Microbial Nutrition and Growth Sofronio Agustin Professor Sofronio Agustin Professor LECTURES IN MICROBIOLOGY LECTURES IN MICROBIOLOGY LESSON 5

2 Lesson 5 Topics  Microbial Nutrition  Environmental Factors  Microbial Growth

3 Microbial Nutrition  Based on intake: (a)Macronutrients (CHONPS) (b)Micronutrients (trace elements)  Based on carbon content: (a)Organic nutrients- contain carbon (b)Inorganic nutrients- simple atom or molecule without carbon

4 Chemical Composition Bacteria are composed of different elements and molecules, with water (70%) and proteins (15%) being the most abundant.

5 Essential Nutrients  Carbon source  Energy Source  Growth Factors

6 Carbon Source  Autotrophs - obtain carbon from inorganic molecules like CO 2  Heterotrophs - obtain carbon from organic matter from other life forms (e.g. sugar, proteins and lipids)

7 Energy Source  Photoautotrophs and photoheterotrophs obtain energy from sunlight  Chemoautotrophs derive electron energy from reduced inorganic compounds  Chemoheterotrophs obtain electron energy from hydrogen atoms of organic compounds

8 Nutritional Categories Summary of different nutritional categories of microbes based energy and carbon sources

9 Methanogens  Methanogens are chemoautotrophic microbes  Example: methane producing Archaea

10 Extracellular Digestion

11 Cell Membrane  Phospholipid bilayer with integral and peripheral proteins  “Fluid mosaic” model - phospholipids and proteins move laterally  Exhibits “selective permeability”

12 Membrane Transport  Passive: (a)Simple diffusion (b)Facilitated diffusion (c)Osmosis  Active: (a)Permease (b)Group translocation (c)Endocytosis

13 Simple Diffusion  Net movement of solute from area of high concentration to a low concentrated area  No energy is expended  Down the concentration gradient (like a river flowing downstream)

14 Diffusion A cube of sugar will diffuse from a concentrated area into a more dilute region, until an equilibrium is reached.

15 Facilitated Diffusion  Transport of polar molecules and ions across the membrane down their concentration gradients  No energy is expended (passive)  Carrier protein facilitates the binding and transport -Specificity -Saturation -Competition

16 Facilitated Diffusion Facilitated Diffusion: The Process

17 Osmosis  Diffusion of solvent (usually, water) through a permeable but selective membrane  Water tends to move toward higher solute concentrated areas

18 Tonicity Fate of cells in different osmotic conditions - isotonic, hypotonic, and hypertonic solutions

19 Active Transport  Transport of molecules against its concentration gradient  Requires energy and transport protein (Ex. Permeases and protein pumps transport sugars, amino acids, organic acids, phosphates and metal ions)  Group translocation transports and modifies specific sugars

20 Endocytosis  Large substances are taken in by the cell but are not transported through the membrane.  Requires energy (active)  Common in eukaryotes - Phagocytosis - Pinocytosis

21 Active Transport Example of permease, group translocation and endocytosis

22 Cellular Transport : Summary

23 Environmental Factors  Temperature  Gas  pH  Osmotic pressure  Other factors  Microbial association

24 Temperature  Psychrophiles – (cold loving) 0 to 15 °C  Psychrotrophs - (food spoilage) grow between 20 to 30 °C  Mesophiles- (most human pathogens) 20 to 40 °C  Thermophiles- (heat loving) 45 to 80 °C  Themoduric - (contaminants of heated food) survive in short exposures to high temp  Hyperthermophiles - (Archaea)

25 Temperature Tolerance

26 Gas Requirements  Two gases that influence microbial growth: (1)Oxygen  Respiration - terminal electron acceptor  Oxidizing agent - toxic forms (2)Carbon dioxide

27 Oxygen Metabolites  Superoxide radical - O 2 -  Singlet oxygen - O 2 with single electron in its valence shell  H 2 O 2 All are toxic byproducts of metabolism neutralized by enzymes SOD (superoxide dismutase), peroxidase and catalase.

