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ISOLATION OF
ALGAE (INDOOR)
K R MICRO NOTES 1
INTRODUCTION
K R MICRO NOTES 2
CONDITIONS REQUIRED FOR THE CULTIVATION
OF ALGAE
• For the successful growth of algae in a culture, the
environment must be conditioned to meet as many of the
intrinsic requirements of that organism as possible.
• The environmental factors may be either physical, such as
temperature and light, or chemical, which provides all the
raw materials used for the structural and protoplasmic
synthesis of the algal cell.
K R MICRO NOTES 3
CONDITIONS REQUIRED FOR THE CULTIVATIONOF
ALGAE
LIGHT:
• Light is an important source of energy for photosynthetic algae
• It helps in the conversion of inorganic carbon into organic matter
• Duration of artificial illumination should be minimum 18hrs/day.
K R MICRO NOTES 4
CONDITIONS REQUIRED FOR THE CULTIVATIONOF
ALGAE
TEMPERATURE:
• The optimum temperature for algae culture is 16-27 C, it can vary
with species
• Temperature lower than 16 C will slow down growth and above 35 C
is lethal.
K R MICRO NOTES 5
CONDITIONS REQUIRED FOR THE CULTIVATIONOF
ALGAE
AERATION AND AGITATION:
• Aeration is required as air contains carbon source for photosynthesis
in the form of Co2
• Proper agitation ensures exposure of all cells towards light and
nutrients
• It also improves gas exchange between culture medium and air.
K R MICRO NOTES 6
CONDITIONS REQUIRED FOR THE CULTIVATIONOF
ALGAE
SALINITY:
• Salinity is the saltiness or dissolved salt concentration in water
• Marine algae grow best in salinity habitat obtained by diluting sea
water
• Salinity of 20-24g/L is optimum
pH:
• For the cultivation of algae pH is also an important requirement,
many algal species have a pH range of 7-9
• The optimum pH being 8.2-8.7
K R MICRO NOTES 7
CULTURE MEDIA
• Under natural conditions, most algae grow as mixed communities
which include various species and genera
• In 1896, Molisch had observed that the mineral nutrition of the algae
was not different from that of higher plants.
K R MICRO NOTES 8
CULTURE MEDIA
• The nutrients in the media are classified into two major groups:
– Macronutrients
– Micronutrients
• The major absolute requirements include
• Carbon, Nitrogen, Phosphorus, Sulfur, Potassium and
Magnesium
• Elements like iron and manganese are required in small amounts.
• Various other elements like Cobalt, Zinc, Boron, Copper and
molybdenum are the trace elements.
K R MICRO NOTES 9
CULTURE MEDIA
• In addition to these basic minerals, several algae like heterotrophs
(heterotrophs: organisms that need organic substrates synthesized by
other organisms.) require additional organic substrate like vitamins,
nucleic acids, growth factors for their growth.
K R MICRO NOTES 10
MACRONUTRIENTS
CARBON SOURCE - Algae require an inorganic carbon source to
carry out photosynthesis.
• CO2 enriched air is supplied to provide the required carbon.
• CO2 sources like ethanol, waste gas from industrial combustion
processes, diesel engines, and fermentation plants are being used.
K R MICRO NOTES 11
MACRONUTRIENTS
SUGAR AND MOLLASES – Source of the organic carbon is
molasses which is available in sugarcane growing countries.
• Using this growth rate is up to about 150 mg/l.
• ACETIC ACID - utilized as organic carbon source in algal plants
especially in producing Chlorella as a health food.
K R MICRO NOTES 12
MACRONUTRIENTS
NITROGEN - Cyanobacteria are capable of assimilating nitrogen in
its elemental form, from the atmosphere, preferred nitrogen supply is
in the form of ammonia and urea.
• A low nitrogen supply results in a low respiration rate and an
increase in the lipid reserves in several algae.
K R MICRO NOTES 13
MACRONUTRIENTS
PHOSPHOROUS - It is essential for almost all cellular processes
• The major form in which algae acquire phosphorous is as inorganic
phosphate either as H2PO4
- or HPO4
2-.
