Chapter 5 Microbial Nutrition and Culture medium Nutrition
Chapter 5 Microbial Nutrition and Culture medium
Nutrition: Nutrition is a process by which chemical substances called nutrients are required from the environment and used for the cellular activities of metabolism and growth. Nutrient:the substance could be used by microbes for metabolism or reproduction of cells.
Nutrient provide the structural substance , energy, metabolic regulate substance and appropriate physiological environment. Some microbes could use nonmaterial energy---light.
Section 1 Six kinds of essential nutrient for microbes I. cellular chemical constitutes It’s much alike in the life in the element. The main elements are C、H、O、N (90%---97% to total dry cell weight), C is about 50%, the others are inorganic nutrient. C/N is 5: 1 usually.
element ØMacronutrient :C、H、O、N、P、 S Øothers element:K、Na、Ca、Mg、Fe、Mn、 Cu、Co、Zn、Mo, et al Existence form: Ø organic:protein、sugar、lipoid、 nucleic acid、vitamin、decomposition substance、metabolic medial products Ø inorganic salt ash Ø Water---70% ~ 90% to wet cell weight
Table 1. The macronutrient content in microbes (% to dry cell weight) element bacteria yeast mould C 50 49. 8 47. 0 N 15 7. 5 5. 2 H 8 5. 7 6. 7 O 20 31. 1 40. 2 P 3 1. 5 1. 2 S 1 0. 3 0. 2
Microbes is omnivorous. The substance utilized by generic species could be utilized by microbes too. The substance couldn’t be utilized by generic species could be utilized by microbes. Some kinds of substance harmful to generic species also could be utilized by microbes.
II. Main nutrient and the function Carbon source Nitrogen source Energy source Growth factor Inorganic salt Water
Functions: vconstitute the cells of the microbes vprovide the energy needed in the physiological activity to microbes vthe source to form the microbial metabolic products Nutrient is the substance base for microbial metabolism and living activity. If lose this base, the life will die.
1. Carbon sources The nutrient provide the carbon element for microbies q. Organic carbon sources: protein, nucleic acid, starch, glucose, et al q. Inorganic acid: CO 2 , Na 2 CO 3 , Ca. CO 3 et al Heterotrophic microbe: is an organism must obtain its carbon in an organic form. Autotrophic microbe: is an organism that uses CO 2, an inorganic gas, as its carbon source.
the carbon sources could be utilized by microbes (compound) saccharide: glucose,fructose,maltose,sucrose, starch,galactose,mannancellulose, hemicellulose,lignin,et al organic acid: lactic acid,citric acid,,short chain fatty acid,long chain fatty acid ,amino acid, et al alcoholic aldehyde: ethanol, et al fat: fat,lecithoid, et al hydrocarbon: natural gas,petroleum,olefin,et al CO 2 carbonate: Na. HCO 3, Ca. CO 3, chalk, et al others: cyanide,protein,peptide,nucleic acid
2. Nitrogen sources The nutrient provide the nitrogen element for microbes. Normally, nitrogen source couldn’t be used as energy sources. Øorganic nitrogen sources :peptone、soybean powder、 maize slurry Øinorganic nitrogen sources:NH 4 NO 3、(NH 4)2 SO 4 Øgas nitrogen sources:N 2 in the atmosphere Øquick nitrogen sources Øslow nitrogen sources
3. Energy sources energy sources are the substances or radiant energy that could provide primal energy sources. Heterotrophs’s carbon sources could also be its energy sources. Organic: energy to chemoheterotrophs Chemical substances Inorganic: energy to chemoautotrophs Energy sources table Radiant energy:energy to photoautotrophs and photoheterotrophs.
one kind of nutrient could provide more than one kind of function mono-function : radiant energy • di-function: reductive inorganic nutrient, such as NH 4+ is the energy sources as well as the nitrogen sources of nitrate bacterial. • triple function: the nutrients including N 、C 、H 、O are energy sources、 carbon sources and nitrogen sources heterotrophs microbes.
