ALGAE CULTURING TECHNIQUES

ALGAE CULTURING TECHNIQUES

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Introduction

• For algae culturing techniques, the artificial algae culture medium is supplemented with various chemicals so that it resembles the natural environment.
• For the initiation of the aquaculture process, the isolation of the axenic culture of algae is the first step.
• Axenic culture should be preserved for a longer period of time.
• The algal reservoir should be free of various contaminants and bacteria.
• Algal culture media are divided into two broad classes viz Freshwater culture media and marine culture media.
• Samples that are collected for algae culturing techniques are not pure.
• Below are the algae culturing techniques.

STERILIZATION

• Sterilization is usually done to develop an aseptic culture environment by killing the microorganisms.
• Autoclaving (heat sterilization) is commonly practiced for algal cultures.
• Some precautions should be taken before culturing algae
• Laminar flow should be turned on before starting the culture work.
• The working surface should be cleaned with 70 % ethanol.
• The Bunsen burner should be used.
• After organizing all requirements under the laminar flow, hands should be cleaned with 70 % ethanol.
• All the sterile pipette, loop, or any material which are going to be used in culture should be flamed and it should be cooled before used.
• All the rim of the test tubes, Petri plates should be flamed and cooled down before use.
• The loop or pipette should be cleaned after every use.

PREPARATION OF STOCK SOLUTIONS

• Algae culture medium is generally composed of three components macronutrients, trace elements and vitamins. They are termed as stock solutions.
• Concentrated stock solutions for these components are prepared and then subsequently diluted to the final media concentration.
• Stock solutions are generally prepared in quantities of 100 mL to 1 liter.
• The sock solutions are stored at 4 °C in a tightly sealed glass bottle so that the final concentration of the solution does not change.

TYPES OF ALGAE CULTURE MEDIUM

1. Bold Basal Medium – Used for culturing fresh algae.

• Prepared using the macronutrients, EDTA, Iron, Boron and Trace Metal Solution.
• The pH of 6.5 is maintained in the algae culture medium for optimal growth.

2. Chu 10 medium – Used for the culture of various algae such as green algae, diatoms and cyanobacteria.

• It is an antificial medium.
• No chelators, vitamins and trace metals are added.
• The pH of the culture is maintained at 6.5.

3. Medium for diatoms – Mainly for the culture of various diatom species

• Soil extract is used for this medium.
• All the components are dissolved in 900 mL of dH₂O except vitamins.
• The final volume is brought up to 1 liter using dH₂
• Then it is sterilized.
• Vitamins are added after cooling the stock solution.
• Then the pH is adjusted to 6.7.
• The vitamin solution is prepared by adding thiamine HCl into 950 mL of dH₂
• 1 mL each from all the stock solution is used and is brought to 1 liter.
• The solution is then frozen for storage.

 

4. Medium for volvox culturing – Mainly used for the culture of Volvox species.

• Also used for the culture of some strains of Eudorina, Pandorina and Gonium.
• Calcium nitrate and glycylglycine are dissolved in 900 mL of dH₂O followed by other stock solutions.
• Then vitamins are added.

5. Medium for blue-green algae (BG11) – Mainly for the culture of Cyanobacteria.

• Used for freshwater, soil and marine organisms which do not require high ionic strength.
• pH of the culture was maintained at 7.5 for optimal growth.
• Then it is autoclaved.

6. Medium for Spirulina – Mainly for Spirulina

• Two separate solutions are prepared.
• Then autoclaved separately.
• The two solutions are combined aseptically and after that 1 mL of cyanocobalamin (B₁₂ ) is added.

ISOLATION OF ALGAL SAMPLES

• Beijerinck first started the techniques for the isolation of microalgae.
• Water samples are collected in clean bottles and kept at a lower temperature so that the cells remain viable.
• Many techniques such as filtration, differential centrifugation, micro pipetting, serial dilution, etc are used for isolation and purification of the algal samples.

ANTIBIOTIC TREATMENT

• Antibiotic treatment is sometimes necessary to obtain pure unialgal cells.
• Penicillin, streptomycin and gentamycin are the common antibiotics used in the microalgal culture.
• The principle of this treatment is, it diminishes the bacterial growth without affecting the algal growth.
• A range of 50–500 mg/l concentrations of antibiotics is generally used.
• The antibiotics prepared for the use in the algal culture are preserved in frozen condition until use.

MODERN MICROALGAL ISOLATION METHOD

• Automated isolation method for microalgae has been developed.
• Flow cytometry is used in the automatic isolation method.
• The main principle in Flow cytometry is scattering of light, excitation and emission of fluorochrome molecules to generate specific multi-parameter data from particles and cells.
• The light source used in flow cytometry is mainly lasers.
• Lasers have a very small size hole to focus light.
• Hence, large single cells are excited thereby reducing the chance of more than one cell.

OPEN POND SYSTEM FOR ALGAE CULTIVATION

• Open ponds are of two types namely natural lakes, lagoons or ponds and artificial systems.
• The major benefit of an open system is that they are easy to handle than the other systems.
• At the same time mixing the culture is very difficult in open systems thereby producing less biomass.

CLOSED CULTURE SYSTEM

• This is carried out by using photobioreactors.
• Photobioreactors are of various types such as a tubular, flat panel or column reactors.
• They used external light supplies for their growth.
• Tubular photobioreactors for algal cultures – long, transparent tubes which can be horizontal vertical or as a helix
• Airlifts create the pumping force which allows CO 2 and O 2 to be exchanged between the liquid medium and the aeration gas.

• Flat Panel Photobioreactors – the algal culture is mixed across the flat panel system.
• Algae which are above the culture absorbs the light.
• Good for immobilization of algae.
• It provides high photosynthetic efficiencies.

• Column Photobioreactors – They are in vertical position or can be bubbled from below.
• The light passes through the culture from the transparent walls.
• These bioreactors give a good mixing of the culture.
• They provide a very high rate of gas transfer.

MACROALGAE CULTIVATION

• marine algae are macroscopic
• Also called seaweeds.
• They include green, brown and red algae.
• Seaweeds are brought to the laboratory after collecting form field.
• The epiphytes and epifauna are removed manually.
• Vegetative tissue or spores are used for maintaining the culture of seaweeds.
• For large scale cultivation, the macroalgae are usually cultured in natural seawater.
• Nutrients and other trace metals are supplied for laboratory culture for growth.
• Macroalgae are cultured in a sterilized glass or Petri dishes filled with enriched medium.
• They are covered with parafilm.
• Algae culture medium is changed depending on the material and temperature.
• The algal thalli are allowed to acclimatize for 4–5 days in sterilized seawater with desired salinity before starting the culture work.
• The thalli are weighed and are cut into small pieces of approximately 2–3 cm.
• After cutting the pieces are transferred to 500 mL of the cleaned conical flask containing 200 mL of sterilized liquid media.
• F/2 medium and PES medium are the most common culture media used.

DISINFECTANT TREATMENT OF ALAGL THALLI

• In order to make the culture free from contamination, the thalli are treated with various disinfectant series.
• The thalli are treated with a 1 % IKI solution for 1 min to eliminate surface microbes.
• The broad-spectrum antibiotic mixture is used for sterilization to prevent any kind of bacterial growth.
• Inside 300 mL of autoclaved seawater in a flask the thalli are cultured.
• 1 % of GeO₂ (Germanium dioxide) is added for the prevention of diatoms growth.