• Phosphorous is the key ingredient in the energy rich ATP molecule
K R MICRO NOTES 14
MACRONUTRIENTS
SULFUR - Algae like chlorella, utilizes organic sulfur sources such as
sulfur containing amino acids under certain conditions.
CALCIUM - It plays an important role in the maintenance of
cytoplasmic membranes.
• Calcium is involved in the formation of skeletons of certain algae.
K R MICRO NOTES 15
MACRONUTRIENTS
SODIUM & POTASSIUM - Sodium is necessary for all marine,
halophilic algae & Cyanobacteria for transformation of nitrogen to
ammonia.
• Potassium is a cofactor for several enzymes and involved in protein
synthesis and osmotic regulation.
• Under potassium deficient conditions, growth and photosynthesis are
reduced and respiration is high.
K R MICRO NOTES 16
MACRONUTRIENTS
MAGNESIUM - The key function of the magnesium is the
aggregation of ribosomes into the functional units and for the
formation of catalase.
K R MICRO NOTES 17
MICRONUTRIENTS
IRON - It plays important role nitrogen assimilation as a functional
part of ferredoxin and affects the synthesis of phycocyanin and
chlorophyll.
• Bleaching and yellow coloring of the algae cultures are often an
indication of iron deficiency of the medium.
K R MICRO NOTES 18
TRACE ELEMENTS
Influences growth in representative number of species
• Have a positive effect on total growth
• Show a direct physiological effect on the alga growth.
• The major trace elements in algal growth are Mn, Ni, Zn, Co, Cu, Mb
etc.
K R MICRO NOTES 19
COMMONMEDIAUSEDFOR CULTURING OF ALGAE
CHU MEDIUM Beneck’s Media
K R MICRO NOTES 20
COMMONMEDIAUSEDFOR CULTURING OF ALGAE
Allen’s Media Allen and Arnon’s
Media
K R MICRO NOTES 21
INDOORCULTIVATION
Algae when grown in a closed room or lab conditions is termed as
indoor cultivation.
There are 3 different methods of cultivation
• Balanced aquarium
• Nutrient solution
• Soil water medium
K R MICRO NOTES 22
Balancedaquarium
• Algae found in stagnant water, troughs, damp, soil etc grow
in such medium
• Here the container is partly filled with water and all the
nutrient requirements are added to it with small amount of
inoculum (algae). Cover the lid and add water from time to
time to compensate evaporation.
K R MICRO NOTES 23
Nutrient solution
• Here the medium is rich in nutrients which is supplied artificially
i.e. a specific media is used for the growth of algae in lab
conditions which can provide enough amount of nutrients for the
inoculum which is added.
K R MICRO NOTES 24
Soil water medium
• Garden soil with little amount sand from manure compost or decay
leaf is made a thin layer with 0.5grams of precipitated calcium
carbonates and steamed at normal atmospheric pressure in autoclave
and inoculated.
K R MICRO NOTES 25
Culture vessels
The vessels which are use for
cultivation are:
• Flat bottomed
depressed flasks or
Erlenmeyer flasks
• Roux flask
• Glass culture tube
and Carboys
K R MICRO NOTES 26
Culture vessels
K R MICRO NOTES 27
ROUXFLASK
• These are flat flasks in which higher irradiance (surface area)
can be achieved. A glass air tube can be inserted to the bottom
of the flask for agitating the medium by aeration.
• The advantage of this flask is an increased ratio of surface to
volume so that a more uniform and intense illumination of the
algal culture can be obtained.
K R MICRO NOTES 28
GLASS CULTURE TUBE
• The tube has an inner
diameter of 3.5 cm and a
length of 40 cm holding about
250 -300 ml of algal
suspension & are mainly used
in laboratories.
• Here, through a glass pipe
CO2 enriched air is bubbled
through the culture to supply
carbon and to settling of the
algae & the tube is closed
with a porous cellulose
stopper covered by an
aluminum cap.
