Chapter 4: Nutrition and Media 第四章 : 微生物营养和培养基

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1 Chapter 4: Nutrition and Media 第四章 : 微生物营养和培养基

2 The whole of nature, as has been said, is a conjugation of the verb to eat, in the active and passive. - William Ralph Inge

3 Content 1. Nutrient requirement for microorganism 2. Transferring of substance into cells 3. Different medium for specific purpose

4 Nutrition 营养 Nutrition 营养 ( 作用 ) include: 1. Substance 物质 2. Energy 能量 Nutrient 营养 ( 物 ): Substance of nutritional values

5 Nutritional requirements at elementary level 在元素水平上的营养需求 Revealed by cell's elemental composition Macro-: C, H, O, N, S. P, K, Mg, Fe, Ca Micro-: Mn, Zn, Ni, Co 钴, Cu, Mo 钼 Forms: Water, inorganic ions 无机离子, small molecules, and macromolecules Either a structural or functional role

6 Major elements, their sources and functions in bacterial cells Element % dry weight Source Function Carbon 50 organic compounds or CO 2 Main constituent of cellular material Oxygen 20 Nitrogen 14 Hydrogen 8 H 2 O, organic compounds, CO 2, and O 2 NH 3, NO 3, organic compounds, N 2 H 2 O, organic compounds, H 2 Constituent of cell material and cell water; O 2 is electron acceptor in aerobic respiration Constituent of amino acids, nucleic acids nucleotides, and coenzymes Main constituent of organic compounds and cell water Phosphoru s 3 inorganic phosphates (PO 4 ) Constituent of nucleic acids, nucleotides, phospholipids, LPS, teichoic acids Sulfur 1 SO 4, H 2 S, So, organic sulfur compounds Potassium 1 Potassium salts Magnesiu m Calcium 0.5 Magnesium salts 0.5 Calcium salts Iron 0.2 Iron salts Constituent of cysteine, methionine, glutathione, several coenzymes Main cellular inorganic cation and cofactor for certain enzymes Inorganic cellular cation, cofactor for certain enzymatic reactions Inorganic cellular cation, cofactor for certain enzymes and a component of endospores Component of cytochromes and certain nonheme iron-proteins and a cofactor for some enzymatic reactions

7 Nutrient Requirements of Microbes 微生物的六种营养要素 1. Carbon source 碳源 2. Nitrogen source 氮源 3. Energy 能量 4. Growth factor 生长因子 5. Inorganic substance 无机物 6. Water 水

8 1. Carbon Sources 碳源 Carbon requirements: A wide range, may also be energy source Organic carbon, CO 2 or CO, methane 甲烷 Heterotrophs 异养生物 : organic carbon Autotrophs 自养生物 : CO 2 as primary or sole carbon source

9 Nutrient Requirements of Microbes Heterotrophy 异养 Autotrophy 自养 Carbon source Nitrogen source Energy Sugar, ethanol, organic acid 有机酸 Protein and its metabolite, organic nitride 氮化物, N 2 The same as carbon source CO 2, carbonate 碳酸盐 N 2, inorganic nitride 无机氮化物 Oxidize inorganic substance / sunlight Growth factor Some need vitamin 维生素 none Inorganic metals Yes Yes water (H 2 O) Yes Yes

10 Carbohydrate Catabolism 碳水化合物分解代谢 Carbohydrates oxidized as their primary source of energy 糖类作为主要能源 Glucose: most common energy source 葡萄糖是最普遍的能源 Respiration 呼吸作用 Fermentation 发酵作用

11 2. ENERGY 能量 1. Light (the sun or lamps) 2. Chemical compounds 1. Inorganic substances Sulfur 硫, CO or ammonia 氨 2. Preformed organic matter Sugar, protein, fats etc.

12 Energy Sources for Bacterial Growth All living organisms require energy source Phototrophs 光能自养菌 : radiant energy (light) Heterotrophs 异养生物 = chemo(hetero)trophs 化能异养 : use (oxidize) organic carbon form Lithotrophs 无机营养生物 = chemoautotrophs 化能自养 : oxidize inorganic compounds

13 4 major nutritional types of procaryotes on the basis of carbon and energy sources 原核生物的四种营养类型 Nutritional Type Energy Source Carbon Source Examples Photoautotrophs 光能自养 Photoheterotrophs 光能异养 Chemoautotrophs 化能自养 or Litho(auto)trophs chemoheterotrophs or Heterotrophs 化能异养

14 CRITICAL THINKING QUESTION How would you go about isolating a microbe that used gasoline 汽油 for a food?

15 Exxon Valdez Oil Spill Approximately 11 million gallons of oil were spilled into the waters of Prince William Sound during the 1989 Exxon Valdez Oil Spill Prince William Sound Oil Spill Recovery Institute Pseudomonas spp. N and P to be added: why?

