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Spirulina – From growth to nutritional product: A review
Accepted Manuscript Spirulina – From growth to nutritional product: A review Ruma Arora Soni, K. Sudhakar, R.S. Rana
PII:
S0924-2244(17)30218-2
DOI:
10.1016/j.tifs.2017.09.010
Reference:
TIFS 2086
To appear in:
Trends in Food Science & Technology
Received Date: 12 April 2017 Revised Date:
18 June 2017
Accepted Date: 25 September 2017
Please cite this article as: Soni, R.A., Sudhakar, K., Rana, R.S., Spirulina – From growth to nutritional product: A review, Trends in Food Science & Technology (2017), doi: 10.1016/j.tifs.2017.09.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Spirulina – from growth to nutritional product: A Review
2
Ruma Arora Soni1,, K.Sudhakar1,3*, R.S.Rana2 1
3 4
2
Energy Centre, Maulana Azad National Institute of Technology, Bhopal (M.P), India
Department of Mechanical Engineering, Maulana Azad National Institute of Technology, Bhopal (M.P), India
5 3,
Faculty of Mechanical Engineering, Universiti Malaysia Pahang, 26600 Pahang, Malaysia
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E mail: [email protected] ;[email protected]
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Abstract
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Background
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Spirulina is multicellular and filamentous cyanobacteria that have achieved a considerable
11
popularity in the health sector, food industry and aquacultures. It develops and grows in
12
water, can be harvested and processed easily. It has very high content of macro and
13
micronutrients, essential amino acids, proteins, lipids, vitamins, minerals and anti-oxidants.
14
Spirulina is considered as a complete food supplement to fight against malnutritional
15
deficiencies in developing countries. Spirulina is deemed safe for human consumption as
16
evident by its long history of food use and latest scientific findings. In recent years, Spirulina
17
has gathered enormous attention from research fraternity as well as industries as a flourishing
18
source of nutraceutical and pharmaceuticals.
19
Scope and Approach
20
The primary objective of this paper is to review the utilization of Spirulina as a dietary
21
supplement in the food industry. In the present work, the three main area of Spirulina
22
research: growth, harvesting and potential application are presented.
23
Key findings and conclusion
24
The important growth parameters have been studied to enhance Spirulina biomass
25
productivity qualitatively and quantitatively. This review provides useful information on
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commercially viable technology for Spirulina cultivation. Mass cultivation and Innovative
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formulations are further needed to fortify conventional foods with Spirulina based protein
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system.
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Keywords: Spirulina; Pharmaceutical; nutritional use; dietary supplement; open pond; PBR
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ACCEPTED MANUSCRIPT Nomenclature
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(PX)
Cell productivity
33
(TC)
Cultivation time
34
Γ
Productivity of the system
35
µ
Specific growth rate
36
x
Biomass concentration
37
(Xm- Xi)
Cell concentration
38
N0
Initial population size
39
t
Amount of time that has past
40
Nt
Population size at time
41
G
Generation time
42
N.S –
Not Specified
43
1. Introduction
44
Algae are photosynthetic organisms that convert light energy from the sun into the chemical
45
energy by the process of photosynthesis. Algae possess simple reproductive structure. The
46
biomass of algae contains various compounds with diversified structures and functions. Algal
47
biotechnology is divided into microalgae, macroalgae and cyanobacteria with its unique
48
specificity (Becker.2007). Sometimes cyanobacteria are also included in microalgae.
49
Microalgae classification includes prokaryotic and eukaryotic unicellular and multicellular.
50
Microscopic are microalgae, Cyanobacteria, are prokaryotic. The Spirulina is Earth's oldest
51
living plant approximately 3.6 billion years ago and a first photosynthetic life form that has
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created our oxygen atmosphere so all life could evolve. Blue-green algae are the evolutionary
53
bridge between green plants and bacteria. At present the main directions in macroalgal
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biotechnology are biofuels, agricultural biostimulants for crop plants, waste water treatment
55
etc. Microalgal biotechnologies refer to the production of different products as phycocyanin,
56
carotenoids, fatty acids and lipids for application in health food, cosmetics, food supplements,
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pharmaceuticals and fuel production. Microalgal groups of major importance are chlorophyte,
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bacillariophytes, while macroalgae are harvested from natural habitats. Algae that is currently
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cultivated for its maximum protein content is the cyanobacterium species Athrospira, which
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is commonly known as Spirulina.
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ACCEPTED MANUSCRIPT Spirulina was first discovered by Spanish Scientist Hernando Cortez and Conquistadors in
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1519. Cortez observed that Spirulina was eaten at the tables of the Aztecs during his visit in
65
Lake Texcoco in the Valley of Mexico. Pierre Dangeard discovered the health benefits of
66
Spirulina who observed that flamingos were surviving by consuming blue-green algae.
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Botanist Jean Leonard supported the findings of Dangeard and people soon started to
68
commercialize Spirulina to reap its benefits (Ugwu et al 2008).The first Spirulina processing
69
plant, Sosa Texcoco, was set up in 1969 by the French.
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Spirulina is the most nutritious, concentrated food that is known to mankind containing
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antioxidants, phytonutrients, probiotics, and nutraceuticals.Spirulina is fast emerging as a
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complete answer to the varied demands due to its imposing nutrient composition which can
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be used for therapeutic uses. The United Nations world at food conference declared that
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Spirulina as the best food for future, and it is gaining popularity nowadays (Pulz and Gross
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2004). World Health Organization has described spirulina as Mankind’s best health product.
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According to UNESCO, spirulina is most ideal food for tomorrow. According to NASA and
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European Space Agency, it is one of the primary foods that can be cultivated in long-term
78
space missions in space. FDA validated it as “One of the best protein source”.
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Intergovernmental institution permitted for the use of Micro-algae Spirulina against
80
Malnutrition (IIMSAM).
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The two most important species of Spirulina are Spirulina maxima and Spirulina platensis. It
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has a considerable high content of micro and macronutrients.Its dry weight chemical
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composition includes 60-70% proteins, carbohydrates, vitamins like provitamin A, vitamin C,
84
vitamin E, minerals such as iron, calcium, chromium, copper, magnesium, manganese,
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phosphorus, potassium, sodium and zinc. Essential fatty acids γ-linolenic acid (GLA),
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pigments like chlorophyll a, phycocyanin and carotenes are also present. Spirulina is also
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used in cosmetics, medicines and waste water treatment. Its cell wall consists of
88
polysaccharides that have a digestibility of 86%, and can be easily absorbed by the human
89
body (Sjors 2010). These microalgae contain chlorophyll a, like higher plants; therefore it is
90
classified as microalgae according to botanists belonging to Cyanophyceae class; and
91
bacterium due to its prokaryotic structure according to bacteriologists (Koru 2009;
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Sudhakar and Premlatha 2015).
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Spirulina is a planktonic photosynthetic cyanobacterium that forms huge populations in
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tropical as well as subtropical bodies of water which contain a high amount of salts such as
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carbonate and bicarbonate with alkaline pH 9.5(Sjors 2010; Habib et al 2008). Generally,
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microalgae have higher growth rates, higher CO2 fixation efficiency and larger quantities of
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ACCEPTED MANUSCRIPT high-value products, such as dietary supplements for human along with animals (Zeng et al
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2011; Anupama 2000).Cost effectiveness and composition of cultivation media along with
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growth rate needs to be managed properly for commercially viable production. From ancient
100
times different media have been used for cultivation of Spirulina and monitoring its growth
101
rate i.e. Zarrouk's media (Zarrouk 1966), Rao's media, CFTIR media, OFERR media,
102
revised media (Raoof et al 2006).
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Past few decades have seen considerable progress in spirulina cultivation for nutritional
104
use however there is no substantial argument on the nutritional productivities, best
105
cultivation method, and ideal growth conditions. This review addresses these issues based
106
on prior publications and the author’s prior work in the large scale cultivation of spirulina
107
for nutritional products. The article starts with the illustration of spirulina growth chain
108
from identifying suitable strain to the final product.
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The present study focuses on growth rate, productivity, growth parameters, different
110
cultivation systems (outdoor and indoor systems), harvesting and drying techniques of
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Spirulina. This review focuses on following aspects:
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•
Strain selection and cultivation of Spirulina.
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•
Optimum parameters for growth of Spirulina.
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•
Harvesting and drying techniques of Spirulina.
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•
Commercial applications of Spirulina as pharmaceutical and nutraceuticals product.
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2. Review of growth system
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Cultivation of algae can be done in open systems like ponds, lakes or lagoons or in a closed
119
system (Singh and Sharma 2012). Presently, two major technologies are being considered
120
for the cultivation of Spirulina: closed photobioreactors (PBR) and open ponds. Both
121
approaches are used commercially to produce high-value products.
122
2.1 Open pond system
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Cultivation of algae in open ponds has been extensively studied (Vardaka et al 2016, Zhang
124
L et al 2015, Madhu, G. M et al 2015, Choi et al 2003, Vega 2005). Open ponds can be
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categorized into natural waters as lakes, lagoons, ponds and artificial ponds or containers.
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The most commonly used systems are shallow big ponds, circular ponds, tanks and raceway
127
ponds. Open systems are easier in construction and operation, results in low production and
128
operating cost (Ugwu et al 2008). The major drawback in open ponds includes poor light
129
utilization by the cells, evaporative losses, diffusion of carbon dioxide to the atmosphere, and
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ACCEPTED MANUSCRIPT requirement of large acres of land. Also, due to inefficient aeration in open cultivation
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systems, their mass transfer rates are very poor resulting in less biomass productivity. The
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growth also depends on location, season, temperature, pH level, nutrient and carbon - dioxide
133
supply (Cuaresma et al 2011).The other major drawback of open pond system is the
134
contamination by fauna and other fast growing heterotrophs. To expel the problems
135
associated with an open system, researchers have tried for closed systems (Singh and
136
Sharma 2012).
137
Table 1 summarizes the advantages and limitations of open ponds, photobioreactors and
138
hybrid system. Large quantities of algae can be grown but they are difficult to grow outdoor
139
as they easily get contaminated. This can be rectified by growing algae in greenhouses, which
140
protect them from foreign particles in the air. The optimally designed algae greenhouse and
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controlled environment systems can increase productivity 10 fold compared to outdoor
142
growth. Construction of greenhouse includes design and optimizing for improved biomass
143
yield. Controlled environment algae facilities are gaining momentum due to improved yields
144
and reduced contamination. The internal systems to control the internal humidity,
145
temperature, and carbon dioxide through the use of fans, vents, evaporative cooling, and
146
climate zoning is done (Sierra et al 2008). pH, nutrients, and bacteria are regulated in the
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water system through fertigation, oxygenation and also sterilization. Integrating the climatic
148
conditions, water, and nutrient systems with simulation allows us to provide exactly what that
149
algae facility needs, resulting in optimized yields. The open roof greenhouses design provides
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complete protection against undesirable weather conditions, while the full vertical vent
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promotes optimum light and air movement.
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158 159 160 161 162 163 Page 5 of 42
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Species Selection
Phase- I
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166 167
Macroalgae
Microalgae
Cyanobacteria
(a)
168
Growth System of Spirulina
170 Phase- II
171 172
174
Open Raceway Ponds
Photo bioreactors
(b)
176
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Growth Parameters of Spirulina
Phase- III
177 178 179 180
Mother culture
Media Selection
Mixing & Aeration
Phase- IV
184
191 192
Centrifugation
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190
Normal Filter
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189
Growth Rate& Productivity
Harvesting System of Spirulina
183
188
Light Intensity
(c)
182
187
Temp. & pH
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186
Hybrid System
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(d) Drying
Grinding/Powdering
Pellets/ Capsules
193
(e)
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Figure 1 : Different phases of Spirulina cultivation system (a) strain selection (b) Growth systems (c) growth parameters (d) Harvesting system (e) Final product as capsules or pellets.
