Microbial Growth

1. Microbial Growth Microbial growth is an increase in the population of microbes (number of cells), rather than an increase in size.Microbial growth is an increase in the population of microbes (number of cells), rather than an increase in size.

2. Microbial Growth Increase in number of cells rather than size Growth of most microorganisms occurs by the process of binary fission DNA replication Double amount of macromolecules, monomers, and inorganic ions Growth of membrane and cell wall Division Generation time varies (Typical 1 - 3 hours) Dependent on nutritional and genetic factors E. coli= 20 minutes to divide ? optimal conditions

4. Cell division and chromosome replication Regulated by Fts proteins (filamentous temperature sensitive) Essential for cell division in all prokaryotes Fts proteins interact to form a division apparatus in the cell called the divisome. FTSz Forms ring around center of cell Directs cell division at the central plane of cell ZipA Anchor that connects FtsZ ring to cytoplasmic membrane FtsA Helps connect FtsZ ring to membrane and also recruits other divisome proteins

7. 6.2 - Fts Proteins and Cell Division DNA replicates before the FtsZ ring forms Location of FtsZ ring is facilitated by Min proteins Direct the placement of FTSz between 2 nucleoids FtsK protein mediates separation of chromosomes to daughter cells GTP Used as fuel source for FTSz polymerization/depolymerization

8. Cell Division Cycle

10. 6.4 - Peptidoglycan Synthesis and Cell Division Production of new cell wall material is a major feature of cell division In cocci, cell walls grow in opposite directions outward from the FtsZ ring In rod-shaped cells, growth occurs at several points along length of the cell

11. Cell Wall Formation Preexisting peptidoglycan needs to be severed to allow newly synthesized peptidoglycan to form Begins at the FtsZ ring Autolysins (enzymes that are similar to lysozyme) breaks glycosidic bonds creating small openings

12. Cell Wall Formation New (M-G-pep) created in cytoplasm New cell wall material is added across the openings Bactoprenol?a hydrophobic alcohol that facilitates transport of new glycan units through the cytoplasmic membrane to become part of the growing cell wall Wall band: junction between new and old peptidoglycan Glycolases A process of spontaneous cell lysis called autolysis can occur unless new cell wall precursors are spliced into existing peptidoglycan to prevent a breach in peptidoglycan integrity at the splice point.

13. Transpeptidation Final step in cell wall synthesis Form cross links between NAM in adjacent chains of peptidoglycan Inhibited by penicillin

14. Population Growth Growth rate= change in cell number or cell mass of population A generation is the interval of two cells from one Generation time (doubling time) Time it takes to produce two new cells Time for cell mass or # to double Varies greatly Type of organism Temperature Nutrients Other conditions Norm= 1-3 hours Exponential growth (Log phase growth) When population doubles/ unit of time Let’s take look at animation http://www.biology.arizona.edu/biomath/tutorials/Applications/Population.html

15. Bacteria grow exponentially

16. Plotting bacterial growth

17. Growth Calculations If you start with 1 cell how many do you have after 4 generations? No = initial number of cells N= # cells after n generations n=number of generations Formula?N= No(2n) N=1(16)=16 cells What if you start with 100 cells? What if you start with 100 cells and go for 5 generations?

18. Growth Calculations E. coli has a generation time of 20 minutes. If you start with 1 E. coli cell how many do you have after 2 hours? g=generation time and t=time Formula?n=t/g n=(2 hours x 60minutes/hour)/20 minutes= ? N= No(2n) N=1(26)=64 cells 5 hours? N=32,768 cells

20. Realistic Growth Calculations How do you determine n if you know N and No only? You start with 2 cells and end up with 2,000 after 2 hours; so how many generations? What is generation time? n=3.3(logN-logNo) So n=3.3(log (2000)– log (2)) n=3.3(3.3-0.3)=9.9 generations g=t/n g=120 minutes/9.9 generations=12.12 minutes per generation

21. More Growth Calculations K is the growth rate constant or the number of generations per unit time for a given organism under a given set of conditions K is used to optimize growth conditions; the faster the growth the larger the K K=ln2/g Example Generation time 30 minutes (k=0.023) Generation time 60 minutes (k=0.011)

22. Summary The faster the growth the greater the k (growth constant) greater the slope when plotting cell concentration per unit time smaller the g (generation time)

23. Recall This Question Again E. coli has a generation time of 20 minutes. If you start with 1 E. coli cell how many do you have after 24 hours? We determined: 4.72 x 1021 cells Theoretically this is correct if cells didn’t die, run out of nutrients, sit in a pool of their own waste for several hours, etc. The growth calculations you learned pertain to EXPONENTIAL PHASE ONLY!

