Effect of pH on microbial growth and distribution

pH is a measure of the hydrogen ion activity of a solution. pH is defined as the negative logarithm of the hydrogen ion concentration (expressed in terms of molarity).

pH = ^-log [H⁺] = log(1/[H⁺])

The pH scale extends from pH 0.0 to pH 14.0.

The habitats in which microorganisms grow vary widely-from pH 0 to 2 at the acidic end to alkaline lakes and soil that may have pH values between 9 and 10.

pH strongly affects microbial growth. Each species has a definite pH growth range and pH growth optimum.

• Acidophiles have their growth optimum between pH 0 and 5.5; 
• Neutrophiles, between pH 5.5 and 8.0; 
• Alkalophiles prefer the pH range of 8.0 to 11.5. 
• Extreme alkalophiles have growth optima at pH 10 or higher.

In general, different microbial groups have characteristic pH preferences. Most bacteria and protists (protozoa and algae) are neutrophiles. Most fungi prefer more acidic surroundings, about pH 4 to 6; photosynthetic protists also seem to favour slight acidity.

Many archaea are acidophiles. For example, the archaeon Sulfolobus acidocaldarius is a common inhabitant of acidic hot springs; it grows well around pH 1 to 3 and at high temperatures. The archaea Ferroplasma acidarmanus and Picrophilus oshimae can grow at pH 0, or very close to it.

Although microorganisms will often grow over wide ranges of pH and far from their optima, there are limits to their tolerance

Maintenance of pH

Drastic variations in cytoplasmic pH can harm microorganisms by disrupting the plasma membrane or inhibiting the activity of enzymes and membrane transport proteins. Most procaryotes die if the internal pH drops much below 5.0 to 5.5. Changes in the external pH also might alter the ionization of nutrient molecules and thus reduce their availability to the organism.

Microorganisms respond to external pH changes using mechanisms that maintain a neutral cytoplasmic pH. Several mechanisms for adjusting to small changes in external pH have been proposed.

Ø  The plasma membrane is impermeable to protons.

Ø  Neutrophiles have an antiport transport system to exchange potassium for protons.

Ø  Extreme alkalophiles like Bacillus alcalophilus maintain their internal pH closer to neutrality by exchanging internal sodium ions for external protons.

Ø  Internal buffering also may contribute to pH homeostasis.

•      If the external pH becomes too acidic, other mechanisms come into play.

ü  When the pH drops below about 5.5 to 6.0, Salmonella enterica serovar Typhimurium and E. coli synthesize an array of new proteins as part of their acidic tolerance response (ATR).

ü  A proton-translocating ATPase is activated and makes more ATP or  pump protons out of the cell and thus contributes to this protective response, 

ü  If the external pH decreases to 4.5 or lower, chaperone proteins such as acid shock proteins and heat shock proteins are synthesized. These prevent the acid denaturation of proteins and aid in the refolding of denatured proteins.

Microorganisms frequently change the pH of their own habitat by producing acidic or basic metabolic waste products. Fermentative microorganisms form organic acids from carbohydrates, whereas chemolithotrophs like Thiobacillus oxidize reduced sulfur components to sulfuric acid. Other microorganisms make their environment more alkaline by generating ammonia through amino acid degradation. Because microorganisms change the pH of their surroundings, buffers often are included in media to prevent growth inhibition by large pH changes.

Phosphate is a commonly used buffer and is a good example of buffering by a weak acid (dihydrogen phosphate, H₂PO₄^-) and its conjugate base (monohydrogen phosphate, HPO₄ ^2-).

H⁺ + HPO4 ^2-⎯⎯→ H₂PO₄^- 

OH^- + H₂PO₄^-   ⎯⎯→ HPO₄ ^2- + HOH

If protons are added to the mixture, they combine with the salt form to yield a weak acid. An increase in alkalinity is resisted because the weak acid will neutralize hydroxyl ions through proton donation to give water.

Peptides and amino acids in complex media also have a strong buffering effect.

 

pH	[H+] Molarity		Environmental examples	Microbial examples
0	10–0	Increasing acidity	Concentrated nitric acid	Ferroplasma, Picrophilus oshimae
1	10–1		Gastric contents, acid thermal springs	Dunaliella acidophila
2	10–2		Lemon juice Acid mine drainage	Cyanidium caldarium, Thiobacillus thiooxidans, Sulfolobus acidocaldarius
3	10–3		Vinegar, ginger ale Pineapple
4	10–4		Tomatoes, orange juice Very acid soil
5	10–5		Cheese, cabbage Bread	Physarum polycephalum, Acanthamoeba castellanii
6	10–6		Beef, chicken Rain water Milk, Saliva	Lactobacillus acidophilus,        E.coli, Pseudomonas aeruginosa, Euglena gracilis, Paramecium bursaria
7	10–7	Neutrality	Pure water, Blood	Staphyloccus aureus
8	10–8	Increasing alkalinity	Seawater	Nitrosomonas spp.
9	10–9		Strongly alkaline soil,  Alkaline lakes
10	10–10		Soap	Microcystis aeruginosa, Bacillus alcalophilus
11	10–11		Household ammonia
12	10–12		Saturated calcium hydroxide solution
13	10–13		Bleach, Drain opener
14	10–14

                         The pH scale and microorganisms with their growth optima.

