Microbiology: Freeze-Dried Cultures

Chapter II: Isolation, Preservation, and Improvement of Industrial Microorganisms

• Industrial microorganisms are originally isolated from nature, but their quality is improved via genetic manipulation.
• Genetic manipulation methods include mutagenesis and selection, nuclear fusion, protoplast fusion (for fungi, streptomycetes, and actinomycetes), and recombinant DNA technology.

Characteristics of Industrial Organisms

• Nutritional Characteristics: The organism must be able to grow in inexpensive or pre-defined media.
• Optimum Temperature: The ideal temperature for maximum productivity is above 40°C.
• Availability in Pure Culture: The organism must be available in pure culture.
• Genetic Stability: The organism should be genetically stable, but amenable to genetic manipulation.
• High Productivity: The organism must have high productivity of desired metabolites or products.
• Ease of Handling: The organism should be easy to handle (e.g., produce spores for easy inoculation and separation from the product for recovery).
• Rapid Growth: The organism should grow rapidly and produce products quickly in large-scale cultures.
• Non-harmful: The organism should not be harmful to humans, plants, or animals.

Sources of Microbes

• Microbes are obtained from culture collections such as ATCC (American Type Culture Collection).
• Standard organisms (from culture collections) are mainly used for assays, training, and educational purposes, not for production.
• These organisms usually offer low yields of the desired product.

Isolation from Nature

• Isolation is commonly from soil due to its high microbial content.
• Methods involve enrichment liquid cultures based on selecting microorganisms with the desired traits.
• Enrichment cultures increase the number of a specific organism relative to others in the original sample.
• Selective forces (e.g., adding specific substances to enrichment liquid cultures) can be used to isolate desired organisms.
• Environmental conditions (e.g., air-drying soil) can be altered to favor the survival of specific organisms before culturing.

Preservation of Industrial Microorganisms

• Industrial cultures need to be viable and free from contamination to maintain productivity.
• To eliminate genetic change, cultures are stored at reduced temperature or dehydrated.

1. Storage at Reduced Temperature

• Agar Slopes: Cultures grown on agar slopes can be stored in a refrigerator (5°C) and subcultured at approximately 6-month intervals. Storing slopes covered with sterile mineral oil extends the time to a whole year.
• Broth: Cultures grown in broth can be stored at -20°C after adding glycerol (10-20%).
• Liquid Nitrogen: This is a universal method reducing metabolic activity by storage at very low temperatures (-150°C to -196°C). This involves growing cultures to maximum stationary phase, resuspending cells in a cryoprotective agent (e.g., 10% glycerol), and freezing the suspension in sealed ampoules. Fungi, bacteriophages, viruses, and algae can also be stored this way. Though freezing and thawing causes some loss of viability, there is little loss during storage. This method is useful for long-term preservation of cells that are unable to survive drying. The equipment, however, is expensive and liquid nitrogen must be replenished regularly to prevent loss of the collection.

2. Storage in a Dehydrated Form

• Drying: Dried soil cultures are widely used for culture preservation, mostly for fungi, actinomycetes, and spore-forming bacteria. Moist soil is inoculated with medium, incubated for several days for growth, allowed to dry at room temperature, and kept in a dry atmosphere or refrigerator.
• Lyophilization (Freeze-Drying): This process involves freezing a culture, then drying it under vacuum to sublimate cell water. Cultures are grown to the maximum stationary phase, resuspended in protective media (e.g., milk, serum, sodium glutamate), transferred to ampoules, frozen, and subjected to vacuum until sublimation is complete. Ampoules are then sealed and stored in a refrigerator. Cultures can stay viable for 10 years or more. While very convenient and stable for service culture collections, freeze-dried cultures are tedious to open and revitalize and may require multiple subcultures. This is not suitable for animal or plant cells.

Quality Control of Preserved Stock Cultures

• Samples from every batch are routinely checked for purity, viability, and productivity (at least 3% of ampoules are tested).
• If even one sample fails quality control, the whole batch is discarded.

Increasing Yield of Products

• Improving the quality of industrial microorganisms: Mutations can create desired characteristics.
• Improving production conditions: Physical and nutritional conditions can be optimized.

Improvement the Quality of Industrial Microorganisms

• Mutations: Mutagenic agents (UV/ionizing radiation, chemicals like nitrous acid, nitrosoguanidine) can cause mutations and lead to changed characteristics; some may be desired mutations.

Primary Metabolites

• Essential for cell growth and associated with cell activities/energy production.
• Production correlates with cell growth and concentration.
• Examples: Amino acids, fatty acids, vitamins, and alcohol.

Secondary Metabolites

• Not essential for cell growth or energy production.
• Production depends on media components and occurs after growth.
• Produced at high concentrations during idiophase.
• Examples: Antibiotics and some vitamins.

Selection of Mutant Producing Improved (Higher) Level of Primary Metabolites

• Glutamic acid and lysine are significant commercially available primary metabolites.
• Glutamic acid (glutamate) is an excitatory neurotransmitter used by the brain as fuel to increase neuron firing, and is used in treatments for muscular dystrophy Parkinson's Disease, Schizophrenia, etc.
• Corynebacterium glutamicum is the main organism for Glutamic acid production. Two conditions are integral: the lack of an enzyme needed to convert alpha-ketoglutaric acid to succinic acid, and an auxotroph nature due to a biotin deficiency. This results in higher glutamic acid production as well as increased release outside the cell due to disrupted membrane permeability.

Selection of Secondary Metabolite Producing Strains

• Strains can be isolated from nature, and improved via mutations, creating strains producing significantly more of the desired secondary metabolites.
• Autotoxic resistance is a key trait for production of secondary metabolites. This strains are resistant to relatively high concentrations of the antibiotic in which they are used to produce.

Techniques used to improve strain qualities

• Mutations of already existing enzyme-encoding genes
• Genetically engineering by inserting a cloned gene, such as for 6'N.Kanamycin-acetyl transferase, to achieve desired characteristics in cells.

This quiz explores the advantages and disadvantages of using freeze-dried cultures in microbiology. Participants will answer questions regarding sub-culturing, preservation techniques, and the optimal conditions for maintaining these cultures. Test your understanding of this important preservation method in microbial biology.