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Downstream processing of microalgae for pigments, protein and carbohydrate

in industrial application: A review

Article  in  Food and Bioproducts Processing · February 2018

DOI: 10.1016/j.fbp.2018.02.002

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Accepted Manuscript

Title: Downstream processing of microalgae for pigments,

protein and carbohydrate in industrial application: A review

Author: Saumyakanti Khanra Madhumanti Mondal Gopinath

Halder O.N. Tiwari Kalyan Gayen Tridib Kumar Bhowmick

PII: S0960-3085(18)30010-5
DOI: https://doi.org/doi:10.1016/j.fbp.2018.02.002
Reference: FBP 935

To appear in: Food and Bioproducts Processing

Received date: 24-10-2017

Revised date: 1-2-2018
Accepted date: 5-2-2018

Please cite this article as: Khanra, S., Mondal, M., Halder, G., Tiwari, O.N., Gayen,
K., Bhowmick, T.K.,Downstream processing of microalgae for pigments, protein and
carbohydrate in industrial application: A review, Food and Bioproducts Processing
(2018), https://doi.org/10.1016/j.fbp.2018.02.002

This is a PDF file of an unedited manuscript that has been accepted for publication.
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 Downstream processing of microalgae for pigments, protein and

carbohydrate in industrial application: A review

Saumyakanti Khanraa, Madhumanti Mondal b, Gopinath Halder b, O N Tiwaric, Kalyan

Gayena,* and Tridib Kumar Bhowmickd, *

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a
Department of Chemical Engineering, NIT Agartala, West Tripura - 799046, India

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b
Department of Chemical Engineering, NIT Durgapur, West Bengal -713209, India
c

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ICAR-Indian Agricultural Research Institute (IARI), New Delhi-110012, India
d
Department of Bioengineering, NIT Agartala, West Tripura - 799046, India

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Corresponding author. Tel.: +91 8974727421(KG), Tel.: +91 8413061175 (TKB).
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Email: kgayen123@gmail.com, kalyan.chemical@nita.ac.in (KG); tbhowmick@gmail.com

(TKB),
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Fax: +91 3812346360

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Short title: Downstream of microalgal products

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Page 1 of 72
Contents
Abstract
1 Introduction
2 Products from microalgae
2.1 Pigments

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2.1.1 β-carotene

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2.1.2 Chlorophyll

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2.2 Microalgal Protein
2.3 Carbohydrate extracted from microalgal source for industrial application

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2.3.1 Agar
3.3.2 Carrageenan

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2.3.3 Alginate
2.3.4 Fucodian
2.4 Biopolymers
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3 Pre-treatment for extraction of microalgal based products
3.1 Drying
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3.2 Particulate Size Reduction

3.3 Cell Disruption
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4. Pigment extraction
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4.1 Chlorophyll
4.1.1 Organic solvent extraction
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4.1.2 Supercritical Fluid Extraction (SFE)

4.2 Carotenoids
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4.2.1 Extraction of carotenoid using subcritical dimethyl ether (DME)

4.2.2 Supercritical CO2
4.3 Extraction and purification of chlorophyll
5. Protein extraction
5.1 Organic solvent extraction
5.2 Sonication
5.3 Protein Purification

Page 2 of 72
6. Carbohydrate extraction
6.1 Alginate
6.1.1 Alkali extraction
6.2 Agar
6.3 Carrageenan

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6.3.1 Alcohol process

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6.3.2 Potassium chloride process
6.3.3 Drum drying process

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7. Current patents and patent applications related to the downstream processing of
microalgae

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8. Current scenario on global algal product market
9. Conclusion and future perspectives

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Acknowledgements
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Page 3 of 72
Abstract
Numbers of high valued and low volume products from microalgae are already in the market

and more number of products is yet to be launched. Downstream processing is the key steps

to maintain the quality of the products. Therefore, this review is focused on the downstream

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processing of the microalgae and cyanobacterial cells for commercial production of

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biomolecules such as pigments (chlorophyll and carotenoid), protein, carbohydrate (agar,

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carageenan, alginate, fucoidan) and biopolymers. It also covers the most recent preferred

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downstream processes for the industries for accomplishing the desired quality of the product

and recent important filed patents on downstream processing of microalgae. Further, this

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article highlights the recent investments made by the industries towards commercial

production of microalgal-based biofuels and bio-products worldwide.

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Keywords: Microalgae; Cyanobacteria; Downstream processing; Pigments; Carbohydrates;

Proteins
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Page 4 of 72
 1. Introduction

Microalgae (microalgae mean both microalgae and cyanobacteria) are considered as potential

source of high-value nutrients such as pigments, proteins, carbohydrates and lipid molecules

(Ghosh et al., 2016; Pignolet et al., 2013; Ursu et al., 2014). Industrial scale production of

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microalgae has evolved worldwide due to human consumption of microalgae as nutritional

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supplements (Spolaore et al., 2006). In the past, successful industrial scale production of

microalgae was made using green algal species Chlorella and cyanobacterial species

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Spirullina (Ciferri and Tiboni, 1985; Liu and Chen, 2014). Large production of microalgal

biomass is costly and required a high volume of seed culture to ensure purity of the cultures

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(Chisti, 2016; Das et al., 2015). However, research is still continuing on the sustainable,

economical production of microalgae biomass at large scale (Su et al., 2008). Harvesting
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processes of microalgal biomass at large scale, are also costly and energy intensive.

Harvesting processes include several techniques such as centrifugation, foam fractionation,

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chemical flocculation, electro-flocculation, membrane filtration, and ultrasonic separation etc

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(Zeng et al., 2016). Some other harvesting process like gravity sedimentation required low
p

energy, but economically non-viable in the industry due to very slow rate (Pittman et al.,
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2010). Selection of harvesting technique for the processing of microalgae is largely depend

on the type of strains, culture condition and the cell density in the harvesting medium (Singh
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et al., 2010). Therefore, finding an efficient harvesting process is still under research for the

industrial application for the production of primary and secondary metabolites from the

microalgae (Ghosh et al., 2017; Ummalyma et al., 2016; Vandamme et al., 2013; Wan et al.,

2015).

Apart from biomass, microalgae produces variety of pigments molecules like chlorophyll,

carotenoids and β-carotene that are used as colorants in cosmetic and food industry. Algae
5

Page 5 of 72
strains, Chlorella sp., Dunaliella sp., and Scenedesmus sp., and cyanobacterial strains

(Spirulina sp. and Nostoc sp.) are used as sources of fine chemicals and nutrient rich food

suppliments (Kay, 1991; Sanmartin et al., 2010; Seo et al., 2013). Further, pigments from the

microalgae are used in cosmetics industry as anti-aging cream, refreshing or regenerating

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care products for healing and repairing of damaged skin with nourishments (Tominaga et al.,

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2012; Zhu, 2015). For example, the microalgae, Haematococcus pluvialis, is known as the

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natural source of keto-carotenoid astaxanthin. Red pigment astaxanthin is the precursor

molecule of vitamin A and this pigment play important role in embryo development and cell

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reproduction in poultry and aquaculture firms (Goto et al., 2015; Lorenz and Cysewski,

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2000). Moreover, astaxanthin has superior antioxidant properties compared to those of β-

carotene, α-carotene, lutein, lycopene, canthaxanthin, and vitamin E and therefore, is

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becoming popular as a human dietary supplement (Guerin et al., 2003; Johnson and An,

1991). Consequently, a number of industries, such as Cyanotech, Seambiotic, Mera

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Pharmaceuticals and Fuji Chemical, are producer of microalgae biomass for value added
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products in cosmetics, nutritious feed and pharmaceuticals (Becker, 2007; Zhu, 2015).

Selection of suitable process for pigment extraction from the microalgae depend on the
p

several factors like biochemical features of pigments, choice of solvents for extraction,
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extraction yield, duration of extraction, reproducibility, denaturation and degradation of

molecules, cost and easy operation (Cuellar-Bermudez et al., 2014). Industry often use
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classical organic solvent extraction techniques like soaking, percolation, counter-current

extraction, pressurized liquid extraction, and soxhlet extraction (Sun et al., 2012). However,

these processes have certain disadvantages and limited for industrial application due to large

amounts of solvent requirement, environmental pollution, risk of health hazards and

denaturation of biomolecules (Peshin et al., 2002). Efficient extraction process of the

biomolecules from the microalgae is the growing demand for the industries. A number of
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Page 6 of 72
processes like ultra high pressure extraction, use of supercritical carbon dioxide for

extraction, microwave processing, combination of techniques such as soaking in liquid

nitrogen followed by buffer extraction, combination of solid-phase and supercritical fluid

extraction are currently being exploited and under research for further development to

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establish energy efficient, low cost extraction technique for the pigments (Abrahamsson et

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al., 2012; Biller et al., 2013; Klejdus et al., 2009; Lowrey et al., 2015).

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Microalgae also considered as reliable rich source of vegetable protein (Becker, 2007).

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Nutritional studies on different microalgae have demonstrated that microalgae produced high

amount and high quality proteins, which are the source of essential amino acids (Galland-

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Irmouli et al., 1999; Gutierrez-Salmean et al., 2015; Hosseini et al., 2013). Recently,

microalgae shows potential as an alternative expression host for recombinant protein

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production (Specht et al., 2010). Certain algae species like red algae (Rhodophyta) and

cyanobacterial strain produce a group of accessory photosynthetic pigment protein complexes

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for light harvesting purpose are called phycobiliproteins (Belford et al., 1983; Gantt and
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Conti, 1966). These proteins have high demand in pharmaceutical industries and specific
p

application in the biological field as fluorophore (Kuddus et al., 2013; Rijgersberg and
ce

Amesz, 1980; Yaakob et al., 2015). Conventional methods utilized for cell disruption of

microalgae before the extraction of the proteins includes bead milling, high pressure and high
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speed homogenization, ultrasonication, microwave treatment, pulsed electric field treatment,

pre-heat treatment, enzymatic treatment etc. (Demuez et al., 2015; Gerde et al., 2012;

Gunerken et al., 2015; Prabakaran and Ravindran, 2011). Protein extraction from microalgae

is done using aqueous, acidic, and alkaline methods, followed by centrifugation,

ultrafiltration, precipitation, or chromatography techniques for the recovery of the protein

molecules (Sari et al., 2015). Industrial scale extraction and purification of proteins from

Page 7 of 72
microalgae is not studied widely and scalable downstream processes of microalgae for

efficient extraction of proteins are still in very high demand. Diversity in microalgal species,

variation in the cell structure, variation in the intracellular protein content, release of protein

degrading (protease) enzymes from the cells are major obstructions for up-scaling of the

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protein extraction process. Some novel extraction techniques such as pulsed electric field,

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microwave-assisted extraction and ultrasound-assisted extraction are employed for successful

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extraction of proteins from microalgae.

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Manipulation in growth conditions can enrich microalgae with high amount of carbohydrate

or polysaccharide molecules. Major components of the cell wall of algae are cellulose and

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hemicellulose. Other than the cell wall, algae also store polysaccharide molecules also in the

cytoplasm. Marine algae produce complex sulfated cell wall polysaccharides, which have
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many biomedical applications (Domozych et al., 2012). Some cyanobacterial strains (e.g.

Nostoc sp., Spirulina sp., Porphyridium sp.) are surrounded by a matrix of polymeric
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substance mainly constituted by polysaccharides, which form a protective layer between the
te

cell and the immediate environment (Colica and Philippis, 2013). Biotechnological potential
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of the cyanobacterial extracellular polymeric matrix are attracting increasing attention to the
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pharmaceutical, bio plastic and food industries. Conventional extraction of extracellular

polymeric matrix from cyanobacterial strain Porphyridium sp. involves alcoholic

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precipitation followed by dialysis and membrane filtration (Patel et al., 2013). However,

research for the scalable operations to extract polysaccharides from microalgae is in urgent

need to facilitate the applications of microalgae for the industries. Novel extraction

technologies such as enzyme-assisted extraction, microwave assisted extraction, ultrasound

assisted extraction, supercritical fluid extraction, and pressurized liquid extraction are

currently being applied for the extraction of bioactive molecules from microalgae (Kadam et

Page 8 of 72
al., 2013). These extraction technologies are attracting interest from the industries because of

its advantages (higher yield, reduced treatment time, and lower cost) compared to the

conventional solvent extraction techniques.