K R MICRO NOTES 29
CARBOYS
• When the cultures in the
small glass tubes start
growing after few days, as
evidenced by thickening of
culture and development of
an intense blue green color
it can be diluted into larger
glass carboys which has a
capacity of 5 -10 liter and is
aerated by compressed air
through sintered stones and
shaken a few times
everyday.
K R MICRO NOTES 30
ISOLATION OF ALGAE
The separation of a single clone form a mixed population is achieved
by various isolation techniques like
• Streak plate method.
• Density centrifugation method.
• Serial dilution method.
• Micromanipulator.
K R MICRO NOTES 31
STREAK PLATE METHOD
• This method is used for the isolation of pure culture of algae.
• Here mixed culture is streaked across the agar these streaks thin out
the inoculums & micro-organisms are separated from each other.
• Each colony is the progeny of a single microbial cell thus
representing clone of pure cultures.
K R MICRO NOTES 32
Isolationtechniques
K R MICRO NOTES 33
DENSITYCENTRIFUGATION
• Repeated low-speed centrifugation (500 - 1000 rpm) is done to
remove large amount of diverse micro organisms.
• These results the algae to float at the upper phase in suspended
material.
K R MICRO NOTES 34
SERIAL DILUTIONMETHOD
• This method is commonly used to obtain pure cultures of those
microorganisms in liquid media.
• The inoculums is subjected to serial dilution in a sterile liquid
medium and a large number of tubes of sterile liquid medium are
inoculated with aliquots of each successive dilution.
K R MICRO NOTES 35
Isolation techniques
K R MICRO NOTES 36
MICROMANIPULATOR
• It is a instrument which
permits one to pick out a single
cell from a mixed culture.
• A series of hanging drops of a
diluted culture are placed on a
sterile cover slip by a
micropipette.
• Now a hanging drop is
searched which contains only
single microorganism cell.
• This cell is now drawn into a
micropipette by suction and
then transferred to a large drop
of sterile medium on another
sterile cover slip.
K R MICRO NOTES 37
PHOTOBIOREACTOR
• A photobioreactor is a bioreactor that utilizes a light source to
cultivate phototrophic microorganisms
• These organisms use photosynthesis to generate biomass from light
and carbon dioxide and include plants, mosses, macro algae,
microalgae, cyanobacteria and purple bacteria.
• Within the artificial environment of a photobioreactor, specific
conditions are carefully controlled for respective species.
• Thus, a photobioreactor allows much higher growth rates and purity
levels than anywhere in nature or habitats similar to nature.
K R MICRO NOTES 38
PHOTOBIOREACTOR
K R MICRO NOTES 39
PHOTOBIOREACTOR
• These reactors are made of stainless steel having a cylindrical shape
with diameter between 20 and 60cm and height between 35 and
100cm.
• On top they are covered by a flat steel lid, the lower part is covered
by a double walled steel cylinder, in which water can be circulated to
maintain constant temperature.
• Most of the reactors are equipped with an internal illumination
system.
K R MICRO NOTES 40
PHOTOBIOREACTOR
• Fluorescence tubes are also present which can easily be removed
without opening the reactor.
• After inoculation the algal suspension is agitated by means of a stirrer.
• Sterile air, enriched with Co2 is passed via the central tube into the
blades through capillary holes.
K R MICRO NOTES 41
Harvesting
• Harvesting the algae which is usually a highly energy intensive step
can also be carried out rapidly and efficiently by the above stirrer.
• When the algae have reached the stationary growth phase, both the
aeration and the stirrer are turned off so that the algae settle on the
bottom of the reactor.
K R MICRO NOTES 42
Harvesting
• The stirrer is connected to a suction pump via its central tube and set
into very slow rotation.
• The thick algal suspension formed at the bottom of the reactor is
harvested through suction slits.
K R MICRO NOTES 43
PHOTOBIOREACTOR
• The reactor remains closed during this process.
• The harvested slurry can be further concentrated by centrifugation.
