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

Summary of Growth Phases • 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

Logarithmic Growth in Cell Count From Binary Fission

Generation Number Cell Count 0 1 1 2 2 4 3 8 4 16 5 32 10 1,024 20 1,048,576

View: http://www.cellsalive.com/qtmovs/ecoli_mov.htm 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

Open (“continuous”) Culture Systems Chemostat: growth rate = dilution rate (D = f/V); constant dilution rate with nutrient limiting growth.

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

Flow (f)

Volume (V)

Wash out!

Generation Time Under Optimal Conditions (at 37oC)

Organism Generation Time (min) Bacillus cereus 28 Escherichia coli 12.5 Staphylococcus aureus 27-30 (causes many infections: toxic shock syndrome one example) Mycobacterium tuberculosis 792 – 932 (agent of Tuberculosis) Treponema pallidum (agent of Syphilis) 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

Microbes & Temperature Proteins 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

95oF 77oF 40oF

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 0°C.

• Pschrotrophs – organisms that can grow at 0-7°C but temp. optima is between 20-30°C

• Mesophiles - organisms with an optimum temperature between 20-40°C.

• Thermophiles - Organisms with an optimum temp. Between 45°C and 70°C.

• Hyperthermophiles - an optimum temp. of 80°C or higher and a maximum temp. as high as 115°C

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 don’t 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

Helicobacter pylori

• Gram-negative, microaerophilic, and acidophilic bacterium.

• 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.

Meet the Microbe!

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 Optimum pH Maximum pH Thiobacillus thiooxidans 0.5 2.0-2.8 4.0-6.0 Sulfolobus acidocaldarius 1.0 2.0-3.0 5.0 Bacillus acidocaldarius 2.0 4.0 6.0 Zymomonas lindneri 3.5 5.5-6.0 7.5 Lactobacillus acidophilus 4.0-4.6 5.8-6.6 6.8 Staphylococcus aureus 4.2 7.0-7.5 9.3 Escherichia coli 4.4 6.0-7.0 9.0 Clostridium sporogenes 5.0-5.8 6.0-7.6 8.5-9.0 Erwinia caratovora 5.6 7.1 9.3 Pseudomonas aeruginosa 5.6 6.6-7.0 8.0 Thiobacillus novellus 5.7 7.0 9.0 Streptococcus pneumoniae 6.5 7.8 8.3 Nitrobacter sp 6.6 7.6-8.6 10.0

• 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 bacteria…Escherichia coli and Staphylococcus

aureus. • Microaerophilic bacteria – Require oxygen levels lower

that that found under normal atmospheric conditions (Helicobacter pilori – found in stomach).

• Aerotolerant Anaerobes – Don’t use oxygen, but are not killed by it.

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

Microbes & Oxygen

Oxygen and Growth

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

Oxygen and Growth Environment

Group Aerobic Anaerobic O2 Effect

Obligate Aerobe

Growth No growth Required (utilized for aerobic respiration)

Microaerophile Growth if level not too high

No growth Required but at levels below 0.2 atm

Obligate Anaerobe

No growth Growth Toxic

Facultative (An)aerobe

Growth Growth Not required for growth but utilized when available

Aerotolerant Anaerobe

Growth Growth Not required and not utilized

Toxic Forms of Oxygen • 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 don’t make these enzymes cannot exist in the presence 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 availability • 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).

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.

STRUCTURE OF MICOBIAL CELLS

Mannitol Salt