第四章微生物发酵常用菌种及扩大课件.ppt

1,Outline of a fermentation process,Raw Materials,Production microorganism,Fermentation,Product purification,Product,Effluent waste,Chapter 3 Strain Breeding and scale-up,Five Common Characteristics of Microorganisms,1. Small volume, large surface area2. Fast absorption and conversion3. Rapid duplication and growth4. Strong adaptability5. Widespread distribution and diversified species,What types of microorganisms cause fermentation to occur?,BacteriaYeastMoldActinomycetes,Bacteria Bacteria are typically one-celled organisms that multiply by simple division and can be seen only with a microscope.,1. Lactic acid bacteriaLactobacillusyoghourt Streptococcusyoghourt Pediococcus sour picklesBifidobacteriumprobiotics,Lactobacillus,Streptococcus,Pediococcus,Bifidobacterium,2. Acetic acid bacteria Acetobactervinegar,3.Glutamic acid fermentative bacteriumCorynebacteriumglutamicumBrevibacterium flavum,4.Bacillus,Bacillus subtilis- protease ,amylase Bacillus licheniformis -protease , amylase,Yeast Yeasts are unicellular (single-celled) fungi and that ordinarily reproduce by budding.,Happy Yeast,Saccharomyces cerevisiae,1. Saccharomyces Saccharomyces cerevisiaebeer Saccharomyces ellipsoideuswine 2.Candida Candida utilis SCP,Mould Fungi that grow in the form of multicellular filaments, called hyphae. In contrast, microscopic fungi that grow as single cells are called yeasts.,1. Mucorchinese cheese2. Rhizopus R.oryzoe amylase3. Aspergillus A.nigercitric acid, glucoamylase A.oryzaesoy sauce, pasta (sweet)sauces,Fungi,Plant or animals,Actinomycetes,Microbes related to antibiotic producting,Streptomyces -streptomycin,About SCP, which is correct?A. Is a kind of protein which extracted from microbial cells.B. Is microbial cell which produced by fermentation.C. Is antibiotics secreted by microbial cells.D. Could not be regarded as food.,Can antibiotic producting microbes be used in the amino acids production?,Choosing Microorganismsfor Industrial Microbiology 1.The nutritional characteristics of the organism. 2. The productivity of the organism, 3.The stability of the organism and its amenability to genetic manipulation. 4. The ease of product recovery from the culture.,Strain Selection,Purchase from Culture CollectionsScreening of nature circumstances MutationsGenetic engineering,Culture Collections,Steps of new species separation and screening,Screening of nature circumstances Most major sources of microbes for use in industrial microbiology are natural materials,Microbial strain collection 1. soil slurry 2. Enrichment Special media Control condition Inhibit unwanted strain,3. Isolation cultivation Streak plate method Pour plate method Spread plate method,Streak plate method,Bacteria are “streaked” over the surface of an agar plate so as to obtain single colonies. It can also highlight the presence of contaminating micro-organisms.,This is an example of a good streak for isolation using the four corners method.,Performing a Plate Streak I,Performing a Plate Streak II,Pour plate method Bacteria are mixed with melted agar. Poured into an empty plate and allowed to solidify.,Spread plate method Small samples of the diluted bacteria are pipetted onto the surface of agar plates. A sterile, bent-glass rod is then used to spread the bacteria evenly over the entire agar surface.,稀释的,4. Pure cultivationGrow only one kind of microbeMust use aseptic technique to avoid contaminating the cultureTransfer a single colony from agar plate to liquid medium,5. Productivity measurement Primary screening：quantity Secondary screening：quality,If I couldnt find an ideal strain from