4. Growth factor Definition: An organic compound such as an amino acid, nitrogen base, or vitamin that cannot be synthesized by an organism and must be provided as a nutrient is a growth factor. Function:coenzyme or necessary to activation of enzyme. Sources of growth factors in culture medium: yeast extract、maize slurry、wort, et al。
narrow sense: vitamin broad sense:vitamin, amino acid, base, fatty acid , et al qauxoautotrophs qauxoheterotrophs qnutritional deficiency mutant qauxo-excessivetrophs
Vitamin necessary to some bacterials vitamin species thiamin B 1 Bacillus anthracis lactoflavin Clostridium tetani nicotinic acid Brucella abortus vitamin B 6 Lactobacillus spp. biotin Leuconostoc mesenteroides pantothenic acid Proteus morganii folacin Leuconostoc dextranicum B 12 Lactobacillus spp. vitamin K Bacteroides melaninogenicus
5. Inorganic salts Macroelement: need the concentration of the element at 10 -3 -10 -4 M Microelement: need the concentration of the element at 10 -6 -10 -8 M
The physiology function for inorganic salts Normal molecular in cell(P、S、Ca、Mg、Fe) Usual function maintain osmotic pressure (Na+) Physiology modulation material Macroelement enzyme activator(Mg 2+) stablize p. H Energy sourse of chemoautotrophic bacteria (S、Fe 2+、 inorganic salts Especial function Microelement NH 4+、MO 2 -) Hydrogen aceptor during respiration without oxygen (NO 3 -、SO 42 -) Enzyme activator (Cu 2+、Mn 2+、Zn 2+) Molecular of special structure (Co、Mo)
6. Water existed state: dissociated state(solvent)and integrated state(structure composition) Physiological function: Ø Ø Ø cell composition solvent of biochemistry reaction chemical and physiological reaction medium material transferred medium regulate cell temperature maintain osmotic pressure
Section 2 Nutrition types of microbes Classification ◆ based on different energy sources : phototrophs: obtain energy from light chemotrophs: obtain energy by oxidation ◆ based on different carbon sources: heterotrophs: can’t use CO 2 as its mainly or sole carbon energy autotrophs: can use CO 2 as its mainly or sole carbon energy ◆ based on growth factor: prototroph or wild type auxotroph
Nutrition type of microbe Nutrition type photoautotr ophs photohetero trophs Energy source Carbon source sunlight CO 2 and simple organic compounds example Cyanobacterial, Purple bacterial, Green sulfur bacterial, Algae Rhodospirillum Nitrobacterial chemoautot rophs inoganic compounds CO 2 chemoheter otrophs organic compounds Sulfbacterial Ironbacterial Hydrobacteri al Most microbes, Prozotoa
Chemoheterotrophs vparasitism ——live in live organisms. vsaprophytism——live in dead organic. Ø autotrophs:can live normally without any organic compounds. Ø heterotrophs:are dependent on other life forms,can live normally with at least one kind of organic compound,its carbon soueces and hydrogen supplier should be in organic form,energy can be obtained by oxidation and absorption of sunlight. to note that such classification is not absolute,and it may be changed under different conditions。
Section 3 Transport mechanisms for nutrient absorption The absorbtion of nutrient and excretion of metabolized products refer to transportion of molecular、 nutrient absorbed in cell and be used、metabolized products secreted out of the cell, that is the whole process of transportion. The cell wall can only block large molecular solute (>600 Da) out of the cell, while the membrane which have lipid bilayer and integral protein is the main barrier to control the nutrient’s flow.
vpermeability is different from absorbtion vcommonly, large molecular first hydrolyze to small molecular, then be aborbed. vfat-dissolved:easily permeable vionization:the weaker of polar, the faster of the permeable
I. Simple diffusion The rate of diffusion is directly proportional to the concentration gradient of the molecule across the membrane. ; Do not acquire metabolic energy, is of no specificity. Only transformed small molecular, O 2、CO 2、 glycerol、ethanol and some amino acids; Lipohilia molecules make use of the permeability of cell membrane to transport from high concentration to the low concentration.
