Understanding Microbial Nutrition and Growth: Essential Nutrients and Their Roles

1. Lecture 5 Microbial Nutrition & Growth Chapter 7 Foundations in Microbiology

2. Microorganism must Eat!!! • Macronutrients • Required in large amounts • Molecules that contain C, H, S & O • Protein, carbohydrates & lipids • Micronutrients • Required in trace (very low) amounts • Mn, Zn, Ni, plus many others These must be supplied in the growth medium!!!

3. Organic / Inorganic • Organic • Contain C • Usually products of living cells • CH4, glucose, starch, lipids, proteins, nucleic acids • Inorganic • No C (except CO2) • Metals & salts • O2, CO2, H2O, PO4, MgSO4, FeSO4, NH4Cl

4. 92 naturally occurring elements 25 are essential for life 6 major elements make up ~96% of the mass of most living organisms O – Oxygen (for almost all organic comp’s) P – Phosphorus (DNA, RNA and ATP (energy)), membranes (phospholipids) C – Carbon (for all organic comp’ds) S – Sulfur (for proteins) H - Hydrogen (for almost all organic comp’s) N – Nitrogen (for proteins, DNA, RNA)

5. Chemical Composition of E. coliDry weight • Carbon 50% • Oxygen 20% • Nitrogen 14% • Hydrogen 8% • Phosphorus 3% • Sulfur 1% • Potassium 1% • Sodium 1% • Calcium 0.5% • Magnesium 0.5% Water is 70% of total weight Table 7.2 page 189

6. Chemical Composition of E. coliDry weight • Protein 50% • RNA 20% • DNA 3% • Carbohydrates 10%

7. Growth Media Recipes What do we need? • Carbon Source – Glucose • Nitrogen Source – NH4Cl, NaNO3, or protein • Sulfur Source – Na2SO4 or protein • Phosphorus – K2HPO4 and/or KH2PO4 (also acts as buffer-resists change in pH of medium as cells grow) • Trace Metal Solution: Contains Fe, Mg, Mn, Ni, Cu, Co, K and others • Vitamin solution (if necessary)

8. Parasitic Lifestyle • Live in or on a host • Derive nutrition from their host • Pathogens • Inclined to cause disease or even death • Obligate parasite • Unable to grow outside of a living host • Facultative parasite • Able to grow outside a living host • Obligate intracellular parasite • Spend all or part of their life cycle within a host cell

9. Saprophytic Lifestyle • Saprophytic • Decomposer • Dead organic matter • Obligate saprobes • Strictly on dead organic matter • Facultative Parasite • Can infect a living host under certain circumstances • Opportunistic pathogen

10. Temperature and Growth • Minimum temperature - Lowest temperature an organism can grow • Maximum temperature - Highest temperature an organism can grow • Optimal temperature • Fastest growth rate & metabolism • Lowest Doubling Time

12. Low Temperature • Psychrophile • Optimal temp < 15ºC and capable of growth at OºC • Obligate psychrophiles generally can not grow above 20ºC • Psychrotrophs or facultative psychrophiles • Grow slowly at low temp but have an optimum temp > 20ºC

13. Intermediate Temperature • Mesophiles • Optimal growth temperature 20º to 40ºC • Most human pathogens (human body temperature = 37ºC) • Some mesophiles can with stand short exposure to high temperatures (this is why pour plates work!!!)

14. High Temperature • Optimal temp > 40ºC • Moderate thermophiles • Optimal growth temperature between 40 and 80ºC • Hyperthermophiles • Optimal growth temperature > 80ºC • Hot springs or deep sea thermal vents

15. Oxygen (O2) and Growth • Aerobe or aerobic organism • Use O2 & can eliminate H2O2 Obligate aerobe • Cannot live without O2 Facultative aerobes • Capable of growth in the absence of O2 Microaerophile • Requires O2 but at low concentration

16. Anaerobic • Anaerobe • Does not require O2 Strict or Obligate anaerobe • Cannot tolerate any O2 Aerotolerant anaerobes • Do not utilize O2 but survive in its presence Capnophiles (usually anerobic or microaerophilic) • Prefer a high [CO2] ([ ] means concentration)

18. Use of Oxygen (O2) • Required for aerobic respiration • C6H12O6 + O2 CO2 + H2O + ATP • Glucose  Glycolysis  Krebs Cycle  Respiratory Chain • O2 is the final electron acceptor (celled terminal electron acceptor) • Generates a lot of ATP • Anaerobic Respiration • NO3-, SO4-2 or CO3-2 used as terminal electron acceptor • Generates less ATP per amount of terminal electron acceptor Respiratory chain represents a series of proteins usually in the cell membrane of prokaryotes that are electron carriers that ultimate drop off electrons to the terminal electron acceptor

19. Fermentation Anaerobic process • Pyruvate  ethanol • Yeast and bacteria • Pyruvate  lactic acid • Bacteria • Pyruvate  acetic acid • Bacteria • Electron donor and acceptor are organic compounds • No electron transport chain • Less energy than from respiration

20. O2 is EXTREMELY Reactive • Build up of O2 in the cell can be deadly • Destructive by-products of O2 • Superoxide ion O2- • Peroxides • H2O2 • Hydroxyl ion • OH- • There are enzymes that detoxify these products

21. Superoxide dismutase O2- +O2- + 2H+ H2O2 +O2 H2O2 +H2O2  2H2O + O2 Catalase

22. pH and growth • Most bacteria are neutrophilic and their optimal growth pH is between pH 6 and 8 • Acidophiles – optimal pH < 3 • Obligate acidophile • optimal growth pH between 0-1 and can’t grow at 7 Alkalinophiles (alkaliphile) • Optimal growth at high pH (> 8) • Obligate grows at pH > 10 but can’t at 7 Note: Fungi are much more tolerant of acidic pH and the optimum growth pH for many is around 5

