Microbial Growth

Microbial Growth



Concepts:


• Growth is defined as an increase in cellular constituents and may result in an increase in a microorganism’s size, population number, or both.
• When microorganisms are grown in a closed system, population growth remains exponential for only a few generations and then enters a stationary phase due to factors such as nutrient limitation and waste accumulation. In an open system with continual nutrient addition and waste removal, the exponential phase can be maintained for long periods.
• Water availability, pH, temperature, oxygen concentration, pressure, radiation, and a number of other environmental factors influence microbial growth. Yet many microorganisms, and particularly bacteria, have managed to adapt and flourish under environmental extremes that would destroy higher organisms.
• In the natural environment, growth is often severely limited by available nutrient supplies and many other environmental factors.
• Bacteria can communicate with each other and behave cooperatively using population density–dependent signals.

 Growth may be defined as an increase in cellular constituents. It leads to a rise in cell number when microorganisms reproduce by processes like budding or binary fission.

 If the microorganism is coenocytic—that is, a multinucleate organism in which nuclear divisions are not accompanied by cell divisions— growth results in an increase in cell size but not cell number.


The Growth Curve:

A. Lag Phase----When microorganisms are introduced into fresh culture medium, usually no immediate increase in cell number occurs, and therefore this period is called the lag phase.
 The cells may be old and depleted of ATP, essential cofactors, and ribosomes; these must be synthesized before growth can begin.
B. Exponential Phase---- During the exponential or log phase, microorganisms are growing and dividing at the maximal rate possible given their genetic potential, the nature of the medium, and the conditions under which they are growing.

 Rate of growth is constant during the exponential phase; that is, the microorganisms are dividing and doubling in number at regular intervals.
 The population is most uniform in terms of chemical and physiological properties during this phase; therefore exponential phase cultures are usually used in biochemical and physiological studies.
 Exponential growth is balanced growth. That is, all cellular constituents are manufactured at constant rates relative to each other.

C. Stationary Phase

 Eventually population growth ceases and the growth curve becomes horizontal.
 This stationary phase usually is attained by bacteria at a population level of around 109 cells per ml.
 In the stationary phase the total number of viable microorganisms remains constant. This may result from a balance between cell division and cell death
 bacteria in a batch culture may enter stationary phase in response to starvation------------------

 Starving bacteria frequently produce a variety of starvation proteins, which make the cell much more resistant to damage in a variety of ways.

 They increase peptidoglycan cross-linking and cell wall strength.


 The Dps (DNA-binding protein from starved cells) protein protects DNA.

 Chaperones prevent protein denaturation and renature damaged proteins.

 As a result of these and many other mechanisms, the starved cells become harder to kill and more resistant to starvation itself, damaging temperature changes, oxidative and osmotic damage, and toxic chemicals such as chlorine.

 Salmonella typhimurium and some other bacterial pathogens become more virulent when starved.

D. Death Phase
 The death of a microbial population, like its growth during the exponential phase, is usually logarithmic (that is, a constant proportion of cells dies every hour).

The Mathematics of Growth:
 population will double in number during a specific length of time called the generation time or doubling time.

 These observations can be expressed as equations for the generation time.
Let N0 = the initial population number

Nt =the population at time t

n = the number of generations in time t

Then inspection of the results in table 6.1 will show that
Nt = N0 * 2n.

Solving for n, the number of generations, where all logarithms
are to the base 10,

Log Nt = log N0 + n • log 2, and

n = log Nt _ log N0/ log 2= log Nt _ log N0/0.301

 The rate of growth during the exponential phase in a batch culture can be expressed in terms of the mean growth rate constant (k).
 This is the number of generations per unit time, often expressed as the generations per hour.

k = n/ t= log Nt _ log N0/0.301t

 The time it takes a population to double in size—that is, the mean generation time or mean doubling time (g), can now be calculated. If the population doubles (t = g), then

Nt = 2 N0
.
Substitute 2N0 into the mean growth rate equation and solve
for k.
k= log (2NO) _ log NO / 0.301g= log 2 + log N0 - log N0/0.301g

k =1/g

 The mean generation time is the reciprocal of the mean growth rate constant.

g =1/k

 Generation times in nature are usually much longer than in culture.


