algal biotechnology mass cultivation of micro-algal species of ... · algae • primitive aquatic...

Algal Biotechnology

Mass cultivation of micro-algal species of commercial value: Spirulina, Dunaliella, Chlorella and others, Micro-algae for human and animal consumption; and waste-

water treatment.

Mitesh Shrestha

Algae

• Primitive aquatic plants of kingdom Plantae.

• They don't have plant parts like roots, stems, leaves or fruits but have filamentous or nonfilamentous thallus or body.

• Algal classification is based on type of photosynthetic pigment present.

• Three classes of algae are Green, Brown and Red algae.

• Chlorophyll a and βcarotenes are present in all three types but other photosynthesis pigments are different.

• Green algae contain chlorophyll b, and xanthophylls;

• Brown algae contain chlorophyll e and fucoxanthine;

• Red algae have phycocyanin and phycoerythrin as pigments.

Algae • Habitat of green algae (Spirogyra, Oedogonium) is fresh water or

terrestrial

• Brown (Sargassum, Laminaria) and red algae (Porphyra, Gelidium) are

marine forms.

• The reserved food material of green algae is starch;

• Mannitol and laminarin is present in brown algae;

• Red algae have Floridian and starch as stored food.

• Blue green algae (BGA) or cyanobacteria were excluded and placed in

kingdom Monera as they possess prokaryotic cellular organization and

functions.

Biotechnological importance of algae:

• Algae as renewable energy source; Chlorella, Dunaliella, Gracilaria and Sargassum produce fuels like diesel, gasoline, methane, butanol, ethanol and aviation fuel.

• They can grow on land or water (arid/saline/alkaline/marshy) unsuitable for crop cultivation.

• They scavenge green house gases and can be used for carbon dioxide mitigation.

• Algae are cheap source for waste water treatment and biogas production.

• Genetically engineered algae are used to enhance biofuel production and as source of protein and vitamin rich food and fodder.

• Algae are used as biofertilizer for crops as rich source of nitrogen, phosphorous, potassium, iodine, iron, calcium, silica and vitamins.

Biotechnological importance of algae:

• Algae have been recommended for pesticide and heavy metal bioremediation.

• Algae are used in formation of biosolar cells.

• Algae as food; Alaria, Laminaria, Sargassum, Porphyra is popular as food in Japan and Europe.

• Algal storage materials like starch, gelatin and lipids are used as gelling agents in jellies, icecreams, confectioneries and bacteriological media.

• Algae have therapeutic importance; Chlorellin from Chlorella is broad spectrum antibiotic.

• Algal pigments have antioxidant properties and therefore used in formulation of age proofing cosmetics.

Growth in laboratory

• In laboratory, algae are cultivated under aseptic conditions and controlled physical parameters like pH, temperature, light intensity, shaking of growth medium and incubation time.

• Algae being photoautotrophic, they are grown in broth or agar medium supplemented with micronutrients like magnesium, nitrate, calcium and iron.

• The incubation is always carried out in illuminated growth chamber for about 7 - 15 days till algal growth is visualized. Identification is carried out with the help of standard classification manual.

• Algal cultivation is cheap as compared to other economic crops.

• Algae do not require prepared and fertilized land for their growth.

• They can grow on marginal land like salinated or drought affected hard soils.

• They also grow in waste water effluents or sewage or even waste water from nuclear reactors.

• They can be grown in open ponds or bioreactor tanks or closed ponds or tanks covered inside shade nets.

• Algae are cultivated on large scale in photobioreactors. Reactors are plastic pumps containing nutrient water for algal growth. Carbon dioxide is supplied intermittently to enhance algal biomass.

• Algae when grown in closed system are protected from air borne microbial contamination, particularly fungal spores which can be pathogenic to algae.

Algal farming

Commercially important pathways

Micro-algae market potential

Micro-algae: Biochemical composition

Composition Chlorella Spirulina maxima

Haematococcus pluvialis

Aphanizomenon flos-aquae

Protein 61- 67.5% 67.5 48 62

Carbohydrates 10 - 15% 14.6 47 23

Lipids 12 -15% 7.5 15 3

Vitamins Upto 0.1%

Pigments

Minerals Around 2% Around 4%

Chlorella cultivation Why Chlorella?

• Can grow in both aquatic and terrestrial habitats • Model organism • High protein content (upto 70% dry wt basis) • Rich in minerals, vitamins • Can even produce lipids (TAG) under stress conditions • Used as human food and animal feed

Chlorella – mass cultivation Open culture system: Circular or raceway ponds. These can handle cell densities of less than 1 g/L.

• A modified system consisting of inclined thin layer system can sustain cell densities of as high as 35 g/L.

• These systems are cheap to build and operate, and are durable

• These systems have problems in managing culture temperature, improving light availability to the cells, checking water loss due to evaporation, controlling contamination. Furthermore, they are inferior to closed PBRs in terms of cell density and biomass productivity.

