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Cultivation of Microalgae in Shake

Flasks and Laboratory Scale


Photobioreactor
Microalgae
• Microalgae are considered a promising source for various high-value
products, including carotenoids and omega-3 and omega-6
polyunsaturated fatty acids (PUFAs).

• But majority of PUFAs production is performed by heterotrophic


fermentation hence only two microalgal high-value products are
successfully marketed at a relevant scale: beta-carotene from Dunaliella
salina, and astaxanthin from Haematococcus pluvialis.

• In addition, Chlorella and Spirulina biomass are marketed in large volumes


as nutraceuticals, and phycocyanin extracted from cyanobacteria has
gained major market share recently.

• Hence this mentioned species are considered to be important while


studying the lab scale algae cultivation.
Lab Scale Cultivation
• Commonly algae biomass production requires sterile conditions with well-
defined external parameters.

• Microalgae biomass production is based on the simple scheme:


CO2+ H2O + nutrients + light energy →biomass + O2.

• The simplest cultivation vessel is an illuminated flask that is placed on a


shaker.

• More efficient cultivation systems-closed laboratory and pilot scale


photobioreactors.

• The latter can provide contaminant-free environment and make possible the
control of culture pH, temperature, CO2concentration in aeration gas, light
intensity, and other culture variables, hence giving a wider range of relevant
products.
Types of Photobioreactors
Type Sub-type Description
Tubular Reactor • Vertical TR Generally in the TR the microalgae culture is
• Horizontal TR circulated in transparent tubes by a centrifugal pump,
• Helical TR and passes intermittently through a degasser –i.e. an
air- sparged vessel, where the accumulated oxygen is
blown off.
Flat plate reactors Vertical FPR Vertically or horizontally oriented flat, transparent
(FPR) Horizontal FPR vessel in which mixing is carried out directly in the
reactor via air sparging
Stirred tank reactor A vertical cylindrical tank, in which agitation is
STR) provided via an impeller or magnetic stirrer (in
smaller units), every parameter can be controlled to a
higher level than in other reactor types. Illumination
can be provided internally/externally by artificial
light.1
Plastic Bags Various different Plastic bags arranged in different patterns are
patterns according installed with aerator to promote yield.
volume.
Photobioreactor for Lab Scale
• Stirred tank photobioreactor is most preferred PBR for lab scale microalgae
cultivation.

• The advantage of this type of photobioreactor over others for lab scale
cultivation is its better parameter control. This is especially important in
while growing sensitive species such as H. pluvialis.

• But this PBRs are only used for lab scale cultivation due to the problematic
light provision for all cells. The bigger the vessel is, the more critical this
factor gets. At lab scale this factor is controlled by using multiple mixers on
middle axel.

• Another photobioreactor which is used for lab scale cultivation is flat plate
reactor.

• For the lab scale cultivation of less sensitive algal species illuminated flask
and plastic bags are better option because of less capital requirement.
Major Constrains
• Light Intensity:
• Light is absolutely essential for microalgae cultivation as they use it as source
of energy and their growth is highest at optimum light intensity and growth
decreases with increase or decrease in intensity.
• Optimum light intensity for microalgae varies depending on the strain.
• For many microalgae, increase in the light intensity causes photo inhibition
because of disruption of the chloroplast lamellae growth and inactivation of
the enzymes involved in the carbon dioxide fixation.
• Optimization of light intensity during lab scale cultivation is a challenge.

• Temperature:
• Temperature influences the algae growth rate, cell size, biochemical
composition, and nutrient requirements.
• If temperature is higher/lower than the optimum -a significant decrease in
the growth rate can be observed.
• Optimum temperature for microalgae also varies depending on the strain.
• This problem can be conquered by using temperature control equipment or
by selecting proper location.
Scale up
• The aim of the scaling-up is to obtain larger cell/product quantities at a larger
scale with at least the same viability and quality of culture as obtained at a
smaller scale.
Cultivation Experiment
in flask

Laboratory Scale
Photobioreactor

Pilot Scale Cultivation

Large Scale Cultivation


Microalgae Cultivation in Shake Flasks
• Shake flasks experiments provide easy multi-parameter variation in parallel
experiments.
Experimental Setup for shake flask cultivation system
Laboratory-scale stirred tank photobioreactor
• Based on the results obtained in the shake-flasks experiments laboratory-
scale stirred tank photobioreactor is developed.
• Schematic Diagram of photobioreactor:
Laboratory Scale photobioreactor:
Promising Species for Lab Scale Cultivation
Product Algal Species Optimum Conditions Conclusion

Astaxanthin -Haematococcus pluvialis : 30 Temperature: 25°C-28°C for -Haematococcus and other


mg/gm vegetative growth and 35°C astaxanthin producing
-Neochloris wimmeri: 0.6% for Astaxanthin algal species can be
accumulation. cultivated in strictly
monitored conditions.
Light: 600 W MH light
Phycobiliproteins -Cynobacteria Temperature: 35°C-37°C -Spirulina is sucessfully
1. Phycocyanin -Spirulina
(Natural Blue Open pond cultivation
Pigment) and Successful in Tamil Nadu
Proteins
β-carotene and Dunaliella salina: 100 mg/gm Temperature: 22°C
Zeaxanthin and 6 mg/g
Light: 135.3 μmol m-2 s-1

Indian companies are mostly producing Spirulina and Cynobacteria


Materials and Methods for Lab Scale Cultivation
• Growing microalgae in shake flasks and laboratory scale stirred tank
photobioreactor. Different group of experiments are setup to optimize the process
parameters.
Materials Description

Algal Strains -Algal cultures are obtained either from universities or from companies such
UTEX and Algae Analytics.
-The strains are transported and incubated at optimum temperature under
continuous light intensity.
Glassware Conical flask, Culture bottles, Measuring Cylinders, Volumetric flasks etc.

Photobioreactor Custom made stirred tank photobioreactor of capacity 3 liter

LED lights LED cases

Standard Chemicals

Media BBM for H. pluvialis

Other Equipment's Lux meter, Air Compressor, Plastic tubing, Media and air filters, Shaker, etc.

Process Inputs Description

Aeration Air compressor, Plastic tubing and Air controller

Temperature Hot water and Cold water lines


maintenance
Method: Batch Type Cultivation
The media is prepared in 150 ml conical flasks, closed with
cotton plugs and is sterilized at 121°C for 20 min

Inoculation is carried out in 500 ml laboratory flasks,


bottles or in photobioreactor under aseptic conditions in
laminar air flow hood

Stock culture is maintained on both semi-solid and liquid


medium for sub culturing

The inoculated flasks are incubated in culture room under


controlled temperature and light intensity while PBR
maintains optimum conditions by itself.

The CO2 is continuously injected in flasks using narrow neck


flask and in photobioreactor through tubing.

The cell density is continuously monitored during green


phase and dry weight and astaxanthin content is measured
in the end.
Thank You!

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