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