Chlorella – mass cultivation Closed Photo Bioreactors: These closed PBR can overcome the problems associated with open systems, but are costly to operate

• The most common design is the tubular design which consists of clear transparent tubes few centimeter in diameter.

• A large scale PBR installed in germany consisted of 500 km long tubes arranged in north south orientation.

• It was installed in a green house covering an area of 1.2 hectares. It had a capacity of 700 cm3 and had annual productivity of 100 tonnes of chlorella biomass.

Chlorella – mass cultivation • Another common design is flat design which are

arranged vertically or in an inclined position facing the sun.

• This system is superior to tubular system as it can handle high densities of cell biomass, cause , little dissolved oxygen buildup, create lower mechanical shear force, and need less capital and maintenance costs (Hu et al. 1996).

Chlorella – mass cultivation • Third option for mass culture of Chlorella is use of fermenters for

heterotrophic cell culture. These can be used for commercial production of chlorella.

• Heterotrophic fermentation offers some advantages, such as high cell density, high biomass yield, elimination of light requirement, and ease of control for monoculture (Chen, 1996).

• A cell density of up to 100 g L−1 with an average volumetric productivity of 13 g L−1 d−1 was obtained from the fermentation of C. Protothecoides (Yan et al., 2011).

Chlorella – mass cultivation • Due to its high capital and operating costs, however, fermentation of

Chlorella may be economically viable only for high-value specialty products but not for low-value, large-volume commodity products like biofuels.

• Lee et al. (1996) reported successful outdoor mixotrophic cultures of Chlorella in a 10-L tubular PBRs in the presence of sugars. The scaling up to 300 L, however, failed to maintain Chlorella monoculture, indicative of the critical challenge of microbial contamination in mixotrophic culture of Chlorella at scale.

Chlorella – Harvesting and drying • Various harvesting and dewatering approaches and techniques have been

applied to Chlorella cultures, including flocculation, flotation, filtration, gravity sedimentation, and centrifugation.

• Flocculation is a process of increasing the effective particle size for settling by cell aggregation.

• Flocculants with positive charges such as multivalent cations or cationic polymers like chitosan can ease the aggregation of Chlorella cells.

• Electroflocculation without addition of chemical coagulants Previously used for removal of microalgae from drinking water can also be used for harvesting Chlorella.

Chlorella – Harvesting and drying • Floatation is an air-assisted separation process in which algal

cells attach to air bubbles to form flocs that float on the water surface for harvesting. The commonly used floatation techniques are dissolved air floatation and dispersed air flotation.

• Membrane microfiltration and ultrafiltration are other techniques for harvesting of chlorella cells. Lee et al. (2012) demonstrated that a membrane filtration process yielded an algae cake of as high as 25.6% solids but associated with membrane fouling.

• Centrifugation is an accelerated sedimentation. Two basic types of large-scale centrifuges were reported for harvesting Chlorella: tubular and disk centrifuges (Lin, 2005). High capital and operating costs of centrifuges make this method affordable only for production of Chlorella-derived high-value products.

Chlorella – Harvesting and drying • Good harvesting and dewatering processes may produce Chlorella

biomass slurry of the solid content of 20–30%.

• A drying process may be required to further reduce the moisture content of algal pastes.

• Spray drying is the most common method for drying Chlorella, particularly for human food (Lin, 2005). The spray drying process can simultaneously rupture rigid cell walls of Chlorella cells, improving therefore the bioavailability of Chlorella biomass for human and animal consumption.

• Freeze-drying and drum drying may be available for drying Chlorella biomass. The harvest and drying processes may contribute 20–30% of the total cost of algal biomass

Chlorella – uses • Human food and animal feed

• Source of carotenoids

• Biofuel exploration (deprivation of Nitrogen and high light increases lipid production as high as 60%)

• CO2 biomitigation

• Bioremediation of waste water (removal of N and P, heavy metals, etc. from water using immobilized Chlorella cells)

• Recombinant protein production

Spirulina (Arthrospira) – mass cultivation

• Arthrospira is found in tropical and subtropical lakes with high alkalinity (up to 400 meq L−1) and high pH (11). This extreme environment essentially excludes the growth of other contaminant algae and is relatively easy to cultivated compared to other algae.

• The optimum temperature for the growth of Arthrospira (Spirulina) is 35–38C and the minimum temperature to sustain growth is 15 – 20C. Year round production problematic in most places


• Areas where production can take place year-round also happen to have the highest precipitation, which presents problems of dilution of the culture and affects productivity

• Dry and hot climates also result in high evaporation rates and thus freshwater replacements are needed.

• Desert climates offer a stable environment that facilitates a higher yield and consistency of biochemical composition.


Commercial production of Spirulina involves four steps: • growing the algae,

• harvesting the biomass,

• drying the biomass

• packaging the biomass


Growing the algae: • In Raceway ponds of 2000 to 5000 m2 and 30-40

cm deep. Turbulent mixing of ponds by means of paddle wheels at velocities of 5 to 60 cm S-1 increase productivity. Mixing helps avoid photoinhibition of cells at surface and death of cells at bottom.