II. Facilitated diffusion The transported molecular in and out of the cell have different appetency to the carrier protein. characteristic: Require the presence of specific carrier protein, down the concentration gradient Do not acquire metabolic energy Transport SO 4 , PO 3, sugars
Carrier protein: namely permeable enzyme(mostly are induction-enzyme), is specific for one particular molecular or a group of structurally similar moleculars. It can make the transportion faster, but is not able to make it against the concentration gradient.
extracellular membrane intracellular facilitated diffusion
Transportion rate 促进扩散 单纯扩散 Concentration gradient Prapartion of simple diffusion and facilitated diffusion
III. Active transport characteristics : Ø The main mode for microbes to absorb nutrient ØAgainst the concentration gradient , require energy ØNeed barrier protein, is specific ØTransport organic iron、inorgnic iron、amino acid、 lactose and other sugars ØNeed energy to change the conformation of barrier protein ØAppetence change →protein conformational change→consume energy simple diffusion 、 facilitated diffusion and active transport: the transported solute won’t be changed.
1. Use electron transfer to pump the proton to extracellular 2. Expel Na+ out of membrane by reverse transport mechanism 3. Na+ combine with barrier protein complex 4. The form of combination site changed, to combine with the solute (eg: sugar, amino acid) 5. Barrier protein conformational change, release Na+ in theintrecellular, then solute separate from barrier protein. Mechanism of active transport:use the gradient of H+ and Na+ Extracellu lar(outsid r the plasma membrane) election transfer intracellular
IV. Group translocation characteristics characteristic : v belong to active transport v solute molecular have chem-modification directional phosphorylation v need a complicated series of transport enzyme v to transform glucose、fructose、mannan、purin、 nucleotide、fatty acid, et al The membrane has high impermeability to most phosphate compounds. Input each glucose may consume one ATP.
v. Mainly depend on PEPand PTS. 1. Activate the heat stable carrier protein (HPr) PEP+ HPr EI Pyruvic acid+ P-HPr 2. Saccharide transferred into membrane after phosphorylation P-HPr +saccharide EII saccharide-P+HPr
Transport of glucose group translocation Cytoplasm stroma cytoplast 2 kinds of systems of PEP and PTS system. High-energy phosphate transfer from HPr to dissolved EIIA, EIIA combines with EIIB mannan transport system and separate in the glucose transport system. No matter in which form, phosphate will first transfer from EIIA to EIIB, and arrive at glycosyl by the process of transport across membrane.
Section 4 culture medium I. Definition: medium,culture medium:Culture medium is the mixed nutrient suitable for microbe’s growth and breed.
II. Principles and methods to select and make culture medium A. Four principles 1. Definite aims(different microbes have different nutrient requirement): to culture which microbes,to get which products,useless 2. Suitable nutrients: pay attention to the concentration and ratio of nutrients, especially C/N ratio
C/N ratio: mol ratio of carbon atom in carbon sources and nitrogen atom in nitrogen sources. Nitrogen content %: NH 3(82%)>CO(NH 2)2(46%)>NH 4 NO 3(35%)>(NH 4)2 CO 3(29. 2%)>(NH 4)2 SO 4(21%) C/N ratio in most medium: 100/0. 5~ 2.