• Fresh medium can be added to the reactor via a sterile filtration
device making continuo cultivation possible.
K R MICRO NOTES 44
K R MICRO NOTES 45

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ISOLATION OF ALGAE (INDOOR) AND PHOTOBIOREACTOR K R.ppt

  • 3. CONDITIONS REQUIRED FOR THE CULTIVATION OF ALGAE • For the successful growth of algae in a culture, the environment must be conditioned to meet as many of the intrinsic requirements of that organism as possible. • The environmental factors may be either physical, such as temperature and light, or chemical, which provides all the raw materials used for the structural and protoplasmic synthesis of the algal cell. K R MICRO NOTES 3
  • 4. CONDITIONS REQUIRED FOR THE CULTIVATIONOF ALGAE LIGHT: • Light is an important source of energy for photosynthetic algae • It helps in the conversion of inorganic carbon into organic matter • Duration of artificial illumination should be minimum 18hrs/day. K R MICRO NOTES 4
  • 5. CONDITIONS REQUIRED FOR THE CULTIVATIONOF ALGAE TEMPERATURE: • The optimum temperature for algae culture is 16-27 C, it can vary with species • Temperature lower than 16 C will slow down growth and above 35 C is lethal. K R MICRO NOTES 5
  • 6. CONDITIONS REQUIRED FOR THE CULTIVATIONOF ALGAE AERATION AND AGITATION: • Aeration is required as air contains carbon source for photosynthesis in the form of Co2 • Proper agitation ensures exposure of all cells towards light and nutrients • It also improves gas exchange between culture medium and air. K R MICRO NOTES 6
  • 7. CONDITIONS REQUIRED FOR THE CULTIVATIONOF ALGAE SALINITY: • Salinity is the saltiness or dissolved salt concentration in water • Marine algae grow best in salinity habitat obtained by diluting sea water • Salinity of 20-24g/L is optimum pH: • For the cultivation of algae pH is also an important requirement, many algal species have a pH range of 7-9 • The optimum pH being 8.2-8.7 K R MICRO NOTES 7
  • 8. CULTURE MEDIA • Under natural conditions, most algae grow as mixed communities which include various species and genera • In 1896, Molisch had observed that the mineral nutrition of the algae was not different from that of higher plants. K R MICRO NOTES 8
  • 9. CULTURE MEDIA • The nutrients in the media are classified into two major groups: – Macronutrients – Micronutrients • The major absolute requirements include • Carbon, Nitrogen, Phosphorus, Sulfur, Potassium and Magnesium • Elements like iron and manganese are required in small amounts. • Various other elements like Cobalt, Zinc, Boron, Copper and molybdenum are the trace elements. K R MICRO NOTES 9
  • 10. CULTURE MEDIA • In addition to these basic minerals, several algae like heterotrophs (heterotrophs: organisms that need organic substrates synthesized by other organisms.) require additional organic substrate like vitamins, nucleic acids, growth factors for their growth. K R MICRO NOTES 10
  • 11. MACRONUTRIENTS CARBON SOURCE - Algae require an inorganic carbon source to carry out photosynthesis. • CO2 enriched air is supplied to provide the required carbon. • CO2 sources like ethanol, waste gas from industrial combustion processes, diesel engines, and fermentation plants are being used. K R MICRO NOTES 11
  • 12. MACRONUTRIENTS SUGAR AND MOLLASES – Source of the organic carbon is molasses which is available in sugarcane growing countries. • Using this growth rate is up to about 150 mg/l. • ACETIC ACID - utilized as organic carbon source in algal plants especially in producing Chlorella as a health food. K R MICRO NOTES 12
  • 13. MACRONUTRIENTS NITROGEN - Cyanobacteria are capable of assimilating nitrogen in its elemental form, from the atmosphere, preferred nitrogen supply is in the form of ammonia and urea. • A low nitrogen supply results in a low respiration rate and an increase in the lipid reserves in several algae. K R MICRO NOTES 13
  • 14. MACRONUTRIENTS PHOSPHOROUS - It is essential for almost all cellular processes • The major form in which algae acquire phosphorous is as inorganic phosphate either as H2PO4 - or HPO4 2-. • Phosphorous is the key ingredient in the energy rich ATP molecule K R MICRO NOTES 14