Recent studies had shown that microalgal biofuel is still not profusely producing at

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commercial level despite its high potential due to high production cost (Gendy and El-

Temtamy, 2013). In an elongation of previous statement, several molecules extracted from

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microalgae (e.g. astaxanthin, β-carotene, omega-3-fatty acids etc.) have high demand in the

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market and there are opportunities for additional new products like nutraceuticals,

pharmaceuticals (e.g. lutein, zeaxanthin), biopolymers and bio plastics, which can help the

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microalgal products based industry to flourish. In addition to its irreplaceable value as a

substitute of fossil fuel, global market for this high value algae product (cosmetics,
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nutraceuticals and pharmaceuticals) is worth around US$ 2 billion (www.oilgae.com, 2016).

Technological development of efficient downstream processing of microalgal products is one

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of the key factors that determine the economic viability of algae based products. In this
te

direction, a number of review articles are reported based on lipid derived biodiesel production
p

(Roux et al., 2017; Scott et al., 2010). However, limited articles focused on the downstream
ce

processing of microalgal products based carbohydrate, protein and pigments. Hence, this

review article focuses on: i) microalgae based products (based on carbohydrate, protein and
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pigments) at commercial scale with market demand and leading industry involved; ii)

detailed technological advances for downstream processing of microalgal products; iii)

extensive search for patented invention have been reported in relation to product specific

extraction; iv) current scenario on global algal biomass market with future perspectives.

2. Products from microalgae

Page 9 of 72
In general, products from microalgae are classified as pigments (e.g. β-carotene, chlorophyll),

protein, carbohydrate (such as agar, carrageenan, alginate, fucodian), as nutritional

supplements and lipid (e.g. biodiesel, polyunsaturated fatty acids). Recently, biopolymers and

bio plastics are also being produced from microalgae. For example, cyanobacterial strains

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like Spirulina and Synechocystis produce poly-3-hydroxybutyrate (P3HB), which can be used

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as a source of production of biodegradable plastics (www.oilgae.com, 2016). Microalgal

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based products with sources molecular structure and application are summarized in Table 1

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and discussed in the subsequent subsections.

2.1 Pigments

2.1.1 β-carotene

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Carotenoids are lipid soluble natural pigments extracted from plants, algae, and

photosynthetic bacteria and these pigments performed as an important component in

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photosynthesis process (http://www.oilgae.com/non_fuel_products/betacarotene.html, 2015).

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β-carotene belongs to the group of carotenoid pigments and this carotenoid molecule was first

commercialized (http://www.oilgae.com/non_fuel_products/betacarotene.html, 2015). β-

p

carotene is considered as the precursor molecule for vitamin A biosynthesis in the human
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body. Production of β-carotene from halo tolerant Dunaliella salina was preferred, due to the

highest carotenoids content (~ 10% of dry biomass) among all other species (Prieto et al.,
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2010). In the chloroplast, cis and trans isomers of β-carotene is present and the ratio of 9-cis

to all trans isomers is found high in Dunaliella salina (Borowitzka and Borowitzka, 1989).

Dunaliella salina, is considered as a rich source of β-carotene since long past and first pilot

plant of Dunaliella salina cultivation was started in USSR and in 1960 (Oren, 2005). .

Thereafter, commercial production of Dunaliella for β-carotene has been flourished

worldwide in different countries like Australia, Israel, USA, India and China (Garcia-
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Page 10 of 72
Gonzalez et al., 2005; Kleinegris et al., 2011; Leon et al., 2003). Currently, Australia is the

major producer of Dunaliella and worldwide annual production of Dunaliella is estimated at

about 1,200 ton.

The carotenoid world market value was estimated $1.5 billion in 2014 and predicted that it

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could be reached to $1.8 billion in 2019 with a compound annual growth rate (CAGR) of 3.9

% (https://www.reportbuyer.com/product/96628/the-global-market-for-carotenoids.html,

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2015). World market for cyanobacterial based pigments is growing rapidly and is expected to

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meet £1 billion pound mark by 2019 (http://www.synthsys.ed.ac.uk/news/team-create-

molecular-toolbox-boost-cyanobacterial-pigment-production, 2016).

2.1.2 Chlorophyll

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Chlorophyll is the green coloured pigment present in plants, algae and cyanobacteria and it

has important role in photosynthesis. Green microalgae Chlorella sp. is popular as a primary
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source of chlorophyll production and called ‘Emerald food’ because of its high chlorophyll
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content (Bewicke and Potter, 2009). The chlorophyll content in Chlorella is about 7% of the

biomass which is five times than the chlorophyll content of Sprirulina

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(http://www.oilgae.com/non_fuel_products/chlorophyll.html, 2016). A Taiwan based

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industry (Weiwang Company) is known for the first commercial production of Chlorella at

different commercial scales (Bewicke and Potter, 2009). In this Industry, Chlorella cells were
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grown in large translucent plastic tubes for effective use of the natural sunlight during the

growth of the cells (Bewicke and Potter, 2009).

2.2 Microalgal Protein

Algae have already been utilized as food source for centuries for its high protein value. In

1960s, Japan was the first country to commercially produce Chlorella sp. for human

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Page 11 of 72
consumption (Vigani et al., 2015). The protein content from different microalgae species and

cyanobacteria varies from ~ 6-70% of its dry biomass (Becker, 2007). Out of many options,

still now, only few algae strains like Chlorella sp., Scenedesmus sp. and cyanobacterial

strains like Spirulina sp. and Athrospira sp. have been selected for large scale production

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(Becker, 2007). USA based company is selling AlgaVia®, a protein rich whole alga by

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claiming that it is vegan in nature, gluten and allergens free and also containing full of amino

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acids and dietary fibre. According to their product claim, it can be used as beverage, snacks,

cereals and in bakery (http://algavia.com/ingredients/proteins/, 2016). Consumption of

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alternative protein is growing rapidly and expected that extracted protein from other sources

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such as insects, algae, and synthetic biology sources would cover up to 50% market of the

total alternative protein by 2054 (http://www.luxresearchinc.com/news-and-events/press-

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releases/read/alternative-proteins-claim-third-market-2054, 2016).

2.3 Carbohydrate extracted from microalgal source for industrial application

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2.3.1 Agar
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Agar is a complex mixture of polysaccharide molecules, which are found as cell wall
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component of marine red algae (Gracilaria gracilis, Gracilaria dura, Pterocladia Sp.) Table
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1) (Marinho-Soriano, 2001). Agar molecules provide flexible structure in algae, which help

to survive the organism from stresses of wave motion in the sea water. (Craigie, 1990). Agar
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from marine algae is largely consisting with two sugar molecules, namely agarose and

agaropectin. Agarose is the gelling part of the agar molecules, made of alternate repeating

unit of two sugar molecule (β-1,3-linked- D-galactose and α-1,4-linked 3,6-anhydro-L-

galactose) (McCandless, 1981). Agaropectin is also made of di-saccharide molecules with a

few hydroxyl groups of 3,6-anhydro-α-l-galactose residues substituted with sulfoxy, methoxy

residues (Hahn et al., 2011). Size of agarose molecule is larger (100KDa) compared to the
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Page 12 of 72
agaropectin (20KDa) molecule. Whereas, sulphate content (0.15%) in agarose is much lower

than the agaropectin (5-8%) (Armisen and Galatas, 1987). This gelatinous substance is

therefore used in food industry especially in bakery for its large water retaining capacity and

as a thickening agent. In Japan, agar is chiefly used as an ingredient in deserts throughout, but

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now the most important worldwide use of agar is as a gelatin-like medium for growing

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organisms in scientific and medical studies

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(http://www.oilgae.com/non_fuel_products/agar.html, 2015).

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Two algae species namely Gracilaria and Gelidium are popular resources for the agar

extraction at industrial scale. Agar About 9600 tons of agar (US$ 173 million) were

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produced worldwide in 2009 increased from 7500 tons from 1999 which is about 3% growth

per year and therefore, it is expected the production of agar will be about 12,000 tons by 2019
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(Bixler and Porse, 2010). Companies that commercially producing agar are Agar del Pacifico

S.A., Chile, Industrias Roko S.A., Spain, K. Tanaka Corp., Japan, Coast Biologicals Ltd.,
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New Zealand, Acroyali Holdings Qingdao Co. Ltd., China, and Iberagar S.A., Portugal
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(Hallmann, 2007). Manuka group from New Zealand has been producing various algal agar
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products in package size ranging from 1 kg to 1 ton. Their products include Standard Grade
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Agar, Bacteriological Agar, PTC (Plant Tissue Culture) Agar, LVPTC (Low Vitrification

Plant Tissue Culture) Agar and Noble/High Purity Agar, which are produced from red
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seaweed Pterocladia lucida (http://www.nzmanukagroup.com/group/nzm-seaweeds/, 2016).

3.3.2 Carrageenan

Carrageenan is a group of natural, hydrophilic polysaccharides, isolated from rodophyta such

as Gigartina sp., Chrondrus sp. and Euchema sp. (Table1). Carrageenan molecule composed

of sulphated galactans and has both ability of gel formation and viscosity increasing

properties. In addition to thermo-reversible gel formation, this compound is reactive at high

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Page 13 of 72
temperatures under acidic conditions (Hernández-Carmona et al., 2013). Carrageenan is the

cell wall hydrocolloid (polymer of carbohydrate and protein) molecules and is extracted by

neutral or alkaline water under pressure at elevated temperature (100 -140 °C)

(http://www.oilgae.com/non_fuel_products/carrageenan.html, 2016; Wüstenberg, 2014).

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Extraction efficiency of carrageen a molecules from the cell wall of microalgae is controlled

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by applying optimum pH, time and temperature. Carrageenans are used in food and

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pharmaceutical industries as water gel applications, such as fruit gels, fruit juices, gummy

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candies and marmalades etc. (Bixler and Porse, 2010).

A number of red algal strains (e.g., Chondrussp., Gigartina sp. Eucheuma sp., Eucheuma sp.,

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Hypnea sp., and Furcellaran sp.) are used for the production of carrageenan

(http://www.oilgae.com/non_fuel_products/carrageenan.html, 2016). Carrageenan fractions

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are known as the kappa (κ), lamda (λ), and iota (ι) carrageenans, which differ in number and

position of sulphate ester groups and in their content of 3, 6-anhydrogalactose units

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(Wüstenberg, 2014). Kappa carrageenan are made from farmed Kappaphycus alvarezii and
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iota carrageenan farmed from Eucheuma denticulatum

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(http://www.oilgae.com/non_fuel_products/carrageenan.html, 2016). Companies like

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Ingredients Solutions Inc., USA, Ceamsa, Spain, Ina Food Industry Co. Ltd., Japan, FMC

Biopolymer, USA, and CP Kelco ApS, Denmark are presently producing carrageenans at
Ac

commercial level (Hallmann, 2007). Ingredients Solutions Inc., USA is one of world's largest

individual carrageenan producing more than 30 different types of carrageenan product line

for food application like meat, dairy, desserts and beverages with a packaging of 50 lb

(http://www.isi.us.com/carrageenan/, 2016).