195 196
Figure 1 illustrates the flowchart for Spirulina cultivation phases (from Phase a – Phase e)
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from strain selection to pellets formation Page 6 of 42
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2.2 Photobioreactors
200
A photobioreactor can be an enclosed, illuminated culture vessel designed for controlled
201
biomass production. Photobioreactor refers to closed systems that are closed to the
202
environment having no direct exchange of gases and contaminants with the environment. The
203
closed system commonly called as photobioreactors, is closed equipment which provides a
204
controlled environment and also results in high productivity of algae. Photobioreactors
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facilitate better control of culture environments such as carbon dioxide supply, water supply,
206
optimal temperature, efficient light intensity, culture density, pH levels, gas exchange,
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aeration and culture density. Algal culture systems can be illuminated by artificial or natural
208
light or by both. Naturally illuminated algal culture systems with large illumination surface
209
areas include open ponds (Hase et al 2000), flat-plate (Hu et al 1996), horizontal/serpentine
210
tubular airlift (Camacho et al 1999], and inclined tubular photobioreactors (Ugwu et al
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2002). In order to overcome the problems with open ponds, much attention is now focused on
212
the development of suitable closed systems such as flat-plate, tubular, vertical column and
213
internally-illuminated photobioreactor. Generally, laboratory-scale photobioreactors are
214
illuminated artificially internally or externally using fluorescent lamps or other light
215
providers. Some of these photobioreactors include bubble column (Ugwu et al 2002, Degen
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et al 2001, Ogbonna et al 2002), airlift column (Chini et al 2003,Harker et al 1996),
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stirred-tank (Kaewpintong et al 2007), helical tubular (Ogbonna et al 1999) conical (Hall
218
et al 2003), torus (Watanabe and Saiki 1997], and seaweed type (Pruvost et al 2006]
219
photobioreactors. Some photobioreactors can be easily tempered. Large scale outdoor
220
systems mainly tubular photobioreactors cannot be easily tempered without high technical
221
efforts. Efforts have been taken in designing temperature-controlled photobioreactors, such as
222
double-walled internally-lighted photobioreactor with both heating as well as cooling water
223
circuit (Chetsumon et al 1998). Photobioreactors, despite their costs, have several major
224
advantages over open systems (Tsoglin et al 1996).
225
•Photobioreactors minimize the contamination and allow axenic algal cultivation of
226
monocultures.
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• Photobioreactors offer better control over conditions such as pH, temperature, light
228
intensity, carbon dioxide concentration etc.
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• Photobioreactors reduce carbon dioxide loss.
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• Photobioreactors prevent water evaporation.
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• Photobioreactors permit higher cell concentrations.
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ACCEPTED MANUSCRIPT • Photobioreactors enhance the production of complex biopharmaceuticals.
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• PBR permits the cultivation of various microalgal species.
234
• PBR design provides the uniform illumination of the culture surface and the fast mass
235
transfer of carbon dioxide and oxygen.
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• PBR has a minimum non-illuminated part.
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Table 1.Comparison between Spirulina production in open, closed and hybrid system
238
(Roberto 2015).
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Closed systems
Hybrid system
(raceway ponds)
(photobioreactors)
(Open Pond + PBR)
Space required
High
Low
High
Area/volume ratio
Low (5–10 m−1)
High (20–200 m−1)
Evaporation
High
Water loss
Very high
CO2-loss
High
Temperature
Highly variable
Weather dependence
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Open systems
Factor
Variable Minimized
Low
Less
Low
Minimizes
Required cooling
Controlled
High
Low
Low
Process control
Difficult
Easy
Difficult
Cleaning
Easy
Required
Difficult
Reproducible
Better
Medium
High
Medium
Medium
High
High
High
Lower
High
Poor
Good
Better
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No evaporation
Population density Harvesting efficiency
Harvesting cost
Light utilization efficiency Most costly parameters
Mixing
Oxygen and temperature control
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Temp control
ACCEPTED MANUSCRIPT Closed systems
Hybrid system
(raceway ponds)
(photobioreactors)
(Open Pond + PBR)
Difficult
Easy
Easy
Capital investments
Low
High
Low
Productivity
Low
control
Hydrodynamic stress on Spirulina
3–5 times more productive
Very low
Low–high
5-7 times more productive Low
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Contamination
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Open systems
Factor
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2.2.1 Vertical-column PBR
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Vertical-column photobioreactors are easy to operate compact and low-cost (Miron et al
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2002). Various designs and scales of vertical column photobioreactors have been reported for
243
the cultivation of algae (Choi et al 2003, Estrada et al 2005; Kaew et al 2007) which are
244
very promising for large-scale cultivation. It was reported that bubble-column and airlift
245
photobioreactors (up to 0.19 m in diameter) can attain a final biomass concentration and a
246
specific growth rate that are comparable to tubular photobioreactors (Sanchez Miron et al
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2002). Some bubble column photobioreactors are equipped with either draft tubes or
248
constructed as split cylinders.
249
2.2.2 Flat plate PBR
250
For cultivation of photosynthetic microorganisms flat-plate photobioreactors have received
251
much consideration due to their large illumination surface area. The work reported paved a
252
way to use flat culture vessels for the cultivation of algae (Samson and Leduy 1985). A flat
253
reactor was developed and equipped with fluorescence lamps (Ramos and Roux 1986].
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After this, an outdoor flat panel reactor was developed using thick transparent PVC materials
255
(Tredici and Materassi 1992). Later extensive works were reported on various designs of
256
flat plate reactors and vertical panels for mass cultivation of different algae ( Hu Q et al
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1996, Zhang et al 2002, Hoekema et al 2002, Olguin et al 2003). Flat plate
258
photobioreactors are constructed using transparent materials for maximum utilization of solar
259
light.
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photobioreactors is relatively high as compared to flat-plate photobioreactors. It has been
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reported that high photosynthetic efficiencies can be achieved with flat-plate photobioreactors
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Accumulation
of
dissolved
oxygen
concentrations
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in
horizontal
tubular
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(Hoekema et al 2002, Olguin et al 2003). Overview of spirulina productivities reported in
263
the literature for various growth systems is presented in Table 2. Among all culture systems
264
productivity of Spirulina platensis is highest in raceway ponds. The areal productivity is
265
generally based on one-hectare ground surface area.
266
Table 2 Spirulina productivity and Photosynthetic efficiency
system
Light
Photosynt
Productivity Reference
path
hetic
(ton ha-1yr-
Efficiency
1
1.5%
43.1
1.5%
20.0
Raceway
La
0.1-
pond
Mancha,
0.25
)
Raceway
Florence,
pond
Italy
Raceway
Malaga,
pond
Spain
Raceway
Australia
0.035
Tubular
Italy
Flat Panel
US
1992)
1.5%
23.6-30.0
(Jimenez et al 2003)
0.30
1.5%
91.0
(Borowitzka 1999)
30.0
(Tredici and Materassi
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Florence,
(Tredici and Materassi
0.30
pond Horizontal
( Olguin et al 2003)
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Reactor
0.06
3.8%
1992) 22.1
EP
0.10
1.8-3%
(Richmond and Zhang, 2001)
2.2.3 Tubular PBR
268
A tubular photobioreactor is the most suitable types of bioreactors for outdoor mass
269
cultivation (Kaewpington et al 2007). Mostly outdoor tubular photobioreactors are
270
constructed either with glass or plastic tube. They can be horizontal/ serpentine (Chaumont
271
et al 1988; Molina et al 2001; Pirt et al 1983; Watanabe and Saiki 1997], vertical
272
(Tredici and Chini 1998] conical (Lee and Low 1991) inclined (Ugwu et al 2002; Torzillo
273
et al 1986). Mixing and aeration of the cultures in tubular photobioreactors are usually done
274
by air-pump or airlift systems. Mass transfer becomes a problem when tubular
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photobioreactors are scaled up. Many have reported that very high dissolved oxygen (DO)
276
levels are easily reached in tubular photobioreactors [Sanchez et al 2002;Pirt et al
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ACCEPTED MANUSCRIPT 1983;Richmond et al 1993;Ugwu et al 2003; Ugwu et al 2005a]. It is difficult to control
278
culture temperatures in most tubular photobioreactors. They can be equipped with a
279
thermostat to maintain the desired culture temperature and it could be very expensive and
280
there will be difficulties in implementing.
281
2.2.4 Internally-Illuminated PBR
282
These photobioreactors can be internally illuminated with fluorescent lamps. Air and CO2 are
283
supplied to the cultures through the spargers with continuous agitation by impellers. This
284
photobioreactor can also be modified in such a way that it can utilize both solar and artificial
285
light system (Ogbonna et al 1999). The artificial light source is used whenever the solar light
286
intensity decreases below a set value as during cloudy weather or at night. It has been
287
reported, on the use of optic fibers to collect and distribute solar light in cylindrical PBR
288
(Mori 1985, Matsunaga et al 1991). A major advantage of internally-illuminated
289
photobioreactor is that it can be heat-sterilized under pressure and by this contamination can
290
be minimized. A continuous supply of light to the photobioreactor can be maintained both
291
day and night by integrating artificial and solar light devices. Outdoor mass cultivation of
292
algae in this type of photobioreactor would have some technical difficulties. Flat plate
293
photobioreactors are generally more efficient in sunlight utilization than tubular
294
photobioreactors because they have a wider surface area. Most early tubular PBRs used tubes
295
10–30 cm in diameter, but almost all tubular reactors used now have a tube diameter of 4 cm.
296
The narrower tube diameter not only improves the light utilization efficiency, but also
297
provides more mixing, which enhances growth (Tredici, 2004). In photobioreactors (PBRs),
298
the microalgae get adheres to the transparent surfaces which lead to biofouling and along
299
with it reduces the solar radiation penetration the PBR. Light intensity reduction within the
300
PBR reduces the biomass productivity which also reduces the photosynthetic efficiency of the
301
Spirulina cultivation system. Adherence of the cells to wall tubes is very common in tubular
302
photobioreactors.Designing of photobioreactor surfaces with proper materials, functional
303
groups or surface coatings, to prevent microalgal adhesion is essential for solving the
304
biofouling problem. Such a significant advance in microalgal biotechnology would enable
305
extended operational periods at high biomass productivity and depreciate the maintenance
306
costs (Zeriouh et al, 2016).
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Table 3: Prospects and limitations of various cultivation systems (Chojnacka, et al 2004;Vymazal 1990;Ugwu et al 2008;Vree et al 2015, Newsted 2004) Dimensions
systems
Specific
Prospects
Limitations
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Culture
growth rate Variable
0.30day-1
systems
Relatively economical, easy to Little control of culture conditions, clean up after cultivation,
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Open
difficulty in growing algal cultures
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good for mass cultivation of for long periods, algae
poor productivity,
occupy large land mass, limited to few strains of algae,
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cultures are easily contaminated
Vertical
0.2 m
0.015 ±
High mass transfer,
Column
diameter
0.002
good mixing with low shear construction require sophisticated
h )
stress,
EP
and 4 m
materials,
column
low energy consumption,
shear stress to algal cultures,
height[
high potentials for scalability,
the decrease of illumination surface
easy to sterilize,
area upon scale-up
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−1
Small illumination surface area,
good for immobilization of algae, reduced photoinhibition and
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Images
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photo-oxidation 0.07 m
Large
surface Scale-up
PBR
wide,
area,
compartments
1.5 m height
suitable for outdoor cultures,
materials,
, 2.5 m
good for immobilization of
difficulty in controlling culture
length
algae,
temperature,
Volume
good light path, good biomass some degree of wall growth,
250lts
productivities,
Productivity
relatively cheap,
- 1.0 g/L
easy to clean up,
day
readily tempered,
illumination
area,
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dissolved oxygen and CO2 along
suitable for outdoor cultures, fairly
support
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Large
good
productivities,
EP
0.055 h−1
and
stress to some algal strains
the tubes, fouling,
biomass some degree of wall growth,
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PBR
D = 3-10cm
many
the possibility of hydrodynamic
low oxygen buildup Tubular
require
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illumination
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Flat plate
requires large land space
relatively cheap
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Internally
Not
Large
Illuminated
Specified
area,
require some technical efforts.
can utilize both solar and
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PBR
surface Outdoor mass cultivation of algae
illumination
artificial light system, contamination
can
be
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minimized in this system Not
Minimize
microbial Requires large areas of land and
System
Specified
contamination,
maximize some technical efforts
biomass and product yield, Maximize CO2 supply
site flexibility,
Polyethylene bag cultures have a
Low-cost materials, easy scalability,
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0.20day-1
relatively short life because the internal surface attracts culture
optimal light exposure,
debris
EP
D <30cm
and
bacteria,
which
isolation of the crop from collectively reduce light penetration predators, very
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Poly Bags
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Hybrid
high
and are a source of contamination
biomass
concentration,
low energy consumption effective weather protection
Page 14 of 42
ACCEPTED MANUSCRIPT 2.3 Hybrid system
2
A hybrid type of photobioreactor is most widely used to exploit the advantages of the two
3
different types of reactor and overcome the disadvantage of other. Integrated airlift system
4
and external tubular loop placed horizontally in a thermostatic pond of water have been
5
reported (Zittelli 2013). The reactor had a total volume of 200 L. The external loop acts like
6
the light-harvesting unit and gives high surface area to volume ratio and controls the
7
temperature of the culture. The airlift system acts as a degassing system where probes can
8
also be integrated in order to regulate the other culture variables. It has the advantage of
9
better control over culture variables, enabling higher productivities and reducing power
10
consumption (Pohl et al 1988; Singh and Sharma 2012; Ugwu et al 2008; Cuaresma et al
11
2011]. Hybrid systems have the features of open ponds and PBRs (Hoekema et al 2002).
