Chapter 5 Microbial Nutrition and Culture medium.

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.

I. cellular chemical constitutes Its 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. Section 1 Six kinds of essential nutrient for microbes

element Macronutrient C H O N P S others element K Na Ca Mg Fe Mn Cu Co Zn Mo, et al Existence form: organic protein sugar lipoid nucleic acid vitamin decomposition substance metabolic medial products inorganic salt ash Water---70% ~ 90% to wet cell weight

elementbacteriayeastmould C5049.847.0 N157.55.2 H85.76.7 O2031.140.2 P31.51.2 S10.30.2 Table 1.The macronutrient content in microbes (% to dry cell weight)

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. Microbes is omnivorous.

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.

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. 1.Carbon sources The nutrient provide the carbon element for microbies Organic carbon sources: protein, nucleic acid, starch, glucose, et al Inorganic acid: CO 2, Na 2 CO 3, CaCO 3 et al

the carbon sources could be utilized by microbes (compound) saccharide glucose fructose maltose sucrose starch galactose lactose 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 CO2 carbonate NaHCO3, CaCO3, chalk et al others cyanide protein peptide nucleic acid

organic nitrogen sources peptone soybean powder maize slurry inorganic nitrogen sources NH 4 NO 3 (NH4) 2 SO 4 gas nitrogen sources N 2 in the atmosphere 2. Nitrogen sources The nutrient provide the nitrogen element for microbes. Normally,nitrogen source couldnt be used as energy sources. quick nitrogen sources slow nitrogen sources

energy sources are the substances or radiant energy that could provide primal energy sources. Heterotrophss carbon sources could also be its energy sources. 3. Energy sources Inorganic:energy to chemoautotrophs Energy sources table Chemical substances Radiant energy energy to photoautotrophs and photoheterotrophs. Organic: energy to chemoheterotrophs

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. one kind of nutrient could provide more than one kind of function

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. Sources of growth factors in culture medium: yeast extract maize slurry wort,et al 4. Growth factor Function coenzyme or necessary to activation of enzyme.

narrow sense vitamin broad sense vitamin, amino acid, base, fatty acid,et al auxoautotrophs auxoheterotrophs nutritional deficiency mutant auxo-excessivetrophs

vitaminspecies thiamin B 1 Bacillus anthracis lactoflavinClostridium tetani nicotinic acidBrucella abortus vitamin B 6 Lactobacillus spp. biotinLeuconostoc mesenteroides pantothenic acidProteus morganii folacinLeuconostoc dextranicum B 12 Lactobacillus spp. vitamin KBacteroides melaninogenicus Vitamin necessary to some bacterials

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 inorganic salts Macroelement Microelement Usual function Especial function Normal molecular in cell(P S Ca Mg Fe) Physiology modulation material maintain osmotic pressure (Na + ) enzyme activator(Mg 2+ ) stablize pH Energy sourse of chemoautotrophic bacteria (S Fe 2+ NH 4 + MO 2 - ) Hydrogen aceptor during respiration without oxygen (NO 3 - SO 4 2- ) Enzyme activator (Cu 2+ Mn 2+ Zn 2+ ) Molecular of special structure (Co Mo)

Physiological function cell composition solvent of biochemistry reaction chemical and physiological reaction medium material transferred medium regulate cell temperature maintain osmotic pressure 6.Water existed state:dissociated state(solvent)and integrated state(structure composition)

based on different carbon sources heterotrophs: cant use CO 2 as its mainly or sole carbon energy autotrophs: can use CO 2 as its mainly or sole carbon energy Section 2 Nutrition types of microbes based on different energy sources phototrophs: obtain energy from light chemotrophs:obtain energy by oxidation based on growth factor prototroph or wild type auxotroph Classification

Nutrition type of microbe Nutrition typeEnergy sourceCarbon sourceexample photoautotr ophs sunlightCO 2 Cyanobacterial Purple bacterial Green sulfur bacterial Algae photohetero trophs sunlight CO 2 and simple organic compounds Rhodospirillum chemoautot rophs inoganic compounds CO 2 Nitrobacterial Sulfbacterial Ironbacterial Hydrobacteri al chemoheter otrophs organic compounds Most microbes Prozotoa

parasitism live in live organisms. saprophytismlive in dead organic. Chemoheterotrophs to note that such classification is not absolute and it may be changed under different conditions 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. autotrophs can live normally without any organic compounds.

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 (>600Da) out of the cell, while the membrane which have lipid bilayer and integral protein is the main barrier to control the nutrient s flow.

permeability is different from absorbtion commonly,large molecular first hydrolyze to small molecular,then be aborbed. fat-dissolved easily permeable ionization 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,O2 CO2 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 Require the presence of specific carrier protein, down the concentration gradient Do not acquire metabolic energy Transport SO4, PO3, 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.

facilitated diffusion extracellular membrane intracellular

Concentration gradient Transportion rate Prapartion of simple diffusion and facilitated diffusion

simple diffusion facilitated diffusion and active transport the transported solute wont be changed. III. Active transport characteristics characteristics The main mode for microbes to absorb nutrient Against the concentration gradient,require energy Need barrier protein, is specific Transport organic iron inorgnic iron amino acid lactose and other sugars Need energy to change the conformation of barrier protein Appetence change protein conformational changeconsume energy

intracellular Extracellula r(outsidr the plasma membrane) election transfer 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 +

characteristic characteristics belong to active transport solute molecular have chem-modification directional phosphorylation need a complicated series of transport enzyme to transform glucose fructose mannan purin nucleotide fatty acid,et al IV. Group translocation The membrane has high impermeability to most phosphate compounds. Input each glucose may consume one ATP.