Measurement of Microbial Growth

a) Measurement of Cell Numbers:
 Petroff-Hausser counting chambers can be used for counting prokaryotes; hemocytometers can be used for both prokaryotes and eukaryotes.

 Larger microorganisms such as protozoa, algae, and nonfilamentous yeasts can be directly counted with electronic counters such as the Coulter Counter.
 It is not as useful in counting bacteria because of interference by small debris particles, the formation of filaments, and other problems.

The Petroff-Hausser Counting Chamber.Formulla:

Bacteria/mm3 = (bacteria/square) (no of squares) (depth of chamber)


 The hot agar used in the pour-plate technique may injure or kill sensitive cells; thus spread plates sometimes give higher counts than pour plates.

b) Measurement of Cell Mass:

 Cells growing in liquid medium are collected by centrifugation, washed, dried in an oven, and weighed. This is an especially useful technique for measuring the growth of fungi. It is time consuming, however, and not very sensitive. Because bacteria weigh so little, it may be necessary to centrifuge several hundred milliliters of culture to collect a sufficient quantity.




The Continuous Culture of Microorganisms:

 A microbial population can be maintained in the exponential growth phase and at a constant biomass concentration for extended periods in a continuous culture system.

Two major types of continuous culture systems commonly are used:
(1) chemostats
(2) turbidostats.
 A chemostat is constructed so that sterile medium is fed into the culture vessel at the same rate as the media containing microorganisms is removed.
 The rate of nutrient exchange is expressed as the dilution rate (D), the rate at which medium flows through the culture vessel relative to the vessel volume, where f is the flow rate (ml/hr) and V is the vessel volume (ml).

D = f/V
For example, if f is 30 ml/hr and V is 100 ml, the dilution rate is
0.30 hr_1.
 Both the microbial population level and the generation time are related to the dilution rate .
 The generation time decreases (i.e., the growth rate rises) as the dilution rate increases.
 The microbial population density remains unchanged over a wide range of dilution rates.
 If the dilution rate rises too high, the microorganisms can actually be washed out of the culture vessel before reproducing because the dilution rate is greater than the maximum growth rate.
 The limiting nutrient concentration rises at higher dilution rates because fewer microorganisms are present to use it.
 At very low dilution rates, an increase in D causes a rise in both cell density and the growth rate. This is because of the effect of nutrient concentration on the growth rate, sometimes called the Monod relationship.
 The growth rate increases when the total available energy exceeds the maintenance energy.


 The Turbidostat
 Turbidostat, has a photocell that measures the absorbance or turbidity of the culture in the growth vessel.

 The turbidostat differs from the chemostat in several ways.—

 The dilution rate in a turbidostat varies rather than remaining constant, and its culture medium lacks a limiting nutrient.
 The turbidostat operates best at high dilution rates; the chemostat is most stable and effective at lower dilution rates.

The Influence of Environmental Factors on Growth:
 Procaryotes such as Bacillus infernus even seem able to live over 1.5 miles below the Earth’s surface, without oxygen and at temperatures above 60°C.

 Microorganisms that grow in such harsh conditions are often called extremophiles.


I. Solutes and Water Activity:

 Because a selectively permeable plasma membrane separates microorganisms from their environment, they can be affected by changes in the osmotic concentration of their surroundings.

 If a microorganism is placed in a hypotonic solution (one with a lower osmotic concentration), water will enter the cell and cause it to burst
 When microorganisms with rigid cell walls are placed in a hypertonic environment, water leaves and the plasma membrane shrinks away from the wall, a process known as plasmolysis.
 This dehydrates the cell and may damage the plasma membrane; the cell usually becomes metabolically inactive and ceases to grow.