2.3.3 Alginate

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Page 14 of 72
Alginates are structural phyco-colloid and used in food industries (as thickening agent),

cosmetics (as thickener and moisture retainer), and pharmaceutical (as alginic acid) industries

(Table 1). Alginate consists of two monomeric unit (α-L-guluronic acid and β-D-mannuronic

acid) at different molecular ratio. In dairy food industry, alginate provides uniformity and an

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proper appearance to the products due to its moisture retention property. Sodium alginate also

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used as ink paste thickeners for textile prints. In medicine, alginates are used as pill

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disintegrators due to its biodegradable properties (Hernández-Carmona et al., 2013). Several

species of large brown algae (e.g., Macrocystis pyrifera, Ascophyllum nodosum, Laminaria

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hyperborean, Laminaria japonica, Lessonia flavicans), large grey weeds (Lessonia

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nigrescens) and kelp species (e.g., Ecklonia maxima, Durvillea antarctica, Durvillea

potatorum), etc. are now used for alginate production

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(http://www.oilgae.com/non_fuel_products/alginate.html, 2015). Leading alginate producing

global industries are: FMC BioPolymer, previously Kelco and Pronova (Norway); Cargill,
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previously Degussa, and Danisco (France); Kimica and Chemifa Food (Japan), Bright Moon
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(China), and Kimica (Chile). Global production of alginate is 30,000 ton/ year with a gross

market value about $339 million /year (Rhein-Knudsen et al., 2015).

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2.3.4 Fucodian

Fucoidan is another polysaccharide macromolecule, which made of monomeric unit of L-

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fucose sugar and molecular weights are within the range of 13-950 kDa (Table 1). Fucoidans

is associated as a cell wall component of brown algae (Phaeophyceae sp.) and the yield of

fucodian depends on seasonal variation, type of algae species and the sea depth where they

grow. Fucoidan after combining with protein and alginic acid molecules, provide structural

toughness and flexibility in the cell walls of algae by forming amorphous matrix (Fu and

Kim, 2010; Hahn et al., 2011).

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Page 15 of 72
A number of bioactivities (anticoagulant, antitumor, antivirus and antioxidant) of fucoidans

molecules make them attractive for applications in pharmaceutical field (Li et al., 2008).

Research had shown that fucoidans molecules are able to influence anticoagulant property of

blood by enhancing the heparin cofactor II (Hahn et al., 2011; Hoek et al., 1995). It also

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exhibit antitumor property by inhibiting proliferation of the cells and inducing apoptosis via

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caspase activation and regulating cell signal kinase pathways (Hahn et al., 2011; Hoek et al.,

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1995). Further, research has shown that proliferation of virus is inhibited by fucoidans

molecules by slowing penetration of virus into the host cells results. Antiviral property of

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fucoidan molecule provide a new strategy for combating against human immunodeficiency

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virus ( HIV ) and herpes simplex virus type 1 (HSV-1) (Hahn et al., 2011).

Market value of all hydrocolloid molecules (e.g. Agar, Carrageenan, Alginate, Fucodian) are
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increasing rapidly and estimated growth would be continued at a compound annual growth

rate (CAGR) of 5.80% till 2020

d

(http://www.marketsandmarkets.com/PressReleases/hydrocolloid.asp, 2016). Total global

te

market value of all hydrocolloid molecules is expected to raise at $7.56 billion by 2020
p

(http://www.marketsandmarkets.com/PressReleases/hydrocolloid.asp, 2016).
ce

2.4 Biopolymers

Algae are considered as the source of biopolymers and biopolymer production from algae is
Ac

counted after biofuel generation, to ensure alternative sources of revenues along with

biofuels. Cyanobacterial strains (e.g., Nostoc sp, Phormidium sp., Chlamydomonas sp.), and

algae strains (e.g. Chlorella sp., Ulva lactuca, Scenedesmussp., Stichiciccis sp., Anabaena

sp.,) are used in manufacturing biopolymers. Different type of plastics which are made from

the algae feedstock includes hybrid plastics, cellulose based plastics, poly-lactic acid and bio-

polyethylene and it has been summarised in Table 2 (Hempel et al., 2011a;

16

Page 16 of 72
http://www.oilgae.com/non_fuel_products/biopolymers.html, 2016). Although, bio plastic

production from algae is technically feasible, economic feasibility of the production process

is still under investigation. Biopolymers are produced as by-products of the bio-fuel

production from algae. A number of industries such as Petro Sun, Dow Chemicals,

t
Cereplast etc. have started research for algae based biopolymer synthesis (Niaounakis,

ip
2015). Biopolymer namely poly-hydroxyalkanoates (PHA) have the fastest growth with a

cr
compound annual growth rate (CAGR) of 27.7% to 2018 while packaging has the biggest

utilization with anticipated worth of $1.72 billion by 2018

us
(http://www.marketsandmarkets.com/PressReleases/biopolymers-bioplastics.asp, 2016). A

an
few industries [e.g., Braskem S.A. (Brazil), Nature Works LLC (U.S), and Indorama

Ventures Public Co. Ltd. (Thailand)] are already producing biopolymer PHA
M
(http://www.marketsandmarkets.com/PressReleases/biopolymers-bioplastics.asp, 2016).

Now-a-days, synthetic plastic becomes the concern in various solid waste management and
d

therefore, bio-degradable polymer becoming important in this regard. Current, research focus
te

is observed on the synthesis of the biodegradable plastic from the natural sources. Some

biodegradable plastic materials under development such as poly-hydroxyalkonates (PHAs),

p

poly-lactides, aliphatic polyesters, polysaccharides, and co-polymers

ce

(http://www.oilgae.com/non_fuel_products/biopolymers.html, 2016). For example, Soley

Biotechnology institute have already been commercially producing various bio plastics such
Ac

as compostable bio plastic, and four different type of biodegradable bio plastic from Spirulina

sp. (http://www.soleybio.com/products/60-bioplastic.html, 2016). Bayramoglu et al., have

shown that in a study with microalgal species Spirulina (Arthrospira platensis) hybrid

polymer made with polyethyleneimine and amidoxime modified Spirulina can be used for

effective removal of uranium ion from aqueous solution (Bayramoglu et al., 2015). In another

study, hybrid polymer matrix was made with polyvinyl alcohol-sodium alginate and red algae
17

Page 17 of 72
(Jania rubens) and used as polymer material for efficient removal of lead from aqueous

solutions (Kadimpati, 2016). Green filamentous algae Cladophora sp. is the rich source of

cellulose. The robustness of Cladophora sp. derived cellulose make this material interesting

application possibilities in foods, as drug delivery agent, as filler in isocyanate-based foams

t
and reinforcements material in biodegradable plastics and construction materials (Mihranyan,

ip
2010). Hirayama et al., have shown isolated microalgae strain Nannochlorum sp. is suitable

cr
for the production of D-lactic acid and also can be effective for greenhouse gas CO2 fixation

(Hirayama and Ueda, 2004). In another study, Nguyen et al., have shown production of L-

us
Lactic acid from bacterial fermentation (Lactobacillus paracasei) by using freshwater

an
microalga (Hydrodictyon reticulum) as a substrate (Nguyen et al., 2012). Application of

metabolic engineering and synthetic biology on cyanobacteria (Synechocystis sp. and

M
Synechococcus sp.) has shown ethylene can be made directly by transferring the ethylene-

forming enzyme gene from Pseudomonas syringae into those cyanobacterial strains (Veetil et
d

al., 2017).
p te

3. Pre-treatment for extraction of microalgal based products

ce

Cultivation and harvesting are the primary steps for the production of microalgal products.

For large scale production, cultivation are mainly performed in two class of systems namely
Ac

open air system (e.g. shallow big ponds, tanks, circular ponds, and raceway ponds etc.) and

photobioreactors (PBR's) system (e.g. flat-plate, tubular, bubble spurge vertical column, and

airlift PBR) (Brennan and Owende, 2010). Cultivation of microalgae in raceway pond is

better scalable process for commercial production compare to the other mode of cultivation.

On the other hand, conventional harvesting methods include filtration, flotation

centrifugation, and sedimentation. Generally, flocculation is the recommended as a pre-

18

Page 18 of 72
treatment owing to its energy efficiency or in other words, low energy requirement. However,

no universal cultivation and harvesting method is available that can be applied for all

microalgae species. The efficient cultivation and harvesting method is selected based on the

nature of the microalgae and the final product/products. After cultivation and harvesting,

t
drying, particle size reduction, cell disruptions are most commonly used pre-treatment

ip
methods and are selected based on extraction procedure and end product. Pre-treatment is

cr
generally done to rupture the cell wall of algae, which allows increased efficiency of

extraction of biomolecules as well as lipid. Fig 1 portrays the schematic of the key steps

us
involved (cultivation, harvesting, pre-treatment, extraction and purification) with the

an
industrial process related with microalgal bio molecule production. Drying, particle size

reduction and cell disruption are different pre-treatment steps involve before extraction steps,
M
are discussed in subsequent sections. Other available methods for cell disruption are freeze-

thaw, chemical lysis, enzymatic treatment, high pressure homogenization (French press),
d

bead-milling/ball milling etc. (Gunerken et al., 2015; Masarin et al., 2016; Slocombe et al.,
te

2012; Verdel et al., 2000; Viswanathan et al., 2012; Zhang et al., 2004).
p

3.1 Drying
ce

In general, algae are dried and grounded to fine powder before extraction process. Disruption

of the cell wall helps to enhance the efficiency of microalgal extraction. To remove the
Ac

moisture from sample, generally two types of drying are being used over the years: thermal

drying and freeze-drying. Wei et al. have extracted arsenosugars from seaweed red algae

(Porphyra sp.), which was pre-treated in oven drier at 50 °C for 18 h and then grounded in a

stainless steel mill. The final extraction efficiency was achieved by following this process

was around 90-96% (Wei et al., 2003). For thermal drying, wide range of optimum

19

Page 19 of 72
temperature (room temperature to 60 °C) and time (18 - 48 h) are reported. But generally, 60

°C temperature and overnight duration is used (Rubio et al., 2010).

For freeze-drying, the drying temperatures are within -50 to - 80 °C with time duration

around 24 - 48 h. The freezing phase has to maintain critically to avoid the spoiling of the

t
ip
product (https://en.wikipedia.org/wiki/Freeze-drying, 2015). Freeze-drying is used instead of

thermal drying when the final product is a living system or for thermo labile product. In

cr
freeze-drying, structure of the biomolecules remains intact, that’s why the biomolecules can

us
absorb fluid again after the drying process and also has shorter preparation. Freeze drying

operation is high energy consuming, longer time taking and more costly compared to the

an
thermal drying process (http://www.powerofhorsemilk.com/what-is-freeze-drying/the-

difference-between-freeze-drying-and-spray-drying/, 2016). Freeze-dryer can be classified as

M
manifold freeze-dryer, rotary freeze-dryer and tray style freeze-dryer. Tray style freeze-

dryers are typically larger than the manifold dryers and can dry product in bulk while
d

manifold freeze-dryer is usually used in laboratory settings. On the other hand, rotary freeze-
te

dryer achieve a uniform drying due to its cylindrical reservoir, which rotates during drying.
p

Post harvesting stability of astaxanthin molecule in algal species (Haematococcus pluvialis)

ce

was improved with freeze drying (-20 °C) process which led to 41% higher astaxanthin

recovery compared to commonly-used spray drying (37 °C) process (Ahmed et al., 2015).
Ac

Ratty estimated that the energy consumption of industrial freeze drying for biomass would

require 38.88 Wh/g of algal biomass (Ratti, 2001). Freeze-drying has disadvantage for large-

scale operation as scaling up the process is costly.

