蛋白降解率比原始出发菌株D-4提高了18.20%,也大大缩短了生长周期和蛋白降解周期,经15次传代实验表明该菌株遗传稳定性良好。在菌株D-4-29-3降解条件的研究中,探讨了pH、温度、接种量、摇床转速、不同碳源及不同配比发酵培养基成分对蛋白降解率的影响。确定其降解蛋白的最优发酵培养基配比与降解的最佳工艺条件如下:蔗糖1.0%,酪蛋白1.0%,酵母膏0.2%,K2HPO4 0.3%;初始pH 7.0,种龄12h,接种量4.0%,温度32~37℃,摇床转速180 rpm/min,降解24h。在此条件下,其蛋白降解率最高可达97.56%。
    Restaurant wastewater has large emissions and serious pollution,the treament has gradually become the new focus of the sewage treatment of urban life.At home and abroad, the technology to deal with food wastewater through microbial fermentation gradually rises.In the experiment,protein degrading strain was isolated from four protein-contained sewage samples which were collected from slaughterhouse、bean curd manufactory、cloaca of university and restaurant.One bacterial strain D-4 for high-protein degradation was screened.Based on morphology, physiology, biochemistry analysis, this bacterial strain was preliminarily identified as Bacillus megaterium,From the growth curve and protein-degrading curve of D-4,it is showed that D-4 is in the lag phase during 0~4h,in the logarithmic phase during 4-28h and in the stationary phase after 28h;it is also showed that the rate of degradation is up to 81.67% at 36h.On the base of the original strain Bacillus megaterium D-4 from nature,was treated with UV and ion beam. In the UV mutagenesis, the exposure time was 4min (the survival rate was17.10%);and under ion beam implantion, the energy of 30keV and dose of 6×10 ion/cm2, the survival rate was 13.40%. Under these conditions, one mutant named D-4-29-3 was bred by primary screening and second screening,the protein degradation rate of was increased by 18.20%, and also the period of degreding protein and growth period were sharp shorten. The stain has good genetic stability after 15 times of subculture.In the study of protein-degrading conditions of strain D-4-29-3,we dicussed the influence on protein-degrading rate of factors pH value、temperature、inoculum size、dissolved oxygen、carbon nutriments and different ratio of fermentation medium composition. The optimum fermentation medium compositions were determined as: Sucrose 1.0%、casin 1.0%、yeast extract 0.2%、K2HPO4 0.3% and initial pH 7.0;and the better conditions of protein degradation was selected for 24h at 32~37℃on a rotary shaker(180~200r/min) incoulated by 4 percent.In these conditions,the highest rate of degrading protein could reach 97.56%.
        

餐饮废水高效降解蛋白菌株的分离筛选及诱变选育研究

摘要4-5
Abstract5
符号说明7-8
目录8-13
图表清单13-14
1 引言14-15
2 文献综述15-28
    2.1 餐饮废水及其处理研究概况15-21
        2.1.1 餐饮废水的特征15
        2.1.2 餐饮废水对环境的污染及对人们日常生活的危害15-16
        2.1.3 目前国内外餐饮废水的处理方法概况16-20
            2.1.3.1 焚烧17
            2.1.3.2 卫生填埋17-18
            2.1.3.3 堆肥18
            2.1.3.4 泔水生产微生物蛋白饲料技术18-19
            2.1.3.5 泔水生产氢气技术19