24. Growth Cycle Lag phase: time it takes for cell to start growing once inoculated Take in nutrients, synthesize essential components, repair damage, adjust to new media/nutrients, adjust to new concentration of nutrients Varies depending on conditions and nature of culture Exponential or log phase: cells growing exponentially When population doubles/ unit of time Rate increases with each new generation Most metabolically active, but most sensitive Stationary phase: No net increase or decrease in population Nutrients run out or waste build up Metabolism and biosynthesis still occurring Death phase: # cells lysing > # new cells

26. Continuous versus Batch Continuous Chemostat No growth phases Always exponential Flow system with constant volume Fresh media added as depleted media discarded Can control growth rate and population density independently Purpose: Measure growth properties, physiology, microbial ecology Batch Test tube Distinct growth phases Fixed volume of media and no flow Media eventually depleted and no replacement Growth rate is dependent on population density Purpose: growth overnight cultures.

28. Continuous Culture Growth Rate (GR) Increase in cell number per unit time Doubling time decreases as GR increases Growth Yield (GY) Number of cells present at a given time Cell concentration Nutrient concentration and dilution rate affects the growth rate and yield

29. GR vs. GY Growth rate controlled independently from growth yield To increase GR increase dilution rate Yield stays generally the same To increase GY increase concentration of nutrients Rate stays generally the same Industrial microbiologists grow bacteria to obtain a lot of cells in a short amount of time

30. As nutrient concentration increases the GY increases but GR stays steady after steady state reached.

32. Applications Can control GR and GY independently Cells always in exponential phase Most physiological experiments require exponential phase Can determine nutrient effects on population or mimic natural environment By adjusting dilution rate and nutrient levels, the experimenter can obtain dilute, moderate and dense populations growing at slow, moderate or rapid growth rates

33. Factors that affect bacterial growth Temperature pH Osmotic pressure/water availability Oxygen

34. Temperature Cardinal temperatures Minimum growth temperature Lowest temperature at which an organism will grow Below this temp.?nutrient transport difficulty due to the fact that membrane gels and transport too slow Optimum growth temperature Temperature at which an organism grows best Metabolic enzyme reactions occurring at maximum rate Maximum growth temperature Highest temperature at which an organism will grow Above this temp.?protein denaturation; membrane collapse, and lysis All can be modified slightly by other environmental properties Usually a 30º range (C) for prokaryotes Extremophiles live at extreme hot and cold temperatures

36. Temperature Classes Psychrophiles Cold lovers Optimum: 0 -15 ºC (depends on organism—usually around 4 ºC) RANGE: -10 ºC ? 20 ºC (cannot survive at room temp!) Min is typically below zero Found in polar regions, at high altitudes, and in depths of oceans (constant cold) Algae in sea ice and snow fields Psychrotolerant (psychrotroph) Optimum: 20 - 40 ºC RANGE: 1 ºC ? 40 ºC Grows best at refrigerator temperatures, but can grow at low temperatures Typically cannot grow at freezing temps. Found in soils and water and foods in fridge Enzymes sensitive to heat b/c of structure Polar and Hydrophobic amino acids?increase flexibility More a helices and fewer ß sheets?increase flexibility Membranes well suited Increase in unsaturated fatty acids (more fluid)

