Microbial Growth

MICR 1010: Introductory Microbiology and Molecular Biology

Life and Metabolism

All living organisms obtain Nutrients (chemicals) from their environment. Nutrients are needed as building materials for the cell and as a source of energy to do cellular work. Nutrients are metabolized (broken down) into simpler molecules and ATP engery.

GlowingColonyEColi : A false-colored image from fluorescence microscopy of a growing colony of E coli cells. "Aging and Death in E. coli" (2005) PLoS Biol 3(2): e58 doi:10.1371/journal.pbio.0030058.

Bacterial Growth
Microbial growth involves an increase in the number of cells. Growth of most microorganisms occurs by the process of binary fission Generation or doubling time can take anywhere from 20 min to 24 h Phases of growth
Lag phase Exponential (logarithmic [log]) phase Stationary phase Death phase

Compare growth in liquid and on solid media

Microbial Growth
Refers to increase in the ____________ of microbes (reproduction) rather than an increase in ____ of the microbe. The formation of a _____ is the result of microbial growth = aggregation of cells arising from single parent cell. The time required for growth and reproduction is known as the doubling or _____ ______.
GlowingColonyEColi : A false-colored image from fluorescence microscopy of a growing colony of E coli cells. "Aging and Death in E. coli" (2005) PLoS Biol 3(2): e58 doi:10.1371/journal.pbio.0030058.

Bacterial Growth binary fission

Growth of Microbial Populations

Image: Pearson Education Inc. (2004) publishing as Benjamin Cummings

Bacterial growth curve in batch culture

 Lag Phase No significant increase in number Metabolically active - growing in size, synthesizing enzymes and incorporating various molecules from the medium Individual organism increase in size Large quantities of energy, in the form of ATP, is produced Log or Exponential Phase Divide at their most rapid rate Growth is dependent on nutrient availability and incubation conditions Population growth is occurring A regular, genetically determined interval called generation time. The population of organism doubles in each generation time

Summary of Growth Phases

Logarithmic Growth in Cell Count From Binary Fission

Generation Number 0 1 2 3 4 5 10 20
View: http://www.cellsalive.com/qtmovs/ecoli_mov.htm

Cell Count 1 2 4 8 16 32 1,024 1,048,576

Image: GlowingColonyEColi : A false-colored image from fluorescence microscopy of a growing colony of E coli cells. "Aging and Death in E. coli" (2005) PLoS Biol 3(2): e58 doi:10.1371/journal.pbio.0030058.

Summary of Growth Phases

Stationary Phase
Balance between cell division and cell death Total # of viable organisms remain constant Results from nutrient and environmental limitations

Death Phase
Decline in the # of viable cells Death is logarithmic Result from nutrient deprivation and buildup of toxic wastes Medium becomes less and less supportive for cell division, many cells lose there ability to divide and cells die.

Growth Measurements
Growth is measured by the change in the number of cells over time. Cell counts done microscopically measure the total number of cells in a population, whereas viable cell counts (plate counts) measure only the living, reproducing population.

Microbial populations show a characteristic type of growth pattern called exponential growth, which is best seen by plotting the number of cells over time on a semi logarithmic graph

Calculating growth rate

Generation time (g) = t/n; where t = elapsed time (hr) and n= number of generations. n= 3.3(logN-logN0) N = number of cells at the end of the elapsed time N0= number of cells at the beginning

Chemostat: growth rate = dilution rate (D = f/V); constant dilution rate with nutrient limiting growth.

Open (continuous) Culture Systems

Turbidostat: dilution rates varies to maintain constant turbidity (cell density); no limiting nutrient.

Wash out!
Flow

(f)

Volume (V)

Generation Time Under Optimal Conditions

(at 37oC)
Organism Generation Time (min) 28 12.5

Bacillus cereus Escherichia coli

Staphylococcus aureus 27-30 (causes many infections: toxic shock syndrome one example) Mycobacterium tuberculosis (agent of Tuberculosis) Treponema pallidum (agent of Syphilis)
792 932

1,980

Nutrient Concentration Effects in Batch Cultures:

Total growth will increase until limiting nutrients are exhausted (included oxygen for aerobes) or metabolic byproducts accumulate that change environmental conditions to inhibit growth (toxicity). Growth rate will also increase with increasing nutrient concentration up to some maximum value, beyond which there is no effect (transporters are saturated with there substrate.

Environmental factors affecting growth

Five groups based on their optimum growth temperature
Food preservation Temp. and disease

Three groups based on pH optimum: Neutrophils (pH 5 8), acidophiles, and alkalophiles Oxygen requirement varies greatly: obligate aerobes, obligate anaerobes, facultative anaerobes, microaerophiles, and aerotolerant anaerobes Water availability: osmotolerance and halophiles

Proteins

Microbes & Temperature

Three-dimensional shape because of the temperature sensitive hydrogen bonds. These bonds will usually break at higher temperatures, and protein become denatured. Denatured proteins lose function. Lipids Also temperature sensitive. Become brittle if temperature is too low. If temperature too high, lipids will be more liquid in form. Outside membrane cannot preserve the integrity of the cell and it will disintegrate.
Images: Myoglobin: Thomas Splettstoesser Wiki PublicDomain Lipid Bylayer Cholesterol: cellbio.utmb.edu/cellbio/membrane_intro.htm

Effects of Temperature on Growth

40oF

77oF

95oF

Most of our plates are incubated at 37oC (98.6oF).