Selection of suitable nutrient kind example explaiation monosaccharides glucose, maltose, xylose, galactose, fructose used as quick carbon sources, but will speed up breathof microbe, decrease resolved O 2, make mid-metabolized products accumulate. disaccharide saccharose, lactose Used as slow carbon sources, but will speed up breathof microbe, decrease resolved O 2, make mid-metabolized products accumulate. polysaccharide starch, cornmeal, cellulose slow carbon sources, only few microbes canuse cellulose as carbon sources molasses Sugar beet molasses, sugancane molasses. good carbon sources, complicated, usually fermented from yeast in fermentation industry, widely used in the production of antibiotic、 acetone and butanol. usually include saccharose 35%, glucose 9%, fructose 7%, 8 kingsother sugars 4%, other deoxidized materials 3%, 24 kinds amino acid 4. 5%, organic acid 5%, wax、steroids and phosphorous 0. 4%, ash 12%, water 20%. malt budded barley mainly used to culture yeast, can be industry grease cottonseed oil, corn oil, bean oil, sunflower seed oil high热 值 , consume more O 2 when cultured in liquid, usually added as消泡剂 in industry alcohols methanol, glycerol Methanol canbe used to culture yeast hydrocarbon CH 4, C 2 H 4, C 4 H 10, C 12~C 20 high热 值 , consume more O 2 when cultured in liquid. hardly dissolved, don’t too much beating up when cultured in liquid. organic acid acetic acid, lactic acid, citric acid will increase the p. H used to prouduce beer in
Cell yield by different energy source carbon sources stroma glucose methane Normal paraffin hydrocar bon methanol acetate fumarat e cell producing efficiency( g cell/g stroma) 0. 51 0. 62 1. 03 0. 40 0. 68 0. 34 0. 36
Common nitrogen sources for microbe Kinds Suitable microbe Characteristic organic : beaf extract, yeast extract, peanut cake power, soybean poder, cotton cake power, maize slurry, maize protein power, peptone, yeast power, fish meal, silkworm pupa power, wheat bran Most microbes, eg: Penicillium, yea st, E. coli contain plenty of amino acid, good nitrogen sources for microbes inorganic: ammonium Part of microbes(amino acid heterotrophs aren’t able to use it) Nitrate can be used after deoxidization, may regulate p. H meantime. few bacteria (approximately 50 genus, 100 species) Nitrogen fixation is second important only to photosynthesis. nitrate, ammonia N 2 salt,
3. Suitable chemical and physical conditions: Ø p. H Ø osmotic pressure and water ability Ø redox potential
p. H:most suitable p. H v bacteria: p. H 7. 0~8. 0 v actinomyces:p. H 7. 0 ~8. 5 v yeast: p. H 3. 8~6. 0 v mould: p. H 4. 0~6. 0 p. H value in the medium could change in the growing process of microorganisms: v If culture in stroma containing carbohydrate, produce acid, so the p. H will decrease. v If decompose protein and amino acid, produce NH 3, p. H will increase. v If use (NH 4)2 SO 4 as nitrogen sources , SO 4 2 - will be excessive, so the p. H decrease. v If decompose positive iron compounds, eg: Na. NO 3, Na+ will be excessive, so the p. H increase.
methods to maintain medium p. H stable Use phosphorus buffer: K 2 HPO 4 /Na 2 HPO 4 , H 2 PO 4/Na. H 2 PO 4 Use“standby alkali” :Ca. CO 3 、 Ca. HCO 3 Use acidesecent salt:citrate、 lactophosphate et al Use liquid N 2 or HCl.
Osmotic pressure: pressure produced by solutes, when filterring from high concentration to low cocentration。 Hypertonic solution will cause plasmolysis. Hypotonic solution will make cell expand or even break.
Utilized ability of water activity, aw aw:the quantity of free or dissociated water that microbes can utilize under natural environment. It’s eaqual to the ratio of saturated steam pressure of the solution and pure water, in the same temperature and pressure. . aw =P/Po P: steam pressure of the solution Po: steam pressure of pure water aw of pure water is 1. 00, under normal temperature and pressure Suitable aw to microbes’ growth is 0. 6~ 0. 998.
Aw of some solution aw 30% glucose 0. 964 1% glucose +20%glycerol 0. 955 1% glucose +40%saccharose 0. 964 Saturated Na. Cl 0. 78 Saturated Ca. Cl 2 0. 30 Saturated Mg. Cl 2 0. 30 Saturated Li. Cl 0. 11
Eh(redox potential):to show the trend of reducer releasing electron or oxidant accepting eletron in the redox system. § Aerobes:> + 0. 1 V。Generally +0. 3~ +0. 4 V § Anaerobes:< + 0. 1 >+0. 1 aerobic respiration § Facultative anaerobes: < +0. 1 fermentation
4. Appropriate raw material veconomy principle: use the low grade replacing high grade, use the waste replacing normal material, use the simple replacing complex veasily treated, with low cost v produce little waste after treated
Sterilization under high pressure: 1. 05 kg/cm,1210 C,15~ 30 min Sterilization under high temperature may destroy some nutrients and change the p. H.