16 MEOR

17 Microbial enhanced oil recovery (MEOR) 1. In situ generation of CO 2 for pressure and solubilization 增溶 2. microbial production of organic acids 3. conversion of hydrocarbons to lower MW 4. production of surface active agents 5. encouragement of consolidation 凝固 of oil to droplets 6. enzymatic modification of hydrocarbons 7. viscosity improvement

18 3. NITROGEN Source 氮源 N 2, ammonia 氨, nitrate/nitrite 硝酸盐, or a nitrogenous 含氮的 organic compound (protein or nucleic acid) Classification based on AA requirement Amino-acid-autotrophs 氨基酸自养生物 :SCP Amino-acid-heterotrophs 氨基酸异养生物

19 4. Growth factors 生长因子 Definition: Small amounts of certain essential organic compounds unable to be synthesized Required to fulfill specific roles in metabolism Results from either a blocked or missing metabolic pathway 起因于阻断或丢失的代谢途径 1. Auxoautotrophs 生长因子自养型微生物 e.g E. coli 2. Auxoheterotrophs 生长因子异养型微生物 : e.g. Lactobacillus 乳酸杆菌

20 Categories of Growth Factors 1. Purines and pyrimidines 嘌呤与嘧啶 : synthesis of nucleic acids (DNA and RNA) 2. Amino acids 氨基酸 : synthesis of proteins 3. Vitamins 维生素 : as coenzymes and functional groups of certain enzymes

21 5. Inorganic Compounds 无机物 Large amount: 10-3 ~10-4 mol/l P. S. K, Mg, Ca, Na, Fe TRACE metals 微量金属 : 10-6 ~10-8 mol/l, required by some enzymes Include: Cu, Zn 锌, Co 钴, Mn, Mo E.g. K 2 HPO 4, MgSO 4 : supply 4 elements of greatest amount

22 Salmonella plays tug-of-war for iron Cell, 2013, 14(1) Iron regulatory proteins

23 Question? Why are amino acid, purines, and pyrimidines often growth factors, whereas glucose is usually not?

24 Nutritional types of major organisms 主要生物体的营养类型 1. Eukaryotes 真核生物 : photoautotrophic 光能自养 (e.g. plants & algae), heterotrophic 异养 (e.g. animals, protozoa 原生动物, fungi) 2. Lithotrophy 无机自养 : unique to procaryotes 3. Photoheterotrophy: common in purple & green bacteria, only a few eukaryotic algae 4. Phototrophy 光能营养 : not found in Archaea

25 Nutritional requirements Vastly different nutritional requirements due to different biosynthetic capacities E. coli: capable of synthesizing all organic compounds it needs Leuconostoc mesenteroides 肠膜明串珠菌 : extremely fastidious (nutritionally demanding)

26 Fastidious orgnisms 需要复杂营养的生物体 Require PREFORMED organic molecules like vitamins, AA, nucleic acids, carbohydrates; In general bacterial pathogens need more PREFORMED organic molecules than do non-pathogens A simple rule of thumb: "if humans can use something for food, many microbes will also love it". - humans are fastidious. The reverse is not always true

27 CRITICAL THINKING QUESTION Why does it make sense from a nutritional standpoint 营养观点 that most pathogens are nutritionally fastidious?

28 Nutrient Transportation 营养物质进入细胞方式 Cytoplasmic membrane: selective transportation Proteins in the membrane: typically span the whole membrane, contacting with the outside environment and cytoplasm. often require the expenditure of energy

29 Four basic types of transport systems 四种传输方式 1. Simple (passive) Diffusion 单纯 ( 被动 ) 运输 2. Facilitated Diffusion 促进扩散 3. Group Translocation 基团转移 4. Active Transport 主动运输

30 Passive diffusion 单纯 ( 被动 ) 扩散 Directed by laws of simple diffusion. No transport protein involved Nonspecific, energy is not required A concentration gradient not be generated.