12
First can be covered open pond this concept reduces the possibility of contamination,
13
evaporative losses, and CO2 desorption. The other type is a partially filled tubular design
14
widened and inflated to approximate an open pond; this design is mainly aimed at reducing
15
costs (Hoekema et al 2002; Olguin et al 2003; Tredici and Materassi 1992]. Some of the
16
advantages and limitations of various cultivation systems are listed in Table 3.
17
Polybags
18
The cultivation of algae using natural ponds is easy, but turning it into a viable feedstock is
19
very difficult. So to enable higher production levels, least investments and operating costs,
20
greater biomass density, better climatic controlled conditions, and industrial scalability, this
21
technique can be implemented. Thin, floating, flexible, multi-compartment photobioreactors
22
(PBR) can be deployed either on land, in salt water ponds or ditches, or in any water body.
23
The bag floats because its water is relatively less dense than what it is floating in. Density can
24
be controlled in different ways, allowing the bags to be vertical to facilitate harvesting. The
25
productivity results have indicated that growing algae in floating bags can be much more
26
efficient than other cultivation methods. Poly Bags achieve optimal light exposure with good
27
productivity results as they float in a cushion of water. Compared to other closed algae
28
systems, this PBR technology has many advantages, including site selection, optimum
29
temperature, low-cost materials, scalability, optimal light intensity, high biomass
30
concentration, low energy consumption and effective environmental condition (Licamele
31
and White 2011).
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ACCEPTED MANUSCRIPT The diameter of the culture vessel is inversely related to cell density with a fixed level of
33
light penetration. However, these bags are superior in productivity to similar rectangular
34
volume fiberglass reactors or plastic tanks. They are, nevertheless, inefficient when compared
35
with internally illuminated cultures. Polyethylene bag cultures have a relatively short life
36
because the internal surface attracts culture trash and bacteria, which collectively reduces
37
light penetration and also increases contamination. At the end of a culture run it is necessary
38
to renew the bag. Large diameter bags are inefficient but bags less than 30 cm diameter can
39
be effective because the surface area to volume relationship for light penetration is improved
40
(Algae Industry Magazine, 2012).
41
3. REVIEW OF GROWTH PARAMETERS
42
Spirulina growth requirements are similar to terrestrial plants but they use these resources
43
very efficiently to increase biomass productivity with comparatively less water use
44
(Sudhakar et al 2011)
45
3.1 Climatic factors
46
Temperature is an important climatic factor influencing the rate of growth of Spirulina.
47
Below 17°C, growth is practically nil, but Spirulina does not die. The optimum temperature
48
for growth is 35°C, but above 38°C Spirulina growth is inhibited. Light is an important factor
49
but direct sunlight is not recommended, 30% of full sunlight is actually better, except that
50
more may be required to quickly heat up the culture in the morning (Saeid and Chojnacka,
51
2015). Growth takes place only in the light, but illumination 24 hours a day is also not
52
recommended. During dark periods, chemical reactions take place within Spirulina, like a
53
synthesis of proteins and respiration.
54
3.2 Media
55
Different culture media are used to start new cultures according to the water source. The
56
water used should be clean or filtered to avoid growth of other algae. Water often contains
57
enough calcium, but if it is too hard it will cause muds. Portable water is convenient whereas
58
RO treated water is the best to grow Spirulina. The make-up media mainly consist of urea.
59
Carbonate is replaced by bicarbonate. Urea, certain ions may be present as sulphate, chloride,
60
nitrate, and sodium which is more efficient to supply nitrogen but is highly toxic with large
61
concentration. Spirulina can grow on either nitrate or urea alone, but using both at the same
62
time is advantageous. Phosphate, magnesium and calcium cannot be increased much.
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ACCEPTED MANUSCRIPT Potassium concentration can be increased accordingly, provided it does not become more
64
than five times the sodium concentration. If fertilizer grade chemicals are used for cost
65
reduction, they should be of the soluble or crystallized type, not of the slow release,
66
granulated type. There are different media preparations according to the local growing
67
conditions. Most commonly used is zarrouks media (Zarrouk 1966; Pragya et al
68
2013).Chemical compositions of different growth media are compared in Table 4.
69
Table 4: Chemical composition of different growth media ( Madkour et al 2012, Atlas and
70
Parks 1997; Venkataraman etal 2005; Pandey et al 2010)
s
CFTRI
OFERR
Media(g
Media(g
Media(g
George’s
onal
ms/l)
ms/l)
ms/l)
Media(g
growth
ms/l)
Media(g
Media(g ms/l) 16.80
15
K2HPO4
0.50
0.50
NaNO3
2.50
2.50
K2SO4
1.00
NaCl
1.00
H2O
CaCl2·2H2 O
FeSO4·2H2 O H3BO3 MnCl2·4H 2O
0.08
AC C
EDTA
0.20
0.04
0.01 2.86 1.180
Cost Media(g ms/l)
8.0
-
16
16.8
0.5
-
0.02
-
0.235
1.5
-
-
-
-
0.60
1.0
0.5
-
0.5
0.353
0.20
1.0
5.0
-
1.00
0.471
0.04
1.2
0.16
0.02
0.1
-
-
-
-
-
-
0.353
0.008
0.04
-
-
0.1
0.176
-
0.01
0.05
-
-
0.265
-
-
0.052ml
-
-
2.86
-
-
-
-
-
1.81
EP
MgSO4·7
ms/l)
Reduced
4.5
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NaHCO3
SC
Ingredient
Conventi
Rao’s
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Page 17 of 42
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2O
NH4VO3 NiSO4·7H2 O NaWO2 Ti2(SO4)3· 6H2O Co(NO3)2· 6H2O Ferric citrate
0.074 22.9 47.8 17.9 4.4
-
(NH4)2HP
Chelated Iron
-
AC C
O4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
0.222
-
-
0.0177
-
-
0.079
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
0.035
-
-
-
-
-
1.00
-
-
-
-
-
-
2.00
-
-
-
-
-
0.1
-
-
-
-
EP
KNO3
-
-
-
-
-
4.4
Peptone
-
RI PT
CuSO4·5H
0.015
-
SC
Na2MoO3·
-
M AN U
2O
0.222
TE D
ZnSO4·7H
2 squeezes
-
-
(¼
-
teaspoon )
Lime
-
-
-
-
-
0.1
-
NH4NO3
-
-
-
-
-
-
0.118
CO (NH2)2
-
-
-
0.2
-
-
0.088
Page 18 of 42
ACCEPTED MANUSCRIPT Fe EDTA
-
0.20
-
-
-
-
-
A5 solution
-
1ml
-
-
-
-
-
71
3.3 Mother culture
73
For Inoculums preparation and culture maintenance fully grown concentrated Spirulina
74
culture is required. The chosen Spirulina strain must have a high proportion of coiled
75
filaments (< 25% straight filaments, or none), and at least 1 % of gamma-linolenic acid
76
(GLA) based on dry weight. Concentrated Spirulina seed culture can be obtained either from
77
the floating layer of a composed culture, or by diluting a freshly filtered biomass. Colour of
78
the culture should be clearly green.The growth rate is about 30 % /day when the temperature
79
and other climatic conditions are adequate (Pal et al 2011). As the growth is proportional to
80
the area of the culture exposed to light, it is recommended to maximize this area at all times.
81
It is reported that minimum cell population is necessary to initiate and sustain Spirulina
82
cultures.
83
3.4 Mixing and aeration
84
Agitation of the culture is necessary to homogenize and ensure a good distribution of lighting
85
among all the filaments of Spirulina. Mixing plays an important role in the productivity of
86
ultrahigh density cultures. Aeration is very necessary for getting good quality and better
87
yields of Spirulina species. It can be achieved by rotators, which maintain the cells in
88
suspension by gentle agitation of growing cells. The Spirulina species produces high biomass
89
yield when the growth medium is aerated (bubbling with air). Aeration gives a homogenous
90
distribution of the Spirulina filaments throughout the growth system for adequate exposure to
91
illumination. It also helps to distribute carbon dioxide concentration uniformly and removes
92
inhibitory substances as oxygen (Dubey 2006; Richmond and Vonshak 1978). Aeration is,
93
therefore, essential for the cultivation of the Spirulina filaments such as Spirulina platensis
94
(Famelart et al 1987; Powls 1985). Adequate and turbulent mixing is essential for higher
95
biomass productivity(Chisti, 2016). Mixing of raceway pond is effected by means of a paddle
96
wheel. Mixing velocity of 5-60 cm/s has been used by many researchers. Low velocities
97
result in dead zones around corners while high velocities incur high energy cost, and may
98
result in shear stress that damages the algae. It also noted that continuous mixing of the
99
culture medium is required to prevent cell sinking and thermal stratification. It is also
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ACCEPTED MANUSCRIPT required to maintain even nutrient distribution, and to remove excess oxygen. When aeration
101
is not adequate, the efficiency of energy utilization and biomass production will be low.
102
Similarly, if growth medium is not aerated, the cell on the surface of the medium float to the
103
surface due to the presence of air-filled vacuoles. These cells suffer photoinhibition, resulting
104
in low growth or low biomass production. The optimal conditions for spirulina were found to
105
be at a light intensity lower than 200 µmol m−2 s−1, CO2 enriched air flow (0.5%), superficial
106
aeration rate of 0.0056 m s−1 in a NaHCO3-free Zarrouk medium(Zhang, L et al 2015).
107
3.5 Temperature and pH
108
Spirulina can grow at 200C- 370C. The best temperature for Spirulina growth is between 290C
109
- 350C. During night growth of Spirulina is least or almost zero. It is reported that the effect
110
of pH on the algal growth, pigment production and protein content of Spirulina species has
111
the direct effect on the antioxidant system (Vonshak and Guy 1987; Ogbonda et al 2007).
112
The growth may be affected in two ways
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113
•
Available carbon alteration, which may interfere with photosynthesis.
114
•
Through the disruption of cell membrane processes.
This may have a direct impact on the accumulation of antioxidants (Matsunaga et al 1991).
116
Moreover, factors such as nutrient availability, ionization and heavy metal toxicity have large
117
impacts on algal metabolism (Newsted 2004).The fluctuation in atmospheric temperature is
118
the main factor affecting the biomass production rates in outdoor Spirulina cultivation. In the
119
rainy season the culture may become contaminated due to raindrops resulting in lowest dried
120
mass. The physical factors which are not favorable in monsoon can be controlled by using the
121
locally available techniques (Pandey 2010). The warm humid environment causes the
122
bacterial contamination. The main contaminants of the Spirulina culture were protozoan like
123
amoeba and paramecium which ultimately spoils the cultures. During the monsoon season
124
insects also appears in the culture and make it unfit for human consumption. To reduce the
125
effect of the low-temperature Spirulina cultures can be kept in the house made of a plastic
126
sheet. When pH is between 9-11, it indicates a healthy culture. It also assures that other
127
strains are prevented from contaminating the tank as they simply can’t live in the alkaline
128
environment that Spirulina grows in
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129 130
3.6 Light intensity
131
All photoautotrophic organisms including photosynthetic bacteria, cyanobacteria and higher
132
plants, convert light energy into chemical energy through photosynthesis. It is reported that
Page 20 of 42
ACCEPTED MANUSCRIPT light quality, intensity and duration are important factors of algal production (Sudhakar and
134
Premlatha 2012; Lucie et al 2016). In an outdoor cultivation system, natural light or solar
135
radiation is the whole sole source of light. Light availability is totally dependent on
136
geographical area, climatic conditions, seasonality and local atmosphere. Spirulina makes its
137
own food in the presence of optimal light. The requirement of light intensity for growth
138
varies from organism to organism. Spirulina also requires a specific range of intensity for its
139
growth (Sudhakar et al 2012) Zarrouks did the first detailed study on the response of
140
Spirulina maxima to light (zarrouk 1966). The optical density of the culture is directly
141
proportional to the light intensity. Higher the optical density higher is the requirement of light
142
and lower is the optical density, lower is the requirement of light (Samuel et al 2010). The
143
light intensity is an important variable in cyanobacteria cultivation. High values of light
144
intensity promote growth parameters such as maximum specific growth rate, whereas low
145
values result in a biomass that is rich in pigments and proteins.Outdoor algal cultures are
146
exposed to two rhythms of the dark and light regime. These cycles impose a unique
147
physiological regime on the adjustment or acclimatization of outdoor algal cells to light.
148
Increasing the cell concentration of culture, increases the self-shading and results in a
149
decrease of the growth rate of Spirulina.