3.2 Particulate Size Reduction

After drying, microalgal biomass becomes powder (or agglomeration) that can be converted

into different size ranges. This size reduction before extraction process helps to increase
20

Page 20 of 72
extraction efficiency by increasing the the interfacial surface area available for biomass-

solvent contact. However, extremely small size of the microalgal biomass may provide

adverse effects such as higher tendency of lipid re-adsorption, fluid channelling effects (only

for super critical carbon dioxide extraction) in the extraction vessel (Halim et al., 2012).

t
ip
Another method for reducing particle size is cryo-milling. It involves ball milling in presence

of liquid nitrogen. In this method, cell biomass frozen at very low temperatures (-196 °C) will

cr
crack easily under low-impact force. The liquid nitrogen used in the cryo-milling process will

us
evaporate and will not affect the extraction of molecules. In addition, cryo-milling operation

at low temperature would ensure the stability of the molecules by preventing oxidation of

an
biomolecules, thus improving the quality. A comparative study of different grinding

processes (ultrasonication, bead milling, enzymatic lysis and microwave treatment) on

M
microalgae (Chlorella vulgaris) was done by Zheng et al., and shown that grinding in liquid

nitrogen was the most effective method in terms of disruption efficiency and time (Zheng et
d

al., 2011). Owing to its cryogenic nature, intense grinding can reduce original powder to
te

nanoscale level in comparatively short time span. A patent had been granted to Jaouad
p

Fichtali and Anand Sundararajan on the novel method for preparing biomass
ce

(Crypthecodinium cohnii) for extraction. Report describes cryogenic milling of dried

Crypthecodinium cohnii biomass and subsequent extraction offer oil recovery around 47 –
Ac

92% (Fichtali and Sundararajan, 2006).

3.3 Cell Disruption

Cell disruption methods can be performed in mechanical or non-mechanical modes.

Mechanical methods include bead mill, press, high-pressure homogenization, ultrasonication,

autoclave, lyophilization, and microwave. On the other hand, non-mechanical methods are

the use of acids, alkalis, enzymes, or osmotic shocks to break the microalgal cells. Bead mill,
21

Page 21 of 72
high-pressure homogenization, and ultrasonication are three of the most widely used methods

for laboratory-scale microalgal cell disruption (Halim et al., 2012). Bead milling is the

simplest, fast and reliable method but its energy consumption varies with different microalgal

species. A comparative analysis between the current methods used for microalgae pre-

t
treatment was done by Hattab and Ghaly, in order to determine the most economically

ip
efficient method for large scale operation (Hattab and Ghaly, 2015). A number of studies

cr
with different microalgae strains (Nannochloropsis sp., Dunaliella sp, Chlorella sp.,

Chlorella sp and Scenedesmus sp.) were compared based on the cell wall disruption

us
efficiency, operational cost, toxicity, suitability for large-scale use, time, reusability and

an
maintenance. Analysis had shown that cell wall coatings of the diverse microalgal species

play a significant role in the disruption processes. Mechanical pre-treatment methods

M
(sonication, steam explosion and microwave radiation) were found effective for large-scale

operations. Other mechanical pre-treatment methods described in the report as unsuitable for
d

large scale operations due to high operational costs, lengthy treatment times, high
te

maintenance costs and the scale up difficulty. Thermal treatment methods (freeze-drying and

autoclave) and enzymatic pre-treatment were reported as inappropriate for microalgae

p

because of the high costs, scale up difficulty and long processing or treatment times
ce

associated with the processes. In the same report, it was mentioned that steam explosion pre-

treatment was species specific for microalgae and suitable for large scale operation due to
Ac

easy cell wall disruption, high intracellular molecule release, relatively low operational cost

and environmentally friendly.

Hattab and Ghaly, had mentioned in their analytical study, that energy consumption in bead

milling operation can vary (35-810 Wh/kg of biomass) due to cell wall structure difference

among the microalgal species and operating conditions (Hattab and Ghaly, 2015). Energy

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Page 22 of 72
consumption for ultrasonic pre-treatment also can be varied (1.7 - 119 Wh/g of biomass) for

different species (Hattab and Ghaly, 2015). In another study, Guldhe et al., reported that

energy consumption for Scenedesmus sp. was 119 Wh/g of biomass (Guldhe et al., 2014).

Whereas, energy consumption for Nannochloropsis sp. was 16.66 Wh/g of biomass as

t
reported in a different study by Adam et al. (Adam et al., 2012). Ultrasound assisted

ip
extraction of biomolecules from microalgae can be operated at low temperature to limit the

cr
thermal denaturation and the process is more economical than freeze drying method as

mentioned in two different reports by Chemat et al. and Wang et al. (Chemat et al., 2011;

us
Wang and Weller, 2006).

an
Another form of cell disruption is steam explosion. Steam explosion disrupts the cell so that

intracellular components can be easily removed. Its works by exposing the biomass at high
M
temperature (ranging from 160 °C to 260 °C) and vapour pressure (ranging from 1.03 to 3.45

MPa) which then brought to ambient temperature by depressurization which leads to the
d

disruption of the cell. In order to increase the lipid extraction efficiency, steam explosion
te

should be performed at a lower temperature (Hattab and Ghaly, 2015). Lorente et al. had
p

studied different pre-treatment methods (autoclaving, ultrasound, microwave and steam

ce

explosion) for evaluation of the most efficient method for the extraction of lipids from three

microalgal strains (Nannochloropsis gaditana, Chlorella sorokiniana and Phaeodactylum

Ac

tricornutum). Results showed that lipid extraction efficiency was higher in steam explosion

pre-treatment method compared to autoclave, ultrasound and microwave technique (Lorente

et al., 2015). However, steam explosion pre-treatment method is relatively new and currently

under investigation in laboratory scale and mainly used in biogas production (Pandey et al.,

2014).

4. Pigment extraction

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Page 23 of 72
The brief outline of the general pigment extraction processes like organic solvent extraction,

super critical fluid extraction, is represented in the Table 3.

4.1 Chlorophyll

t
4.1.1 Organic solvent extraction

ip
Organic solvents are used in this method, which penetrate the cell membrane to dissolve

cr
lipoproteins of chloroplast along with the lipids into the extract phase. Organic solvents such

us
as diethyl formamide, methanol, ethanol and acetone, are used to extract the chlorophyll.

Before extraction, pretreatment (e.g., homogenization, grinding, sonication or ultrasound)

an
enhances the effectiveness of chlorophyll extraction as it disrupts cells (Hosikian et al.,

2010b). Apart from cell disruption, other parameters like nature of organic solvent used,
M
residence time of extraction process, number of extraction cycles and storage duration of

microalgae, play important role in extraction efficiency (Hosikian et al., 2010a). In a study by
d

Sartory and Grobbelaar, on algae (Selenastrum capricornuturn), had shown alcoholic

te

solvents (methanol and ethanol) were better compared to the other solvents (acetone and

acetone with DMSO) for extraction of chlorophyll a (Sartory and Grobbelaar, 1984). In the
p

same study, it had been shown that complete isolation of chlorophyll a was achieved by
ce

boiling of algal biomass in methanol or 95% acetone for 3 to 5 minutes prior to 24-hour

extraction process(Sartory and Grobbelaar, 1984). Schumann et al., had shown the efficient
Ac

extraction of chlorophyll a can be made from two microalgae strains (Stichococcus sp. and

Chlorella sp.) using dimethyl formamide as a solvent assisted by mechanical homogenisation

treatment (Schumann et al., 2005). Chlorophyll a extracted in the stationary growth phase

was significantly higher compared to the extracted chlorophyll in the logarithmic phase from

same species (Schumann et al., 2005).

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Page 24 of 72
4.1.2 Supercritical Fluid Extraction (SFE)

Organic solvent used for lipophilic extraction of biomolecules from the algae, has

environmental concerns. Therefore, efforts are being made to look for new sustainable

process, which does not include environmentally toxic or harmful solvent. Supercritical fluid

t
ip
extraction is a method where use of carcinogenic solvents can be omitted and high purity of

the extract and less processing steps are the pros’ of this process.

cr
A substance has density of liquid state and viscosity of gaseous state at critical conditions.

us
The supercritical solvent has more affinity to the molecules, which have polar or non-polar

constituents and less affinity with high molecular weights. The advantage of this process is

an
the easy removal of solvent molecules from the extract by increasing the operational pressure.

CO2 is preferred choice as a fluid for SFE as its pretty cheap, readily available, inert and non-
M
flammable (Hosikian et al., 2010a). Chlorophyll being primarily used in food and

pharmaceutical industry, so there are rigorous regulation about its quality and hence by using
d

supercritical CO2 extraction a solvent free pure extract can be easily achievable. Upon
te

decompression, the supercritical CO2 is removed from the extract as it evaporates to the
p

ambiance in its gaseous state (Herrero et al., 2006). Chlorophyll extraction from microalgae
ce

using supercritical CO2 largely depends on the temperature and operating pressure. Macias-

Sanchez et al. had performed a number of studies on chlorophyll extraction using

Ac

supercritical CO2 from microalgae Synechococcus sp.. Results showed that optimum

extraction conditions were 60 °C and 500 bar for Synechococcus sp., which able to extract

0.715 µg of chlorophyll a/mg dry weight of microalgae (Macías-Sánchez et al., 2007).

4.2 Carotenoids

4.2.1 Extraction of carotenoid using subcritical dimethyl ether (DME)

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Page 25 of 72
Dimethyl ether (DME) is characterized as low normal boiling point (−24.8 °C) and have

relative permittivity of 1.08 (gaseous state) and 5.34 (liquid states) at 30.5 °C (Wu et al.,

2011). DME in liquid state has strong affinity for lipid molecules and partially miscible with

water. European Food Safety Authority (EFSA), US Food and Drug Administration (FDA)

t
and Food Standards Australia New Zealand, has approved DME as a safe solvent for

ip
extraction for the use in the production of food and food ingredients(Goto et al., 2015).

cr
An apparatus with semi continuous flow is preferred for DME extraction. The three major

us
components of DME extraction apparatus are extractor, a needle valve to control flow rate,

and storage tank. These components are connected in series. For the extraction, wet algal

an
biomass is loaded along with the colourless glass beads into the extractor. Liquid DME is

able to extract pigments efficiently while passing through the extractor at different time
M
intervals. In the apparatus, colourless glass beads are present above the biomass and at the

end of the extraction process glass beads changes its colour to the olive green. The extracted
d

carotenoid pigments are collected from the glass beads and at the end of the process DME is
te

evaporated by opening the valve of the storage vessel. Goto et al., have shown that by using
p

286 g DME, they have extracted approximately 390 µgg-1 of fucoxanthin from algae U.
ce

Pinnatifida (Goto et al., 2015).

4.2.2 Supercritical CO2

Ac

A number of studies have been performed on different microalgae strains (Nannochloropsis

sp., Synechococcus sp. and Dunaliella sp.) for extracting carotenoid molecules using super

critical CO2 extraction (Goto et al., 2015; Macias-Sanchez et al., 2009; Macias-Sanchez et al.,

2008). Different studies on the carotenoid extraction process using supercritical fluid suggest

that extraction process provide better selectivity of the carotenoid molecules while operating

at 40–80°C and 20–40 MPa pressures.

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Page 26 of 72
4.3 Extraction and purification of chlorophyll

One patent had been granted for the innovative separation process of chlorophyll a and

chlorophyll b from plant origin, including algae by using column chromatography method

(Anderson and Melvin, 1966). This patented invention is an economical method for purifying

t
ip
specific chlorophylls. It has been claimed that polyethylene powder, when used in a

chromatographic column would yield complete separation of chlorophylls from xanthophyll

cr
and carotene. This method described as more economical method for specific chlorophyll

us
purification. After initial purification with a polyethylene filled column further purification

was done with sucrose filled column for the separation of chlorophyll a and chlorophyll b.

an
The chlorophyll a and b thus purified is crystalline and of extremely high purity(Anderson

and Melvin, 1966).