Effect of various environmental factors on microbial growth- Effect of Temperature on the growth of microorganisms

The growth of microorganisms also is greatly affected by the chemical and physical nature of their surroundings. Microorganisms being mostly unicellular and poikilothermic (coldblooded), any change in the environment is easily reflected on them.  An understanding of environmental influences aids in the control of microbial growth and the study of the ecological distribution of microorganisms. 

Procaryotes are present anywhere life can exist. Many habitats in which procaryotes thrive would kill most other organisms. Some microorganisms can adapt to extreme and inhospitable environments. Procaryotes such as Bacillus infernus are even 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.

Effect of Temperature on the growth of microorganisms 

Environmental temperature has a great effect on microorganisms since their temperature varies with that of the external environment.

Temperature has a major effect on microbial growth due to the temperature sensitivity of enzyme-catalysed reactions. Each enzyme has an optimum temperature at which it functions optimally. At temperatures below the optimum, it ceases to be catalytic. As the temperature rises from this low temperature, the rate of catalysis increases, till the optimal temperature is reached. The velocity of the reaction will roughly double for every 10°C rise in temperature. As the rate of each reaction increases, metabolism is more active, and the microorganism grows faster. However, beyond a certain point, further increases actually slow growth, and sufficiently high temperatures are lethal.

Microbial growth thus has distinct cardinal temperatures—minimum, optimum, and maximum growth temperatures. 

Based on their temperature ranges for growth, microorganisms  can be placed in one of five classes.

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, it is an enormous habitat for psychrophiles. Chlamydomonas nivalis is a psychrophilic algae seen in snowfield or glacier turning it pink with its bright red spores. Pseudomonas, Vibrio, Alcaligenes, Bacillus, Arthrobacter, Moritella, Photobacterium, and Shewanella are common psychrophiles  among bacteria. A psychrophilic archaeon, Methanogenium, has been isolated from Ace Lake in Antarctica.

Psychrophilic microorganisms have adapted to their environment in several ways. Their enzymes, transport systems, and protein synthetic mechanisms function well at low temperatures. The cell membranes of psychrophilic microorganisms have high levels of unsaturated fatty acids and remain semifluid when cold. Indeed, many psychrophiles begin to leak cellular constituents at temperatures higher than 20°C because of cell membrane disruption.

2. 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.

3. Mesophiles are microorganisms with growth optima around 20 to 45°C; they often have a temperature minimum of 15 to 20°C. Their maximum is about 45°C or lower. Most microorganisms probably fall within this category. Almost all human pathogens are mesophiles, because their environment is a fairly constant 37°C.

4. Some microorganisms are thermophiles; they can grow at temperatures of 55°C or higher. Their growth minimum is usually around 45°C and they often have optima between 55 and 65°C. The vast majority are procaryotes although a few photosynthetic protists and fungi are thermophilic. These organisms flourish in many habitats including composts, self-heating hay stacks, hot water lines, and hot springs.

5. Hyperthermophiles are Thermophiles which can grow at 90°C or above and with maxima above 100°C. They usually do not grow well below 55°C.  Their growth optima can be between 80°C and about 113°C are called hyperthermophiles. Pyrococcus abyssi and Pyrodictium occultum are examples of marine hyperthermophiles found in hot areas of the seafloor.

High temperatures damage microorganisms by denaturing enzymes, transport carriers, and other proteins. Temperature also has a significant effect on microbial membranes. At high temperatures, the lipid bilayer melts and disintegrates. Thus, when organisms are above their optimum temperature, both function and cell structure are affected. At very low temperatures, membranes solidify.  If temperatures are  low, function is affected but not necessarily cell chemical composition and structure.

Thermophiles differ from mesophiles in many ways.

• They have more heat-stable enzymes and protein synthesis systems, which function well at high temperatures.
• Heat-stable proteins have highly organized, hydrophobic interiors; more hydrogen bonds and other noncovalent bonds strengthen the structure. 
• Larger quantities of amino acids such as proline also make the polypeptide chain less flexible.
• The proteins are stabilized and aided in folding by special chaperone proteins.
• In thermophilic bacteria, DNA is stabilized by special histone like proteins.
• Their membrane lipids are more saturated, more branched, and of higher molecular weight. This increases the melting points of membrane lipids thus making them temperature stable.
• Archaeal thermophiles have membrane lipids with ether linkages, which protect the lipids from hydrolysis at high temperatures. Sometimes archaeal lipids actually span the membrane to form a rigid, stable monolayer.