 Many microorganisms keep the osmotic concentration of their protoplasm somewhat above that of the habitat by the use of compatible solutes,( that are compatible with metabolism and growth when at high intracellular concentrations.) so that the plasma membrane is always pressed firmly against their cell wall.

 Most procaryotes increase their internal osmotic concentration in a hypertonic environment through the synthesis or uptake of choline, betaine, proline, glutamic acid, and other amino acids; elevated levels of potassium ions are also involved to some extent.
 Algae and fungi employ sucrose and polyols—for example, arabitol, glycerol, and mannitol— for the same purpose.
 Polyols and amino acids are ideal solutes for this function because they normally do not disrupt enzyme structure and function.

 A few procaryotes like Halobacterium salinarium raise their osmotic concentration with potassium ions (sodium ions are also elevated but not as much as potassium).
 Halobacterium’s enzymes have been altered so that they actually require high salt concentrations for normal activity
 Since protozoa do not have a cell wall, they must use contractile vacuoles to eliminate excess water when living in hypotonic environments.

 The amount of water available to microorganisms can be reduced by interaction with solute molecules (the osmotic effect) or by adsorption to the surfaces of solids (the matric effect).

 Microbiologists generally use water activity (aw) to express quantitatively the degree of water availability
 It is also equivalent to the ratio of the solution’s vapor pressure (Psoln) to that of pure water (Pwater).

aw = P soln / Pwate

 Water activity is inversely related to osmotic pressure; if a solution has high osmotic pressure, its aw(water potential) is low.
 A microorganism must expend extra effort to grow in a habitat with a low aw value because it must maintain a high internal solute concentration to retain water.
 Some microorganisms can do this and are osmotolerant

• For example, Staphylococcus aureus can be cultured in media containing any sodium chloride concentration up to about 3 M. It is well adapted for growth on the skin.
• The yeast Saccharomyces rouxii will grow in sugar solutions with aw values as low as 0.6.

 The alga Dunaliella viridis tolerates sodium chloride concentrations from 1.7 M to a saturated solution.
 Halophiles have adapted so completely to hypertonic, saline conditions that they require high levels of sodium chloride to grow, concentrations between about 2.8 M
 The archaeon Halobacterium can be isolated from the Dead Sea (a salt lake between Israel and Jordan and the lowest lake in the world), the Great Salt Lake in Utah, and other aquatic habitats with salt concentrations.

 Halobacterium and other extremely halophilic bacteria have significantly modified the structure of their proteins and membranes rather than simply increasing the intracellular concentrations of solutes, the approach used by most osmotolerant microorganisms.


• The enzymes, ribosomes, and transport proteins of these bacteria require high levels of potassium for stability and activity. In addition, the plasma membrane and cell wall of Halobacterium are stabilized by high concentrations of sodium ion. If the sodium concentration decreases too much, the wall and plasma membrane literally disintegrate

II. pH

 Acidophiles have their growth optimum between pH 0 and 5.5; Most fungi prefer slightly acid surroundings, about pH 4 to 6; algae also seem to favor slight acidity.e.g-----
 The Archaea Ferroplasma acidarmanus and Picrophilus oshimae can actually grow at pH 0, or very close to it. the alga Cyanidium caldarium and the archaeon Sulfolobus acidocaldarius are common inhabitants of acidic hot springs; both grow well around pH 1 to 3 and at high temperatures.

 neutrophiles, between pH 5.5 and 8.0;
• Most bacteria and protozoa are neutrophiles.

 alkalophiles prefer the pH range of 8.5 to 11.5.
• Extreme alkalophiles like Bacillus alcalophilus maintain their internal pH closer to neutrality by exchanging internal sodium ions for external protons.

 In bacteria, potassium/proton and sodium/proton antiport systems probably correct small variations in pH.
 When the pH drops below about 5.5 to 6.0, Salmonella typhimurium and E. coli synthesize an array of new proteins as part of what has been called their acidic tolerance response.