M
d

5. Protein extraction
te

Two unicellular microalgal species Spirulina and Chlorella attracted attention as major
p

source of protein, which can be produced in large scale. Nutritional values of the algae and
ce

use of algae as dietary supplements are well established by different research review articles

(Boiko et al., 1963; Wells et al.). However, poor protein digestibility and bioavailability of
Ac

the protein molecules from the unprocessed algae cells are growing concern for the nutrition

scientists and emphasis has been given for whole cell processing, improvement of the

extraction methods of protein from the algae cells (Kose et al., ; Lubitz, 1961; Saleh et al.,

1985). To extract protein, removal of interfering molecules (e.g. lipids, nucleic acids,

carbohydrates, and pigments) is most important. In this section, conventional processes like

27

Page 27 of 72
organic solvent extraction and sonication (Table 5) have been discussed for the purpose of

protein extraction along with new emerging techniques (Fig 1).

5.1 Organic solvent extraction

t
Tricholoracetic acid (TCA)/acetone precipitation is the most common method frequently used

ip
for the extraction of small amount of proteins for proteomic study (Slocombe et al., 2012).

cr
Denaturation and precipitation of the protein occurred due to the very low pH and negative

charges on TCA and presence of the organic acetone in the environment. In this method,

us
denaturation of the protein ensures ceasing of proteolytic activity and other protein degrading

activity of the enzymes. However, in this method, solubilisation of precipitated protein is

an
extremely difficult. In a study, dinoflagellate Alexandrium tamarense cell was broken with

the help of ultrasonic disrupter and treated with 10% TCA/acetone (w/v) and 80% acetone
M
(v/v) to collect the precipitated protein after centrifugation (Wang et al., 2009). Study report

had shown that application of this method would able to extract highest amount of protein
d

(~6.73µg/µl) from 1x 106 cells compared to other methods used in the study. Phenol based
te

protein extraction from red seaweed Eucheuma cottonii also studied by Lim et al. and shown
p

that protein extraction with phenol in addition with lysis buffer produce higher protein yields
ce

(0.027 mg/g) compared to the other methods (Lim and Teo, 2015). Schwenzfeier et al. have

used solvent extraction method to extract protein from the species Tetraselmis sp. and the
Ac

resultant yield of protein was 64 % (w/w) (Schwenzfeier et al., 2011). Although, many

techniques or methods of protein extraction from algae cells are reported in scientific

journals, most of the operations are very small. Scalability of protein extraction is a major

issue in industrial scale operation and needed to overcome before establishing protein based

manufacturing process.

5.2 Sonication
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Page 28 of 72
Efficient extraction of protein from the cells depends on the successful disruption of the cells

to get the accessibility of the molecule. Sonication is one of the techniques available for cell

disruption for small-scale operation (1-6 L of cell culture). Effect on cell disruption was

studied by varying different sonication waves (0.02, 0.4, 1.0, 2.2, 3.3, and 4.3 MHz) to

t
different species of algal (Chaetoceros gracilis, C. calcitrans, and Nannochloropsis sp.) cell

ip
suspension. It had been shown in the report that efficiency of cell disruption was species

cr
specific and highest efficiency was achieved for the Chaetoceros gracilis at 2.2 MHz, for C.

calcitrans at 3.3 MHz and Nannochloropsis sp. at 4.3 MHz (Kurokawa et al., 2016). In

us
another study, Meijer and Wijffels have performed sonic disruption on Chlorella sp for 1 min

an
for 3 cycles using 70W energy in the presence of 1% (w/v) SDS and shown almost all protein

recovery from Chlorella sp (Meijer and Wijffels, 1998). However, this method has been seen
M
is useful for small quantities of cells ( less than 50 g) and has limited use for large quantity of

cells due to the long sonication time requirement to achieve sufficient cell disruption
d

(https://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/
te

cell_lysates_ecoli/, 2015).
p

5.3 Protein Purification

ce

Advancement in genetic engineering brings the opportunity to use microalgae as a platform

for recombinant protein expression. Chlamydomonas reinhardtii is a well studied model also
Ac

considered as an expression system for recombinant protein due to the availability of

complete genome sequence and relatively easy and economical to grow at large scale

(Shamriz and Ofoghi, 2017). A sustainable and economical method to isolate protein of

interest from the cultivated microalgal broth for industrial application is another major

challenge. Recombinant therapeutic protein synthesis using Chlamydomonas reinhardtii,

require highly pure (more than 99%) and bioactive molecules after completion of purification

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Page 29 of 72
process (Rasala et al., 2010). Expression of human antibody using diatom Phaeodactylum

tricornutum and purification of expressed recombinant protein was done by Hempel et al.

(Hempel et al.). In this study, antibody was purified in small scale operation with protein A-

Sepharose beads column and high purity of the eluted protein was confirmed by gel

t
electrophoresis. Several chromatography methods, ion-exchange, size exclusion, affinity and

ip
hydrophobic interaction chromatography are used routinely in the laboratories for purifying

cr
diverse protein molecules from algae (Han et al., 2012; Verdel et al., 2000; Wang et al.,

2014). Wang et al., had purified d-galactose-6-sulfurylase enzyme from microalgae

us
Betaphycus gelatinus using ion exchange chromatography and hydrophobic interaction

an
chromatography and able to isolate monomeric protein (65 kDa) molecule with increasing 4.9

fold purification and 3.7% yield of the cell lysate (Wang et al., 2014). Verdel et al., had
M
isolated monomeric (43 kDa), haloperoxidases enzyme from freshwater microalgae strain

Cladophora glomerata by using ion-exchange and gel filtration chromatography (Verdel et

d

al., 2000). In another study Han et al., had isolated protein (N-acetyl-D-galactosamine-
te

specific protein) molecules (21.4 kDa) from filamentous red alga Aglaothamnion oosumiense,

using affinity chromatography (Han et al., 2012).

p
ce

Apart from the different chromatography methods, membrane based filtration method (such

as ultrafiltration, cross flow filtration) is also very much popular for protein purification from
Ac

microalgae (Henderson et al., 2008; Her et al., 2004; Zhang et al., 2010). Membranes

filtration method has various additional advantages over chromatography, which are high

volumetric throughput, high extract concentration, economical single use membranes

availability and lower cost. However, one of the disadvantages of using membranes for

protein purification is the availability of specific size membrane which is most essential for

accurately target specific protein for purification(Buyel et al., 2015).

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Page 30 of 72
In a detail study on the process economics by Grima et al., had been shown that cost of

metabolite production from algae influenced by the cost of biomass production, amount of

metabolite produced by the cells and purification cost (Molina Grima et al., 2003). In this

study, it had been shown that biomass contributes ~40% and recovery process contributes

t
~60% in the production cost of a molecule like eicosapentanoic acid (EPA).

ip
6. Carbohydrate extraction

cr
Commercially extracted polysaccharides from algae include mainly alginates, agar and

us
carrageenan. . In this section, conventional extraction processes (e.g., alkali extraction,

an
alcohol process, KCl process, drum drying process; Table 4 and Fig. 1) are highlighted which

are used in industries for extracting and processing alginates, agar and carrageenan.
M
6.1 Alginate

6.1.1 Alkali extraction

d
te

Reduction of particle size of the algal biomass is required before extraction process in order

to enhance the extraction process. After size reduction, algal biomass is rehydrated with
p

formaldehyde solution (~ 0.1 %) for 1 to12 h depending on the species to make the biomass
ce

soften and to avoid pigmentation of alginate. The formaldehyde solution reacts with phenolic

compounds leading to polymerization that makes colouring substances insoluble. Afterwards,

Ac

resulting biomass is treated with acids for conversion of the alginate salts (Ca 2+, K +, etc.)

in to insoluble alginic acid. Treatment with acid to remove the external salt is always

required. However, formaldehyde treatment may not require for all the species. Subsequently,

to extract the alginate, the acidified biomass is transferred into extractor and about nine fold

(dry biomass basis) water is added biomass. The biomass-water mixture is heated around 80
0
C by adding sodium carbonate powder to maintain the pH around 10. Extracted alginate
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Page 31 of 72
from biomass comes into liquid phase, which is viscous, and water addition may be required

to increase the efficiency of the process. Thereafter, extracted alginate is filtered using rotary

vacuum drum filter to remove solid particles resulting clear alginate solution. This method is

well established and have been performed on algae such as Sargassum sp., Laminaria

t
digitata, and Sargassum sp. (Hernández-Carmona et al., 2013).

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6.2 Agar

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Extraction of agar is obtained by cooking of the algal biomass in water at boiling

us
temperature. The efficiency of extraction process lies on the precise control of pH (6.3–6.5)

by careful addition of acid. Moreover, the extraction efficiency is enhanced by applying

an
pressure, which increases the yield of agar with less processing time. However, optimum

extraction conditions depend on type of algal species. Filtration is required to to remove the
M
residual biomass in hot condition and thereafter, hot filtrate is allowed to cool for gel

formation. Sometimes, the gel is treated with bleach (usually sodium hypochlorite) for
d

improve the quality by removing colour. Finally, water is removed either by a freeze-thaw
te

process or by squeezing it out using pressure. (Hernández-Carmona et al., 2013).

p
ce

6.3 Carrageenan

6.3.1 Alcohol process

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In this method, carrageenan is separated from other cellular components and then it is

solubilised in an aqueous solution. Firstly, dry algal biomass is washed with running water to

remove salts, shells, sand, or any foreign particles. Thereafter, the extraction is performed in

a mild alkaline condition (pH ~8-9 by adding sodium bicarbonate) at boiling condition for

around two hours with solid: liquid ratio about 1:50. In this process, the carrageenan is

transferred in to solution phase and the cellulose and other insoluble material are discarded
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via different clarification processes such as filter aid on a pressure filter(Hernández-Carmona

et al., 2013). To recover the carrageenan from the solution, clear solution is evaporated under

vacuum to approximately one third of the original volume. Thereafter, precipitation is carried

out using organic solvent (e.g. isopropyl alcohol with solvent: extract ratio of 2:1). This

t
process is commercially applied for processing of C. crispus and different Gigartina species.

ip
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Tuvikene et al., have followed similar procedure to extract carrageenans from the biomass of

the algal community collected from the Kassari Bay (Baltic Sea, Estonia). They used tap

us
water to remove foreign particle and dried at outdoor temperature. Extracting medium was

distilled water and various concentration of KOH or NaOH solutions. At 8 hour extraction

an
time, their extraction yield was varied from 27 – 31 % algal biomass (Tuvikene et al., 2006).
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6.3.2 Potassium chloride process

This process is very similar to alcohol process where the carrageenan is initially extracted in
d

a solution phase. Thereafter, 1% (w/v) KC1 (instead of alcohol) is used for the precipitation
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which is enhanced by lowering the temperature. Dewatering is achieved through a freeze-

p

thaw process or by pressing the gel, which is similar in the agar process. This process is
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popular and cheaper for the production of kappa carrageenan. This extraction process is

effectively used for extraction kappa carrageenan bearing seaweed as Kappaphycus alvarezii
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and other Eucheuma species(Hernández-Carmona et al., 2013).

6.3.3 Drum drying process

In drum drying process, carrageenan is recovered from the aqueous solution by evaporation

using single or double drum dryer instead of precipitation. The efficiency of the evaporation

process is better by applying vacuum during evaporation that also prevents thermal

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breakdown of the carrageenan. This methodology can be applied to extract any type of

carrageenan from microalgae (Hernández-Carmona et al., 2013).

7. Current patents and patent applications related to the downstream

t
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processing of microalgae

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Number of patents have been filed regarding increment of extraction efficiency from ruptured

cell biomass and details of those patents have been presented in Table 7. Sepal Technologies

us
Ltd., was granted for a patent for developing an apparatus and method for

separating microalgae from water without rupturing the algae cells (Borodyanski and

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Konstantinov, 2003). In this method, flocculation, flotation and dehydration steps were used

for efficiently isolate microalgae from water without rupturing cells, in order to obtain dry,
M
concentrated biomass. Green extraction technologies had been assigned a patent application

proposing a novel biomass fractionation apparatus. Another patent was granted to Katz et al.,
d

in the year 2013, for their innovative continuous flocculation deflocculation process for
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harvesting of microalgae from a variety type of waters sources including saltwater, brackish
p

water, fresh water, and treated wastewater (Katz et al., 2013). A reusable composite
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paramagnetic nanoparticle was used in a patented method to efficiently isolate algae from

culture broth using an externally applied magnetic field (Liang, 2015).

