The Common Nutrient Requirements

1. Chapter 5 Microbial Nutrition The Common Nutrient Requirements • macroelements (macronutrients) • C, O, H, N, S, P, K, Ca, Mg, and Fe • required in relatively large amounts • micronutrients (trace elements) • Mn, Zn, Co, Mo, Ni, and Cu • required in trace amounts • often supplied in water or in media components All Organisms Require Carbon, Hydrogen, Oxygen and Electron Source • carbon is backbone of all organic components present in cell • hydrogen and oxygen are also found in organic molecules • electrons play a role in energy production and reduction of CO2 to form organic molecules

2. Nutritional Types of Organisms 自營 異營 光營 化學營 無機營 有機營

4. Requirements for Nitrogen, Phosphorus, and Sulfur • needed for synthesis of important molecules (e.g., amino acids, nucleic acids) • nitrogen supplied in numerous ways • phosphorus usually supplied as inorganic phosphate • sulfur usually supplied as sulfate via assimilatory sulfate reduction Sources of nitrogen, Phosphorus and Sulfur • Nitrogen • organic molecules • ammonia • nitrate via assimilatory nitrate reduction • nitrogen gas via nitrogen fixation • Phosphorus • most organisms use inorganic phosphorus which is directly incorporated into their cells • Sulfur • most organisms use sulfate and reduce it by assimilatory sulfate reduction

5. Growth Factors • organic compounds • essential cell components (or their precursors) that the cell cannot synthesize • must be supplied by environment if cell is to survive and reproduce Classes of growth factors • amino acids • needed for protein synthesis • purines and pyrimidines • needed for nucleic acid synthesis • vitamins • function as enzyme cofactors Practical Applications of growth factors • use of microorganisms in bioassays to quantify growth factor • industrial production of growth factors by microorganisms

7. Uptake of Nutrients by the Cell carrier saturationeffect • diffusion rate • reaches a plateau • when carrier becomessaturated • Some nutrients enter by passive diffusion • Most nutrients enter by: • facilitated diffusion • active transport • group translocation • rate of facilitated • diffusion increases • more rapidly and • at a lower concentration Passive Diffusion • molecules move from region of higher concentration to one of lower concentration because of random thermal agitation • H2O, O2 and CO2 often move across membranes this way

8. Facilitated Diffusion • similar to passive diffusion • movement of molecules is not energy dependent • direction of movement is from high concentration to low concentration • size of concentration gradient impacts rate of uptake • differs from passive diffusion • uses carrier molecules (permeases) • smaller concentration gradient is required for significant uptake of molecules • effectively transports glycerol, sugars, and amino acids • more prominent in eucaryotic cells than in procaryotic cells

9. conformational change of carrier A model of facilitated diffusion

10. Active Transport • energy-dependent process • ATP or proton motive force used • moves molecules against the gradient • concentrates molecules inside cell • involves carrier proteins (permeases) • carrier saturation effect is observed at high solute concentrations ABC transporters • ATP-binding cassette transporters • observed in bacteria, archaea, and eucaryotes

11. Active transport using proton and sodium gradients

12. Group Translocation • chemically modifies molecule as it is brought into cell • best known system: transports a variety of phosphoenolpyruvate (磷酸烯醇丙酮酸): sugar phosphotransferase system (PTS) • sugars while phosphorylating them using phosphoenolpyruvate (PEP) as the phosphate donor • energy-dependent process Bacterial PTS

13. Iron Uptake fungi E. coli • ferric iron is very insoluble so uptake is difficult • microorganisms use siderophores to aid uptake • siderophore complexes with ferric ion • complex is then transported into cell Siderophore ferric iron complexs

14. Culture Media • most contain all the nutrients required by the organism for growth • classification • chemical constituents from which they are made • physical nature • function Types of Media

15. Complex Media Defined or Synthetic Media • contain some ingredients of unknown composition and/or concentration • all components and their concentrations are known

16. Some media components • peptones • protein hydrolysates prepared by partial digestion of various protein sources • extracts • aqueous extracts, usually of beef or yeast • agar • sulfated polysaccharide used to solidify liquid media