• Culture medium used is Zarrouk (1966) mediium with various modifications.

• Strain selection and scaling up


Growing the algae: • Maintenance of pH above 9.5 to avoid

contamination. It is achieved by supplying CO2 gas to the medium. A pH value above 10.5 results in precipitation of CaCO3 and death of cells at the bottom.

• Replenishment of nutrients (to supplement loss of CaPO4 and CaCO3 and other nutrients)

• Control of contaminants (strains of Chlorella, Oocystis and S. Minor), zooplanktons

• Control of underwater light conditions (by paddling wheels)


Harvesting: • The biomass concentration in most outdoor ponds

of an Arthrospira culture is about 1 g L−1. • Harvesting usually done by filtration carried out for

several rounds to make a slurry with 5-20% solid matter.

• Drying is usally done by quick drying method using drum drying, solar drying, vacuum drying, sun drying, spray drying, etc. The water content has to be less than 7% in the final product.

• The final product is kept in sealed air tight packages to avoid loss of antioxidants.


Spirulina (Arthrospira) – Nutritional profile

Dunaliella

Dunaliella cells: Left stressed cells with β-carotene, right non-stressed green cells. Source: Microalgae- A market analysis, 2013

• D. salina, a green alga, is most salt tolerant eukaryote known.

• Can grow at salinity as high as 35% w/v NaCl.

• Cultivated for production of carotenoids, mainly β-carotene.

• Since it grows in brine of high salinity, CO2 in enrichment is essential for high yield. (the solubility of CO2 at 15C in 2 M NaCl is 1.02 g L−1, and at 40C it is only 0.53 g L−1 (Lazar et al., 1983)

Dunaliella

Dunaliella

• Mass cultivation is done in either large unmixed shallow (0.3 m deep) open ponds or in intensive raceway ponds

• The large shallow ponds are cheap to operate but have low biomass productivity (about 0.1 g dry weight L−1).

• However, in the extensive culture systems used in Australia, the high salinity and the high irradiances enhanced the carotenoid content of the algal cells.

Dunaliella- mass cultivation

• In Eilat, Israel and in India and China, D. salina culture is in 20 – 30 cm deep paddle wheel-driven raceway ponds (intensive culture) with individual ponds up to 3000 m2 in area.

• The extensive culture systems used in Australia are not possible because of cost of the land (as in Israel), or unsuitable weather conditions for part of the year (i.e., high rainfall in the monsoon season in India and very cold winters in Inner Mongolia and in Tianjin, China)


• Biomass productivity in raceway ponds is much higher than in the extensive open ponds because of better mixing.

• Ben- Amotz (1999) states that in Israel annual average carotenoid productivities of about 200 mg β-carotene m−2 d−1 can be achieved in 20-cm-deep raceway ponds, meaning that a 5-ha plant produces about 10 kg β-carotene d−1.

• In most of these plants the Dunaliella are harvested by centrifugation, which significantly increases production costs.


• A two-stage cultivation process for D. salina β-carotene production has been studied (Ben-Amotz, 1995).

• Here the alga is first grown in nutrient-rich medium for the rapid production of biomass, and then the algae are transferred to an N-deficient medium to enhance carotenogenesis.

• Two stage cultivation is possible where the intensive raceway cultivation process is used, but is not possible in the extensive large ponds used in Australia.


• The harvesting of D. salina is more difficult than for most other microalgae because of the small cell size, with neutrally or positively buoyant cells in high-specific-gravity, high viscosity, and corrosive brine at low solid concentrations between 0.1 and 0.5 g L−1 dry weight.

• Furthermore, the naked cells are very easily damaged, and this can lead to loss of β-carotene during harvesting due to oxidation.

• Harvesting Methods currently used in commercial production include flocculation and flotation and/or centrifugation.

• From the harvested cells pigments can be extracted by solvent extraction.


• Liu, J. and Hu, Q. 2013. Chlorella: Industrial Production of Cell Mass and Chemicals. In: Richmond A. And Hu, Q. (eds.) Handbook of Microalgal Culture: Applied Phycology and Biotechnology, Second Edition. John Wiley and Sons pp329-338.

• Belay, A. (2013). Biology and Industrial production of Arthorspira (Spirulina). In: Richmond A. And Hu, Q. (eds.) Handbook of Microalgal Culture: Applied Phycology and Biotechnology, Second Edition. John Wiley and Sons pp339-358.

• Borowitzka, M.A. 2013. Dunaliella: Biology, production,and Markets. In: Richmond A. And Hu, Q. (eds.) Handbook of Microalgal Culture: Applied Phycology and Biotechnology, Second Edition. John Wiley and Sons pp359-368.

References

algal biotechnology mass cultivation of micro-algal species of ... · algae • primitive aquatic...

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