Microalgae Culture: Isolation and Growth Methods

• Microalgae culture involves various aspects from interests and applications to isolation and perpetual cultures.
• It encompasses batch and continuous cultures, photobioreactors, outdoor growth, measuring culture growth, and key factors affecting microalgae culturing.
• 'Algae' include protists and cyanobacteria, outlining their phylogenetic relationships to plantae, fungi, animalia, bacteria, and archaea.
• The diagram specifies various algal groups such as green algae, brown algae, red algae, and diatoms.

Historical Notes

• Methods and basic culture medium concepts were mainly developed in the late 1800s and early 1900s.
• Beijerinck first reported pure algae cultures and isolated free-living Chlorella and Scenedesmus in bacteria-free cultures in 1890.
• Miquel first isolated and established pure cultures of freshwater and marine diatoms in 1892.
• Warburg pioneered work to grow microalgae in dense laboratory cultures (especially Chlorella) in 1919.
• Ketchum and Redfield described a method for maintaining continuous cultures of marine diatoms for chemical analyses in 1938.

Applications of Microalgae

• Biodiesel, bioethanol, bioactive compounds, added-value compounds, and carbon sequestration.
• Algal-based bio-energy includes oil, ethanol, methanol, biodiesel, biohydrogen, biogas, and long-chain hydrocarbon.
• Algae are cultivated as staple food & vitamins, containing carbohydrates, phycobiliproteins, beta-carotene, and vitamins.
• Algal-based pharmaceuticals include Gama-Linolenic acid (GLA), Arachidonic acid (AA), Eicosapentaenoic acid (EPA), and Docosahexaenoic acid (DHA) i.e. omega-3 fatty acid.
• Spirulina platensis and maxima serve as food and nutrition with high protein, iron, essential unsaturated fatty acids, source of vitamin B12, anti hypertension properties, and growth of intestinal bacteria.
• Chlorella is used as an active immunostimulator, free radical scavenger, it reduces blood lipids, and prevents gastric ulcers.
• Muriellopsis accumulates carotenoids to treat degenerative diseases
• Chlorella, Tetraselmis, Isochrysis, Pavlova, and other species increase the value of aquaculture as a feed.
• Anabaena and Nostoc are used as fertilizer
• Spirulina and Chlorella can be utilized for cosmetic purposes
• Nannochloropsis oculata is used as a pharmaceutical for its tyrosinase inhibition
• Tolypothrix byssoidea is used as a pharmaceutical for its Tubercidin production
• Chlamydomonas is used as a pharmaceutical for its L-asparaginase

Additional-Value Compounds

• These include B Carotenoids, Polyunsaturated Fatty Acids, Vitamins, Pigments, Glycerol.
• Lipids such as Tri-Glycerides, Hydrocarbons, and Biodiesel.
• Spent Biomass, such as Biohydrogen, Biogas, Bio-ethanol, Bio-methanol, and Fertilizers,
• Isotopic compounds can be used for medicine/research.
• Phycobiliproteins can be used for food.
• Pharmaceuticals can be used as anti cancer antibiotics or as a food supplement
• B-carotene is used as a food or chicken feed.
• Vitamins C and E can be used as a vitamin supplement.
• Polysaccharides can be used as viscosifiers.

Bioactive Compounds

• Health benefits of some bioactive compounds produced by microalgae include; reducing brain damage, anti-inflammatory activity, antioxidant activity, reducing allergy symptoms & cellular damage.
• These Compounds can be used as antimicrobial, cardiovascular, inflammatory, immunological responses, and as dietary supplements.

Biofuels

• Algae oil extraction helps clean the air of greenhouse gas emissions from power plants and other sources.
• Process entails 30% of algae used for fuel and more than 90% of water that can be recycled.
• 70% of the processed algae can be further used for methane production for power, fertilizer, or animal feed.
• Algae grows rapidly, and is is one of the sustainable biofuels
• To produce biofuels extraction of lipids is required, rapid growth rate 2-10x faster than terrestrial plants, all cells are photosynthetic
• Oil content can be 4 to 50% harvested frequency around 24/7
• Can capture CO2 recycle water and nutrients
• No direct competition with food production.
• Algae’s low footprint makes biomass yields 15x higher than normal crops and can use saline water.