31 Figure 8.9 The diffusion of solutes across membranes

32 Figure 8.11 The water balance of living cells shriveled shrink lysed 萎缩的 裂解的 质壁分离 弛缓的 肿胀的

33 Facilitated Diffusion 促进扩散 Involves a protein to transport specific Solutes: not concentrated against gradient Energy: not required Not widely used in prokaryotes

34 Figure 8.13 One model for facilitated diffusion

35 Passive & Facilitated Diffusion

36 Membrane-spanning transporters 穿膜运输蛋白 In prokaryotes, membrane-spanning transporters typically contain 12 alpha helices that align with each other in a circle to form a channel through the membrane. 原核生物的穿膜运输蛋白由 12 个 螺旋形成穿膜的通道

37 Three individual transporters, each showing a different type of transport event. The cotransported molecule is shown in yellow. 有三种运输蛋白, 协同运输分子为黄色 Antiporters Symporters are proteins that transport a substance across along the membrane with another in one direction substance, while a second frequently substance a proton in the (H + ) opposite 同向运输蛋白 direction 反向运输蛋白 Uniporters are proteins that simply transport a molecule in a unidirectional fashion across the membrane 单向运输蛋白 Three individual transporters

38 Group translocation 基团转位 Protein-substrate: chemical modification No actual concentration takes place Most requires energy. Catabolic pathways sometime use Efficient: bring substrate into the cell and begin the breakdown process

39 Glucose across membrane - energy from PEP

40 For The glucose system uptake, in E. the coli system contains consists 24 proteins, of 5 proteins. at least Sequential 4 phosphate transfer occurs from PEP to Enzyme IIc, the latter actually necessary Energy source: transports for one phosphoenolpyruvate (and given phosphorylates) sugar 磷酸烯醇式丙酮酸 the sugar. Mechanism of the phosphotransferase system

41 Active transport 主动运输 Target NOT altered & accumulation of target occurs inside conc. > external conc. Proteins: molecular pumps against a concentration gradient Energy source: ATP or proton motive force (PMF)

42 Figure 8.16 An electrogenic pump

43 Figure 8.17 Cotransport

44 Simple transporter: Lac permease

45 Figure 8.14 The sodium-potassium pump: a specific case of active transport

46 Removal of phospho-head groups of membrane lipids immobilizes voltage sensors of K+ channels Nature 451, (14 February 2008)

47 Figure 8.15 Review: passive and active transport compared

48 Summary of bacterial transport systems (P 109 ) Property Passive Diffusion Facilitated Diffusion Active Transport Group Translocation Carrier Mediated Concentration Against Gradient Specificity Energy Expended Solute Modified During Transport

49 Discussion? Why do microorganisms normally take up nutrients using transport proteins? What advantage is active transport compared to facilitated diffusion? 5 min each

50 Manner to cultivate bacteria 细菌培养方式 Liquid media 液体培养基 : used for growth of pure batch cultures 纯批量培养物生长 : fermentation industry 发酵工业 Solidified media 固化培养基 :1~2% agar Isolation of pure cultures 纯培养物分离 Estimate viable bacterial populations 测活菌数 Semisolid medium 半固体培养基 : 0.5% agar

51 Culture Medium Function: 1. Isolation & maintenance of pure cultures 纯培养的分离和保存 2. Identification 鉴定细菌 Example Basal medium 基底培养基 Minimal medium NNN medium with blood 含琼脂 盐及兔血的培养基 ; for trypanosoma 锥虫属

52 Principles of medium design and selection 培养基设计与选择的原则 Objective 目的明确 Bacterial mass or metabolism products? Balanced nutrition 营养协调 Ten-time-rule: H 2 O > C+energy > N source > P, S > K,Mg > growth factor Appropriate physical & chemical condition ph: Bacteria: 7.0 ~ 8.0; Acetenomycets: 7.5 ~ 8.5 Yeast: 3.8 ~ 6.0; Mould: 4.0 ~ 5.8 Osmotic pressure & a w : optimal a w 0.6~0.998 Economic 经济节约

53 Agar 琼脂 - the usual gelling agent 胶凝剂 For solid or semisolid medium A hydrocolloid 水状胶体 derived from red algae Unique physical properties: 1. Melts at 100 degrees 2. Remains liquid until cooled to 45C at which it gels 3. Not metabolized by most bacteria relatively inert 4. Simply holds (gels) nutrients in aqueous solution. 5. Nontoxic to most organisms

54 Categories of Culture medium based on the composition 培养基分类 1. Chemically-defined(synthetic)medium 组合培养基 Exact chemical composition is known For growing fastidious organisms & nutritional requirements 2. Complex (undefined) medium 天然复合培养基 Composition is not known contain biological origin (blood, milk, yeast or beef extract) - full range of growth factors For most bacteria and fungi 3. Semi-defined medium 半组合培养基

55 Medium for some specific bacteria Pathogenic bacteria of animals: complex medium 复合培养基 A few fastidious pathogens Treponema pallidum 梅毒螺旋体, Mycobacterium leprae 麻风分支杆菌 : artificial culture media & conditions not been established.