  • 15. MACRONUTRIENTS SULFUR - Algae like chlorella, utilizes organic sulfur sources such as sulfur containing amino acids under certain conditions. CALCIUM - It plays an important role in the maintenance of cytoplasmic membranes. • Calcium is involved in the formation of skeletons of certain algae. K R MICRO NOTES 15
  • 16. MACRONUTRIENTS SODIUM & POTASSIUM - Sodium is necessary for all marine, halophilic algae & Cyanobacteria for transformation of nitrogen to ammonia. • Potassium is a cofactor for several enzymes and involved in protein synthesis and osmotic regulation. • Under potassium deficient conditions, growth and photosynthesis are reduced and respiration is high. K R MICRO NOTES 16
  • 17. MACRONUTRIENTS MAGNESIUM - The key function of the magnesium is the aggregation of ribosomes into the functional units and for the formation of catalase. K R MICRO NOTES 17
  • 18. MICRONUTRIENTS IRON - It plays important role nitrogen assimilation as a functional part of ferredoxin and affects the synthesis of phycocyanin and chlorophyll. • Bleaching and yellow coloring of the algae cultures are often an indication of iron deficiency of the medium. K R MICRO NOTES 18
  • 19. TRACE ELEMENTS Influences growth in representative number of species • Have a positive effect on total growth • Show a direct physiological effect on the alga growth. • The major trace elements in algal growth are Mn, Ni, Zn, Co, Cu, Mb etc. K R MICRO NOTES 19
  • 20. COMMONMEDIAUSEDFOR CULTURING OF ALGAE CHU MEDIUM Beneck’s Media K R MICRO NOTES 20
  • 21. COMMONMEDIAUSEDFOR CULTURING OF ALGAE Allen’s Media Allen and Arnon’s Media K R MICRO NOTES 21
  • 22. INDOORCULTIVATION Algae when grown in a closed room or lab conditions is termed as indoor cultivation. There are 3 different methods of cultivation • Balanced aquarium • Nutrient solution • Soil water medium K R MICRO NOTES 22
  • 23. Balancedaquarium • Algae found in stagnant water, troughs, damp, soil etc grow in such medium • Here the container is partly filled with water and all the nutrient requirements are added to it with small amount of inoculum (algae). Cover the lid and add water from time to time to compensate evaporation. K R MICRO NOTES 23
  • 24. Nutrient solution • Here the medium is rich in nutrients which is supplied artificially i.e. a specific media is used for the growth of algae in lab conditions which can provide enough amount of nutrients for the inoculum which is added. K R MICRO NOTES 24
  • 25. Soil water medium • Garden soil with little amount sand from manure compost or decay leaf is made a thin layer with 0.5grams of precipitated calcium carbonates and steamed at normal atmospheric pressure in autoclave and inoculated. K R MICRO NOTES 25
  • 26. Culture vessels The vessels which are use for cultivation are: • Flat bottomed depressed flasks or Erlenmeyer flasks • Roux flask • Glass culture tube and Carboys K R MICRO NOTES 26
  • 27. Culture vessels K R MICRO NOTES 27
  • 28. ROUXFLASK • These are flat flasks in which higher irradiance (surface area) can be achieved. A glass air tube can be inserted to the bottom of the flask for agitating the medium by aeration. • The advantage of this flask is an increased ratio of surface to volume so that a more uniform and intense illumination of the algal culture can be obtained. K R MICRO NOTES 28
  • 29. GLASS CULTURE TUBE • The tube has an inner diameter of 3.5 cm and a length of 40 cm holding about 250 -300 ml of algal suspension & are mainly used in laboratories. • Here, through a glass pipe CO2 enriched air is bubbled through the culture to supply carbon and to settling of the algae & the tube is closed with a porous cellulose stopper covered by an aluminum cap. K R MICRO NOTES 29
  • 30. CARBOYS • When the cultures in the small glass tubes start growing after few days, as evidenced by thickening of culture and development of an intense blue green color it can be diluted into larger glass carboys which has a capacity of 5 -10 liter and is aerated by compressed air through sintered stones and shaken a few times everyday. K R MICRO NOTES 30