17. Functional Types of Media Blood agar • supportive or general purpose media • support the growth of many microorganisms • e.g., tryptic soy agar • enriched media • general purpose media supplemented by blood or other special nutrients • e.g., blood agar • selective media • favor the growth of some microorganisms and inhibit growth of others • e.g., MacConkey agar • selects for gram-negative bacteria Chocolate agar

18. differential media • distinguish between different groups of microorganisms based on their biological characteristics • e.g., blood agar • hemolytic versus nonhemolytic bacteria • e.g., MacConkey agar • lactose fermenters versus nonfermenters

19. Isolation of Pure Cultures • pure culture • population of cells arising from a single cell • spread plate, streak plate, and pour plate are techniques used to isolate pure cultures The Spread Plate and Streak Plate • involve spreading a mixture of cells on an agar surface so that individual cells are well separated from each other • each cell can reproduce to form a separate colony (visible growth or cluster of microorganisms)

20. Spread-plate (平板塗佈) technique 4. spread cells across surface 1. dispense cells onto medium in petri dish 2. - 3. sterilize spreader Appearance of a Spread Plate

21. Streak (條紋) plate technique

22. The Pour Plate (倒皿法) • sample is diluted several times • diluted samples are mixed with liquid agar • mixture of cells and agar are poured into sterile culture dishes

23. Bacterial Colony Morphology Bacillus subtilis

24. Colony growth • most rapid at edge of colony • oxygen and nutrients are more available at edge • slowest at center of colony • in nature, many microorganisms form biofilms on surfaces

25. Growth Chapter 6Microbial Growth • increase in cellular constituents that may result in: • increase in cell number • e.g., when microorganisms reproduce by budding (出芽生殖) or binary fission (二分裂法) • increase in cell size • e.g., coenocytic microorganisms have nuclear divisions that are not accompanied by cell divisions • microbiologists usually study population growth rather than growth of individual cells

26. The Procaryotic Cell Cycle • cell cycle is sequence of events from formation of new cell through the next cell division • most bacteria divide by binary fission • two pathways function during cycle • DNA replication and partition (分開) • cytokinesis (細胞質分裂)

27. The Cell Cycle in E. coli • E. coli requires ~40 minutes to replicate its DNA and 20 minutes after termination of replication to prepare for division

28. Chromosome Replication and Partitioning • most procaryotic chromosomes are circular • origin of replication – site at which replication begins • terminus– site at which replication is terminated • replisome – group of proteins needed for DNA synthesis; parent DNA spools through the replisome as replication occurs • MreB – an actin homolog plays role in determination of cell shape

29. Cytoskeletal Proteins - Role in Cytokinesis • process not well understood • protein MreB • similar to eucaryotic actin • plays a role in determination of cell shape and movement of chromosomes to opposite cell poles • protein FtsZ, • similar to eucaryotic tubulin • plays a role in Z ring formation which is essential for septation MinCD: inhibitor of Z-ring Formation of the cell division apparatus in E. coli.

30. DNA Replication in Rapidly Growing Cells • cell cycle completed in 20 minutes • 40 minutes for DNA replication • 20 minutes for septum formation and cytokinesis • look at timing-how can this happen? • Second, third or fourth round of replication can begin before first round of replication is completed The Growth Curve • observed when microorganisms are cultivated in batch culture • culture incubated in a closed vessel with a single batch of medium • usually plotted as logarithm of cell number versus time • usually has four distinct phases

31. Microbial growth curve in a closed system population growth ceases maximal rate of division and population growth decline in populationsize no increase

32. Lag Phase • cell synthesizing new components • e.g., to replenish spent materials • e.g., to adapt to new medium or other conditions • varies in length • in some cases can be very short or even absent Exponential Phase • also called log phase • rate of growth is constant • population is most uniform in terms of chemical and physical properties during this phase Balanced growth • during log phase, cells exhibit balanced growth • cellular constituents manufactured at constant rates relative to each other