56 A chemically-defined medium for growth of heterotrophic bacterium (ph 7.0) 异养菌 Component Amount Function of component sucrose 10.0 g C and energy source K 2 HPO g ph buffer; P and K source KH 2 PO g ph buffer; P and K source (NH 4 ) 2 HPO g ph buffer; N and P source MgSO 4 7H 2 O 0.20 g S and Mg ++ source FeSO 4 7H 2 O 0.01 g Fe ++ source MnSO 4 H 2 O g Mn ++ Source water 985 ml

57 Defined enrichment medium 富集培养基 for Thiobacillus 硫杆菌属 thiooxidans, a lithoautotrophic bacterium (ph 3.0) 无机自养菌 Component Amount Function of component NH 4 Cl 0.52 g N source KH 2 PO g P and K source MgSO 4 7H 2 O 0.25 g S and Mg ++ source CaCl 2 2H 2 O 0.07 g Ca ++ source Elemental Sulfur 1.56 g Energy source C0 2 5%* C source water 1000 ml *Aeratemedium intermittently with air containing 5% CO 2. 间歇性地将培养基暴露于空气

58 Complex medium for the growth of fastidious bacteria (ph 6.6) Component Amount Function of component Beef extract 1.5 g Source of vitamins and other growth factors Yeast extract 3.0 g Source of vitamins and other growth factors Peptone 6.0 g Source of amino acids, N, S, and P Glucose 1.0 g C and energy source Agar 15.0 g Inert solidifying agent water 1000 ml

59 Commonly used medium Bacteria: LB 肉汤培养基 Actinomycets: 高氏培养基 Fungi: 孟加拉红培养基 PDA 琼脂 高盐察氏琼脂 自生固氮菌或硅酸盐细菌 : 无氮培养基

60 Selective Media 选择型培养基 Inhibits some bacteria while selecting others Example: Brilliant Green Agar 碱性亮绿琼脂 Dyes: inhibit the growth of Gram (+) Selects for G (-) bacteria: most G.I. Tract infections Coliform 大肠杆菌 : ferment lactose and/or sucrose, acidic (yellow colony)

61 Examples of Selective Media EMB (Eosin Methylene Blue 伊红美兰培养基 ) Dyes inhibit Gram (+) bacteria Selects Gram (-) bacteria, especially E. coli G.I. Tract infections caused by Gram (-) 深紫色, 绿色金属闪光

62 Differential Media 鉴别培养基 for Hemolytic Streptococci 溶血链球菌 Blood agar plates (TSA 大豆胰蛋白胨琼脂 with 5% sheep blood) 血琼脂平板 α lysis of RBC s or not Alpha: Incomplete lysis Beta: Complete lysis Gamma: No lysis β

63 Selective and Differential Media (I) 选择和鉴别培养基 Mannitol Salt Agar 甘露醇盐琼脂 Identify Staphylococcus aureus 金黄色葡萄球菌 High salt conc. (7.5%) inhibits most bacteria Sugar: Mannitol ph indicator ( yellow when acid)

64 Selective & Differential Media (II) MacConkey s Agar used to identify Salmonella 沙门菌 : ph Indicator Bile salts 胆盐 & crystal violet: inhibits G+ Lactose 乳糖 Many Gram (-) enteric 小肠的 non-pathogenic bacteria can ferment lactose, Salmonella can not Coliform 大肠杆菌 : acidic (red) Salmonella, Shigella 志贺菌, Citrobacter 柠檬酸杆菌 (typical): alkaline (white)

65 Differentiation of C. diphtheriae 白喉杆菌 Black on tellurit 亚碲酸盐 agar White on blood agar

66 Selective enrichment medium for extreme halophiles 耐盐性生物富集培养基 (ph 7.4) Component Amount Function of component Casamino acids 7.5 g Source of amino acids, N, S and P Yeast extract 10.0 g Source of growth factors Trisodium citrate 柠檬酸钠 3.0 g C and energy source KCl 2.0 g K + source MgSO 4 7 H 2 O 20.0 g S and Mg ++ source FeCl g Fe ++ source NaCl 250 g Na + source for halophiles and inhibitory to nonhalophiles water 1000 ml

67 TYPES OF CULTURE MEDIA TYPE PURPOSE Chemically defined Complex Reducing Selective Differential Enrichment

68 Key Points 1. Six major types of nutrients for microorganism 六大营养要素 2. Four major nutritional types of procaryotes on the basis of carbon and energy sources 3. Growth factor and its major categories 4. Four basic types of nutrient transport systems 5. Difference between synthetic medium and complex medium 合成与天然培养基的区别 6. Application of selective medium and differential medium 选择与鉴别培养基的应用

69 EXPERIMENT DESIGN How to screen bacillus and yeasts of specific function from environment Tip Sampling location: Environment rich in the target microbes Screening method: Method for obtaining pure culture Medium Checking metabolite

70 Thanks!