  • 31. ISOLATION OF ALGAE The separation of a single clone form a mixed population is achieved by various isolation techniques like • Streak plate method. • Density centrifugation method. • Serial dilution method. • Micromanipulator. K R MICRO NOTES 31
  • 32. STREAK PLATE METHOD • This method is used for the isolation of pure culture of algae. • Here mixed culture is streaked across the agar these streaks thin out the inoculums & micro-organisms are separated from each other. • Each colony is the progeny of a single microbial cell thus representing clone of pure cultures. K R MICRO NOTES 32
  • 34. DENSITYCENTRIFUGATION • Repeated low-speed centrifugation (500 - 1000 rpm) is done to remove large amount of diverse micro organisms. • These results the algae to float at the upper phase in suspended material. K R MICRO NOTES 34
  • 35. SERIAL DILUTIONMETHOD • This method is commonly used to obtain pure cultures of those microorganisms in liquid media. • The inoculums is subjected to serial dilution in a sterile liquid medium and a large number of tubes of sterile liquid medium are inoculated with aliquots of each successive dilution. K R MICRO NOTES 35
  • 36. Isolation techniques K R MICRO NOTES 36
  • 37. MICROMANIPULATOR • It is a instrument which permits one to pick out a single cell from a mixed culture. • A series of hanging drops of a diluted culture are placed on a sterile cover slip by a micropipette. • Now a hanging drop is searched which contains only single microorganism cell. • This cell is now drawn into a micropipette by suction and then transferred to a large drop of sterile medium on another sterile cover slip. K R MICRO NOTES 37
  • 38. PHOTOBIOREACTOR • A photobioreactor is a bioreactor that utilizes a light source to cultivate phototrophic microorganisms • These organisms use photosynthesis to generate biomass from light and carbon dioxide and include plants, mosses, macro algae, microalgae, cyanobacteria and purple bacteria. • Within the artificial environment of a photobioreactor, specific conditions are carefully controlled for respective species. • Thus, a photobioreactor allows much higher growth rates and purity levels than anywhere in nature or habitats similar to nature. K R MICRO NOTES 38
  • 40. PHOTOBIOREACTOR • These reactors are made of stainless steel having a cylindrical shape with diameter between 20 and 60cm and height between 35 and 100cm. • On top they are covered by a flat steel lid, the lower part is covered by a double walled steel cylinder, in which water can be circulated to maintain constant temperature. • Most of the reactors are equipped with an internal illumination system. K R MICRO NOTES 40
  • 41. PHOTOBIOREACTOR • Fluorescence tubes are also present which can easily be removed without opening the reactor. • After inoculation the algal suspension is agitated by means of a stirrer. • Sterile air, enriched with Co2 is passed via the central tube into the blades through capillary holes. K R MICRO NOTES 41
  • 42. Harvesting • Harvesting the algae which is usually a highly energy intensive step can also be carried out rapidly and efficiently by the above stirrer. • When the algae have reached the stationary growth phase, both the aeration and the stirrer are turned off so that the algae settle on the bottom of the reactor. K R MICRO NOTES 42
  • 43. Harvesting • The stirrer is connected to a suction pump via its central tube and set into very slow rotation. • The thick algal suspension formed at the bottom of the reactor is harvested through suction slits. K R MICRO NOTES 43
  • 44. PHOTOBIOREACTOR • The reactor remains closed during this process. • The harvested slurry can be further concentrated by centrifugation. • Fresh medium can be added to the reactor via a sterile filtration device making continuo cultivation possible. K R MICRO NOTES 44
  • 45. K R MICRO NOTES 45