1、4. 微生物培养基 Cultural media,A cultural medium is the substance in which a specific organism lives and grows. It must contain the essential nutrients needed for the microbe to grow.A good medium is very important to the success of an industrial fermentation. The medium supplies nutrients for growth, ene
2、rgy, building of cell substance, and biosynthesis of fermentation products.,4.1 培养基的类型及功能 Classifications of media and their functions,按纯度分类： -合成培养基 chemically defined media: compound identity and concentration of all components are known. Synthetic media -天然培养基 chemically undefined media: ingredien
3、ts include one or more chemically complex substances such as protein hydrolysates and extracts. Crude media,The choice of defined or undefined medium is dependent upon its application. -Chemically defined media are useful in biochemical or metabolic studies of organisms. -General laboratory growth m
4、edia and industrial media are often chemically complex. Such as 花生饼、蛋白胨等。一般不需外加微量元素 (trace elements)、维生素等。,按状态分类： -Solid media：suitable to culture and preserve species and spores; cultivating fungi. -Liquid media: 80%-90%water, industrial fermentation media. -Semi-solid media: liquid medium + agar 主
5、要用于鉴定细菌、观察细菌运动特征等。,按用途分类 孢子培养基、种子培养基和发酵培养基1.孢子培养基 spore media 要求：使菌体迅速生长，产生较多的优质孢子，不易引起菌种发生变异 (variation)。 基本配制要求： not rich in nutrition, especially organic nitrogen soure. optimal mineral salt concentration optimal pH and humidity,生产上常用孢子培养基： 麸皮、大米、小米培养基；由葡萄糖、蛋白胨、牛肉膏和食盐等配制的琼脂斜面培养基。大米和小米常用作霉菌孢子培养基，因
6、它们含氮少、疏松、表面积大，是较好的孢子培养基。水分控制在2125。,种子培养基 inoculum media: 种子培养基是供孢子发芽、生长和大量繁殖菌丝体，并使菌丝体长得粗壮，成为活力强的种子。 Inoculum media are usually less nutritious than are production media, and they usually contain a considerably lower level of the main nutritive carbon source.,一般种子培养基常包括有机氮源和无机氮源最后一级的种子培养基的成分最好接近发酵培养基
7、。发酵培养基 production media 发酵培养基是供菌体生长、繁殖和合成产物之用。 要求：既要使种子接种后能迅速生长，达到一定的菌丝浓度，又要使长好的菌体迅速合成所需产物。,配制要求：其组成除有菌体生长所必需的元素和化合物外，还需有合成产物所需的特定元素、前体和促进剂等。In addition to the carbon and nitrogen compounds, a medium contains inorganic salts, vitamins, growth factors, precursors of fermentation products.,Microbial g
8、rowth under industrial fermentation conditions usually utilizes a “luxury metabolism” of the organisms. Thus, good yields of fermentation products occur only if relatively large amounts of carbon, and possibly nitrogen, are channeled through the metabolic pathways of the microorganism.,4.2 发酵培养基的成分及
9、来源Composition of media and their sources,The particular composition of a fermentation medium can be simple to complex depending on the particular microorganism and its fermentation. Autotrophic(自养的) microorganisms require only the simplest of inor-ganic media.Heterotrophic (异养的) microorganisms lack
10、the ability to synthesize many of their sustenance and growth requirements.,4.2.1 碳源 carbon sources,Carbon source is one of main composition of media.The functions are: providing carbons and energy; the components for synthesis of products. These can include simple sugars, complex carbohydrates, alc
11、ohols, amino and other organic acids, and short-chain lipids. 常用的碳源有糖类、油脂、有机酸和低碳醇。蛋白质、氨基酸也可用作碳源。,4.2.1.1 糖类 sugars,Sugars used mainly: glucose, molasses (糖蜜), dextrin (糊精).Glucose: most microbes can utilize it. 速效碳源、 high concentrationspeed respiration upDO no enoughintermediates (中间产物）accumulation
12、inhibiting growth,molasses (糖蜜)Beet(甜菜) and cane molasses are by-products of the sugar industry. containing 50% -75% fermentable sugars, mainly sucrose. Also containing nitrogenous compounds, vitamins and minerals. Cheaper,Starch and dextrin (糊精),Starch: -widely used in fermentation industry- starch