Conversion C to F = 1.8xC + 32 http://www.sciencemadesimple.com/conversions.html
Image: Pearson Education Inc. (2004) publishing as Benjamin Cummings

Effect of Temperature on Growth

Temperature is a major environmental factor controlling microbial growth. The cardinal temperatures are the minimum, optimum, and maximum temperatures at which each organism grows

Effect of temperature

Image: Brock :Biology of Microorganims. (2006) 11th edition Madigan/Martiko

Temperature Ranges for Microbial Growth

Microorganisms can be grouped by the temperature ranges they require, there are 5 temperature classes: Psychrophiles - cold-loving organisms, defined by their ability to grow at 0C. Pschrotrophs organisms that can grow at 0-7C but temp. optima is between 20-30C Mesophiles - organisms with an optimum temperature between 20-40C. Thermophiles - Organisms with an optimum temp. Between 45C and 70C. Hyperthermophiles - an optimum temp. of 80C or higher and a maximum temp. as high as 115C

Temperature optima of bacteria

Meet the Microbe! Psychrophilic Algae: Chlamydomonas nivalis

Images: ChlamydomonasNivalis: Green algae psychrophile, www-es.s.chiba-u.ac.jp/.../snowalgae_ak.html WatermelonSnow: Will Beback, Wiki WatermelonSnowball: http://exviking.net/mflowers/small/Chlamydomonas_nivalis.htm WatermelonSnowFootprint : https:/.../seki/planyourvisit/wheretoeat.htm

Extreme Thermophile
Thermophiles produce some of the bright colors (the orange colors) seen in hot springs. How do they tolerate the high temperatures? cell membranes dont contain fatty acids special enzymes fold their DNA into special heat-stable coils enzymes themselves are heat stable with extra bonds between amino acids.

Grand Prismatic Spring, Yellowstone National Park

Microbial Growth at Low or High pH

The acidity or alkalinity of an environment can greatly affect microbial growth. Some organisms have evolved to grow best at low or high pH, but most organisms grow best between pH 6 and 8. The internal pH of a cell must stay relatively close to neutral even though the external pH is highly acidic or basic. Organisms that grow best at low pH are called acidophiles; those that grow best at high pH are called alkaliphiles.

pH Ranges for Microbial Growth

Acidophiles - microorganisms which grow at an optimum pH well below neutrality (7.0) between pH 0 and 5.5 Neutrophiles - those which grow best at neutral pH, between pH 5.5 and 8.0 Alkaliphiles - those that grow best under alkaline conditions, between pH 8.5 and 11.5

Bacterial growth at various pH

pH
As with temperature, bacteria have minimum, optimum and maximum pH ranges. Neutrophiles Protozoans and most bacteria have an optimum pH range of 6.5 to 7.5. pH range of human organs and tissues. Acidophiles Most fungi & some bacteria grow best in acid niches. Obligate acidophiles have to live in an acidic environment. Acid- tolerant Microbes will survive in an acid environment, but do not prefer that.
Images: HelicobacterPylori : Electron micrograph of H. pylori possessing multiple flagella. Yutaka Tsutsumi, M.D. Professor Department of Pathology Fujita Health University School of Medicine

Meet the Microbe!

Helicobacter pylori

and acidophilic bacterium.

Gram-negative, microaerophilic,

Can thrive in the stomach and upper small intestines and cause ulcers. However, many who are infected do not show any symptoms. Helicobacter spp. only known microorganisms to thrive in highly acidic environment of stomach.

pH : Alkaliphiles
Meet the Microbe!

Vibrio cholerae (Cholera

bacteria) will grow outside the body at a pH of 9.0.

Gram negative, vibrio-shaped, alkaliphile in class Gammaproteobacteria. In environment, prefers warm, salty alkaline water and many Vibrio species can multiply in shellfish. Vibrio cholerae can thrive in fresh water.

pH profiles for some prokaryotes

Organism Minimum pH 0.5 1.0 2.0 3.5 4.0-4.6 4.2 4.4 5.0-5.8 5.6 5.6 5.7 6.6 Optimum pH 2.0-2.8 2.0-3.0 4.0 5.5-6.0 5.8-6.6 7.0-7.5 6.0-7.0 6.0-7.6 7.1 6.6-7.0 7.0 7.8 7.6-8.6 Maximum pH 4.0-6.0 5.0 6.0 7.5 6.8 9.3 9.0 8.5-9.0 9.3 8.0 9.0 8.3 10.0

Thiobacillus thiooxidans Sulfolobus acidocaldarius Bacillus acidocaldarius Zymomonas lindneri Lactobacillus acidophilus Staphylococcus aureus Escherichia coli Clostridium sporogenes Erwinia caratovora Pseudomonas aeruginosa Thiobacillus novellus Nitrobacter sp

Streptococcus pneumoniae 6.5

Microbes & Oxygen

Obligate Aerobes Need oxygen to stay alive.
Aerobic respiration = Use of O2 to break down food into useable energy.