150
The attenuation coefficient was observed to scale linearly with microorganism density.
151
The irradiance attenuation coefficient at wavelength λ, αλ, is calculated according to,
152
Gλ(z)/Gλ(0) = e −αλz
153
The Spectral Irradiances at different depths z is calculated according to above equation
154
knowing the value of αλ,
155
The spectral attenuation cross section, Aλ, is defined as, Aλ = αλ/X
156
Using the irradiance attenuation coefficients for each culture, αλ,
157
, Gλ(z) = Gλ(0)e −αλz
158
Where Gλ(z) = spectral irradiance at depth z
159
Gλ(0)= Incident spectral irradiance just below the culture surface.
160
X = microorganism density in grams of dry biomass per liter (g/l).
161
It has been observed that decreasing the depth of a pond from 20 cm to 10 cm achieve the
162
targeted biomass density of 0.19 grams dry biomass per liter (g/l).The shallower ponds
Eq.1
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Eq.2
Eq.3
Page 21 of 42
ACCEPTED MANUSCRIPT achieve greater biomass densities with a decrease in monetary costs of dewatering and
164
harvesting the resultant biomass.
165
Bezerra et al , 2011 reported that the maximum cell concentration (Xm) increased from 5200
166
to 5800 mg L−1 when the light intensity was increased from 36 to 72 µmol photons m−2 s−1,
167
highlighting growth limitation by light intensity within this irradiance level. On the other
168
hand, an additional increase in light intensity up to 108 µmol photons m−2 s−1 led only to a
169
reduction in the cultivation time from 8 to 6 days. Similar results were obtained by Danesi et
170
al. (2004) using urea as a nitrogen source in the light intensity range of 2–5 klux. This
171
behavior suggests that, at a relatively high light intensity (108 µmol photons m−2 s−1), cell
172
growth was accelerated by the faster photosynthetic production of ATP and NADPH; but,
173
when cell concentration reached 5800 mg L−1, the growth stopped likely due to photo
174
saturation or shadowing.
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175
3.7 Growth rate & productivity
177
Salinity or nutrient concentration affects the growth rate of algae. Specific growth rates of
178
Spirulina were reported to be lower in increased salinity concentrations. The highest growth
179
was achieved at the lowest salinity ratio for studies performed with various concentrations of
180
NaHCO3 and NaCl salts. The growth rate of Spirulina undergoes simple cell division. Thus,
181
under normal growth conditions the specific growth rate is described by the following
182
equation:
EP
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176
µ=
t dx x dt
Eq. 4
Calculation of specific growth rate has been described in many ways. Most commonly used
185
formula is
186
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184
µ=
ln x2 − ln x1 t 2 − t1
Eq. 5
187
Where x1 and x2 are biomass concentration at time interval t1 and t2 The simple equation that
188
combines the specific growth are (µ) and the doubling time or the generation time (g) of the
189
culture is:
190
g=
ln 2
µ
==
0.693
µ
= d .t
Eq. 6
191
Cell productivity (PX) is a function of the independent variable, which is described as the
192
lowest difference in the cultivation time (TC). Page 22 of 42
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194
biomass productivity is the nutritional content of the culture medium. The use of certain
195
nutrients can alter production costs and affect growth or biomass composition (Grobbelaar
196
2007; Sassano et al 2007). Annual biomass production of Spirulina in PBRs is 3000 tonnes
197
which are maximum when compared to other microalgae species (Jayati et al 2015,
198
Bharathiraja et al 2015) Productivity is a measure of how much algal biomass is produced
199
per area per unit of time. Production up to 127,000 kg ha-1 yr-1 can be achieved in high-rate
200
raceway ponds. Productivity rates between 20 and 30 gm-2day-1 (73–109,000kg ha-1yr-1) are
201
in the range of usual open raceway performance (Bharathiraja et al 2015).
202
The productivity of the system γ is defined as
SC
γ =µx
203
RI PT
193
Eq. 7
Where µ is the specific growth rate in units of reciprocal of time and x is the biomass
205
concentration.
206
The cell productivity (PX) is calculated as the ratio of the variation in cell concentration (Xm-
207
Xi) to the cultivation time (TC)
208
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Eq. 8
As demonstrated in the earlier work (Miron et al 1999; Neils et al 2011), there is an optimal
210
biomass concentration which corresponds to the highest productivity.
211
Masojı´dek et al. (2003) applied a peristaltic pump as circulation apparatus to cultivate
212
Spirulina platensis and obtained a cell productivity of 0.5 g/ L/day, which was considered a
213
relatively high value in open pond cultivation system.
214
Toyoshima et al 2015 reported the maximum biomass productivities of Spirulina platensis in
215
the warm temperature habitat. 9 g dry biomass m−2 day−1 in summer and in the subtropical
216
habitat 10 g dry biomass m−2 day−1 in autumn and. 6 g dry biomass m−2 day−1 in winter in the
217
closed bioreactor. The maximum specific growth rate of 0.141 was found at 320C for
218
Spirulina platensis and that of 0.144 was found at 37 0 C for Spirulina fusiformis.Maximum
219
biomass production of 2.4 g l-1 and chlorophyll a production of 16.6 mg l -1 were observed at
220
320C for Spirulina platensis. Maximum biomass production of 2.3 g l
221
production of 14.2 mg l -1 were observed at 370C for Spirulina fusiformis ( Rafiqul Islam, M
222
et al 2003, Allen, K. A 2016). Spirulina, (Arthrospira platensis) is normally cultivated in
223
high salinity (>100 g/L) media or in high bicarbonate (16 g/L alkalinity) waters to allow
224
stable growth and reduce the harmful bacteria and fungi invasions. The maximum
225
productivity of biomass Spirulina is in the range of 21 -13.2 g m2 /d (Vonshak A
226
1997).Maximum biomass yield of Spirulina reported in the large open pond is lower than
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-1
and chlorophyll - a
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other species.Spirulina biomass yield of 35 tonnes/ hectare/yr has been reported in a
228
commercial open mass cultivation pond at Siam Algae, Bangkok (Habib et al 2008)
229 230
4. Review of harvesting system
231
The best time for harvesting is early morning for following reasons •
Percentage of proteins in the Spirulina is highest in the morning.
233
•
Cool temperature makes the work easier.
234
•
More sunshine hours will be available to dry the product.
•
culture medium,
237 238
•
Removals of the residual culture medium to obtain the fresh Spirulina biomass, containing about 20 % dry matter.
239 240
Filtration - to obtain a biomass containing about 10 % dry matter and 50 % residual
SC
236
Harvesting is carried out in two steps:
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Different harvesting techniques used are •
filtration,
242
•
flotation,
243
•
centrifugation,
244
•
precipitation,
245
•
ion exchange,
246
•
Electrolytic and
247
•
Ultrasonic vibration.
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Harvesting of microalgae Spirulina is done using a filter or mesh cloth of at least 50 microns
249
to efficiently collect Spirulina from its medium..
250
4.1 Centrifugation
251
Centrifugation is a method to separate Spirulina algae from the media. Centrifugation and
252
chemical precipitation are economically feasible, where centrifugation being in appreciably
253
better A centrifuge is an equipment, driven by a motor, that puts an object in rotation around
254
a fixed axis, applying a force perpendicular to the axis. This method is reasonably efficient,
255
but sensitive algal cells may be damaged by pelleting against the rotor wall. Centrifugation
256
and drying are currently considered too expensive for personal use, though viable on a
257
commercial and industrial scale. The centrifuge works using the sedimentation principle,
258
where the centripetal acceleration is used to evenly distribute substances of greater and lesser
259
density.
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261
During commercial production processes filtration devices are used for harvesting. These are
262
of two types, i.e. inclined or vibrating screens. Inclined Screens are 380-500 mesh with a
263
filtration area of 2-4 m2 per unit and are capable of harvesting nearly about 10-18 m3 of
264
Spirulina culture per hour (Ogbonna et al 1999). Efficiencies of biomass harvesting are very
265
high which nearly 95%. Inclined, stationary screen is considered as a better solution for
266
harvesting Spirulina. Vibrating screens filter the same volume per unit time as the inclined
267
screens, but require one-third of the area. Their harvesting efficiencies are often very high.
268
The combination of both inclined filter and a vibrating screen is used. In the process of
269
pumping the algal culture, the Spirulina filaments may be damaged physically. Repeated
270
harvesting leads to the increasing enrichment of the culture with unicellular microalgae or
271
short filaments of Spirulina, which can pass through the screen easily. According to the work
272
reported in large-scale production of Spirulina the vibrating screen may not be the optimum
273
device for harvesting. Next step is the washing of excess salts from the biomass. The washed
274
cake is frequently homogenized before being dried.
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275
4.3 Drying
277
Though Spirulina can be consumed fresh, it has to be used after slight drying (Ankita et al
278
2013). Spirulina should be consumed within 6 hours of its harvest although it can be
279
preserved for later consumption for a period of up to one or more year by sun drying or in
280
greenhouses or in a solar drier.
281
Spirulina is relatively easily digestible in its fresh form (Richmond and Vonshak 1978).
282
Health and nutrition companies have tried to minimize the nutrients lost during drying and
283
maximizing the pure microalgae biomass recovered, while still keeping cost effective (
284
Sierra et al 2008). Different drying methods include sun drying, freeze drying, spray drying,
285
drum drying and cooking. Since Spirulina has a thin, fragile cell wall so, sun drying is
286
sufficient to sterilize the algae and make it consumable. Sun drying is the most popular
287
drying method, but requires a few precautions. Direct sun drying must be very quick,
288
otherwise the chlorophyll will be destroyed and the dry product will appear blue.
289
industries spray drier is used for Spirulina which flash dries fine droplets at very high
290
temperature and yields an extremely fine powder of low apparent density. Although freeze
291
drying considered as the best way of drying but far too expensive and complicated. The
292
biomass to be dried must be thin enough to dry before it starts fermenting. Fundamentally
293
two types of shapes are used, thin layers of rather fluid biomass laid on a plastic film, and
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In
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295
the film, while in the later case it flows vertically through the tray. The rod shape is
296
theoretically better as evaporation can take place all around; rods are obtained by extrusion to
297
a diameter of 1 to 2 mm. But rods must be sturdy enough to maintain their shape, so this type
298
of drying is restricted to biomass that can be dewatered by pressing. The total duration of the
299
drying should not be less than 2 hours. Drying temperature should be limited to 68°C and
300
drying time is limited to 7 hours. For better preservation under storage, moisture should not
301
exceed 3-4%. During the drying process as well as afterward the product must be protected
302
against contaminations from dust and insects and should not be touched by hands. Incipient
303
fermentation during drying can be detected by smelling during the drying process as well as
304
afterward. For long time storage of Spirulina, it is vacuum dried and packed air‐tight where it
305
sustains its nutritional qualities for at least five years. The best storage is in heat sealed,
306
aluminized plastic bags.
307
As far as drying treatment is concerned, significant amounts of energy are needed to
308
evaporate water from the high moisture containing biomass. The evaporation energy
309
for 1 kg of water is 2.257 kJ, while depending on the drying equipment the efficiency of
310
the process varies. In the case of solar drying the efficiency is considered to be around
311
50 % since the material is exposed to open air, while for vacuum drying the efficiency
312
can rise up to 80 %. Taking into consideration the final moisture content that can be
313
achieved, specifically 4 % and 2.5 % for solar and vacuum drying, respectively, the
314
amount of cultivated and harvested biomass that it is needed to acquire 1 kg of dried
315
material differs. (Papadaki et al 2017)
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4.4 Grinding/powdering
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The dry chips or rods are usually converted to powder by grinding to increase their apparent
319
density. Spirulina is used as a whole food/ dietary supplement which is available in tablet,
320
flake and powder form (Figure 2). Spirulina can be directly ground to ultra fine powder
321
form. It is also used as a feed supplement in the aquaculture, aquarium and poultry industries.
322
Commercial Spirulina is most often sold as a deep green-coloured powder or a tablet. It is
323
used as an ingredient in packaged health food snacks and drinks. The strained Spirulina algae
324
paste is laid out and triple-washed with potable water for salt removal before it goes into a
325
drying vessel that converts it into powder form. The dried Spirulina flakes are crushed using
326
high impact ultrafine grinding mill. Grinding is continued for about 6 to 10 hours, till the
327
average powder size reaches 200 ~ 800nm. The two most common forms of commercially
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available Spirulina are powder and tablets. It is also an ingredient in some protein and
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energy-boosting powder mixes.