13、 hydrolyze using microbial amylase dextrin glucose-Most common used starches: corn, wheat, potato starches. Also using corn flour. -advantages: 缓效碳源、cheaper than glucose,油和脂肪 oils and fats,Oil and fats are also used as carbon sources because some microbes contain lipase (脂肪酶)Fat glycerol(甘油) and fat
14、ty acids oxidize CO2+ water, releasing much energyProviding enough O2 is important.常用的油：豆油、菜子油、猪油、鱼油、棉子油等。,有机酸 organic acids,Some microbes can use organic acids as carbon sources, such as lactic acid, citric acid, acetic acid. pH of cultural medium will increase. CH3COONa + O2 2CO2 + H2O + NaOH,烃和醇类
15、 hydrocarbon and ethanol,石油及裂解产物如正烷烃也可用作 carbon sources.自然界中能同化乙醇的微生物和能同化糖质的微生物一样普遍。,4.2.2 氮源 nitrogen sources,氮源主要用于构成细胞物质（氨基酸、蛋白质、核酸等）和含氮代谢物。有机氮源 organic nitrogen sources 常用的: 花生饼、棉子饼、玉米浆、玉米蛋白粉、蛋白胨、酵母粉、鱼粉、蚕蛹粉、尿素、废菌丝体和酒糟等。 organic nitrogen sources microbial proteinase amino acids metabolized by mic
16、robes.,organic nitrogen sources contain rich proteins, peptides, amino acids, vitamins and growth factors.在含有机碳源的培养基中，微生物常表现出生长旺盛、菌丝或细胞浓度增长迅速的特点。有些微生物对氨基酸有特殊的需求。如缬氨酸可提高红霉素的发酵单位。,玉米浆 Cornsteep Liquor,Cornsteep liquor is the water extract by-product resulting from the steeping (浸泡) of corn during the
17、commercial production of corn starch and other corn products.Of the 50% solids of cornsteep liquor, nearly half is lactic acid. The rest includes amino acids, glucose and other reducing sugars, salts, vitamins, and precursors (前体)such as those for the penicillin molecule.,The high lactic acid conten
18、t of cornsteep liquor results from the growth of lactic acid bacteria during its manufacture.This variation in composition, at times, can lead to poor reproducibility of an industrial fermentation.,尿素 ureaUrea is also a commonly used nitrogen source.Its feature: -single component, no nutritional fea
19、ture of complex nitrogen sources; -cheaper, often used in production of penicillin and glutamic acid.,yeast extractpeptones有机氮源除了作为菌体生长繁殖的营养外，有的还是产物的前体。缬氨酸、半胱氨酸和-是合成青霉素和头孢菌素的主要前体；甘氨酸可作为L-丝氨酸的前体等。,无机氮源 inorganic nitrogen source,常用的：氨水(ammonia)、铵盐(ammonium)和硝酸盐(nitrate)Microbes can utilize it faster.A
20、fter it is utilized, the pH of medium will be changed. (NH4)2SO4 2NH3 + H2SO4 NaNO3 + 4H2 NH3 + 2H2O +NaOH,生理酸性物质：NH4 酸性物，硫酸铵生理碱性物质： NH4 碱性物，硝酸铵正确使用生理酸碱性物质Except using as a nitrogen source, ammonia is often used to adjust pH of media. Stirring is necessary.,4.2.3 无机盐及微量元素 mineral salts and trace ele
21、ments,Mineral salts and trace elements are needed when microbial cells grow and reproduce. Most common mineral salts and trace elements: 磷(phosphorus)、镁 (magnesium)、硫 (sulphur)、钾(potassium)、钠(sodium)、铁(iron)、氯(chlorine)、锰(manganese)、锌(zinc)、钴(cobalt)、 钙等,作为微生物细胞生理活性物质组成或生理活性作用的调节物。When their concent
22、ration is low, they show a stimulation function for cell grow and product synthesis. When their concentration is high, they will inhibit the cell growth. Their optimal concentrations depend on the species and strains.,无机盐成分一般所用的浓度范围,These elements are added into cultural media in the form of their s
23、alts. Except synthetic media, cobalt钴, copper, iron, manganese锰, zinc, molybdenum钼 are not added into crude media.,Phosphorus element is one of nucleic acids and proteins composition, one component of ATP, energy transfer. Phosphorus can stimulate microbial growth and metabolism. But it inhibits cel
24、l growth when its concentration is high. 许多次级代谢过程对磷酸盐浓度的承受限度比生长繁殖过程低，故必须严格控制。,Magnesium does not involve the cell institution. But Mg is the activator for many important enzymes.如己糖磷酸化酶、柠檬酸脱氢酶等镁离子可提高抗生素产生菌的抗生素耐受能力，如链霉素、卡那霉素等产生菌。常以硫酸镁的形式加入培养基。,硫存在于细胞的蛋白质中， 是含硫氨基酸的组分和某些辅酶的活性基，如辅酶 A (coenzyme A), 谷胱甘肽等
