Anaerobic respiration and culturing methods

 

Organisms capable of living in the presence of atmospheric oxygen are classified as aerobes, while those that grow in its absence are known as anaerobes. In the aerobic organism, oxygen plays a critical role in aerobic respiration to produce energy and also for biosynthesis of sterols and unsaturated fatty acids. In aerobic respiration, oxygen serves as the final electron acceptor in the electron transport chain, for production of energy.

Anaerobic bacteria produce energy through fermentation or anaerobic respiration in absence of oxygen relies on alternative exogenous electron acceptors, which are typically inorganic compounds. Common examples of these alternative electron acceptors include nitrate, sulfate, carbon dioxide, ferric ions, selenate and others. Anaerobes cannot produce sterols and unsaturated fatty acids (UFAs) for themselves, so these are required to be added to the medium for anaerobic growth.

Types of Anaerobes

1. Facultative Anaerobes: These organisms can grow without oxygen but are also capable of growing in its presence. When oxygen is available, they primarily utilize aerobic respiration for energy production e.g. Escherichia coli and Staphylococcus.
2. Aerotolerant Anaerobes: These can survive and grow in the presence of oxygen, but they are not dependant on oxygen for their metabolic processes. These organisms essentially "ignore" oxygen and continue to function as anaerobes such as Enterococcus faecalis naturally found in the intestine.
3. Strict (Obligate) Anaerobes: Obligate anaerobes, such as Bacteroides, Fusobacterium, Clostridium, and Methanococcus, cannot tolerate oxygen. Exposure to oxygen is dangerous for these organisms. For instance, Bacteroides gingivalis, a strict anaerobe, thrives in the oxygen-deprived crevices around teeth in the human mouth. Tetanus causing Clostridium tetani produces neurotoxin that causes muscle-spasm (Lock-Jaw) and breathing problems.

Oxygen Toxicity and Cellular Damage: 

Oxygen accepts electrons during cellular processes and is rapidly reduced by flavoproteins and other cellular components. This reduction generates reactive oxygen species (ROS), including superoxide radicals, hydrogen peroxide, and hydroxyl radicals. These ROS are highly toxic and act as potent oxidizing agents, causing damage to cellular structures and components. 

Microorganisms must defend themselves against toxic oxygen-derived compounds (ROS) to survive. Failure to do so can result in their destruction.

Enzymes Protecting Against Oxygen Toxicity

Aerobes and facultative anaerobes typically produce enzymes such as superoxide dismutase (SOD) and catalase. These enzymes neutralize harmful oxygen species:

1. Superoxide dismutase (SOD) converts superoxide radicals into hydrogen peroxide and oxygen: ​
2. Catalase then breaks down hydrogen peroxide into water and oxygen:Strict anaerobes, however, lack these enzymes or have them in insufficient quantities, making them unable to survive in oxygen-rich environments.

Culturing Aerobic bacteria vs. Anaerobic bacteria

The oxygen requirements of microbes necessitate distinct cultivation techniques for aerobes and anaerobes:

Culturing Aerobic Bacteria

Aerobic bacteria require oxygen for growth, so their cultivation involves ensuring adequate oxygenation:

1. Shaking the culture vessel: Agitation increases oxygen diffusion into the medium.
2. Aerating the medium: Sterile air is pumped into the culture vessel to maintain oxygen levels.

Culturing Anaerobic Bacteria

The media used for culturing anaerobic microbes are boiled to remove dissolved oxygen commonly used media are thioglycolate medium, cooked meat medium, anaerobic blood agar and tryptic soy anaerobic medium.

Anaerobic microbes require the absence of oxygen, and specific methods are employed to exclude it:

1. Using reducing agents in media: Anaerobic media, such as those containing thioglycollate or cysteine, include reducing agents to remove dissolved oxygen. Boiling the medium during preparation helps eliminate oxygen, ensuring anaerobic conditions below the surface.
2. Vacuum and gas flushing systems: Air is removed using a vacuum pump, and residual oxygen is flushed out with nitrogen gas. Carbon dioxide is often added to the system to meet the growth requirements of some anaerobes.
3. GasPak jar system: A GasPak jar is a convenient method for culturing small quantities of anaerobic microbes. The system includes a gas pack containing calcium carbonate, which releases carbon dioxide and hydrogen. A palladium catalyst promotes the reaction of residual oxygen with hydrogen, forming water and effectively removing oxygen. The water produced is absorbed by a desiccant, maintaining anaerobic conditions within the jar.

GAS-PAK Jar System

These strategies ensure appropriate environmental conditions for the growth of aerobes or anaerobes, based on their oxygen tolerance or requirements.

Use of anaerobic bacteria

1. Treatment of Industrial waste-water,
2. Biodegradation of toxic pollutants,
3. Production of medical products such as vaccines, antibiotics, hormones (insulin), steroids, vitamins, etc,
4. Production of various Enzymes for industry eg. Cellulases for paper, detergent and food industry, Catalase for food preservation etc.
5. Fermentations for alcohol production, fuel, lactic fermented foods etc.