 If the external pH decreases to 4.5 or lower, chaperones such as acid shock proteins and heat shock proteins are synthesized.

Buffers often are included in media to prevent growth inhibition by large pH changes. Phosphate is a commonly used buffer and a good example of buffering by a weak acid (H2PO4 _) and its conjugate base (HPO4 2–).




III. Temperature
 microbial growth occurs at temperatures extending from _20°C to over 100°C.
 Some species (e.g., Neisseria gonorrhoeae) have a small range; others, like Enterococcus faecalis, will grow over a wide range of temperatures.


1. Psychrophiles------ grow well at 0°C and have an optimum growth temperature of 15°C or lower; the maximum is around 20°C.

 They are readily isolated from Arctic and Antarctic habitats; because 90% of the ocean is 5°C or colder.
 The psychrophilic archaeon Methanogenium has recently been isolated from Ace Lake in Antarctica.
 The cell membranes of psychrophilic microorganisms have high levels of unsaturated fatty acids and remain semifluid when cold.that is the reason fo their surviving.




2. psychrotrophs or facultative psychrophiles.

 Many species can grow at 0 to 7°C even though they have optima between 20 and 30°C, and maxima at about 35°C. These are called psychrotrophs or facultative psychrophiles.

 Psychrotrophic bacteria and fungi are major factors in the spoilage of refrigerated foods e.g--------Pseudomonas fluorescens


3.Mesophiles are microorganisms with growth optima around 20 to 45°C;

 Almost all human pathogens are mesophiles, as might be expected since their environment is a fairly constant 37°C.
e.g Escherichia coli, Neisseria gonorrhoeae,

4. Thermophiles; they can grow at temperatures of 55°C or higher

 The vast majority are procaryotes although a few algae and fungi are thermophilic


 Thermophiles differ from mesophiles in having much more heat-stable enzymes and protein synthesis systems able to function at high temperatures. Their membrane lipids are also more saturated than those of mesophiles and have higher melting points; therefore thermophile membranes remain intact at higher temperatures.



5. Hyperthermophiles. Procaryotes that have growth optima between 80°C and about 113°C are called hyperthermophiles.
 They usually do not grow well below 55°C. Pyrococcus abyssi and Pyrodictium occultum are examples of marine hyperthermophiles found in hot areas of the seafloor.




Oxygen Concentration
 Almost all multicellular organisms are completely dependent on atmospheric O2 for growth—that is, they are obligate aerobes.

 Oxygen serves as the terminal electron acceptor for the electron- transport chain in aerobic respiration. In addition, aerobic eukaryotes employ O2 in the synthesis of sterols and unsaturated fatty acids.

 Facultative anaerobes do not require O2 for growth but do grow better in its presence. In the presence of oxygen they will use aerobic respiration.
 Aerotolerant anaerobes such as Enterococcus faecalis simply ignore O2 and grow equally well whether it is present or not.
 In contrast, strict or obligate anaerobes (e.g., Bacteroides, Fusobacterium, Clostridium pasteurianum, Methanococcus) do not tolerate O2 at all and die in its presence.

 Aerotolerant and strict anaerobes cannot generate energy through respiration and must employ fermentation or anaerobic respiration pathways for this purpose.

 Finally, there are aerobes such as Campylobacter, called microaerophiles, that are damaged by the normal atmospheric level of O2 (20%) and require O2 levels below the range of 2 to 10% for growth.

The nature of bacterial O2 responses can be readily determined by growing the bacteria in culture tubes filled with a solid culture medium or a special medium like thioglycollate broth, which contains a reducing agent to lower O2 levels

Fungi are normally aerobic, but a number of species— particularly among the yeasts—are facultative anaerobes


Algae are almost always obligate aerobes.
Many microorganisms possess enzymes that afford protection against toxic O2 products. Obligate aerobes and facultative anaerobes usually contain the enzymes superoxide dismutase (SOD) and catalase, which catalyze the destruction of superoxide radical and hydrogen peroxide, respectively. Peroxidase also can be used to destroy hydrogen peroxide.