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A patent was granted to Polish, Jagiellonian University, claiming the efficient extraction

process of highly pure pigments (diatoxanthin and diadinoxanthin) from diatom

(Phaeodactylum tricornutum) (KuczyŃSka and JemioŁA-RzemiŃSka, 2016). This

patented process, consisting five different steps such as cultivation of diatom species,

pigment extraction, partitioning of chlorophylls and carotenoids, alkalization of

chromatography gel and open column chromatography, allows obtaining highly pure
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Page 34 of 72
diatoxanthin and diadinoxanthin molecules from the diatoms. Fucoxanthin is also a pigment

(xanthophyll) is found in the chloroplasts of many brown algae (Phaeodactykum sp.,

Isochrysis sp., Amphora sp., Naviculla sp. and Chaeotocerous sp.). Fucoxanthin extraction

method was patented by Dr. Oran Ayalon and claimed that extraction (1.5% of the dry

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biomass) was made from harvested biomass of algae by using solvent extraction and

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supercritical fluid (CO2) extraction method (Ayalon, 2017). Chlorophyll and carotenoids are

cr
valuable compounds used as ingredients in food and neutraceutical products. USA based

platform technology company, Heliae Development LLC was granted a patent for the

us
production method of chlorophyll and carotenoids along with lipids from the algal biomass

an
(Aniket, 2012). Chlorophylls, carotenoids and neutral lipids are extracted from the

dehydrated algal biomass followed by the separation of the carotenoids and chlorophylls
M
using adsorption or membrane diafiltration method.

Protein extraction method from the biomass of microalgae was patented and claimed that
d

high quality purified protein (below 5 kDa) was obtained from different species of
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Chlorella by adopting multi-step processing of the biomass (Patinier, 2017). This process
p

includes washing of the harvested biomass, thermal permeabilization of the biomass at a

ce

temperature between 50 and 150 °C; elimination of permeabilized biomass by a solid-liquid

separation technique (frontal or tangential filtration, flocculation and multi-stage

Ac

centrifugation) and ultrafiltration of the soluble fraction of protein on a membrane with a cut-

off threshold lower than five kDa. Heliae Development LLC also invented a multi-step

extraction method of globulin protein from intact freshwater algae (Nannochloropsis sp.)

biomass (Aniket, 2013). In this process, freshwater algal biomass was mixed with salt water

and heated to a temperature below the boiling point of the extraction mixture, comprised of a

liquid fraction enriched in globulin proteins and a biomass fraction. Thereafter, the biomass

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fraction was treated with different solvents (e.g. methanol, ethanol, isopropanol, acetone,

ethyl acetate, acetonitrile) at different step and liquid fraction enriched in globulin proteins

was separated from the biomass fraction in each step. Another patented method was

published in the year 2013, on the method of protein fractionation along with lipids from

t
algae biomass (Kumar and Hatcher, 2013). In this method 60% of a total protein was

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extracted from the solids biomass of algae into the aqueous liquid phase after keeping the

cr
biomass in a reactor, maintaining subcritical temperature (200-350°C), followed by

us
separating the reactor effluent into solids and liquid.

In 2012, one Colombian company (Ecopetrol S. A.) was granted a patent for improved

an
method for extracting fermentable sugars based on microalgae (Garzon et al., 2012). This

method claimed that treatment of algal biomass with sulphuric acid at elevated temperature
M
(100 – 200 °C) and high pressure (101.14 - 303.42 kPa), disrupt the algal (Chlorella vulgaris,

Chlamydomonas sp., Scenedesmus Basilensis) cell wall and facilitates the extraction of the
d

carbohydrate. Another patent was granted for the process of extracting carbohydrates and
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lipids from the algae and use the extracted carbohydrates as carbon source in fermentation
p

processes for the production of alcohols (e.g., ethanol, butanol) (Massetti et al., 2014). In this
ce

process, algal semisolid biomass was produced by increasing the pH (higher than or equal to

10) and adding one anionic flocculant (polyacrylamides, polyacrylates, polymethacrylates,

Ac

polycarboxylates) in the aqueous cell suspension. Afterwards, carbohydrate and lipids both

were extracted from the concentrated semisolid biomass by solvent extraction and acid

hydrolysis method. University of Toledo had assigned a patent claiming enzymatic (fungal

acid protease) digestion of algae (Chlorella sp., Schizochitrium limacium) biomass was

helpful for efficient extraction of the macromolecules (lipid, carbohydrate and protein) from

the cells (Vadlamani et al., 2014).

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A patent was granted to Utah state university in the year 2013, for the method of producing

bio plastics from algae (Miller et al., 2013). The method describes the processing of algae to

yield an aqueous phase containing glycerol, and fermenting the aqueous phase with bio

plastic-producing bacteria to yield bio plastics. Another patent was granted on the

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hydrothermal liquefaction method of algal (Scenedesmus sp Nannochloropsis, Spirulina sp.)

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biomass for the production of viscoelastic oil (15 - 30 wt.% of the wet biomass) (Chailleux et

cr
al., 2015).

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A patent was granted on extraction of important molecules from algal species (Cyclotella sp.,

Tetraselmis sp.) to the Synthetic Genomics Inc., in the year 2016 (Domaille et al., 2016). In

an
this process, biomolecules are extracted under elevated pressure, which made extraction

process more efficient than earlier conventional methods. A method of fractionating algal
M
biomass was patented by Czartoski et al., in the year 2011 (Czartoski et al., 2010). Renewable

processing of algae and recovery of cells and cell derived biomolecules from non-polar
d

solvent solution was claimed as invention in this method.

p te

8. Current scenario on global algal product market

ce

For the production of nutraceuticals and high-valued low-volume food supplements about
Ac

9,000 tonnes of algal biomass is getting produced commercially (Darzins et al., 2010). In the

EU, the biggest algae investment is the £26 million by the UK Carbon Trust to build one

large algae farm in Northern Africa by 2020 (Jha, 2008). Carbon trust launched a new £8

million research programme, Algae Biofuels Challenge (ABC) in 2009, to support such

development. The ABC is now led by a number research teams from 11 institutions including

Universities of Manchester, New Castle, and Southampton, the Plymouth marine laboratory,

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Page 37 of 72
and Scottish association for marine sciences. A Spanish renewable energy company Aurantia

and Green Fuel Tech of Massachusetts (USA) formed a partnership through a $92 million

project in 2007 to produce algae oil. An Italian energy company, Eni, has installed a 1 ha

pilot facility for algae oil production in Gela, Sicily (Singh and Gu, 2010). Details of the

t
companies that are currently producing algae based biofuel and other value added products

ip
can be found in Table 6. The global algae biomass market is worth between US$ 5- 7 billion.

cr
From this total, the health food sector accounts for US$2 billion and the aquaculture

applications account for US$ 0.7 billion(www.oilgae.com, 2016). Currently being

us
commercially produced algae based products, its uses, market value have been mention in

an
Table 8. Companies using algae as a raw material for algal products are producing some

diverse product than only biofuel. While company like Sapphire Energy is mainly focussing
M
on green crude oil, companies like Algenol Biofuels and Phycal are also focussing on jet fuel

along with algal biodiesel. Few companies like BioProcess Algae, Heliae and Earthrise
d

Nutritional are totally focusing on nutritional, animal feedstock, therapeutic and organic
te

products without producing any biofuel related products, which seems to recent catchy trend.

Most companies (e.g. Aurora Algae, Cellana, and Solazyme) have been producing other
p

products like nutricals, health supplements along with the biofuel. Other than that,
ce

collaboration between Duke Energy and University of Kentucky are working on the CO2

remediation from environment. In term of production, Algenol Biofuels are producing

Ac

10,000 gallons per acre per year in 36-acre land whereas Cellana producing 100,000 metric

tons of biomass per year in 6 acre land.

9. Conclusion and future perspectives

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Downstream processing of microalgal biomass is a combination of sequential steps that

consists of the cultivation of microalgal biomass, harvesting of biomass, extraction of

molecules, and purification of extracted molecules for commercialization. Among all these

steps, extraction is most important and comparatively expensive process (Kim et al., 2013).

t
There are various technological and economic obstacles like development of energetically

ip
efficient and cost effective extraction process, absence of scalable extraction process, are

cr
present of microalgal bio-active molecules extraction. The selection of a robust microalgal

strain, which has optimum bio-active molecule content, high growth rate, and is immune

us
towards invasion by local microbes, still remain a major challenge (Halim et al., 2012).

an
Another challenge is the development of an optimum process for successful outdoor large-

scale cultivation of microalgae. One possible solution to these problems is to develop a single
M
optimum process where production of bio-active molecule will subsequently be done along

with the production of biodiesel from microalgal biomass. For examples, particular strain
d

having high chlorophyll, protein and carbohydrate contents should be processed optimally to
te

extract all the components instead of single product. Another strain having high carotenoid,

extracellular polysaccharides and carbohydrate should be opted different downstream process

p

than previous one to extract all the products.

ce

A flow diagram at industrial scale for downstream processing of algal biomass has been
Ac

shown in Fig 2. This flow chart depicted a combined biochemical conversion processes

where biomass of algae can undergo a number of sequential extraction processes for

extraction of high value protein, fuel esters, omega-3-esters, pigments and carbohydrates. In

this bio-refinery approach selection of suitable solvent is crucial factor for extraction from the

algal dry or wet biomass. Wet biomass (Chen et al., 2012) can be used for the extraction of

lipid rich fraction (both polar and neutral lipid). This lipid rich fraction can be further

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Page 39 of 72
processed into fuel, fuel additives, Omega 3 ester, and pigments. After lipid extraction,

remaining biomass can be processed for protein extraction. Conventional solvents such as

hexane, chloroform, isopropanol or combination of solvents at specific concentration

(dichloromethane/methanol (2:1 v/v), isopropanol/hexane (2:1 v/v) etc.) also can be used for

t
the extraction of protein molecules (Ansari et al., 2017; Ansari et al., 2015; Cheng et al.,

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2011; Choi et al., 1987; Hernandez et al., 2014; Hidalgo et al., 2015). Rest of the biomass can

cr
be used as carbohydrate rich molecules which have wider applications in bio-plastic, agar,

sugars and other value added chemicals. However, strain specific bio-refinery approach may

us
be the best option available for industrial application.

an
Energy industries are looking for the breakthrough technology for the production of

economical sustainable biofuels from the biomass of microalgae with minimum waste
M
generation at large scale. A hydrothermal liquefaction method with or without involvement

of catalyst is under current research focus for direct conversion of wet algal biomass into the
d

biofuel products and value added chemicals (Eboibi et al., 2014; Kim et al., 2017; Lopez
te

Barreiro et al., 2013). In this process, biomass of microalgae can be converted into liquid
p

biofuel precursors (biocrude oil), biogas and minimum solid residue. This process can be
ce

completed within 30 minutes of time period and can be operated around 220 bar pressure and

250 ºC to 374 ºC (supercritical point of water) temperature. However, economic feasibility of

Ac

this process is growing concerns before it actually implemented at large scale (Tzanetis et al.,

2017). Residence time of the hydrothermal liquefaction was drastically reduced from 30 min

to 10 second with a new technology name as flash hydrolysis (Garcia-Moscoso et al., 2013).

In this process, protein can be extracted (more than 60 wt% of total nitrogen) efficiently from

microalgae (Scenedesmus sp.) biomass when temperature kept at above 240 ºC.