37. Psychrotrophs

38. Temperature Classes Mesophiles Optimum: 37-40 ºC (body temp) RANGE: 12 ? 48 ºC Most common Most pathogens E. coli Thermophiles Heat loving Optimum: 45-80 ºC (depending on organism) RANGE: 40 ? 85ºC Compost, soils, hot water heaters, some hot springs Hyperthermophiles Optimum: 90-121 ºC RANGE: 89 ? 120 ºC Steam vents, hot springs, volcanoes Mostly Archaea Results of studies of different organisms Prokaryotes can grow at higher temps than Eukaryotes Most thermophiles (hyperthermophiles) are archaea Phototrophs tend not to grow at higher temps

39. Temperature Requirements

40. How can thermophiles and hyperthermophiles thrive at high temperatures? Enzymes more heat stable Only a few key amino acids are different from mesophiles Increase in salt bridges (ionic bonds) between amino acids Densely packed hydrophobic interiors Example of heat stable enzyme = Taq polymerase used in PCR, isolated from Thermus aquaticus Membranes are more heat stable Bacteria - saturated fatty acids (dec. fluidity) and stronger hydrophobic environment (greater interaction of fatty acid tails) Archaea contain isoprene units?lipid monolayer and ether linkage

41. Physical Requirements pH Most natural environments pH 5-9 Most bacteria produce organic acids as they grow and metabolize When growing bacteria, pH can change during growth so buffers are added to moderate the pH pH should be near normal on inside of cell Acidophiles Grow at low pH (<5) Fungi in general and some bacteria (obligate – must grow at low pH) If pH is increased, membranes are destroyed and cells lyse Thiobacillus and acid mine drainage (pH 1) Alkaliphiles Grow at high pH (>10-11 pH) Soda lakes, high carbonate soils

42. Preserving Food Most bacteria grow best between pH 6.5 – 7.5 Neutrophiles - pH 5.4 - 8.5 Foods can be preserved by acid pH

43. Osmotic Effects on Microbial Growth Osmosis Positive water balance Normally, cytoplasm has higher solute concentration than environment (positive water balance) Water activity (aw) = vapor pressure of air to water Low aw = hypertonic Hypotonic environments What happens? Plasmolysis Caused by hypertonic environments Use of salt as a preservative

44. Salt Lovers Halophiles Specific requirement for Na Can grow at high salt concentration without negative water balance. Mild: require 1-6% NaCl Moderate: require 6-15% NaCl Extreme: require 15-30% Halotolerant: can tolerate low aw, but not optimal for growth How can a cell exist in salty environment? Compatible solutes do not inhibit cell activity Increase in internal solute concentration Synthesis versus transport of a compatible solute

46. Others Osmophiles Tolerates high sugar concentrations which cause low aw Xerophiles Tolerate dry environments

47. Chemical Requirements Oxygen Variation in need to metabolize O2 Divided into several groups: Obligate (strict) aerobes Aerobic metabolism (requires O2 to make energy) Growth at 21% O2 Detoxify products of metabolism Microaerophiles Aerobic metabolism (requires O2 in small amounts for energy) Growth at reduced O2 levels Facultative anaerobe (E. Coli) In presence of O2 uses aerobic metabolism to make energy (faster) In absence of O2 will ferment (less energy produced) Obligate (strict) anaerobe (Clostridium) Anaerobic metabolism or fermentation No O2 metabolism and killed by O2 Aerotolerant Anaerobic metabolism or fermentation (no benefit from oxygen) No O2 metabolism, but tolerates O2

48. Toxic Forms of Oxygen Products of O2 metabolism?toxic Singlet oxygen: O2 boosted to a higher-energy state Superoxide free radicals: O2– Peroxide anion: O22– Hydroxyl radical (OH?)

49. Toxic Forms of Oxygen Organisms that use aerobic metabolism must detoxify these products Catalase enzyme: 2 H2O2?2 H2O + O2 Peroxidase enzyme: H2O2?2 H+ + H2O Superoxide dismutase enzyme: detoxifies O2-and OH• Obligate anaerobes lack these enzymes

50. How are anaerobic organisms grown? They grow at the bottom of tubes, away from oxygen Reducing agents added to media of anaerobes Resazurin: reduce O2 ? H2O Anaerobic jars and chambers (air replacement)

51. Chemical Requirements Oxygen (O2)