ALGAE CULTURING TECHNIQUES

ALGAE CULTURING TECHNIQUES

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.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.

ALGAE CULTURING TECHNIQUES

 

  1. 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.

  1. 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.

 

  1. 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.

  1. 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.

ALGAE CULTURING TECHNIQUES

  1. 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.

ALGAE CULTURING TECHNIQUES

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.

ALGAE CULTURING TECHNIQUES

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.

ALGAE CULTURING TECHNIQUES

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.

ALGAE CULTURING TECHNIQUES

  • 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.

ALGAE CULTURING TECHNIQUES

  • 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.

ALGAE CULTURING TECHNIQUES

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 a 300 mL of autoclaved seawater in a flask the thalli are cultured.
  • 1 % of GeO2 (Germinium dioxide) is added for the prevention of diatoms growth.