Obligate Anaerobes Die in presence of oxygen. It is poisonous to them.

Anaerobic respiration = break down food into useable energy without the use of O2.

Facultative Anaerobes Not strict aerobes or anaerobes.

Many yeasts and enteric bacteriaEscherichia coli and Staphylococcus aureus.

Microaerophilic bacteria Require oxygen levels lower that that found under normal atmospheric conditions
(Helicobacter pilori found in stomach).

Aerotolerant Anaerobes Dont use oxygen, but are not killed by it.

(Lactobacilli - This genus will make pickles from cucumbers and cheese from milk.)

Oxygen and Growth

1: Obligate aerobic bacteria gather at top of test tube to absorb maximal amount of oxygen. 2: Obligate anaerobic bacteria gather at bottom to avoid oxygen. 3: Facultative anaerobes gather mostly at the top, since aerobic respiration is most beneficial; but as lack of oxygen does not hurt them, they can be found all along the test tube. 4: Microaerophiles gather at upper part of test tube, not at top. Require O2, but at low concentration. 5: Aerotolerant bacteria are not affected by oxygen, and they are evenly spread along the test tube.

Aerobic and anaerobic bacteria can be identified by growing them in liquid culture:

Oxygen and Growth

Environment

Group
Obligate Aerobe

Aerobic
Growth

Anaerobic O2 Effect
No growth No growth Required (utilized for aerobic respiration) Required but at levels below 0.2 atm Toxic Not required for growth but utilized when available Not required and not utilized

Microaerophile Growth if level not too high Obligate Anaerobe Facultative (An)aerobe Aerotolerant Anaerobe No growth Growth

Growth Growth

Growth

Growth

 Using oxygen (1/2 O2) in metabolism creates toxic waste. Several toxic forms of oxygen can be formed in the cell, but enzymes are present that can neutralize most of them. Superoxide in particular seems to be a common toxic oxygen species. Microbes that dont make these enzymes cannot exist in the presence of oxygen.

Toxic Forms of Oxygen

Oxygenation and enzymic capacity

Anaerobic growth chambers

Physical Effects of Water

Water is important reactant in many metabolic reactions.

Most cells die in absence of water. - Some have cell walls that retain water. What genus comes to mind? - Endospores and cysts can cease most metabolic activity for years.

What organisms make endospores? Which make cysts?

Cell walls of bacteria prevent them from exploding in a hypotonic environment, but most bacteria are vulnerable in hypertonic environments.

 Water is solvent for biomolecules, and its availability is critical for cellular growth The availability of water depends upon its presence in the atmosphere (relative humidity) or its presence in solution or a substance (water activity, (Aw) Aw pure (100%). H2O is 1.0; affected by dissolved solutes such as salts or sugars. Aw =1/[solute] Microorganisms live over a range of Aw from 1.0 to 0.7. The Aw of human blood is 0.99; seawater = 0.98; maple syrup = 0.90; Great Salt Lake = 0.75. Water activities in agricultural soils range between 0.9 and 1.0. Osmotolerant organisms will grow over a wide range of water activity or osmotic concentration.
Xerophiles are organisms which live in dry environments (made dry by lack of water).

Water availability

Effect of salt on growth

Halophiles are microorganisms that require high levels of NaCl for growth
Mild halophiles require 1-6% salt. Moderate halophiles require 7-15% salt. Extreme halophiles that require 15-30% NaCl for growth are found among the Archaea

Halotolerant organisms are able to grow at moderate salt concentrations, even though they grow best in the absence of NaCl.

Effect of salt on growth

Osmotic Effects on Microbial Growth

When microorganisms are placed in hypertonic environments , water would leave the cell. As a result the plasma membrane shrinks away from the cell wall, this process is called plasmolysis.

(What happens to the organism?)

To counteract this situation, organisms produce or accumulate intracellular compatible solutes that maintain the cell in positive water balance. The solutes are compatible with metabolism and growth even at high intracellular concentrations.
Photo: Journal of Bacteriology http://jb.asm.org/content/vol188/issue10/cover.shtml

Glycocalyx & Osmotic Pressure

Some bacteria have an additional layer outside of the cell wall called the glycocalyx. One type of glycocalyx is called a Slime Layer. glycoproteins loosely associated with the cell wall. cause bacteria to adhere to solid surfaces and help prevent the cell from drying out Meet the Microbe! The slime layer of Staphylococcus epidermidis allows it to exist on the salty environment of the skin.

Mannitol Salt
STRUCTURE OF MICOBIAL CELLS