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Powdered Spirulina
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(a)
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Figure 2 :Dried Spirulina (a) Spirulina flakes (b) Powdered form of Spirulina
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(b)
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4.5 Pellets/ capsules
335
Spirulina powder is pressed together into a tablet or granule shape ( Ogbonda et al 2007) for
336
improved acceptance and performance. It is formulated as a completely balanced diet which
337
provides optimum growth and health (Slade et al 2013). It contains proteinated trace
338
minerals for higher stability, biological availability and overall human health.
339
Advantages of Spirulina pellets are as follows
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Excellent water stability
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Easily consumable
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Contains extra levels of preservative and antioxidants
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Longer shelf life.
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4.6 Spirulina products
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Spirulina fights against aging, oxidative stress, diabetes, cardiovascular diseases,
347
hypertension, arthritis, infertility and cancer. Spirulina is considered as a superfood as it is
348
the best food supplement. Different healthcare industries make Spirulina products. Major
349
companies which are involved in cultivating spirulina globally are :
350
Earthrise Nutritionals (USA California) (earthrise.com)
351
DIC Lifetec Spirulina (Japan) (dlt-spl.co.jp/business/en/spirulina/)
352
Cyanotech Spirulina (USA Hawaii) (cyanotech.com)
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Boonsom Spirulina Farm (Thailand) (boonsomfarm.com)
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FEBICO (Far East Bio-Tec Co.) (Taiwan) (febico.com)
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Spirulinea (France/Laos) (spirulinea.com)
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ACCEPTED MANUSCRIPT Spiruline de Burkina (Burkina Faso) (spirulineburkina.org)
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Green Valley (Germany) (greenvalley.de)
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Natesis Spirulina (France) (natesis.com)
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Spirulina.PL (Poland) (spirulina.pl)
360
All Seasons Health (United Kingdom) (allseasonshealth.com)
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NaturKraftWerke Spirulina (Switzerland) (naturkraftwerke.com)
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Sanatur Spirulina (Germany) (sanatur.de)
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Marcus Rohrer Spirulina (Netherlands) (spirulina.nl)
364
Taiwan Chlorella (Taiwan) (taiwanchlorella.com)
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RBC Life Sciences (USA) (rbclifesciences.com)
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Gerophyta Nutraceuticals Company in Tamil Nadu, India offers a wide range of products,
367
as Spirulina Powder, Spirulina Capsules, Spirulina tablets, Spiruvita-C, Dr. Spirulina Diavita-
368
C, Spirulina herbal face pack. Other companies also offers a wide range of products as
369
spirulina bar, spirulina green tea, Spirulina personal care products, Spirulina chocolates,
370
spirulina drinks, spirulina honey etc.
371
5. Spirulina benefits
372
5.1 Nutritional composition of Spirulina
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Spirulina is a microalga that has been consumed for decades due to its high nutritional value
374
and reported health benefits. Today Spirulina is endorsed as a secret, potent superfood, also
375
considered as the miracle that grows naturally in oceans and salty lakes in subtropical
376
climates. Spirulina contains practically all the components found in the ideal complete food.
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A considerable proportion of proteins, vitamins, mineral salts, carbohydrates, pigments, trace
378
elements, and essential fatty acids are present. Unlike other algae, Spirulina is easier to
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consume
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Protein
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Spirulina is the richest source of proteins.Spirulina is abundant in plant protein, which makes
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up 60%-70% of its weight (Balasubramani, R et al 2016). Soya flour, contains about 35%
384
protein. Qualitatively, Spirulina provides complete proteins as it contains the full range of
385
essential amino acids which is 47% of total protein weight.
386 387
Vitamins
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ACCEPTED MANUSCRIPT The vitamins naturally found in Spirulina are ß-carotene, B1, B2, B12, E. Its ß-carotene
389
content is unusually high which is about 30 times higher than found in a carrot. Spirulina is
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also exceptionally rich in vitamin B12 cobalamin. This vitamin is, most difficult to get from a
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vegetarian diet because no fruit, vegetable, grain, or legume contains it. Spirulina has four
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times as much vitamin B12 than raw liver, which was considered to be the best source of this
393
nutrient. Spirulina is also recognized as an excellent source of vitamin E comparable to those
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found in wheat gram(Yin, C et al 2017). The primary antioxidant vitamins contained in
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Spirulina are ß-carotene, carotenoids, and vitamin E.
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Minerals
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Spirulina contains mineral such as iron, magnesium, calcium, and phosphorus. Spirulina is a
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splendid source of iron which contains 20 times more iron than wheat gram. Iron is a mineral
400
that is mainly present in foods from animals, such as meat, and fish (Roberto 2015;
401
Balasubramani, R et al 2016). Spirulina is very advantageous for athletes, vegetarians,
402
pregnant women, and teenagers. Average nutritional analysis of Spirulina per 100gm is
403
shown in Table 5.
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Table:5: Average nutritional analysis of Spirulina per 100g (Roberto 2015) Nutritional Value(in
mgs)
mgs) Calcium
1000
Phosphorus
800
2200
Magnesium
400
Minerals
8000
Iron
58
Dietary Fibre
7000
Zinc
3
Vitamin A
212
Copper
1.2
Chlorophyll
600
Manganese
0.5
Fat
22000
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63000
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Chromium
0.03
Vitamin B1
3.5
Potassium
1.4
Vitamin B2
0.4
Gamma-linoleic acid
1
Vitamin B3
1.3
Vitamin B8
0.005
Vitamin B5
0.2
Vitamin B9
Vitamin B6
6
Vitamin B12
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5.2 Pharmaceuticals and nutraceutical applications
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Spirulina is the best complete nutritional food source of protein, beta carotene, GLA, B
409
Vitamins, minerals, chlorophyll, sulfolipids, glycolipids, superoxide dismutase, phycocyanin,
410
enzymes, RNA, DNA, and supplies many nutrients that are lacking in most of the people's
411
diets. Nutraceutical food products supplement the diet as well as facilitate the prevention or
412
treatment of a disease or disorder. There are many Nutraceutical and Functional food
413
products which are commercially available with researched and approved health benefits. The
414
current estimated global market size for nutraceuticals products is approximately 30 to 60
415
billion dollars, which is primarily in the United States, Japan, and Europe. Spirulina products
416
have a potential short-term growth market demand of over 197 billion dollars. As the demand
417
for nutraceuticals and food supplements is increasing, organisms that can rapidly produce
418
nutritional compounds are in demand. The ability of Spirulina as a potent for anti-viral, anti–
419
cancer, hypocholesterolemic and health improvement agent is getting attention as a
420
nutraceutical and pharmaceutical.
421
Spirulina has the following health benefits
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•
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•
Nourishes people with digestion, assimilation & elimination
424
•
Prevents diabetes
425
•
Aids in reducing stress
426
•
Prevents depression
427
•
Concentrated impressive nutrients to weight loss
Helps athletes with long lasting energy and vitality
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•
Improves memory and mental clarity
429
•
Stimulates immune system to destroy invading disease organisms and carcinogens
430
•
Enhance the immune system with its antiviral, anti-tumor and interferon inducing effects
431
•
Promotes tissue repair in wounds and burns and also has the anti-infectious properties
433
•
Decreases cholesterol levels and helps to lower the risk of cardiovascular disease
434
•
Functions as an anti-inflammatory agent
435
•
Reduce the inflammation characteristic of arthritis
436
•
Govern the appetite and helps to stimulate the metabolism
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Hailed by health obsessives as the superfood to conquer viruses, prevent aging and even ward
438
off cancer, Spirulina may be able to play another, much more significant role as a way to
439
combat malnutrition in developing countries.In light of Spirulina’s gamut of nutritional
440
goodness, a number of individuals and organizations are developing Spirulina programmes to
441
address malnutrition. Aloni et al 2016 reported that the administration of Spirulina at a dose
442
of 10 g per day seemed to significantly and quickly improve the nutritional status of
443
undernourished children in the intervention group when compared to the control group.
444
Indeed, the rate of global acute malnutrition decreased from 30% before the Spirulina
445
supplements to 20% at day 30. According to The Hindu Survey Spirulina powder ranges
446
from 1000 – 4500INR /Kg. Spirulina capsules range from 250- 900INR / 60 capsules.
447
Spirulina face packs vary from 360 – 900INR / 100gm.
448
6. Future outlook
449
Spirulina is a promising food source with protein content about 65-70%.However the
450
maximum protein content reported in literature till date is 59%. Proper designing of
451
cultivation system, growth efficient techniques and use of organic fertilizer may be adopted
452
to maximize the protein content of Spirulina.
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The food processing technique including drying of Spirulina biomass is an
important step to retain the nutrition and active compound.
455
•
Further efforts should be made to increase protein content and biomass yield.
456
•
Open raceway pond is economical but the annual production of only 0.8gms liter -1 day-1 has been reported.
457 458 459
•
Open pond cultivation system has many drawbacks such as improper light intensity, contamination and requires large acres of land. Page 31 of 42
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•
Aeration is very necessary for getting good quality and better yields of
461
Spirulina species. Aeration should be done every 3-4 hrs, to avoid clump
462
formation.
463
•
It has been reported on a dry weight basis for Spirulina, productivity and total biomass production at the end of a production cycle in PBR were 30 mg L-1
465
day -1 and 0.9 g.L-1.
466
•
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Highest productivity with Spirulina plantesis was reported in raceway pond at
467
Australia with a photosynthetic efficiency of 1.5% and areal productivity of 91
468
ton ha-1yr-1. •
The daily production system according to the work reported is greater with
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PBR and lesser with the open raceway ponds, So, qualitatively as well as
471
quantitatively when measured, PBR systems are more efficient.
472
•
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Hybrid reactors are fortunate outcomes of groundbreaking strategies and technology advancements of Spirulina cultivation. Productivity is expected to
474
increase in the case of the hybrid system.The hybrid system proves to be a
475
better solution to overcome the drawbacks of open pond and PBR. Poly bags
476
can be a good option if economically tested.
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Climatic factors play an important role in Spirulina cultivation where the optimum temperature is very important. So, in very hot or cold or less humid
479
conditions greenhouse can be used for Spirulina cultivation. Different
480
greenhouse designs have been studied to design for Spirulina.
481
•
485 486
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Various harvesting system has been reported among them centrifugation may not be suggested for Spirulina as it is expensive and it may also break
482 483
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Spirulina cells on separation.
•
Normal filter or mesh cloth of 30µ - 450µ is highly used and it may separate
Spirulina very easily and efficiently.
•
Greenhouse-based solar drying is preferred over open drying in order to
487
maintain the nutritional quality. Microwave or oven drying method can also be
488
used as an alternate method.
489 490
•
Dried powder may be transformed to easy and consumable form than pellets, powder and capsules which are already available in the market.
491
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493
The main purpose of this review is to call attention for different cultivation systems, growth
494
parameters, and productivity of Spirulina in various climatic conditions. The present review
495
had revealed that significant studies have been carried out on various growth techniques to
496
increase the protein productivity of Spirulina.
497
The following conclusions are drawn from the study.
498
•
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Spirulina is mainly reported to be grown in open raceway ponds for commercial or industrial purposes. New hybrid techniques such as poly bags and greenhouse can
500
also be implemented to increase cost economics and the annual spirulina biomass
501
productivity. •
Commercial Industrial scale cultivation of spirulina uses inorganic chemicals which
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are expensive and requires optimization of media concentration to avoid intrusion of
504
prevention of facultative pathogens.
505
•
Climatic factors, light intensity and aeration are very important in Spirulina growth system. The culture conditions also influence the growth phases of Spirulina
507
platensis, causing changes in its composition. Growth rate and doubling time can be
508
increased by bringing variations in used growth media.
509
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There has been a significant change in functional properties of Spirulina under stressed conditions. Environmental stresses like high pH, light, salinity and
511
temperature affect growth and nutrient productivity. •
carbohydrates, essential fatty acids, vitamins, minerals, carotenes, chlorophyll a and
513
phycocyanin to fight against malnutrition. So it can be used in nutraceuticals and
514
pharmaceuticals applications.
515 516
Spirulina is contended to have several health benefits as it contains essential proteins,
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Innovative formulations are required to fortify conventional foods with Spirulina and
517
more scientific, clinical and toxicological research has to be carried out for extensive
518
usage of Spirulina in food and pharma industry.
519 520
•
Development of various Spirulina fortified foods is required to create nutritional awareness and increase the acceptance level in developing countries.
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•
Despite their evident use as a nutritional product, Industrial production of Spirulina
522
are still more or less confined to the limited natural areas. Mass cultivation of
523
spirulina has to be encouraged globally to avoid food shortages in near future. Acknowledgement
525
We are very thankful to the Honourable Ex-Director, Dr. K.K.Appukuttan, Maulana Azad
526
National Institute of Technology Bhopal, India for his continued support and guidance to
527
complete this research work. This research did not receive any specific grant from funding
528
agencies in the public, commercial, or not-for-profit sectors.
