Lecture 4-1 Nutrition, Laboratory Culture and Metabolism of Microorganisms

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1 Lecture 4-1 Nutrition, Laboratory Culture and Metabolism of Microorganisms CHAPTER 4 in BROCK BIOLOGY OF MICROORGANISMS Zhao Liping, Chen Feng School of Life Science and Biotechnology

2 What does a cell need to be a successful chemical machine? The cell needs energy Chemical transformations Movement The cell needs building materials Precursors for monomers Growth factors etc.

3 I. Nutrition and Culture of Microorganisms A key feature of cells is their ability to direct chemical reactions and organize molecules into specific structures: metabolism. Metabolic reactions are either energy releasing, called catabolic reactions, or energy requiring, called anabolic reactions. 代谢包括了异化作用 ( 释放能量 ) 和同化作用 ( 需要能量 ) 两种.

4 4.1 Microbial Nutrition Carbon 碳 Many prokaryotes require an organic compound as carbon source. Some prokaryotes are autotrophs 自养型, use CO 2 as C source. Nitrogen 氮 A typical bacterial cell is about 12% nitrogen (by dry weight) Nitrogen is an important element in proteins, nucleic acids, and several other constituents in the cell.

5 Other Macronutrients: Phosphorus 磷 : is required for synthesis of nucleic acids and phospholipids. Sulfur 硫 : is required in amino acids cysteine and methionine, and present in a number of vitamins and coenzyme A. Potassium 钾 : required by enzymes, including those involved in protein synthesis. Magnesium 镁 : stabilize ribosomes, cell membranes, and nucleic acids and is also required for the activity of many enzymes. Calcium 钙 : stabilize cell wall and plays a key role in the heat stability of endospores. Is not essential for the growth of many microorganisms. Sodium 钠 : required by some organisms. Its needs often reflects the habitat of the organism.

6 Iron 铁 Plays a major role in cellular respiration, being a key component of the cytochromes and iron-sulfur proteins involved in electron transport. 在细胞的呼吸中有重要作用, 是电子传递链中细胞色素和铁硫蛋白的重要组分. Micronutrients (trace Elements) 微量元素 They are metals, many of which play a structural role in various enzymes.

7 Growth Factors 生长因子 Are organic compounds that are required in very small amounts and only by some cells. Include vitamins, amino acids, purines, and pyrimidines.

8 Nutritional requirements of different microbes Different microorganisms can have vastly different nutritional requirements because they have different biosynthetic capacities E. coli: capable of synthesizing all organic compounds it needs Leuconostoc mesenteroides: need many externally provided organic compounds-extremely fastidious (nutritionally demanding) 肠膜明串珠菌 ( 需要极复杂营养的 )

9 4.2 Culture media 培养基 Culture media are the nutrient solutions used to grow microorganisms in the laboratory. Chemically defined(the exact chemical composition is known) 化学限定的培养基 Synthetic media 合成培养基 Minimal media 限制性培养基 Basal media 基础培养基 Chemically undefined 非化学限定的培养基 Complex media 复合培养基 Natural media 天然培养基

10 Prepare 1L LB Medium (Luria-Bertani Medium): Per liter: Bacto-tryptone: Bacto-yeast extract: NaCl: H 2 O: Autoclave 10g 5g 10g To 1 L

11 PDA Medium (Potato Dextrose Agar) Autoclave 200 grams of sliced potato in 1000 ml of tap water. Filter through a hankerchief. To the turbid filtrate add 20 grams of glucose and 10 grams of agar. Add water to make 1 liter total volume. Autoclave PDA slants give good growth of most fungi, Agrobacterium, Nocardia, and many others.

12 SSW Medium (Synthetic Seawater Medium) MgSO 4.7 H 2 O g NaCl g MgCl2.H2O g CaCl 2.2 H 2 O g Yeast extract g Peptone g Glycerol... 3 ml Water ml This medium is excellent for growing most bacteria from the oceans.

13 Two most important nutritional factors Energy source 能量来源 Chemical energy Light energy Carbon source 碳源 Organic compounds Inorganic compounds CO 2 or carbonate

14 4.3 Laboratory Culture of Microorganisms Pure Culture: a culture containing only a single kind of microorganism. 纯培养 Solid culture media: culture media prepared in a semisolid form by the addition of a gelling agent to liquid media. 固体培养基

15 Aseptic Technique 无菌 ( 操作 ) 技术 the series of steps used to prevent contamination during manipulations of cultures and sterile culture media. 无菌操作 : 在培养基消毒及培养过程中防止 ( 微生物 ) 污染的一系列步骤.

16 Figure 4.4 Aseptic transfer (a) Loop is heated until red-hot and cooled in air briefly. (b) Tube is uncapped. (d) Sample is removed on sterile loop. (e) After removing sample on loop, the tube is reflamed and the sample transferred to a sterile medium. (f) The tube is recapped. Loop is reheated before being taken out of service (c) Tip of tube is run through the flame.