Aerotolerant microorganisms may lack catalase but almost always have superoxide dismutase. The aerotolerant Lactobacillus
plantarum uses manganous ions instead of superoxide dismutase to destroy the superoxide radical

All strict anaerobes lack both enzymes or have them in very low concentrations and therefore cannot tolerate O2.

 How to grow anaerobes???????/

1) Special anaerobic media containing reducing agents such as thioglycollate or cysteine may be used.
2) Often CO2 as well as nitrogen is added to the chamber since many anaerobes require a small amount of CO2 for best growth.

(3) One of the most popular ways of culturing small numbers of anaerobes is by use of a Gas- Pak jar . In this procedure the environment is made anaerobic by using hydrogen and a palladium catalyst to remove O2 through the formation of water

Pressure:
 Some bacteria in the gut of deep-sea invertebrates such as amphipods and holothurians are truly barophilic—they grow more rapidly at high pressures.
 These gut bacteria may play an important role in nutrient recycling in the deep sea
 One barophile has been recovered from the Mariana trench near the Philippines (depth about 10,500 m) that is actually unable to grow at pressures below about 400 to 500 atm when incubated at 2°C.

 Thus far, barophiles have been found among several bacterial genera (e.g., Photobacterium, Shewanella, Colwellia). Some members of the Archaea are thermobarophiles (e.g., Pyrococcus spp., Methanococcus jannaschii)

Radiation

 Many forms of electromagnetic radiation are very harmful to microorganisms.
 ionizing radiation, radiation of very short wavelength or high energy, which can cause atoms to lose electrons or ionize. Two major forms of ionizing radiation are
(1) X rays, which are artificially produced, and
(2) Gamma rays, which are emitted during radioisotope decay.


 Low levels of ionizing radiation will produce mutations and may indirectly result in death, whereas higher levels are directly lethal.

 Ionizing radiation can be used to sterilize items. Some procaryotes (e.g., Deinococcus radiodurans) and bacterial endospores can survive large doses of ionizing radiation.



 Ultraviolet (UV) radiation, mentioned earlier, kills all kinds of microorganisms due to its short wavelength (approximately from 10 to 400 nm) and high energy. The most lethal UV radiation has a wavelength of 260 nm, the wavelength most effectively absorbed by DNA.

• The primary mechanism of UV damage is the formation of thymine dimers in DNA . Two adjacent thymines in a DNA strand are covalently joined to inhibit DNA replication and function.
• This damage is repaired in several ways. In photoreactivation, blue light is used by a photoreactivating enzyme to split thymine dimers.
• A short sequence containing the thymine dimer can also be excised and replaced. This process occurs in the absence of light and is called dark reactivation.
• Damage also can be repaired by the recA protein in recombination repair and SOS repair.


Microbial Growth in Natural Environments

Growth Limitation by Environmental Factors

 Liebig’s law of the minimum states that the total biomass of an organism will be determined by the nutrient present in the lowest concentration relative to the organism’s requirements. This law applies in both the laboratory and in terrestrial and aquatic environments.

 Shelford’s law of tolerance states that there are limits to environmental factors below and above which a microorganism cannot survive and grow, regardless of the nutrient supply.


Quorum Sensing and Microbial Populations
 More recently it has become clear that many bacteria can communicate with one another and behave cooperatively.
 A major way in which this cooperation is accomplished is by a process known as quorum sensing or autoinduction.
 Quorum sensing was first discovered in gram-negative bacteria and is best understood in these microorganisms.

 The most common signals in gram-negative bacteria are acyl homoserine lactones (HSLs). These are small molecules composed of a 4- to 14-carbon acyl chain attached by an amide bond to homoserine Lactone.
 Some well-studied examples are-----
(1) bioluminescence production by Vibrio fischeri,
(2) Pseudomonas aeruginosa synthesis and release of virulence factors
(3) conjugal transfer of genetic material by Agrobacterium tumefaciens
(4) antibiotic production by Erwinia carotovora and Pseudomonas aureofaciens.