25、。硫是某些产物如青霉素、头孢菌素等分子的组成部分，在培养基中加入Na2SO4等含硫化合物作硫源。,铁(iron)是细胞色素、细胞色素氧化酶和过氧化氢酶的成分，因此铁是菌体有氧氧化必不可少的元素。In iron-made fermentor, generally iron ion concentration can be 30 g/ml. So iron containing compounds are not necessary for industrial media.,Chlorine does not have nutritional functions for most microor
26、ganisms, but it is necessary for cell growth of halophile.一些产含氯代谢物如金霉素和灰黄霉素等的发酵中，除天然含有外，通常还需加入0.1%氯化钾。在啤酒生产中，2060mg/ml 的氯对酶和酵母有一定的促进作用。,Sodium, potassium, calcium are not components of cell constitution, they are still necessary composition for cultural media. 钠、钾离子与维持细胞的渗透压有关。钾离子是许多酶的激活剂，能促进糖代谢。钙是某
27、些酶(如蛋白酶)的激活剂，还参与细胞膜通透性的调节。培养基中钙盐过多时，会形成磷酸钙沉淀。,锌、镁、钴、锰等是某些酶的辅基或激活剂。镁离子还可提高抗生素生产菌对自己所产生抗生素的耐性；钴既是一些酶的激活剂，又是VB12的组成元素，发酵中加入一定量的钴盐，能使VB12的产量提高数倍；锰对于羧化作用是必需的，糖代谢中许多酶的活性都与锰有关。,4.2.4 Water,Main composition of mediaFunctions: -join directly some metabolisms -solvents -providing a physiological environments
28、for cell growth and synthesis of products.Effect of water quality on fermentation,4.2.5 生长因子、前体、产物促进剂,(1) 生长因子 growth factorsDefinition: small amount of organic compounds necessary for microbial growth. Features: no synthesis by itself. Examples：amino acids, purines, vitaminsSources: normally organi
29、c nitrogen source. Cornsteep liquor.,(2) 前体 precursors,Definition: substances added prior to or simultaneously with the fermentation, which are incorporated without any major change into the molecule of the fermentation product, and which generally serve to increase the yield or improve the quality
30、of the product.,前体是指某些化合物加入到发酵培养基中，能直接被微生物在生物合成过程中结合到产物分子中去，而其自身的结构并没有多大变化，但是产物的产量却因加入前体而有较大的提高。Examples：苯乙酸青霉素G 苯氧乙酸青霉素V,发酵过程中所用的一些前体物质,(3) 产物促进剂,产物促进剂 product stimulator Definition: substances are neither nutrients nor precursors, and increase product yield after adding them. Examples: Tween (0.1%
31、) amylase(淀粉酶), cellulase(纤维素酶) 大豆酒精提取物 cellulase,Mechanisms: not very clearPossible reasons: -enzyme inducers 诱导剂 -improvement of cell permeability 渗透性 -protection on enzyme activity,4.3 培养基的设计及优化media design and its optimization,Importance of media designCurrent situations of media design: many fa
32、ctors empirical 经验 + scientific 4.3.1 培养基成分选择的原则 (1) 菌体的同化能力： large molecules + enzymes small molecules,Selection of carbon sources: glucose, starches,不同糖化工艺所得糖液质量的比较,Selection of nitrogen sources: -If the microbe produces proteolytic enzymes, a variety of crude nitrogen sources such as soybean meal
33、 can be used. -If the microbe lack of proteases, the organic nitrogen source should be hydrolyzed. 豆饼粉water + hydrochloric acid 盐酸, pH1.0 100C amino acids.,(2) 代谢物的阻遏和诱导 -葡萄糖效应 - combination of organic and inorganic nitrogen/carbon sources -enzyme agent production: For many induced enzymes, starch a
34、nd dextrin are common carbon source.,碳源对生长和产酶的影响,-age of microorganisms -inducement and inhibition of nitrogen source for some products. (3) 合适的C、N比 -The ratio of C、N have greatly effects on microbial growth and product synthesis -一般工业发酵培养基的C、N比为100: (0.22.0) - Many factors influence the optimal rat
35、io,(4) pH的要求,A important environment factorFluctuation (波动) of cultural pHOptimal media formulation is the key factor to avoid fluctuation of cultural pH除考虑营养需求，也要考虑其代谢后对培养体系pH缓冲的贡献，使pH 处于较适宜的状态,4.3.2 培养基的优化 media optimization,The best media formulation is decided by tests.A general procedure of med
36、ia optimization: (1) 根据前人的经验和培养基成分选择的原则，初步确定可能的培养基成分； (2) decision of best media with single factor test (3) optimization of each component concentration by some test design.,The criterion for a best industrial medium,1. 培养基能够满足产物的最经济合成2. By-product concentration as low as possible 3. The raw materials are cheaper, easy to transport and store. The supply is steady. 4. 能满足总体工艺的要求，如不影响通气、提取、纯化及废物处理,