B. Four methods Ø Ecological simulation Ø Lookup literature Ø Elaborate Ø Experiment Comparison
Category of Culture medium v Based on the component of the culture medium: Complex medium ( or Undefined medium ) Complex medium is one kind of culture mediums, which utilizes animal/ plant/micro-organism or the extract from these substance, and the components are undefined, e. g. broth peptone medium , which is a growth medium for bacteria; malt extract medium , which is a growth medium for yeast. Advantages:convenience; be rich in nutrition; variety
Defined medium ( or Synthetic medium ) Defined medium is one kind of the culture mediums, which is compounded by many pure chemical reagent, and the components ( including the microelement ) are defined exactly, e. g. Glu-NH 4+ medium, which is a growth medium for bacteria; Starch-NO 3 - medium, which is a growth medium for actinomycete. Expensive, be used in research( metabolism analysis/ genetic analysis) Advantages :The component is exact; Well-repeat Semi-defined medium
v Base on the physical condition of culture medium: Solid medium: add solidifying agents; the content of solidifying agents is 1~ 2%. Condition of being the solidifying agents: Can’t be utilized or decomposed by microbe; can keep the solid condition in the growth temperature range; It isn’t harmful for microbe at solidification point temperature; can’t be destoried by sterilization; good-diaphaneity; convenience; cheap. Solidifying agents that we always use: Agar and Glutin Usage: isolation/identification/conservation of the strain (Solid medium allows the surface to form mono-colony )
Semi-solid medium: the content of solidifying agents is 0. 5% Usage: observe the motion of bacteria; mensurate the titer of bacteriophage. Liquid medium : no solidifying agent Usage: microbe culture ;the research on physiology /Metabolism
The Difference between Agar and Glutin Chemistry Component Nourish -ment Resolv -ability Melt temperature Solidification temperature Concentration Diaphaneity Adhesion value Tolerance of Sterilization under high pressure Agar polysaccharide polymer none infrequent 96℃ 40℃ 1. 5~2 % high big high Glutin protein nitrogen source easy 25℃ 20℃ 5~12 % high big low
EMB (Eosin Methylene Blue) Culture medium identified enteric bacterial E. Coli growed in EMB (Eosin Methylene Blue) Culture, can decompose lactose and produce much mixed acid. The thallus carry with H+, so acidic chromophore Eosin is attracted to the positively charged molecules of E. coli. Eosin can combine with Methylene Blue, which makes the colonies purple. Green metallic luster is observed from the surface glisten of colonies. Colonies that ferment lactose are red, and colonies that produce acid weakly are brown, whereas non-fermenters are transparent.
EMB (Eosin Methylene Blue) Culture medium
Brief Summary 1、Microorganisms need about 10 kinds of macroelement,part of them are used to form carbohydrate 、 lipid 、 protein 、 nucleic acid, others are bittly used as coenzyme and prosthetic group. 2、All the microorganisms can be classifed on the basis of their needness of carbon courses 、energy sources 、hydrogen atom and electron. 3、Nutrients can’t permeated through cytoplasm membrane persistently by passive transport, they must choose one kind of active transport mechanism to do so. Medium are necessary to culture microbe. You may need to filtrate or separate special microbes in microorganisms identification 、water quality appraisal 、food analysis and so on, remember different aim determines different medium.
Reviewing question of this chapter 1、 What are the macroelement and microelement? 2、 How many kinds of macroelements are needed by microbes? 3、 What is nutrition, what is nutrient, What physiological function does the nutrient have? 4、 Try to compare the similarities and differences about nutrients and nutriention mode among animals 、plants and microorganisms. 5、 What are carbon sources? List some carbon sources commonly used by microbes. 6、 Find the differences on the using of carbon sources by microbes. 7、 What are nitrogen sources? List some nitrogen sources commonly used by microbes.
8、 What are the characteristic of the using of nitrogen sources by microbes? 9、 What is amino acid autotrophs, what is amino acid heterotrophs? 10、 Try to compare the differences of the way that nutrients use to get into cell. 11、 What is culture medium and what is complex medium? 12、 What is semi-solid medium? 13、What is differential medium? Give an example of this kind of medium and explain its principle. 14、What is the C 、N ratio? 15、What are the basic principles to make medium?
References: Zhou Deqin,Microbiological Tutorial,2002, second edition,higher education press Kathleen Park Talaro, fundations in microbiology, 2005, fifth edition, higher education press
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