• Gram-positive bacteria also regulate activities by quorum sensing, often using an oligopeptide signal.


 An interesting and important function of quorum sensing is to promote the formation of mature biofilms by the pathogen Pseudomonas aeruginosa, and it may play a role in cystic fibrosis.


SUMMARY:

1. Growth is an increase in cellular constituents and results in an increase in cell size, cell number, or both.
2. When microorganisms are grown in a closed system or batch culture, the resulting growth curve usually has four phases: the lag, exponential or log, stationary, and death phases .
3. In the exponential phase, the population number doubles at a constant interval called the doubling or generation time . The mean growth rate constant (k) is the reciprocal of the generation time.
4. Exponential growth is balanced growth, cell components are synthesized at constant rates relative to one another. Changes in culture conditions (e.g., in shift-up and shift-down experiments) lead to unbalanced growth. A portion of the available nutrients is used to supply maintenance energy.
5. Microbial populations can be counted directly with counting chambers, electronic counters, or fluorescence microscopy. Viable counting techniques such as the spread plate, the pour plate, or the membrane filter can be employed.
6. Population changes also can be followed by determining variations in microbial mass through the measurement of dry weight, turbidity, or the amount of a cell component.
7. Microorganisms can be grown in an open system in which nutrients are constantly provided and wastes removed.
8. A continuous culture system is an open system that can maintain a microbial population in the log phase. There are two types of these systems: chemostats and turbidostats.
9. Most bacteria, algae, and fungi have rigid cell walls and are hypertonic to the habitat because of solutes such as amino acids, polyols, and potassium ions. The amount of water actually available to microorganisms is expressed in terms of the water activity (aw).
10. Although most microorganisms will not grow well at water activities below 0.98 due to plasmolysis and associated effects, osmotolerant organisms survive and even flourish at low aw values. Halophiles actually require high sodium chloride concentrations for growth .
11. Each species of microorganism has an optimum pH for growth and can be classified as an acidophile, neutrophile, or alkalophile.
12. Microorganisms can alter the pH of their surroundings, and most culture media must be buffered to stabilize the pH.
13. Microorganisms have distinct temperature ranges for growth with minima, maxima, and optima—the cardinal temperatures. These ranges are determined by the effects of temperature on the rates of catalysis, protein denaturation, and membrane disruption.
14. There are five major classes of microorganisms with respect to temperature preferences: (1) psychrophiles, (2) facultative psychrophiles or psychrotrophs, (3) mesophiles, (4) thermophiles and (5) hyperthermophiles.

15. Microorganisms can be placed into at least five different categories based on their response to the presence of O2: obligate aerobes, facultative anaerobes, aerotolerant anaerobes, strict or obligate anaerobes, and microaerophiles .
16. Oxygen can become toxic because of the production of hydrogen peroxide, superoxide radical, and hydroxyl radical. These are destroyed by the enzymes superoxide dismutase, catalase, and peroxidase.
17. Most deep-sea microorganisms are barotolerant, but some are barophilic and require high pressure for optimal growth.
18. High-energy or short-wavelength radiation harms organisms in several ways. Ionizing radiation—X rays and gamma rays—ionizes molecules and destroys DNA and other cell components.
19. Ultraviolet (UV) radiation induces the formation of thymine dimers and strand breaks in DNA. Such damage can be repaired by photoreactivation or dark reactivation mechanisms.
20. Visible light can provide energy for the formation of reactive singlet oxygen, which will destroy cells.
21. Microbial growth in natural environments is profoundly affected by nutrient limitations and other adverse factors. Some microorganisms can be viable but unculturable and must be studied with special techniques.
22. Often, bacteria will communicate with one another in a density-dependent way and carry out a particular activity only when a certain population density is reached. This phenomenon is called quorum sensing.

JSR