Isolation Techniques

• Heat sterilization, autoclaving is the common method
• High Tempretures (>100 c) must be used
• Autoclaves are heavy wall to withstand pressure to sterilize liquids
• Filter Serilization is needed for Fragile components
• Rapidity is key to use a filter

Main Isolation Factors

• Some Species are “weeds” or easy and common, while others are seemingly impossible to catch, or hard to grow
• understanding and Mimicing natural occuring environmental conditions.
• Toxonomic knowledge (eg diatoms and silica)
• Eliminating contaminants

Isolation Methods

• Single cell isolation by Micropipette.
• Isolation via the use of Agar.
• Dilution Techniques for abundant organisms.
• Flow Cytometry automatic isolation.

Perpetual cultures

• Culture collections: repositories of diversity.
• Continuous subculturing helps in maintain cultures and to lose features.
• The important factors are:
  • Choice of culture medium.
  • Light & Temp.
  • Transfer Interval.
  • Culture Containers.
• Cryopreservation helps preserve storage of a living organism, or a portion thereof, at an ultralow temperature below -130*C.
• Empirical science of the cell when injured during freezing, and thawing is not fully understood
• Important steps to help algae minimize stress while cooling the species
• An auqeusous Suspension is important, as the risk of cryoinjury can impact the organism.

Batch cultures

• Culture maintains initial cell culture, to remain in the bioreactor.
• Initiated by the transfer of a small protion of a culture, giving an increase in biomass
• Batch cultures are low cost, easy to manipulate, and have high yield.
• There are a lot of cycles within a batch, this contains the growth curve: containing Log phase, lag phase, stationary phase and decline.
• The Log phase cells acclimate, Exponential phase for fastest growth, Stationairy phase for stable nutrient medium is used, decline phase cell nutrient decreases, and growth/death

Continuous Cultures

• Cell growth is maintained through fresh medium.
• Constant environment-cells at constant steady state.

Continuous VS Semicontinuous Cultures

• Cell count is in log scale.
• Generations nutrient concentration.
• Semicontinous variations in nutrient level over time
• Chemostat: fixed nutrient, constant nutrient limitation, cultures seen just before exhaustion
• Turbidostat: Dilution at their max generates constant population.

Turbidostat VS Chemostat

• Turbidostat regulates attenutation, chemostat uses a nutrient inflow.

Photobioreactors

• Photobioreactors are closed vessel for large scale production under controlled conditions that can be optimized.
• There must be access to light, if not fermentors are used for heterotrophic growth

Factors of Consideration for Photobioreactors

• Most Important Design of a photobiorecator, there are issues with light inhibition, and that optical thin containers must attenuate light, and self shading occurs.
• Mix for even flow and to even out the PH and temperature
• Gase Exchange needed/air/must be taken to ensure that Co2 input does not adversely PH

Types of Photobioreactors and Outdoor Ponds

• Outdoor ponds cheaper culture process but limited to smaller species, commericially sensitive
• Open ponds, or deep or standard

Growth Rates

• During exponential groth increasing cell rates is important
• If T is expressed in days then r can be divided by the natural log base 2
• Must consider carrying capacity, a competion for limiting
• Batch Culture, and Growth, as wells as Doubles will all impact growth rates
• Continusus Culture Growth will always lead to exponential growth indefinitely

Measuring Growth Rates

• Any estimates require a time series for change in biomass
• To estimate cell count and microscope viewing is used
• Proxies, such as optical density need to be used
• All come down to optical density, fluorescence for light.

Growth Media and Growth Conditions

• Media must be a specific Algal group, while abosrbing different Regions
• Macronturients like macronuriens.
• There must also be light spectra available since it is key factor
• Light intensity and illumination is important for any specific requirement
• Most alge groups have different photosynetic pigments such as Chlorophyl A

Macronutrients - marine Cultures

• Natural Seawater 50 elements
• Macronutrients like silicon, Nirtrogen, Phosphorus
• PH buffers to man stain constant overtime during growth
• Articial seawater using the main salts