33. Unbalanced growth • rates of synthesis of cell components vary relative to each other • occurs under a variety of conditions • change in nutrient levels • shift-up (poor medium to rich medium) • shift-down (rich medium to poor medium) • change in environmental conditions Effect of nutrient concentration on growth

34. Stationary Phase • total number of viable cells remains constant • may occur because metabolically active cells stop reproducing • may occur because reproductive rate is balanced by death rate Possible reasons for entry into stationary phase • nutrient limitation • limited oxygen availability • toxic waste accumulation • critical population density reached

35. Starvation responses • morphological changes • e.g., endospore formation • decrease in size, protoplast shrinkage, and nucleoid condensation • production of starvation proteins • long-term survival • increased virulence Death Phase • two alternative hypotheses • Cells are Viable But Not Culturable (VBNC) • Cells alive, but dormant • programmed cell death • Fraction of the population genetically programmed to die (commit suicide)

36. Loss of Viability

37. Prolonged Decline in Growth • bacterial population continually evolves • process marked by successive waves of genetically distinct variants • natural selection occurs

38. The Mathematics of Growth • generation (doubling) time • time required for the population to double in size • Varies depending on species of microorganism and environmental conditions • range is from 10 minutes for some bacteria to several days for some eucaryotic microorganisms

39. Generation time determination

40. Measurement of Microbial Growth • can measure changes in number of cells in a population • can measure changes in mass of population Measurement of Cell Numbers • direct cell counts • counting chambers • electronic counters • on membrane filters • viable cell counts • plating methods • membrane filtration methods

41. Counting chambers • easy, inexpensive, and quick • useful for counting both eucaryotes and procaryotes • cannot distinguish living from dead cells Electronic counters • useful for large microorganisms and blood cells, but not procaryotes • microbial suspension forced through small orifice • movement of microbe through orifice impacts electric current that flows through orifice • instances of disruption of current are counted

42. Direct counts on membrane filters • cells filtered through special membrane that provides dark background for observing cells • cells are stained with fluorescent dyes • useful for counting bacteria • with certain dyes, can distinguish living from dead cells Viable Counting Methods • spread and pour plate techniques • diluted sample of bacteria is spread over solid agar surface or mixed with agar and poured into Petri plate • after incubation the numbers of organisms are determined by counting the number of colonies multiplied by the dilution factor • results expressed as colony forming units (CFU)

43. Viable Count Method - Membrane filtration especially useful for analyzing aquatic samples

44. Plating methods… • simple and sensitive • widely used for viable counts of microorganisms in food, water, and soil • inaccurate results obtained if cells clump together Measurement of Cell Mass • dry weight • time consuming and not very sensitive • quantity of a particular cell constituent • e.g., protein, DNA, ATP, or chlorophyll • useful if amount of substance in each cell is constant • turbidometric measures (light scattering) • quick, easy, and sensitive

45. Turbidometric measures (light scattering) more cells  more light scattered  less light detected

46. The Continuous Culture of Microorganisms • growth in an open system • continual provision of nutrients • continual removal of wastes • maintains cells in log phase at a constant biomass concentration for extended periods • achieved using a continuous culture system

47. Dilution Rate and Microbial Growth dilution rate – rate atwhich medium flowsthrough vesselrelative to vessel size cell densitymaintained at widerange of dilutionrates Chemostat dilution rate and microbial growth

48. The Chemostat (化學恆定) The Turbidostat • rate of incoming medium = rate of removal of medium from vessel • an essential nutrient is in limiting quantities • chemostat operates best at low dilution rate • regulates the flow rate of media through vessel to maintain a predetermined turbidity or cell density • dilution rate varies • no limiting nutrient • turbidostat operates best at high dilution rates

49. Importance of continuous culture methods • constant supply of cells in exponential phase growing at a known rate • study of microbial growth at very low nutrient concentrations, close to those present in natural environment • study of interactions of microbes under conditions resembling those in aquatic environments • food and industrial microbiology The Influence of Environmental Factors on Growth • most organisms grow in fairly moderate environmental conditions • extremophiles • grow under harsh conditions that would kill most other organisms