nature, how would I do?,Spontaneous (natural) mutationInduced mutation,mutation,Technique for inducing mutation,Physical mutagens e.g. X-rays, g-rays, UVChemical mutagens e.g. base analogs, nitrous acid, alkylating agents,碱基类似物,亚硝酸,烷化剂,Steps:,How are mutants detected by scientists?VisibleNutritional (auxotrophic)What is replica plating, and how is it used to detect auxotrophic mutants?Resistance mutations (plate on media containing the chemical),营养缺陷型,影印培养法,降解纤维素产生透明圈,摇床复筛,Replica-plating technique to screen for mutant strains of a colony-forming microorganism.,Q: How would you select for protease(amylase, celluase or lase)-producing mutant strain?,Cell Biology Techniques,ProtoplastRemoving the cell wall with lytic enzymes in the presence of osmotic stabilizers.In the presence of fusogenic agent such as polyethylene glycol (PEG), protoplasts are induced to fuse and form transient hybrids or diploids.Regeneration of viable cells from the fused protoplasts.,融合剂,二倍体,Genetic Engineering,Various high value added products have been produced fromgenetic engineeringmethods.,Culture preservation,dry,oxygen deficit,low temperaure,Culture preservation,Oligotrophe,菌种保藏的方法很多，一般有下面几种：A ：斜面冰箱保藏法B：沙土管保藏法 C：石蜡油封存法 D：真空冷冻干燥保藏法E：液氮超低温保藏法,If fermentation in a large tank receive only one loop of inoculum, a prolonged period would result.energy consumingcontamination,Necessity of inoculum development,Inoculum Development The preparation of a population of microorganisms from a dormant stock culture to an active state of growth that is suitable for inoculation in the final production stage.,休眠的,So the inoculum volume has to be quite largeA seed fermenter is usually required to produce the inoculum volumeThe seed fermenters purpose is not to produce product but to prepare inoculum,Aeration: 通风, 通气,In inoculum developmentInoculum level: approximately 0.5 to 5 percent inoculum by volume from the preceding step.Incubation period: short, in log growth phase, little fermentation productInoculum media: balanced for rapid cell growth and not for product formation.,The process of inoculum development,实验室阶段,1、培养物选择的原则,目的：种子扩培到一定的量和质，根据菌种的特点最终的培养物可分为两类：,对于不产孢子和芽胞的微生物 获得一定数量和质量的菌体,对于不产芽孢和孢子的微生物，实验室阶段的种子扩培最终是获得一定数量和质量的菌体，如谷氨酸的种子培养。,获得一定数量和质量的孢子,菌丝体培养步骤少，因而更容易获得量和质稳定的种子，但操作繁琐。,对于产孢子的微生物,获得一定数量和质量的菌丝体,便于操作，但需要更仔细的控制。,2、培养基选择的原则,培养基的选择应该是有利于菌体的生长，对孢子培养基应该是有利于孢子的生长。,在原料方面，实验室种子培养阶段，规模一般比较小，因此为了保证培养基的质量，培养基的原料一般都比较精细。,3、起始接种物的传代问题,细菌,保藏斜面 活化斜面,产孢子,保藏 母斜面 子斜面,目的：使菌种的传代次数尽可能的少。,母瓶：活化、纯化，使保藏菌种生长，并去除变异株。 所以接种时要稀一点、便于纯化生长到单菌落。,子瓶: 大量繁殖，得到大量孢子。 接种：从母斜面上点接种，选取生长好的 单菌落 接种时密一点，得到大量的孢子。,孢子培养时注意湿度，子斜面使用一般不超过1个月,孢子培养,生产车间阶段,1、培养物的选择原则,在生产车间阶段，最终一般都是获得一定数量的菌丝体。,缩短发酵时间,有利于获得好的发酵结果,菌丝体比孢子要有利：,Inoculation of a Fermenter from another Seed Tank,2、培养基选择的原则,目的：获得一定数量和质量的菌体，因此培养基的选择应首先考虑的是有利于孢子的发育和菌体的生长.,在原料方面：不如实验室阶段那么精细，而是基本接近于发酵培养基，这有两个方面的原因: 一是成本 二是驯化,薯干粉,成分 斜面 种子罐 发酵麦芽汁 麦汁 薯干粉 10% 22-28%(NH4)2SO4 0.5%琼脂 2%,3、发酵级数的确定,谷氨酸：三级发酵,一级种子（摇瓶）二级种子 （小罐）发酵,青霉素：三级发酵,一级种子 （小罐）二级种子（中罐）发酵,一般由菌丝体培养开始计算发酵级数，但有时，工厂从第一级种子罐开始计算发酵级数,发酵级数确定的依据,级数受发酵规模、菌体生长特性、接种量的影响,级数大，难控制、易染菌、易变异，管理困难，一 般2-4级。,在发酵产品的放大中，反应级数的确定是非常重要 的一个方面,Aeration: 通风, 通气,Industrial Fermentation Setting,Review Questions1. Cite some micro-organisms used widely in food industry and their corresponding products.2. How to get an ideal strain for fermentation industry? 3. Whats the purpose of inoculum development ? Cite the process of it.,3. Why can not the micro-organisms product excessive metabolites spontaneously? By which methods can we get the excessive metabolites?4.How to reduce the concentration of end products in fermentation?5.How to improve the permeability of cell membrane?,