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Algal biomass also can be processed through the thermo-chemical method (e.g. pyrolysis,

gasification) for producing biocrude oil and syngas (e.g. CO, H2, CH4, CO2 etc.) at large scale

(Khoo et al., 2013; Sanchez-Silva et al., 2013; Wang et al., 2017). In the pyrolysis process,

dry biomass of microalgae used as feedstock and converted into the intermediate product of

t
biofuel (biocrude oil) through combustion at 400- 600 ºC in absence of oxygen or air.

ip
Pyrolysis of algal biomass also produces char product along with the biocrude oil. A fast

cr
pyrolysis (a few seconds residence time and high heating rate) allow producing high oil yield

from the feedstock. Very recently microwave assisted pyrolysis process became popular for

us
maximizing the oil yield (Hong et al., 2017). Oil yield can be varied between 30-70%

an
depending on the process parameters (Chiaramonti et al., 2015).

Algal biomass can be a resource for energy industries for the producing syngas at large scale
M
through gasification process in presence of oxygen or air. In this process, biomass of

microalgae is partially combusted at very high temperature (800- 1000 ºC) for producing
d

syngas (Beneroso et al., 2013; Hong et al., 2017; Hu et al., 2013). Supercritical water
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gasification of microalgae is showing promising result for production of syngas at laboratory

p

scale. In this process, gasification of three different micro algae strains (Botryococcus
ce

braunii, Nannochloropsis oculata and Tetraselmis chuui) was studied at 673 K temperature

and 25 MPa pressure, leads to the production of different mixture of gases (CO, H2, CH4,
Ac

CO2, C2H6, C2H4, etc.) (Krishnan et al., 2016). Study report also showed that this process

became more efficient when nickel was used as a catalyst.

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Acknowledgements

Authors would like to convey their sincere thanks to the Department of Biotechnology, India

for financial assistance (Project No. - BT/322/NE/TBP/2012) and National Institute of

Technology, Agartala for their support of this work.

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p te
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Wang, A., Islam, M.N., Qin, X., Wang, H., Peng, Y., Ma, C., 2014. Purification, identification, and
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characterization of d-galactose-6-sulfurylase from marine algae (Betaphycus gelatinus).

Carbohydrate Research 388, 94-99.
Wang, D.-Z., Lin, L., Chan, L.L., Hong, H.-S., 2009. Comparative studies of four protein preparation
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methods for proteomic study of the dinoflagellate Alexandrium sp. using two-dimensional
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Wang, L., Weller, C.L., 2006. Recent advances in extraction of nutraceuticals from plants. Trends in
Food Science & Technology 17, 300-312.
Wang, X., Sheng, L., Yang, X., 2017. Pyrolysis characteristics and pathways of protein, lipid and
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Wei, C., Li, W., Zhang, C., Van Hulle, M., Cornelis, R., Zhang, X., 2003. Safety Evaluation of
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Wells, M.L., Potin, P., Craigie, J.S., Raven, J.A., Merchant, S.S., Helliwell, K.E., Smith, A.G., Camire,
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International Publishing, Cham, pp. 77-89.
Zhang, C., Sanders, J., Bruins, M., 2014. A process for isolating proteins from solid protein-containing
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Zhang, M.M., Pan, G., Yan, H., Chen, H., 2004. A method to extract algae toxin of microcystin-LR.

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Zhang, X., Hu, Q., Sommerfeld, M., Puruhito, E., Chen, Y., 2010. Harvesting algal biomass for biofuels
using ultrafiltration membranes. Bioresource Technology 101, 5297-5304.
Zheng, H., Yin, J., Gao, Z., Huang, H., Ji, X., Dou, C., 2011. Disruption of Chlorella vulgaris Cells for the

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Release of Biodiesel-Producing Lipids: A Comparison of Grinding, Ultrasonication, Bead Milling,
Enzymatic Lysis, and Microwaves. Applied Biochemistry and Biotechnology 164, 1215-1224.
Zhu, L., 2015. Biorefinery as a promising approach to promote microalgae industry: An innovative

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framework. Renewable and Sustainable Energy Reviews 41, 1376-1384.

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Fig. 1. Schematic of cultivation, harvesting, pretreatment, extraction of various industrial

microalgal products (pigments: chlorophyll, β-carotene; carbohydrate: alginate, agar,
carrageenan; protein). The cultivation, harvesting are common for all the products.
Pretreatment and extraction method depends on the specific products. Pretreatment method
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broadly classified as cell disruption, drying and particulate size reduction. Extraction of
pigment, carbohydrates and proteins can be obtained by chemical methods of pre-treated
biomass.

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Fig. 2. Schematic for the downstream processing of harvested microalgal biomass for the
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extraction of various industrial microalgal products (lipids, protein, carbohydrate and
pigments).
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Table 1 Industrial Products (other than lipid based products) from Microalgae

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Product Product Name Source Structure Application Reference
Categor
y
1. β-carotene Spirulina platensis, β-carotene is found to be useful in the (http://www.oilg
Pigment

Caulerpa taxifolia. following cases: ae.com/non_fuel

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Prevention against cancer and heart _products/betaca
disease, To slow the progression of rotene.html,
cataracts,To prevent macular 2015;
degeneration, To boost immunity, To https://en.wikipe
protect the skin against sunburn, dia.org/wiki/Beta

ed
pt Asthma, Depression. -Carotene, 2015)
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2.Chlorophyll Green algae Structure of Chlorophyll a In the food industry, chlorophyll is used (http://www.oilg
as a natural pigment ingredient in ae.com/non_fuel
processed foods. _products/chloro

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Have antioxidant activity. phyll.html, 2016;
https://en.wikipe
dia.org/wiki/Chlo
rophyll, 2015)

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Structure of Chlorophyll b
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Structure of Chlorophyll C

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Protein Powder Microalgae. Exp: Nutritional benefits (Becker, 2007;
Protein

or tablets Chlorella sp. and Used as feedstock for animals and https://en.wikipe
Cyanobacteria. Exp: poultry dia.org/wiki/Spir

an
Athrospira sp. ulina_(dietary_su
pplement), 2016)

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Spirulina powder at 400x, unstained wet mount. Pic from

ed
https://en.wikipedia.org/wiki/Spirulina_(dietary_supplement)
1.Agar Rhodophyta (red Consists of 70% agarose and 30% agaropectin. Best known application of agar is the (Hahn et al.,
Carbohydrate

algaes) preparation of culture media in Petri 2011)

Exp.: Gelidium , dishes for the growth of micro -
Gracilaria and organisms.
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Pterocladia Sp. In the food industry agar is used as
thickening agent and can be applied as
substitute for pectin, gelatin and starch.
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2.Carrageenan Red seaweeds Exp.: Generally two types found: Common food additives due to their (Usov, 2011)
Chondrus crispus and thickening, gelling and emulsion
Mastocarpus stellatus . stabilizing properties in the food

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industry.
Application in the pharmaceutical and
cosmetic industry.

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3.Alginate Algae, Exp.: Alginate is a linear polysaccharide consisting of two types of Widely applied as a stabilizing, (Hahn et al.,
Macrocystis pyrifera anionic monomers. thickening or emulsifying agent in the 2011)
Ascophyllum nodosum β - d - mannuronic acid (M) and α - l - guluronic acid (G) food, cosmetic, paper and dye

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Laminaria sp. are linked via 1,4 - glycosidic bonds in homopolymeric MM - industries.
Ecklonia sp. and GG - blocks respectively. In medical fields alginates
are used for example as dental
impression materials, for tissue

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engineering applications or as cell
encapsulation material.

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4.Fucoidan Brown seaweed Fucoidans are sulphated polysaccharides mainly consisting of l Exhibit anticoagulant abilities by (Hahn et al.,
Exp.: Fucus - fucose with molecular weights ranging from 13 to 950 kDa. enhancing the heparin cofactor II, 2011)
vesiculosus, so they may become an alternative to

an
Pelvetia canaliculata heparin due to their herbal origin.
Antitumor efficacy, owing to their
ability to inhibit proliferation and to
induce apoptosis via caspase and extra -

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cellular signal -regulated kinase
pathways.
Their ability to hamper the replication
of viruses, or their penetration into host
cells results in antiviral activities and

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yields positive effects for example,
against human immunodeficiency virus
( HIV ) andherpes simplex virus type 1
(HSV - 1).
pt
1.Biopolymers( Nostoc sp, Phormidium Repeating group of polyethylene which can be derived from Thickening agents for mobility control (http://www.oilg
PLA, Bio- mucicola , Chlorella algal Biomass. in waterflood oil recovery, Food ae.com/non_fuel
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Polyethylene stigmaaphora, additives, Flocculants useful in waste _products/biopol

Organic Plastic

etc.) Chlorella vulgaris water treatment, Soil conditioning, ymers.html,

Drilling mud extenders, Pet food,Farm 2016;
feed stabilizers https://en.wikipe
dia.org/wiki/Poly
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ethylene, 2015)

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Table 2 Composition and possible source of bio polymers from microalgae

Sl. Name Composition Source Reference

No.

1. Hybrid Plastics Denatured algae Spirulina (Bayramoglu

biomass conjugated with (Arthrospira et al., 2015;
petroleum based plastics platensis) biomass/ http://www.oil

t
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like polyurethane and Red algae (Jania gae.com/non_f
polyethylene as fillers rubens)/ Filamentous uel_products/b
green algae of the iopolymers.ht

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order Cladophorales ml, 2016;
Kadimpati,

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2016)

2. Cellulose based Plastics derived from Cellulose laden algal (http://www.oi

Plastics cellulose of lipid biomass of any lgae.com/non_

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extracted algal biomass microalga strain/ fuel_products/
Filamentous biopolymers.ht
green algae ml, 2016;
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Cladophora sp. Mihranyan,
2010)

3. Poly Lactic Lactic acid and its Nannochlorum sp. / (Hirayama and
d

Acid (PLA) polymer poly lactic acid Bacterial Ueda, 2004;

(PLA) are used as a fermentation of algal Nguyen et al.,
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biodegradable biomass 2012)

alternative (Hydrodictyon
p

reticulum)
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4. Bio Ethylene monomer Bacterial digestion of (http://www.oi

Polyethylene which is being used in algal biomass, or lgae.com/non_
the production of directly from algae/ fuel_products/
polyethylene biopolymers.ht
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Cyanobacteria ml, 2016;

(Synechocystis sp. Veetil et al.,
and Synechococcus 2017)
sp.)

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Table 3 Pigment Extraction

Sl. Method Merits Demerits Reference

No. Name
1. Organic Extraction efficiency depends on the species Fire, health, and (Hosikian
Solvent used, volume of the extractor, reaction time, environmental hazards; et al.,
method sample volume, moisture content, types of regulatory issues 2010b)
lipids present, and in case of solvent-based
methods, choice of the solvents, solvent ratios,

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etc

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2. Supercritical Rapid process, no organic solvent or acid very expensive equipment, (http://ww

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Fluid needed, lipids can be used for further analysis. complex equipment, supply w.biomed
Extraction of CO2 needed central.co
m/content
/suppleme

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ntary/219
0-4715-
24-13-
S1.PDF,

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2015)
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Table 4 Carbohydrate Extraction

Method Name Merits Demerits

Alkali Extraction Easy and effective method Quality of product
sometimes low
Pressure method Reduces processing time, Potentially destructive,
increases yield Presence of residual

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biomass

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Alcohol Process It can be used to produce disadvantage of this
any kind of carrageenan. method is the high cost
involved in the distillation

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of
large volumes of alcohol
and the purchase of the
necessary explosion-proof

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equipment.
KCl process This process is cheaper than
the alcohol-based method, it is
exclusive to the production of

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kappa carrageenan.
Drum Drying This process is cheaper and The economy of the
uses much less alcohol than process is influenced by
the the alcohol recovery
aforementioned alcohol efficiency.
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precipitation method, can be
applied to the production of
any carrageenan type.
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Table 5 Protein Extraction
Sl. Method Name Merits Demerits Reference
No.
1. Solvent extraction not very labour intensive lipids Expensive, not all lipids extracted. (http://www
suitable for further Analysis, Various mixtures of solvents, .biomedcent
techniques take out environmental temperatures and pressures needed for ral.com/cont

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contaminants specific samples to ensure that all free fat ent/supplem
is extracted, drying of samples required entary/2190

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-4715-24-
13-S1.PDF,
2015)

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2. Sonication High yield Energy intensive, Poor product quality (R et al.,
due to the damage during the process 2015)

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Table 6 Details of prominent companies producing algae based biofuel and other value added
products

Sl. Name of Investme Land Production of Products Strategic

No. the nt used Biofuel Partners
company
1 Algenol $ 190 36-acre 10,000 gallons per acre Ethanol BioFields S.A.P.I. de
Biofuels million per year ULS Diesel C.V.