529
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Highlights Various Methods of spirulina cultivation are appraised.
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Growth conditions and Spirulina yield are reviewed.
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Spirulina growth for nutritional use is highlighted.
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Recommendations of spirulina as super food is suggested
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PII:
S0924-2244(17)30218-2
DOI:
10.1016/j.tifs.2017.09.010
Reference:
TIFS 2086
To appear in:
Trends in Food Science & Technology
Received Date: 12 April 2017 Revised Date:
18 June 2017
Accepted Date: 25 September 2017
Please cite this article as: Soni, R.A., Sudhakar, K., Rana, R.S., Spirulina – From growth to nutritional product: A review, Trends in Food Science & Technology (2017), doi: 10.1016/j.tifs.2017.09.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Spirulina – from growth to nutritional product: A Review
2
Ruma Arora Soni1,, K.Sudhakar1,3*, R.S.Rana2 1
3 4
2
Energy Centre, Maulana Azad National Institute of Technology, Bhopal (M.P), India
Department of Mechanical Engineering, Maulana Azad National Institute of Technology, Bhopal (M.P), India
5 3,
Faculty of Mechanical Engineering, Universiti Malaysia Pahang, 26600 Pahang, Malaysia
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E mail: [email protected] ;[email protected]
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Abstract
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Background
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Spirulina is multicellular and filamentous cyanobacteria that have achieved a considerable
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popularity in the health sector, food industry and aquacultures. It develops and grows in
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water, can be harvested and processed easily. It has very high content of macro and
13
micronutrients, essential amino acids, proteins, lipids, vitamins, minerals and anti-oxidants.
14
Spirulina is considered as a complete food supplement to fight against malnutritional
15
deficiencies in developing countries. Spirulina is deemed safe for human consumption as
16
evident by its long history of food use and latest scientific findings. In recent years, Spirulina
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has gathered enormous attention from research fraternity as well as industries as a flourishing
18
source of nutraceutical and pharmaceuticals.
19
Scope and Approach
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The primary objective of this paper is to review the utilization of Spirulina as a dietary
21
supplement in the food industry. In the present work, the three main area of Spirulina
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research: growth, harvesting and potential application are presented.
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Key findings and conclusion
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The important growth parameters have been studied to enhance Spirulina biomass
25
productivity qualitatively and quantitatively. This review provides useful information on
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commercially viable technology for Spirulina cultivation. Mass cultivation and Innovative
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formulations are further needed to fortify conventional foods with Spirulina based protein
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system.
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Keywords: Spirulina; Pharmaceutical; nutritional use; dietary supplement; open pond; PBR
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ACCEPTED MANUSCRIPT Nomenclature
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(PX)
Cell productivity
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(TC)
Cultivation time
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Γ
Productivity of the system
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µ
Specific growth rate
36
x
Biomass concentration
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(Xm- Xi)
Cell concentration
38
N0
Initial population size
39
t
Amount of time that has past
40
Nt
Population size at time
41
G
Generation time
42
N.S –
Not Specified
43
1. Introduction
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Algae are photosynthetic organisms that convert light energy from the sun into the chemical
45
energy by the process of photosynthesis. Algae possess simple reproductive structure. The
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biomass of algae contains various compounds with diversified structures and functions. Algal
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biotechnology is divided into microalgae, macroalgae and cyanobacteria with its unique
48
specificity (Becker.2007). Sometimes cyanobacteria are also included in microalgae.
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Microalgae classification includes prokaryotic and eukaryotic unicellular and multicellular.
50
Microscopic are microalgae, Cyanobacteria, are prokaryotic. The Spirulina is Earth's oldest
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living plant approximately 3.6 billion years ago and a first photosynthetic life form that has
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created our oxygen atmosphere so all life could evolve. Blue-green algae are the evolutionary
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bridge between green plants and bacteria. At present the main directions in macroalgal
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biotechnology are biofuels, agricultural biostimulants for crop plants, waste water treatment
55
etc. Microalgal biotechnologies refer to the production of different products as phycocyanin,
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carotenoids, fatty acids and lipids for application in health food, cosmetics, food supplements,
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pharmaceuticals and fuel production. Microalgal groups of major importance are chlorophyte,
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bacillariophytes, while macroalgae are harvested from natural habitats. Algae that is currently
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cultivated for its maximum protein content is the cyanobacterium species Athrospira, which
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is commonly known as Spirulina.
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ACCEPTED MANUSCRIPT Spirulina was first discovered by Spanish Scientist Hernando Cortez and Conquistadors in
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1519. Cortez observed that Spirulina was eaten at the tables of the Aztecs during his visit in
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Lake Texcoco in the Valley of Mexico. Pierre Dangeard discovered the health benefits of
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Spirulina who observed that flamingos were surviving by consuming blue-green algae.
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Botanist Jean Leonard supported the findings of Dangeard and people soon started to
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commercialize Spirulina to reap its benefits (Ugwu et al 2008).The first Spirulina processing
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plant, Sosa Texcoco, was set up in 1969 by the French.
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Spirulina is the most nutritious, concentrated food that is known to mankind containing
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antioxidants, phytonutrients, probiotics, and nutraceuticals.Spirulina is fast emerging as a
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complete answer to the varied demands due to its imposing nutrient composition which can
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be used for therapeutic uses. The United Nations world at food conference declared that
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Spirulina as the best food for future, and it is gaining popularity nowadays (Pulz and Gross
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2004). World Health Organization has described spirulina as Mankind’s best health product.
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According to UNESCO, spirulina is most ideal food for tomorrow. According to NASA and
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European Space Agency, it is one of the primary foods that can be cultivated in long-term
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space missions in space. FDA validated it as “One of the best protein source”.
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Intergovernmental institution permitted for the use of Micro-algae Spirulina against
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Malnutrition (IIMSAM).
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The two most important species of Spirulina are Spirulina maxima and Spirulina platensis. It
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has a considerable high content of micro and macronutrients.Its dry weight chemical
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composition includes 60-70% proteins, carbohydrates, vitamins like provitamin A, vitamin C,
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vitamin E, minerals such as iron, calcium, chromium, copper, magnesium, manganese,
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phosphorus, potassium, sodium and zinc. Essential fatty acids γ-linolenic acid (GLA),
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pigments like chlorophyll a, phycocyanin and carotenes are also present. Spirulina is also
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used in cosmetics, medicines and waste water treatment. Its cell wall consists of
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polysaccharides that have a digestibility of 86%, and can be easily absorbed by the human
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body (Sjors 2010). These microalgae contain chlorophyll a, like higher plants; therefore it is
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classified as microalgae according to botanists belonging to Cyanophyceae class; and
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bacterium due to its prokaryotic structure according to bacteriologists (Koru 2009;
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Sudhakar and Premlatha 2015).
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Spirulina is a planktonic photosynthetic cyanobacterium that forms huge populations in
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tropical as well as subtropical bodies of water which contain a high amount of salts such as
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carbonate and bicarbonate with alkaline pH 9.5(Sjors 2010; Habib et al 2008). Generally,
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microalgae have higher growth rates, higher CO2 fixation efficiency and larger quantities of
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ACCEPTED MANUSCRIPT high-value products, such as dietary supplements for human along with animals (Zeng et al
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2011; Anupama 2000).Cost effectiveness and composition of cultivation media along with
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growth rate needs to be managed properly for commercially viable production. From ancient
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times different media have been used for cultivation of Spirulina and monitoring its growth
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rate i.e. Zarrouk's media (Zarrouk 1966), Rao's media, CFTIR media, OFERR media,
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revised media (Raoof et al 2006).
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Past few decades have seen considerable progress in spirulina cultivation for nutritional
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use however there is no substantial argument on the nutritional productivities, best
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cultivation method, and ideal growth conditions. This review addresses these issues based
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on prior publications and the author’s prior work in the large scale cultivation of spirulina
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for nutritional products. The article starts with the illustration of spirulina growth chain
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from identifying suitable strain to the final product.
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The present study focuses on growth rate, productivity, growth parameters, different
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cultivation systems (outdoor and indoor systems), harvesting and drying techniques of
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Spirulina. This review focuses on following aspects:
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Strain selection and cultivation of Spirulina.
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Optimum parameters for growth of Spirulina.
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Harvesting and drying techniques of Spirulina.
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Commercial applications of Spirulina as pharmaceutical and nutraceuticals product.
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2. Review of growth system
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Cultivation of algae can be done in open systems like ponds, lakes or lagoons or in a closed
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system (Singh and Sharma 2012). Presently, two major technologies are being considered
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for the cultivation of Spirulina: closed photobioreactors (PBR) and open ponds. Both
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approaches are used commercially to produce high-value products.
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2.1 Open pond system
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Cultivation of algae in open ponds has been extensively studied (Vardaka et al 2016, Zhang
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L et al 2015, Madhu, G. M et al 2015, Choi et al 2003, Vega 2005). Open ponds can be
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categorized into natural waters as lakes, lagoons, ponds and artificial ponds or containers.
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The most commonly used systems are shallow big ponds, circular ponds, tanks and raceway
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ponds. Open systems are easier in construction and operation, results in low production and
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operating cost (Ugwu et al 2008). The major drawback in open ponds includes poor light
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utilization by the cells, evaporative losses, diffusion of carbon dioxide to the atmosphere, and
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ACCEPTED MANUSCRIPT requirement of large acres of land. Also, due to inefficient aeration in open cultivation
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systems, their mass transfer rates are very poor resulting in less biomass productivity. The
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growth also depends on location, season, temperature, pH level, nutrient and carbon - dioxide
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supply (Cuaresma et al 2011).The other major drawback of open pond system is the
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contamination by fauna and other fast growing heterotrophs. To expel the problems
135
associated with an open system, researchers have tried for closed systems (Singh and
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Sharma 2012).
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Table 1 summarizes the advantages and limitations of open ponds, photobioreactors and
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hybrid system. Large quantities of algae can be grown but they are difficult to grow outdoor
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as they easily get contaminated. This can be rectified by growing algae in greenhouses, which
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protect them from foreign particles in the air. The optimally designed algae greenhouse and
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controlled environment systems can increase productivity 10 fold compared to outdoor
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growth. Construction of greenhouse includes design and optimizing for improved biomass
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yield. Controlled environment algae facilities are gaining momentum due to improved yields
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and reduced contamination. The internal systems to control the internal humidity,
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temperature, and carbon dioxide through the use of fans, vents, evaporative cooling, and
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climate zoning is done (Sierra et al 2008). pH, nutrients, and bacteria are regulated in the
147
water system through fertigation, oxygenation and also sterilization. Integrating the climatic
148
conditions, water, and nutrient systems with simulation allows us to provide exactly what that
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algae facility needs, resulting in optimized yields. The open roof greenhouses design provides
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complete protection against undesirable weather conditions, while the full vertical vent
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promotes optimum light and air movement.
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Species Selection
Phase- I
164
166 167
Macroalgae
Microalgae
Cyanobacteria
(a)
168
Growth System of Spirulina
170 Phase- II
171 172
174
Open Raceway Ponds
Photo bioreactors
(b)
176
M AN U
175
Growth Parameters of Spirulina
Phase- III
177 178 179 180
Mother culture
Media Selection
Mixing & Aeration
Phase- IV
184
191 192
Centrifugation
AC C
Phase- V
190
Normal Filter
EP
185
189
Growth Rate& Productivity
Harvesting System of Spirulina
183
188
Light Intensity
(c)
182
187
Temp. & pH
TE D
181
186
Hybrid System
SC
173
RI PT
169
(d) Drying
Grinding/Powdering
Pellets/ Capsules
193
(e)
194
Figure 1 : Different phases of Spirulina cultivation system (a) strain selection (b) Growth systems (c) growth parameters (d) Harvesting system (e) Final product as capsules or pellets.