23. Osmotic Pressure (Water Activity) • Water Activity (aw) – For pure water aw = 1.000 – Affects growth strongly and selects for particular organisms - Human blood – 0.995 - Bread – 0.950 - Maple Syrup – 0.900 - Salt Lakes, Salted Fish – 0.750 - Cereals, Dried Fruit – 0.700

24. Osmotic Pressure (Water Activity) • Water Activity (aw) – For pure water aw = 1.000 • Halophiles – a type of extremophile • Osmophile • Hypertonic / hypersaline environments • Salt lakes, salt ponds • Obligate halophile • Optimal growth ≥ 25% NaCl but requires at least 9% NaCl • Facultative halophiles • Resistant to salt but don’t normally reside in high salt environments… Staphylococcus aureus (8% salt)

27. Ecological Associations • Symbiotic • Two organisms live in close association • Required by one or both individuals Mutualism • Mutually beneficial relationship Commensalism • One species derives benefit without harming the other Parasitism • One species derives benefit and the other is harmed

28. Other Associations • Synergism • Interrelationship • 2 or more free-living organisms • Benefits all but is not necessary for survival • Antagonism • One species secretes a substances that inhibits or destroys another species • Antibiotics • Antimicrobial proteins

29. Cell Division & Endospores Binary fission (budding) vs. Sporulation Binary fission and budding are forms of reproduction Sporulation is, in most cases, not a form of reproduction but is used for survival of the cell under harsh conditions. There are some exceptions Note: Endospores are found only in Gram-positive bacteria Implicated in disease… examples Bacillus anthracis, Clostridium botulinum, Clostridum tetani

30. Binary Fission

31. Binary / Transverse Fission and Budding • One cell becomes two • Division plane forms across the width of the cell • Parent cell enlarges • Replication of the chromosome • Transverse septum • Continuous

32. Sporulation • Dormant bodies • Resting structure of some Gram + • Bacillus, Clostridium & Sporoscarcina • Vegetative cycle • Endospore cycle • Unfavorable environmental conditions • Heat, irradiation, desiccation, disinfectants • Thick impervious cortex • Long lived • 250 mya spore

33. Protein filaments migrate from the middle of the cell to opposite poles Two rings form near each pole Production of a spore only occurs at one pole Forespore or prespore forms Spore matures within the “mother cell”… cell lyses and pore is feed Spore withstands extreme environmental conditions

34. Other Spore Formers • Epulopiscium spp. • Unusually large, bacterial symbiont of the intestinal tract of marine surgeon fish • Surgeon fish are herbivores & detritivorous • Some strains of Epulopiscium do not reproduce vegetatively • Viviparity

35. Viviparity

37. Estimating the Number of Bacterial Cells in a Sample (e.g., water, food, soil) Main method is the plate count method which we will go over here…will count only live cells in a sample (Viable count) but not all live cells may form colonies Other methods are microscopic methods but there are limitations… cells could be live or dead unless a “vital” stain is used; and flow cytometry which is expensive…

38. Plate Count Method • Dilute a sample with sterile saline until the microbial cells can be counted accurately • A broad range of dilutions is used since the exact number of bacteria is not known • Plates should have between 30 and 300 colonies • Fewer than 30 colonies is not statistically accurate • Too Few To Count (TFTC) • More than 300 colonies is simply to difficult to count • Colonies are too close together • Too Numerous To Count (TNTC)

39. Colony Forming Units (cfu) • Each viable cell will develop into a colony • There are two assumptions: • Each colony arose from one cell • Can’t be certain that two cells close together produced one colony • Random sample from the population • Statistically accurate

40. Calculate Colony Forming Units • Count the bacterial colonies on the plates the have between 30 & 300 colonies • Divide the number of colonies by the dilution factor • There are 130 colonies on the 10-6 dilution • 130 / 10-6 = 1.3 x 108 or 130,000,000 • Report cfu / mL or cfu / gram

41. Large number of cells 0.1 0.01 0.1 0.01 0.01 10-7 10-8 10-6 10-2 10-4

42. The 1st plate has TNTC • The 3rd plate has TFTC • The 2nd plate has 21 colonies and will be used for the calculations • The concentration of the cells is • 21 / 0.1 mL = 210 cells / mL • 210 X 107 = 2.1 X 109

43. Population Growth

44. Measurement of Microbial Population • Viable plate count method • Counting colonies on agar medium • Spectrophotometric analysis • Measure of turbidity

45. Growth Curve

46. Growth Curve • Lag phase – “flat” period of adjustment, enlargement; little growth • Exponential growth phase – a period of maximum growth will continue as long as cells have adequate nutrients & a favorable environment • Stationary phase – rate of cell growth equals rate of cell death cause by depleted nutrients & O2, excretion of organic acids & pollutants • Death phase – as limiting factors intensify, cells die exponentially in their own wastes

47. Spectrophotometer • Useful laboratory tool - inexpensive • Electronically compares the amount of light transmitted through a sample with that transmitted through a blank. • The ratio of the amount of light transmitted through a sample to that transmitted through a blank is called the transmittance

48. Spectrophotometer light transmitted through a sample light transmitted through the blank t = %T = t x 100%

50. Absorbance • A = -log10(t) • A = Optical Density or O.D. • O.D.400 nm or A400 nm