 

The cardinal temperatures for a particular species are not rigidly fixed but often depend to some extent on other environmental factors such as pH and the available nutrients. For example, Crithidia fasciculate, a flagellated protist living in the gut of mosquitoes, will grow in a simple medium at 22 to 27°C. However, to grow at 33 to 34°C extra metals, amino acids, vitamins, and lipids are required.

The cardinal temperatures vary greatly between microorganisms. The temperature optimum is always closer to the maximum than to the minimum. The growth temperature range for a particular microorganism usually spans about 30 degrees. Some species (e.g., Neisseria gonorrhoeae) have a small range (stenothermal); others, like Enterococcus faecalis, will grow over a wide range of temperatures(eurythermal).

The major microbial groups differ from one another regarding their maximum growth temperatures. The upper limit for protists (protozoa/algae) is around 50°C. Some fungi can grow at temperatures as high as 55 to 60°C. Procaryotes can grow at much higher temperatures than eucaryotes.

Optima usually range from 0°C to 75°C, whereas microbial growth occurs at temperatures extending from less than ^-20°C to over 120°C. Some archaea can even grow at 121°C (250°F), the temperature normally used in autoclaves.

Effect of oxygen on the growth of microorganisms

 Based on the effect of oxygen on growth, microorganisms occupy different regions when grown  in a culture tube, as demonstrated in the figure.

The different relationships with O₂ appear due to 

•  the inactivation of proteins 
•  the effect of toxic O₂ derivatives.

Enzymes can be inactivated when sensitive groups like sulfhydryls are oxidized. An example is the nitrogen-fixation enzyme nitrogenase, which is very oxygen sensitive.

Oxygen accepts electrons and is readily reduced because its two outer orbital electrons are unpaired. Flavoproteins, several other cell constituents, and radiation promote oxygen reduction. The result is usually some combination of the reduction products superoxide radical, hydrogen peroxide, and hydroxyl radical.

O₂ + e– → O₂ ^– (superoxide radical)

O₂ ^– + e^– + 2H⁺ → H₂O₂ (hydrogen peroxide)

H₂O₂ + e^– + H⁺ → H₂O + OH^– (hydroxyl radical)

These reactive oxygen species (ROS) are extremely toxic because they are powerful oxidizing agents and rapidly destroy cellular constituents. 

Neutrophils and macrophages use these toxic oxygen products to destroy invading pathogens.

A microorganism must be able to protect itself against such oxygen products or it will be killed. 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.

2O₂ ^– + 2H+ O₂ → H₂O₂ (superoxide dismutase)

 2H₂O₂ → 2H₂O + O₂ (catalase)

H₂O₂ + NADH + H⁺ → 2H₂O + NAD (peroxidase)

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. 

Although strict anaerobes are killed by O₂, they may be recovered from habitats that appear to be aerobic. In such cases they associate with facultative anaerobes that use up the available O₂ and thus make the growth of strict anaerobes possible. For example, the strict anaerobe Bacteroides gingivalis lives in the mouth where it grows in the anaerobic crevices around the teeth.

Different approaches must be used when growing aerobes and anaerobes since aerobes need O₂ and anaerobes are killed by O₂. When culturing aerobic microorganisms, either the culture vessel is shaken to aerate the medium or sterile air must be pumped through the culture vessel.

With anaerobes, all O₂ must be excluded using

(1) Special anaerobic media containing reducing agents such as thioglycollate or cysteine may be used. The reducing agents will eliminate any dissolved O₂ remaining within the medium so that anaerobes can grow beneath its surface.

(2) The medium is boiled during preparation to dissolve its components; boiling also drives off oxygen very effectively.

(3) Oxygen also may be eliminated from an anaerobic system by removing air with a vacuum pump and flushing out residual O₂ with nitrogen gas or CO₂. Many anaerobes require a small amount of CO₂ for best growth.

(4) One of the most popular ways of culturing small numbers of anaerobes is by use of a GasPak/Gas generator envelope. Water is added to chemicals in envelope to generate Hydrogen and carbon dioxide. Carbon dioxide promotes more rapid growth of microorganisms. The palladium catalyst catalyzes the formation of water from hydrogen and oxygen, thereby removing oxygen if at all it is present.

(5) Plastic bags or pouches can be used when only a few samples are to be incubated anaerobically. These have a catalyst and calcium carbonate to produce an anaerobic, carbon-dioxide rich atmosphere. A special solution is added to the pouch’s reagent compartment; petri dishes or other containers are placed. Anaerobic indicator strip Methylene blue becomes colorless in absence of O₂.

 Reference: Prescott's Microbiology