            2.1.3.6 泔水生产生物柴油技术19-20
            2.1.3.7 其他技术20
        2.1.4 特殊微生物技术在废水处理中的应用20-21
    2.2 紫外线诱变技术在环境工程中的应用21-22
    2.3 离子注入微生物诱变育种22-27
        2.3.1 离子注入的生物学机理23-25
            2.3.1.1 电荷交换效应23
            2.3.1.2 表面刻蚀和容积损伤23-24
            2.3.1.3 产生自由基24
            2.3.1.4 辐射细胞近旁效应24
            2.3.1.5 离子注入生物效应进程24-25
        2.3.2 离子注入微生物诱变育种的特点及诱变程序25
        2.3.3 离子束诱变技术在微生物育种中的应用25-27
    2.4 本项研究的目的与内容27-28
3 餐饮废水蛋白降解菌株的分离、筛选及鉴定28-37
    3.1 实验材料28-30
        3.1.1 分离样品28
        3.1.2 部分试剂28
        3.1.3 培养基28-29
        3.1.4 部分溶液配方29
        3.1.5 实验仪器设备29-30
    3.2 实验方法30-31
        3.2.1 蛋白含量的测定30
        3.2.2 富集30
        3.2.3 分离30
        3.2.4 初筛方法30
        3.2.5 复筛方法30-31
        3.2.6 菌种鉴定31
            3.2.6.1 形态特征的鉴定31
            3.2.6.2 生理生化特征鉴定31
    3.3 结果与分析31-36
        3.3.1 蛋白含量测定的标准曲线31-32
        3.3.2 初筛结果32
        3.3.3 复筛结果32-33
        3.3.4 菌种初步鉴定33-35
            3.3.4.1 形态特征33
            3.3.4.2 生理生化特征33-35
        3.3.5 生长与降解特性35-36
            3.3.5.1 生长曲线35
            3.3.5.2 降解曲线35-36
    3.4 本章小结36-37
4 餐饮废水高效降解蛋白菌株的诱变选育37-48
    4.1 实验材料37
        4.1.1 菌种37
        4.1.2 培养基37
        4.1.3 实验仪器设备37
    4.2 实验方法37-39
        4.2.1 蛋白降解菌株的紫外诱变选育37-38
            4.2.1.1 诱变出发菌株生长曲线37
            4.2.1.2 菌悬液的制备37-38
            4.2.1.3 诱变最佳时间的选择38
            4.2.1.4 初筛方法:琼脂块法38
            4.2.1.5 复筛方法38
        4.2.2 蛋白降解菌株的离子注入诱变选育38-39
            4.2.2.1 诱变出发菌株生长曲线38-39
            4.2.2.2 离子注入诱变流程39
            4.2.2.3 诱变最佳剂量的选择39
            4.2.2.4 初筛方法39
            4.2.2.5 复筛方法39
    4.3 结果与分析39-47
        4.3.1 蛋白降解菌株的紫外诱变选育39-42
            4.3.1.1 出发菌株诱变菌龄的选择39
            4.3.1.2 紫外线诱变剂量的选择39-40
            4.3.1.3 初筛结果40-41
            4.3.1.4 复筛结果41-42
        4.3.2 蛋白降解菌株的离子注入诱变选育42-47
            4.3.2.1 出发诱变菌株的菌龄选择42-43
            4.3.2.2 离子束诱变剂量的选择43
            4.3.2.3 初筛结果43-44
            4.3.2.4 复筛结果44-45
            4.3.2.5 D-4-29-3菌株的生长与降解特性45-47
    4.4 本章小结47-48
5 餐饮废水高效降解蛋白诱变菌株降解条件的初步研究48-57
    5.1 实验材料48
        5.1.1 菌种48
        5.1.2 培养基48
        5.1.3 实验仪器设备48
    5.2 实验方法48-50
        5.2.1 发酵过程中pH的变化48
        5.2.2 初始pH对蛋白降解率的影响48
        5.2.3 温度对蛋白降解率的影响48
        5.2.4 溶氧量对蛋白降解率的影响48-49
        5.2.5 接种量对蛋白降解率的影响49
        5.2.6 不同碳源对蛋白降解率的影响49
        5.2.7 发酵培养基的正交实验49
        5.2.8 菌种保存49-50
            5.2.8.1 斜面保存49-50
            5.2.8.2 二甲基亚砜(DMSO)保存50
    5.3 结果与分析50-56
        5.3.1 发酵过程pH的变化50
        5.3.2 初始pH对蛋白降解率的影响50-51
        5.3.3 温度对蛋白降解率的影响51-52
        5.3.4 溶氧量对蛋白降解率的影响52-53
        5.3.5 接种量对蛋白降解率的影响53-54
        5.3.6 不同碳源对蛋白降解率的影响54
        5.3.7 发酵培养基的正交实验结果54-56
    5.4 本章小结56-57
6 结论57-58
参考文献58-63
个人简历 在学期间发表的学术论文与研究成果63-64
致谢64
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