195 196
Figure 1 illustrates the flowchart for Spirulina cultivation phases (from Phase a – Phase e)
197
from strain selection to pellets formation Page 6 of 42
ACCEPTED MANUSCRIPT 198
2.2 Photobioreactors
200
A photobioreactor can be an enclosed, illuminated culture vessel designed for controlled
201
biomass production. Photobioreactor refers to closed systems that are closed to the
202
environment having no direct exchange of gases and contaminants with the environment. The
203
closed system commonly called as photobioreactors, is closed equipment which provides a
204
controlled environment and also results in high productivity of algae. Photobioreactors
205
facilitate better control of culture environments such as carbon dioxide supply, water supply,
206
optimal temperature, efficient light intensity, culture density, pH levels, gas exchange,
207
aeration and culture density. Algal culture systems can be illuminated by artificial or natural
208
light or by both. Naturally illuminated algal culture systems with large illumination surface
209
areas include open ponds (Hase et al 2000), flat-plate (Hu et al 1996), horizontal/serpentine
210
tubular airlift (Camacho et al 1999], and inclined tubular photobioreactors (Ugwu et al
211
2002). In order to overcome the problems with open ponds, much attention is now focused on
212
the development of suitable closed systems such as flat-plate, tubular, vertical column and
213
internally-illuminated photobioreactor. Generally, laboratory-scale photobioreactors are
214
illuminated artificially internally or externally using fluorescent lamps or other light
215
providers. Some of these photobioreactors include bubble column (Ugwu et al 2002, Degen
216
et al 2001, Ogbonna et al 2002), airlift column (Chini et al 2003,Harker et al 1996),
217
stirred-tank (Kaewpintong et al 2007), helical tubular (Ogbonna et al 1999) conical (Hall
218
et al 2003), torus (Watanabe and Saiki 1997], and seaweed type (Pruvost et al 2006]
219
photobioreactors. Some photobioreactors can be easily tempered. Large scale outdoor
220
systems mainly tubular photobioreactors cannot be easily tempered without high technical
221
efforts. Efforts have been taken in designing temperature-controlled photobioreactors, such as
222
double-walled internally-lighted photobioreactor with both heating as well as cooling water
223
circuit (Chetsumon et al 1998). Photobioreactors, despite their costs, have several major
224
advantages over open systems (Tsoglin et al 1996).
225
•Photobioreactors minimize the contamination and allow axenic algal cultivation of
226
monocultures.
227
• Photobioreactors offer better control over conditions such as pH, temperature, light
228
intensity, carbon dioxide concentration etc.
229
• Photobioreactors reduce carbon dioxide loss.
230
• Photobioreactors prevent water evaporation.
231
• Photobioreactors permit higher cell concentrations.
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Page 7 of 42
ACCEPTED MANUSCRIPT • Photobioreactors enhance the production of complex biopharmaceuticals.
233
• PBR permits the cultivation of various microalgal species.
234
• PBR design provides the uniform illumination of the culture surface and the fast mass
235
transfer of carbon dioxide and oxygen.
236
• PBR has a minimum non-illuminated part.
237
Table 1.Comparison between Spirulina production in open, closed and hybrid system
238
(Roberto 2015).
RI PT
232
Closed systems
Hybrid system
(raceway ponds)
(photobioreactors)
(Open Pond + PBR)
Space required
High
Low
High
Area/volume ratio
Low (5–10 m−1)
High (20–200 m−1)
Evaporation
High
Water loss
Very high
CO2-loss
High
Temperature
Highly variable
Weather dependence
M AN U
SC
Open systems
Factor
Variable Minimized
Low
Less
Low
Minimizes
Required cooling
Controlled
High
Low
Low
Process control
Difficult
Easy
Difficult
Cleaning
Easy
Required
Difficult
Reproducible
Better
Medium
High
Medium
Medium
High
High
High
Lower
High
Poor
Good
Better
EP Variable
AC C
Biomass quality
TE D
No evaporation
Population density Harvesting efficiency
Harvesting cost
Light utilization efficiency Most costly parameters
Mixing
Oxygen and temperature control
Page 8 of 42
Temp control
ACCEPTED MANUSCRIPT Closed systems
Hybrid system
(raceway ponds)
(photobioreactors)
(Open Pond + PBR)
Difficult
Easy
Easy
Capital investments
Low
High
Low
Productivity
Low
control
Hydrodynamic stress on Spirulina
3–5 times more productive
Very low
Low–high
5-7 times more productive Low
SC
Contamination
RI PT
Open systems
Factor
239
2.2.1 Vertical-column PBR
241
Vertical-column photobioreactors are easy to operate compact and low-cost (Miron et al
242
2002). Various designs and scales of vertical column photobioreactors have been reported for
243
the cultivation of algae (Choi et al 2003, Estrada et al 2005; Kaew et al 2007) which are
244
very promising for large-scale cultivation. It was reported that bubble-column and airlift
245
photobioreactors (up to 0.19 m in diameter) can attain a final biomass concentration and a
246
specific growth rate that are comparable to tubular photobioreactors (Sanchez Miron et al
247
2002). Some bubble column photobioreactors are equipped with either draft tubes or
248
constructed as split cylinders.
249
2.2.2 Flat plate PBR
250
For cultivation of photosynthetic microorganisms flat-plate photobioreactors have received
251
much consideration due to their large illumination surface area. The work reported paved a
252
way to use flat culture vessels for the cultivation of algae (Samson and Leduy 1985). A flat
253
reactor was developed and equipped with fluorescence lamps (Ramos and Roux 1986].
254
After this, an outdoor flat panel reactor was developed using thick transparent PVC materials
255
(Tredici and Materassi 1992). Later extensive works were reported on various designs of
256
flat plate reactors and vertical panels for mass cultivation of different algae ( Hu Q et al
257
1996, Zhang et al 2002, Hoekema et al 2002, Olguin et al 2003). Flat plate
258
photobioreactors are constructed using transparent materials for maximum utilization of solar
259
light.
260
photobioreactors is relatively high as compared to flat-plate photobioreactors. It has been
261
reported that high photosynthetic efficiencies can be achieved with flat-plate photobioreactors
AC C
EP
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240
Accumulation
of
dissolved
oxygen
concentrations
Page 9 of 42
in
horizontal
tubular
ACCEPTED MANUSCRIPT 262
(Hoekema et al 2002, Olguin et al 2003). Overview of spirulina productivities reported in
263
the literature for various growth systems is presented in Table 2. Among all culture systems
264
productivity of Spirulina platensis is highest in raceway ponds. The areal productivity is
265
generally based on one-hectare ground surface area.
266
Table 2 Spirulina productivity and Photosynthetic efficiency
system
Light
Photosynt
Productivity Reference
path
hetic
(ton ha-1yr-
Efficiency
1
1.5%
43.1
1.5%
20.0
Raceway
La
0.1-
pond
Mancha,
0.25
)
Raceway
Florence,
pond
Italy
Raceway
Malaga,
pond
Spain
Raceway
Australia
0.035
Tubular
Italy
Flat Panel
US
1992)
1.5%
23.6-30.0
(Jimenez et al 2003)
0.30
1.5%
91.0
(Borowitzka 1999)
30.0
(Tredici and Materassi
TE D
Florence,
(Tredici and Materassi
0.30
pond Horizontal
( Olguin et al 2003)
M AN U
Mexico
RI PT
Location
SC
Reactor
0.06
3.8%
1992) 22.1
EP
0.10
1.8-3%
(Richmond and Zhang, 2001)
2.2.3 Tubular PBR
268
A tubular photobioreactor is the most suitable types of bioreactors for outdoor mass
269
cultivation (Kaewpington et al 2007). Mostly outdoor tubular photobioreactors are
270
constructed either with glass or plastic tube. They can be horizontal/ serpentine (Chaumont
271
et al 1988; Molina et al 2001; Pirt et al 1983; Watanabe and Saiki 1997], vertical
272
(Tredici and Chini 1998] conical (Lee and Low 1991) inclined (Ugwu et al 2002; Torzillo
273
et al 1986). Mixing and aeration of the cultures in tubular photobioreactors are usually done
274
by air-pump or airlift systems. Mass transfer becomes a problem when tubular
275
photobioreactors are scaled up. Many have reported that very high dissolved oxygen (DO)
276
levels are easily reached in tubular photobioreactors [Sanchez et al 2002;Pirt et al
AC C
267
Page 10 of 42
ACCEPTED MANUSCRIPT 1983;Richmond et al 1993;Ugwu et al 2003; Ugwu et al 2005a]. It is difficult to control
278
culture temperatures in most tubular photobioreactors. They can be equipped with a
279
thermostat to maintain the desired culture temperature and it could be very expensive and
280
there will be difficulties in implementing.
281
2.2.4 Internally-Illuminated PBR
282
These photobioreactors can be internally illuminated with fluorescent lamps. Air and CO2 are
283
supplied to the cultures through the spargers with continuous agitation by impellers. This
284
photobioreactor can also be modified in such a way that it can utilize both solar and artificial
285
light system (Ogbonna et al 1999). The artificial light source is used whenever the solar light
286
intensity decreases below a set value as during cloudy weather or at night. It has been
287
reported, on the use of optic fibers to collect and distribute solar light in cylindrical PBR
288
(Mori 1985, Matsunaga et al 1991). A major advantage of internally-illuminated
289
photobioreactor is that it can be heat-sterilized under pressure and by this contamination can
290
be minimized. A continuous supply of light to the photobioreactor can be maintained both
291
day and night by integrating artificial and solar light devices. Outdoor mass cultivation of
292
algae in this type of photobioreactor would have some technical difficulties. Flat plate
293
photobioreactors are generally more efficient in sunlight utilization than tubular
294
photobioreactors because they have a wider surface area. Most early tubular PBRs used tubes
295
10–30 cm in diameter, but almost all tubular reactors used now have a tube diameter of 4 cm.
296
The narrower tube diameter not only improves the light utilization efficiency, but also
297
provides more mixing, which enhances growth (Tredici, 2004). In photobioreactors (PBRs),
298
the microalgae get adheres to the transparent surfaces which lead to biofouling and along
299
with it reduces the solar radiation penetration the PBR. Light intensity reduction within the
300
PBR reduces the biomass productivity which also reduces the photosynthetic efficiency of the
301
Spirulina cultivation system. Adherence of the cells to wall tubes is very common in tubular
302
photobioreactors.Designing of photobioreactor surfaces with proper materials, functional
303
groups or surface coatings, to prevent microalgal adhesion is essential for solving the
304
biofouling problem. Such a significant advance in microalgal biotechnology would enable
305
extended operational periods at high biomass productivity and depreciate the maintenance
306
costs (Zeriouh et al, 2016).
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Page 11 of 42
ACCEPTED MANUSCRIPT
Table 3: Prospects and limitations of various cultivation systems (Chojnacka, et al 2004;Vymazal 1990;Ugwu et al 2008;Vree et al 2015, Newsted 2004) Dimensions
systems
Specific
Prospects
Limitations
RI PT
Culture
growth rate Variable
0.30day-1
systems
Relatively economical, easy to Little control of culture conditions, clean up after cultivation,
SC
Open
difficulty in growing algal cultures
M AN U
good for mass cultivation of for long periods, algae
poor productivity,
occupy large land mass, limited to few strains of algae,
TE D
cultures are easily contaminated
Vertical
0.2 m
0.015 ±
High mass transfer,
Column
diameter
0.002
good mixing with low shear construction require sophisticated
h )
stress,
EP
and 4 m
materials,
column
low energy consumption,
shear stress to algal cultures,
height[
high potentials for scalability,
the decrease of illumination surface
easy to sterilize,
area upon scale-up
AC C
PBR
−1
Small illumination surface area,
good for immobilization of algae, reduced photoinhibition and
Page 12 of 42
Images
ACCEPTED MANUSCRIPT
photo-oxidation 0.07 m
Large
surface Scale-up
PBR
wide,
area,
compartments
1.5 m height
suitable for outdoor cultures,
materials,
, 2.5 m
good for immobilization of
difficulty in controlling culture
length
algae,
temperature,
Volume
good light path, good biomass some degree of wall growth,
250lts
productivities,
Productivity
relatively cheap,
- 1.0 g/L
easy to clean up,
day
readily tempered,
illumination
area,
M AN U surface Gradients of pH,
dissolved oxygen and CO2 along
suitable for outdoor cultures, fairly
support
TE D
Large
good
productivities,
EP
0.055 h−1
and
stress to some algal strains
the tubes, fouling,
biomass some degree of wall growth,
AC C
PBR
D = 3-10cm
many
the possibility of hydrodynamic
low oxygen buildup Tubular
require
SC
illumination
RI PT
Flat plate
requires large land space
relatively cheap
Page 13 of 42
ACCEPTED MANUSCRIPT
Internally
Not
Large
Illuminated
Specified
area,
require some technical efforts.
can utilize both solar and
RI PT
PBR
surface Outdoor mass cultivation of algae
illumination
artificial light system, contamination
can
be
SC
minimized in this system Not
Minimize
microbial Requires large areas of land and
System
Specified
contamination,
maximize some technical efforts
biomass and product yield, Maximize CO2 supply
site flexibility,
Polyethylene bag cultures have a
Low-cost materials, easy scalability,
TE D
0.20day-1
relatively short life because the internal surface attracts culture
optimal light exposure,
debris
EP
D <30cm
and
bacteria,
which
isolation of the crop from collectively reduce light penetration predators, very
AC C
Poly Bags
M AN U
Hybrid
high
and are a source of contamination
biomass
concentration,
low energy consumption effective weather protection
Page 14 of 42
ACCEPTED MANUSCRIPT 2.3 Hybrid system
2
A hybrid type of photobioreactor is most widely used to exploit the advantages of the two
3
different types of reactor and overcome the disadvantage of other. Integrated airlift system
4
and external tubular loop placed horizontally in a thermostatic pond of water have been
5
reported (Zittelli 2013). The reactor had a total volume of 200 L. The external loop acts like
6
the light-harvesting unit and gives high surface area to volume ratio and controls the
7
temperature of the culture. The airlift system acts as a degassing system where probes can
8
also be integrated in order to regulate the other culture variables. It has the advantage of
9
better control over culture variables, enabling higher productivities and reducing power
10
consumption (Pohl et al 1988; Singh and Sharma 2012; Ugwu et al 2008; Cuaresma et al
11
2011]. Hybrid systems have the features of open ponds and PBRs (Hoekema et al 2002).