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Jet Fuel

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Gasoline
2 Sapphire $135 million - 100 barrels of green Green Crude Oil Monsanto
Energy crude per day The Linde Group;
Tesoro Refining and

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Marketing Company
LLC
Institute of System
Biology

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3 BioProcess $50 million - 1 metric ton of biomass Animal Feed
Algae per day Nutritionals
Fuels
4 Heliae - 20-acre Nutrition Mars Family
Therapeutics Salim Group

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Health & Beauty Clarecastle Group
Agrosciences
5 Aurora - - 15 tons of biomass per Biofuel Oak Investment
Algae month Omega-3 Essential Partners
Fatty Acids Noventi Ventures
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Protein and animal Gabreil Venture
feed Partners
6 Cellana $ 20 million 6-acre 100,000 metric tons of Omega-3 Nutritional U.S. Department of
biomass per year Oils Energy
Food and Feed Department of
d
Supplements Agriculture
Biocrude Oil
7 Duke Energy $200,000 - 26,000 Liters CO2 Remediation KY Department of
te

and the Services Energy Development

University of Algal Biomass and Independence
Kentucky Fuel and Aquaculture US-China Energy
Feeds Research Center
p

Advanced Coal
Technology Consortia
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ENN Group
Pittsburgh State
University
8 Earthrise - 108-acre 600 tons of Spirulina Spirulina and Spirulina -
Nutritionals powder are produced based formulated
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each year green products

9 Phycal $65 million 21,000 - Algal Oil US Department of
square Bioproducts Energy
feet Jet Fuel National Energy
Technology Laboratory
10 Solazyme - - 400,000 gallons of fuel Fuel Sustainable Oils
to the Air Force and Health supliments (camelina-based
190,000 gallons to the Chemicals biofuel)
U.S. Navy Nutrition Honeywell subsidiary
UOP
11 Seambiotic - 5-hectare Its 1000-square-meter Biofuel -
commerc facility produces Food additives
ial plant roughly 23,000 grams Animal and fish feed
of algae per day--three
tons of algal biomass
would yield around 100
to 200 gallons of biofuel
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Table 7 Details of patents related with microalgae based products and process (based on pigments, protein and carbohydrate)

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Sl. Title Priority Assignee Publication Inventors Strain of microalgae Patent Numbaer Reference
No number/ Date/ Grant /Application
. Date Date number
1 Microalgae 2003-02-25 Sepal 2003-02-25 Genady Generic application US 6524486 B2 (Borodyansk
separator Technologies Borodyanski /US09748249 i and

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apparatus and Ltd Irina Konstantinov Konstantino
method v, 2003)

2 Continuous 2011-10-20 Board Of 2013-04-25 Lynn E. Nannochloropsis, WO 2013059754 A1/ (Katz et

flocculation Regents, The Katz, Kerry A. Chlorella, Dunaliella, al.,

ed
deflocculation University Of Kinney, Jinyong Scenedesmus, US20130102055 2013)
process for Texas System Choi, Eric Chen Selenastrum,
efficient Oscillatoria,
harvesting Phormidium, Spirulina,
of microalgaef Amphora, and
pt
rom aqueous Ochromonas
solutions
3 Harvesting 2010-01-22 Colorado 2015-01-04 Hongjun Liang Generic application US 20150152376 A1/ (Liang,
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micro algae School Of US9464268/ US2011 2015)

Mines 0201076
4 Microalgae ext 2015-04.- 0 Micoperi Blue 2016-11-03 Guido EMILIANI Phaeodactylum WO 2016174646 A1 (Emilia
ract for Growth S.R.L. tricornutum, Arthrospir ni,
agricultural aplatensis (Spirulina), E 2016)
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use uglenagracilis and Porp

hyridium cruentum.
5 A process of 2015-04-29 Uniwersytet 2016-11-03 Phaeodactylum WO 2016175670 A1 (Kuczy
isolation and Jagiellonski Paulina tricornutum ŃSka
purification of Kuczyńska, Małgor and
diatoxanthin zata Jemioła- JemioÅ
and Rzemińska A-
diadinoxanthin RzemiÅ
ƒSka,
2016)
6 Improved 2015-08-28 Oran Ayalon 2017-03-09 Oran Ayalon Phaeodactykum sp., WO 2017037692 A1 (Ayalon,
process for Isochrysis sp., Amphora 2017)
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Sl. Title Priority Assignee Publication Inventors Strain of microalgae Patent Numbaer Reference
No number/ Date/ Grant /Application
. Date Date number

an
producing sp., Naviculla lensi,
fucoxanthin Naviculla incerta and
and/or Chaeotocerous sp.
polysaccharide

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s
from microalg
ae
7 Methods of 2010-04-06 Heliae Dev 2012-02-14 Aniket KALE - US8115022B2 (Aniket,
producing LLC 2012)

ed
biofuels,
chlorophylls
and
carotenoids
pt
8 Method for PCT/FR201 Roquette 2017-06-01 Samuel Patinier Chlorella US20170152294 (Patinier,
extracting 5/051940, Freres 2017)
soluble Jul 18, 2014
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proteins from
microalgal
biomass
9 A protein rich 2015-07-24 Synthetic 2017-02-02 George C. RUTT, - WO2017019125A1 (Rutt et al.,
food Genomics, Inc. James H. Flatt , 2015)
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ingredient Peter Domaille ,

from biomass
Gerardo V. Toledo
and methods
of production
10 A process for 2012-09-21 Wageningen 2014-03-27 Zhang CHEN , - WO2014046543A1 (Zhang et
isolating Universiteit Johan Pieter al., 2014)
proteins from Marinus Sanders ,
solid protein- Marieke Elisabeth
containing BRUINS
biomass
selected from
vegetable
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Sl. Title Priority Assignee Publication Inventors Strain of microalgae Patent Numbaer Reference
No number/ Date/ Grant /Application
. Date Date number

an
biomass,
algae, seaweed
and
combinations

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thereof

11 Selective 2010-04-06 Heliae Dev 2014-06-03 Aniket KALE Nannochloropsis US8741629B2 (Aniket,
heated LLC 2014)
extraction of

ed
globulin
proteins from
intact
freshwater
algal cells
pt
12 Selective 2010-04-06 Heliae Dev 2013-11-05 Aniket KALE - US8574587B2 (Aniket,
heated LLC 2013)
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extraction of
albumin
proteins from
intact
freshwater
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algal cells
13 Fractionation o 2011-12-09 Old Dominion 2013-01-13 Sandeep Scenedesmus sp., WO2013086302 A1 (Kumar and
f proteins and University Kumar, Patrick G. Chlorella sp., Euglena Hatcher,
lipids from Research Hatcher sp., Chlamydomonas 2013)
microalgae Foundation sp., Spirulina sp.,
Porphyridium sp.
14 Improved 2010-12-23 Ecopetrol S.A. 2012-06-28 Fuentes Laura - WO2012085689 A1 (Garzon et
method for Liliana al., 2012)
obtaining garzon, Kafarov
fermentable viatcheslav, Solano
sugars based Andrés Fernando
on microalgae Barajas, Isaza

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Sl. Title Priority Assignee Publication Inventors Strain of microalgae Patent Numbaer Reference
No number/ Date/ Grant /Application
. Date Date number

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and Manuel Laureano
macroalgae Nuñez,
15 Process for the 2013-11-19 Eni S.P.A. 2015-05-28 Felicia Massetti WO2015075630A1 (Massetti et
extraction of Federico Capuano al., 2014)

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lipids and Roberto Medici
sugars from Roberta Miglio
algal biomass

16 Enzymatic 2013-08-30 The University 2015-03-05 Agasteswar Chlorella sp., WO2015031762A1 (Vadlamani

ed
digestion of Of Toledo Vadlamani, Schizochitrium et al., 2014)
microalgal David J. Taylor, limacium
biomass for Patricia Relue,
lipid, sugar, Sridhar Viamajala,
and protein Heng Shao,
pt
recovery Sasidhar Varanasi

17 Methods of bio 2012-06-08 Utah State 2013-12-26 Charles Scenedesmus sp. US20130344550 A1 (Miller et
ce

plastic University Miller, Asif al., 2013)

production Rahman, Ronald
Sims, Ashik
Sathish, Renil
Anthony
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18 Viscoelastic 2013-09-26 Ifsttar, 2015-04-02 Emmanuel Scenedesmus sp WO2015044891 A1 (Chailleux et

material Algosource, Chailleux et. al. Nannochloropsis, al., 2015)
produced by Universite De spirulina sp.
hydrothermal Nantes, Centre
liquefaction of National De
microalgae La Recherche
Scientifique,
Oniris, Ecole
Des Mines De
Nantes
19 Solvent 2012-02-29 Synthetic 2016-05-19 Cyclotella and US 20160137951 A1 (Domaille et
extraction of Genomics, Inc. Peter Domaille, Joe Tetraselmis al., 2016)

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Sl. Title Priority Assignee Publication Inventors Strain of microalgae Patent Numbaer Reference
No number/ Date/ Grant /Application
. Date Date number

an
products from Toporowski, Judit
algae Bartalis

20 Algae biomass 2009-03-10 Valicor Inc 2011-04-14 Thomas J. US20110086386A1 (Czartoski et

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fractionation Czartoski al., 2010)
(Czartoski Robert Perkins
Thomas Jorge L.
J, Robert Villanueva
Perkins, Villan Glenn Richards

ed
ueva Jorge
L, Glenn
Richards)
pt
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Table 8 Current prominent algae products, uses and its market size and value

Sl. Market
Product Uses Market Size Reference
No. value
Boosts the immune
system, Improve
about 10,000
digestion, Reduce
1. Spirulina metric tons per US$20/kg in 2010
fatigue
year
Build endurance

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Detoxifier

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complete food(high
content of protein,
about 2000 tons US $44 per
2. Chlorella vitamins, and minerals
per year in 2009 kilogram in 2010
including carotenoids

cr
and chlorophyll)
nutraceuticals,
predicted to hit
pharmaceutical
3. Astaxanthin $700 million by US$ 2500/kg.
industries and in food

us
2017
coloration applications.
expected to be
colorant and as a source
4. β-carotene worth $285 -
of provitamin A
million in 2015 (www.oilgae.com,
prevention and treatment

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of several diseases like expected to cross 2016)
Omega-3 Fatty
5. atherosclerosis, $4 billion in -
Acids
thrombosis, arthritis, 2018.
cancers, etc.
face and skin care
M
products (e.g., anti-
aging cream, refreshing expected to reach
6. Algae Cosmetics or regenerated care $13.2 billion by -
products, emollient and 2018
as an anti-irritant in
d
peelers)
Carageenan had
prices between
te

Hydrocolloids
$3.30 billion in $10-12/kg in
(agar, alginates as gelling and thickening
7. 2010 2012.
and agents
Agar had prices
carrageenans)
between $20-
p

23/kg in 2012.
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Highlights

Microalgal products based on pigments, proteins and carbohydrates

Downstream processing of microalgal products
Recent patent applications on downstream processing of microalgal products
Current research trends and market scenario

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