28 Bacterial Types  Obligate aerobe  Facultative anaerobe  Obligate anaerobe

29 Obligate Aerobes  Require oxygen for metabolism  Possesses enzymes that can neutralize the toxic oxygen metabolites: SOD, peroxidase and catalase  Ex: Most fungi, protozoa, and bacteria like Bacillus sp. and Pseudomonas sp.

30 Obligate Anaerobes  Cannot use oxygen for metabolism  Do not possess SOD and catalase  The presence of oxygen is toxic to the cell  Ex: Clostridium sp. and Bacteroides sp.

31 Anaerobiosis Anaerobic culture techniques: (a) anaerobic chamber, (b) anaerobic jar

32 Facultative Anaerobes  Does not require oxygen for metabolism, but can grow in its presence  During minus oxygen states, anaerobic respiration or fermentation occurs  Possess superoxide dismutase and catalase  Ex. E. coli and S. aureus

33 Thioglycolate Broth Thioglycollate broth is used to demonstrate aerotolerance of bacteria. Aerobes, facultative anaerobes, and obligate anaerobes can be detected using this medium.

34 Other Gas Requirements  Microaerophiles - requires less than 10% of atmospheric O2. Ex: Campylobacter jejuni  Capnophiles - requires increased CO2 (5-15%) tension for initial growth. Ex: S. pneumoniae

35 pH  Most cells grow best between pH 6-8  Acidophiles (up to pH 0) - molds and yeast  Alkalinophiles (up pH 10) urea- decomposing bacteria like Proteus sp.

36 Osmotic Pressure  Osmophiles - live in solutions with high solute concentration (e.g. sugar content in jams)  Halophiles - requires high salt concentrations and withstands hypertonic conditions Ex. Halobacterium sp. (Archaea)  Facultative halophiles - can survive high salt conditions but is not required for survival Ex. Staphylococcus aureus

37 Other Factors  Radiation- withstand UV, infrared rays  Barophiles – withstand high pressures  Spores and cysts- can survive dry habitats

38 Microbial Interactions Influence microorganisms have on other microbes:  Symbiotic relationship  Non-symbiotic relationship

39 Symbiotic Relationship Organisms that live together in close nutritional relationships Types:  Mutualism – both organism benefit  Commensalism – only one organisms benefits  Parasitism – typically host-microbe relationship

40 Commensalism  “Satellitism” as a form of commensalism  Staphylococcus aureus provides vitamins and amino acids to Haemophilus influenzae, which grows around colonies of S. aureus.

41 Non-Symbiotic Relationships  Organisms are free-living, and do not rely on each other for survival  Types:  Synergism – shared metabolism enhances growth of both microbes  Antagonism- competition between microorganisms

42 Microbe-Host Interactions  Can be commensal, parasitic, and synergistic  Ex. E. coli produce vitamin K for the host

43 Microbial Growth  Binary fission  Generation time  Growth curve  Enumeration of bacteria

44 Binary Fission  Parent cell enlarges and duplicates its DNA  Septum formation divides the cell into two separate chambers  Complete division results in two identical daughter cells

45 Steps in Binary Fission Rod-shaped bacteria undergoing binary fission

46 Growth Curve  Lag phase  Log phase  Stationary phase  Death phase

47 Phases of Bacterial Growth Growth curve in a bacterial culture.

48 Enumeration of Bacteria  Direct Methods: (a)Microscopic (b)Viable plate count (c)Membrane filtration (d)Most probable number  Indirect Methods: (a)Turbidity (b)Metabolic assay (c)Dry weight determinations

49 Direct Microscopic Count  The direct cell method counts the total dead and live cells in a special microscopic slide containing a premeasured grid.  Petroff-Hausser counting chamber used in dairy industry.

50 Standard Plate Count Serially diluted samples are plated out and bacterial count expressed in CFU/ml.

51 Membrane Filtration Membrane filtration and coliform counts.

52 Turbidimetric Turbidimetric measurements as indicators of bacterial growth. The greater the turbidity the larger the population density.

53 Coulter Counter  The Coulter Counter uses an electronic sensor to detect and count the number of cells.  Rapid automated counting method