Mainly depend on PEPand PTS. PEP+ HPr EI Pyruvic acid+ P-HPr P-HPr +saccharide EII saccharide-P+HPr 1. Activate the heat stable carrier protein (HPr) 2. Saccharide transferred into membrane after phosphorylation

Transport of glucose group translocation 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. cytoplast Cytoplasm stroma

I. Definition: medium culture medium Culture medium is the mixed nutrient suitable for microbes growth and breed. Section 4 culture medium

A. Four principles 1. Definite aims different microbes have different nutrient requirement : to culture which microbes to get which products useless II. Principles and methods to select and make culture medium 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. C/N ratio in most medium:100/0.5 2. 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%)

Selection of suitable nutrient kindexampleexplaiation monosaccharidesglucose,maltose,xylose,galactose, fructose used as quick carbon sources,but will speed up breathof microbe,decrease resolved O 2,make mid-metabolized products accumulate. disaccharidesaccharose,lactoseUsed as slow carbon sources, but will speed up breathof microbe,decrease resolved O 2,make mid-metabolized products accumulate. polysaccharidestarch,cornmeal,celluloseslow carbon sources,only few microbes canuse cellulose as carbon sources molassesSugar 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%, 8kingsother sugars 4%,other deoxidized materials3%,24 kinds amino acid 4.5%,organic acid 5%,wax steroids and phosphorous0.4%, ash 12%,water20%. maltbudded barleymainly used to culture yeast,can be used to prouduce beer in industry grease cottonseed oil, corn oil,bean oil,sunflower seed oil high ,consume more O 2 when cultured in liquid,usually added as in industry alcoholsmethanol,glycerolMethanol canbe used to culture yeast hydrocarbonCH 4,C 2 H 4,C 4 H 10,C 12 ~C 20 high , consume more O 2 when cultured in liquid.hardly dissolved,don t too much beating up when cultured in liquid. organic acidacetic acid, lactic acid,citric acidwill increase the pH

carbon sources stroma glucosemethane Normal paraffin hydrocar bon methanolethanolacetate fumarat e cell producing efficiency( g cell/g stroma) 0.510.621.030.400.680.340.36 Cell yield by different energy source

Common nitrogen sources for microbe KindsSuitable microbeCharacteristic 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,ye ast,E.coli contain plenty of amino acid,good nitrogen sources for microbes inorganic: ammonium salt nitrate, ammonia Part of microbes(amino acid heterotrophs aren t able to use it) Nitrate can be used after deoxidization,may regulate pH meantime. N2N2 few bacteria (approximately 50 genus, 100 species) Nitrogen fixation is second important only to photosynthesis.

pH osmotic pressure and water ability redox potential 3. Suitable chemical and physical conditions:

pH most suitable pH v bacteria: pH7.0~8.0 v actinomyces pH7.0 ~8.5 v yeast: pH3.8~6.0 v mould pH4.0~6.0 pH value in the medium could change in the growing process of microorganisms v If culture in stroma containing carbohydrate,produce acid,so the pH will decrease. v If decompose protein and amino acid, produce NH3,pH will increase. v If use (NH4)2SO4 as nitrogen sources, SO4 2- will be excessive,so the pH decrease. v If decompose positive iron compounds,eg:NaNO3, Na+ will be excessive,so the pH increase.

methods to maintain medium pH stable Use phosphorus buffer K 2 HPO 4 /Na 2 HPO 4, H 2 PO 4 /NaH 2 PO 4 K 2 HPO 4 /Na 2 HPO 4, H 2 PO 4 /NaH 2 PO 4 Usestandby alkali CaCO3 CaHCO 3 Use acidesecent salt citrate lactophosphate et al Use liquid N2 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 water activity, aw aw the quantity of free or dissociated water that microbes can utilize under natural environment. Its 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.

solution awaw 30% glucose 0.964 1% glucose +20%glycerol 0.955 1% glucose +40%saccharose 0.964 Saturated NaCl 0.78 Saturated CaCl 2 0.30 Saturated MgCl 2 0.30 Saturated LiCl 0.11 A w of some solution

Eh redox potential to show the trend of reducer releasing electron or oxidant accepting eletron in the redox system. Aerobes + 0.1V Generally +0.3~ +0.4V Anaerobes < + 0.1 Facultative anaerobes < +0.1 fermentation >+0.1 aerobic respiration

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 Sterilization under high pressure 1.05kg/cm 1210C 15 30min Sterilization under high temperature may destroy some nutrients and change the pH.

B. Four methods Ecological simulation Lookup literature Elaborate Experiment Comparison

Category of Culture medium 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

Solid medium : add solidifying agents; the content of solidifying agents is 1 2 . sterilization; good-diaphaneity; convenience; cheap. Condition of being the solidifying agents: Cant be utilized or decomposed by microbe; can keep the solid condition in the growth temperature range; It isnt harmful for microbe at solidification point temperature; cant 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 ) Base on the physical condition of culture medium:

Semi-solid medium: the content of solidifying agents is 0.5% Usage: observe the motion of bacteria; mensurate the titer of bacteriophage. Usage: observe the motion of bacteria; mensurate the titer of bacteriophage. Liquid medium : no solidifying agent Usage: microbe culture the research on physiology /Metabolism Usage: microbe culture the research on physiology /Metabolism

The Difference between Agar and Glutin ChemistryComponentNourish-mentResolv-abilityMelttemperatureSolidificationtemperatureConcent-rationDiaphaneity Adhesio n value Tolerance of Sterilization under high pressure Agar polysaccha- ride polymernone infrequen t 96 40 1.5~2 % highbighigh Glutinproteinnitrogensourceeasy 25 20 5~12 % highbiglow

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 identified enteric bacterial

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 cant 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

Chapter 5 Microbial Nutrition and Culture medium.

Documents

Transcript of Chapter 5 Microbial Nutrition and Culture medium.