17 Method of making a streak plate to obtain pure cultures. FLASH (a) Loop is sterilized, and then a loopful of inoculum is removed from tube. (b) Streak is made over a sterile agar plate, spreading out the organisms. Following the initial streak, subsequent streaks are made at angles to it, the loop being resterilized between streaks.

18 Method of Pure Culture from soil: Dilution

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21 Method of Pure Culture: Streak over an agar plate

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23 Culture anaerobic microorganisms: 钯 碳酸氢钠与硼氢化钠

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25 II. Energetics and Enzymes

26 4.4 Bioenergetics 生物能学 Energy-ability to do work Chemical energy-energy released when organic or inorganic compounds are oxidized kilocalorie (kcal)-the quantity of heat energy necessary to raise the temperature of 1kg of water 1 o C =4.184 kj

27 Energy Free energy, G 自由能 -the energy released that is suitable to do useful work Free energy of formation G 0 f-the energy yield or energy required for the formation of a given molecule from its constituent elements

28 Energy production Flow of electrons from donor (Glucose) through carriers to acceptor (O 2 ) produce energy for cells 电子流从供体通过载体达到受体的过程产生细胞所需要的能量 ATPase is the electricity generator ATP 酶 High energy compounds (ATP) save energy in a form ready for use by cells as if a battery for the electric car

29 III. Oxidation-reduction and energy-rich compounds

30 4.5 Oxidation-reduction (redox) Oxidation-removal of an electron or electrons from a substance 氧化即从物质中移去电子 Reduction-addition of an electron or electrons to a substance 还原即在物质中加入电子. redox reactions with transfer of hydrogen atom (one electron and one proton) 氧化还原反应即氢原子的转移 ( 一个电子和一个质子 ) Redox

31 4.5.1 Eletron donors and acceptors Electron donor 电子供体 -the oxidized substance half reaction-oxidation (electron donating) H 2 2e - +2H + Electron acceptor 电子受体 -the reduced substance half reaction-reduction (electron accepting) 1/2O 2 + 2e - +2H + H 2 O Redox reaction 氧还反应 involved electrons being donated by an electron donor and being accepted by an electron acceptor H 2 +1/2O 2 H 2 O

32 4.5.2 Reduction potential E 0 氧化还原势 E 0 - tendency to become oxidized or to become reduced 被氧化或被还原的趋势 This potential is measured electrically in volts (V) in reference to a standard substance-h 2

33 4.5.3 Redox couple 氧还对 The same molecule can be either electron donor or acceptor depending on what other molecules they react with, in other words, depending the difference of reduction potential between reactants. 同一个分子与不同物质反应时, 可以是电子供体也可以是受体 When writing a redox couple, the oxidized form is always placed on the left, eg. 2H + /H 2 or 1/2O 2 /H 2 O

34 4.5.4 The Electron Tower Who will donate electrons? Who will accept electrons? The reduced substance of a redox couple whose reduction potential is more negative donates electrons to the oxidized form of a redox couple whose potential is more positive The oxidized form of a redox couple whose reduction potential is more positive accepts electrons from the reduced substance of the redox couple whose reduction potential more negative

35 The electron tower Redox couples from the strongest reductants at the top to the strongest oxidants at the bottom.

36 4.5.5 Electron donor energy source Why does everybody need oxygen? Why does a doctor often give weak patients glucose? How can a bacterial cell survive under anoxic conditions?

37 4.6 NAD as a Redox Electron Carrier Primary electron donor electron carrier terminal electron acceptor In the cell, the transfer of electrons in an redox reaction from donor to acceptor usually involves one or more intermediates referred to as carriers. When such carriers are used, we refer to the initial donor as the primary electron donor and to the final acceptor as the terminal electron acceptor. 细胞中电子由供体到受体的转移时通常包括了一些中间物 ( 载体 ). 如果有载体存在, 则最初的受体为初级电子供体, 最后的受体为最终电子受体

38 Two general classes of electron carriers Diffusible carriers 扩散性载体 Freely diffusible carriers include the coenzymes nicotinamide-adenine dinucleotide (NAD + ) and NADphosphate (NADP + ). NAD + and NADP + are hydrogen atom carriers and transfer two hydrogen atoms to the next carrier in the chain. 烟酰胺腺嘌呤二核苷酸 Non-diffusible carriers 非扩散性载体 firmly attached to enzymes in the cytoplasmic membrane. They include NADH dehydrogenases, flavoproteins, iron-sulfur proteins, and cytochromes.nadh 脱氢酶, 黄素蛋白, 铁硫蛋白, 细胞色素

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40 Schematic example of an oxidation-reduction reaction involving the oxidized and reduced forms of the coenzyme nicotinamide-adenine dinucleotide, NAD+ and NADH.

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42 Energy-rich compounds

43 4.7 Energy storage Bacterial cells can store energy for future needs Energy-rich insoluble polymer Polysaccharides: starch, glycogen 淀粉, 糖原 Lipid: poly- -hydroxybutyrate (PHB) 聚 羟基丁酸 Elemental sulfur 元素硫

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