12
First can be covered open pond this concept reduces the possibility of contamination,
13
evaporative losses, and CO2 desorption. The other type is a partially filled tubular design
14
widened and inflated to approximate an open pond; this design is mainly aimed at reducing
15
costs (Hoekema et al 2002; Olguin et al 2003; Tredici and Materassi 1992]. Some of the
16
advantages and limitations of various cultivation systems are listed in Table 3.
17
Polybags
18
The cultivation of algae using natural ponds is easy, but turning it into a viable feedstock is
19
very difficult. So to enable higher production levels, least investments and operating costs,
20
greater biomass density, better climatic controlled conditions, and industrial scalability, this
21
technique can be implemented. Thin, floating, flexible, multi-compartment photobioreactors
22
(PBR) can be deployed either on land, in salt water ponds or ditches, or in any water body.
23
The bag floats because its water is relatively less dense than what it is floating in. Density can
24
be controlled in different ways, allowing the bags to be vertical to facilitate harvesting. The
25
productivity results have indicated that growing algae in floating bags can be much more
26
efficient than other cultivation methods. Poly Bags achieve optimal light exposure with good
27
productivity results as they float in a cushion of water. Compared to other closed algae
28
systems, this PBR technology has many advantages, including site selection, optimum
29
temperature, low-cost materials, scalability, optimal light intensity, high biomass
30
concentration, low energy consumption and effective environmental condition (Licamele
31
and White 2011).
AC C
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Page 15 of 42
ACCEPTED MANUSCRIPT The diameter of the culture vessel is inversely related to cell density with a fixed level of
33
light penetration. However, these bags are superior in productivity to similar rectangular
34
volume fiberglass reactors or plastic tanks. They are, nevertheless, inefficient when compared
35
with internally illuminated cultures. Polyethylene bag cultures have a relatively short life
36
because the internal surface attracts culture trash and bacteria, which collectively reduces
37
light penetration and also increases contamination. At the end of a culture run it is necessary
38
to renew the bag. Large diameter bags are inefficient but bags less than 30 cm diameter can
39
be effective because the surface area to volume relationship for light penetration is improved
40
(Algae Industry Magazine, 2012).
41
3. REVIEW OF GROWTH PARAMETERS
42
Spirulina growth requirements are similar to terrestrial plants but they use these resources
43
very efficiently to increase biomass productivity with comparatively less water use
44
(Sudhakar et al 2011)
45
3.1 Climatic factors
46
Temperature is an important climatic factor influencing the rate of growth of Spirulina.
47
Below 17°C, growth is practically nil, but Spirulina does not die. The optimum temperature
48
for growth is 35°C, but above 38°C Spirulina growth is inhibited. Light is an important factor
49
but direct sunlight is not recommended, 30% of full sunlight is actually better, except that
50
more may be required to quickly heat up the culture in the morning (Saeid and Chojnacka,
51
2015). Growth takes place only in the light, but illumination 24 hours a day is also not
52
recommended. During dark periods, chemical reactions take place within Spirulina, like a
53
synthesis of proteins and respiration.
54
3.2 Media
55
Different culture media are used to start new cultures according to the water source. The
56
water used should be clean or filtered to avoid growth of other algae. Water often contains
57
enough calcium, but if it is too hard it will cause muds. Portable water is convenient whereas
58
RO treated water is the best to grow Spirulina. The make-up media mainly consist of urea.
59
Carbonate is replaced by bicarbonate. Urea, certain ions may be present as sulphate, chloride,
60
nitrate, and sodium which is more efficient to supply nitrogen but is highly toxic with large
61
concentration. Spirulina can grow on either nitrate or urea alone, but using both at the same
62
time is advantageous. Phosphate, magnesium and calcium cannot be increased much.
AC C
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32
Page 16 of 42
ACCEPTED MANUSCRIPT Potassium concentration can be increased accordingly, provided it does not become more
64
than five times the sodium concentration. If fertilizer grade chemicals are used for cost
65
reduction, they should be of the soluble or crystallized type, not of the slow release,
66
granulated type. There are different media preparations according to the local growing
67
conditions. Most commonly used is zarrouks media (Zarrouk 1966; Pragya et al
68
2013).Chemical compositions of different growth media are compared in Table 4.
69
Table 4: Chemical composition of different growth media ( Madkour et al 2012, Atlas and
70
Parks 1997; Venkataraman etal 2005; Pandey et al 2010)
s
CFTRI
OFERR
Media(g
Media(g
Media(g
George’s
onal
ms/l)
ms/l)
ms/l)
Media(g
growth
ms/l)
Media(g
Media(g ms/l) 16.80
15
K2HPO4
0.50
0.50
NaNO3
2.50
2.50
K2SO4
1.00
NaCl
1.00
H2O
CaCl2·2H2 O
FeSO4·2H2 O H3BO3 MnCl2·4H 2O
0.08
AC C
EDTA
0.20
0.04
0.01 2.86 1.180
Cost Media(g ms/l)
8.0
-
16
16.8
0.5
-
0.02
-
0.235
1.5
-
-
-
-
0.60
1.0
0.5
-
0.5
0.353
0.20
1.0
5.0
-
1.00
0.471
0.04
1.2
0.16
0.02
0.1
-
-
-
-
-
-
0.353
0.008
0.04
-
-
0.1
0.176
-
0.01
0.05
-
-
0.265
-
-
0.052ml
-
-
2.86
-
-
-
-
-
1.81
EP
MgSO4·7
ms/l)
Reduced
4.5
TE D
NaHCO3
SC
Ingredient
Conventi
Rao’s
M AN U
Zarrouk’
RI PT
63
Page 17 of 42
ACCEPTED MANUSCRIPT
2O
NH4VO3 NiSO4·7H2 O NaWO2 Ti2(SO4)3· 6H2O Co(NO3)2· 6H2O Ferric citrate
0.074 22.9 47.8 17.9 4.4
-
(NH4)2HP
Chelated Iron
-
AC C
O4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
0.222
-
-
0.0177
-
-
0.079
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
0.035
-
-
-
-
-
1.00
-
-
-
-
-
-
2.00
-
-
-
-
-
0.1
-
-
-
-
EP
KNO3
-
-
-
-
-
4.4
Peptone
-
RI PT
CuSO4·5H
0.015
-
SC
Na2MoO3·
-
M AN U
2O
0.222
TE D
ZnSO4·7H
2 squeezes
-
-
(¼
-
teaspoon )
Lime
-
-
-
-
-
0.1
-
NH4NO3
-
-
-
-
-
-
0.118
CO (NH2)2
-
-
-
0.2
-
-
0.088
Page 18 of 42
ACCEPTED MANUSCRIPT Fe EDTA
-
0.20
-
-
-
-
-
A5 solution
-
1ml
-
-
-
-
-
71
3.3 Mother culture
73
For Inoculums preparation and culture maintenance fully grown concentrated Spirulina
74
culture is required. The chosen Spirulina strain must have a high proportion of coiled
75
filaments (< 25% straight filaments, or none), and at least 1 % of gamma-linolenic acid
76
(GLA) based on dry weight. Concentrated Spirulina seed culture can be obtained either from
77
the floating layer of a composed culture, or by diluting a freshly filtered biomass. Colour of
78
the culture should be clearly green.The growth rate is about 30 % /day when the temperature
79
and other climatic conditions are adequate (Pal et al 2011). As the growth is proportional to
80
the area of the culture exposed to light, it is recommended to maximize this area at all times.
81
It is reported that minimum cell population is necessary to initiate and sustain Spirulina
82
cultures.
83
3.4 Mixing and aeration
84
Agitation of the culture is necessary to homogenize and ensure a good distribution of lighting
85
among all the filaments of Spirulina. Mixing plays an important role in the productivity of
86
ultrahigh density cultures. Aeration is very necessary for getting good quality and better
87
yields of Spirulina species. It can be achieved by rotators, which maintain the cells in
88
suspension by gentle agitation of growing cells. The Spirulina species produces high biomass
89
yield when the growth medium is aerated (bubbling with air). Aeration gives a homogenous
90
distribution of the Spirulina filaments throughout the growth system for adequate exposure to
91
illumination. It also helps to distribute carbon dioxide concentration uniformly and removes
92
inhibitory substances as oxygen (Dubey 2006; Richmond and Vonshak 1978). Aeration is,
93
therefore, essential for the cultivation of the Spirulina filaments such as Spirulina platensis
94
(Famelart et al 1987; Powls 1985). Adequate and turbulent mixing is essential for higher
95
biomass productivity(Chisti, 2016). Mixing of raceway pond is effected by means of a paddle
96
wheel. Mixing velocity of 5-60 cm/s has been used by many researchers. Low velocities
97
result in dead zones around corners while high velocities incur high energy cost, and may
98
result in shear stress that damages the algae. It also noted that continuous mixing of the
99
culture medium is required to prevent cell sinking and thermal stratification. It is also
AC C
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Page 19 of 42
ACCEPTED MANUSCRIPT required to maintain even nutrient distribution, and to remove excess oxygen. When aeration
101
is not adequate, the efficiency of energy utilization and biomass production will be low.
102
Similarly, if growth medium is not aerated, the cell on the surface of the medium float to the
103
surface due to the presence of air-filled vacuoles. These cells suffer photoinhibition, resulting
104
in low growth or low biomass production. The optimal conditions for spirulina were found to
105
be at a light intensity lower than 200 µmol m−2 s−1, CO2 enriched air flow (0.5%), superficial
106
aeration rate of 0.0056 m s−1 in a NaHCO3-free Zarrouk medium(Zhang, L et al 2015).
107
3.5 Temperature and pH
108
Spirulina can grow at 200C- 370C. The best temperature for Spirulina growth is between 290C
109
- 350C. During night growth of Spirulina is least or almost zero. It is reported that the effect
110
of pH on the algal growth, pigment production and protein content of Spirulina species has
111
the direct effect on the antioxidant system (Vonshak and Guy 1987; Ogbonda et al 2007).
112
The growth may be affected in two ways
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•
Available carbon alteration, which may interfere with photosynthesis.
114
•
Through the disruption of cell membrane processes.
This may have a direct impact on the accumulation of antioxidants (Matsunaga et al 1991).
116
Moreover, factors such as nutrient availability, ionization and heavy metal toxicity have large
117
impacts on algal metabolism (Newsted 2004).The fluctuation in atmospheric temperature is
118
the main factor affecting the biomass production rates in outdoor Spirulina cultivation. In the
119
rainy season the culture may become contaminated due to raindrops resulting in lowest dried
120
mass. The physical factors which are not favorable in monsoon can be controlled by using the
121
locally available techniques (Pandey 2010). The warm humid environment causes the
122
bacterial contamination. The main contaminants of the Spirulina culture were protozoan like
123
amoeba and paramecium which ultimately spoils the cultures. During the monsoon season
124
insects also appears in the culture and make it unfit for human consumption. To reduce the
125
effect of the low-temperature Spirulina cultures can be kept in the house made of a plastic
126
sheet. When pH is between 9-11, it indicates a healthy culture. It also assures that other
127
strains are prevented from contaminating the tank as they simply can’t live in the alkaline
128
environment that Spirulina grows in
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3.6 Light intensity
131
All photoautotrophic organisms including photosynthetic bacteria, cyanobacteria and higher
132
plants, convert light energy into chemical energy through photosynthesis. It is reported that
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134
Premlatha 2012; Lucie et al 2016). In an outdoor cultivation system, natural light or solar
135
radiation is the whole sole source of light. Light availability is totally dependent on
136
geographical area, climatic conditions, seasonality and local atmosphere. Spirulina makes its
137
own food in the presence of optimal light. The requirement of light intensity for growth
138
varies from organism to organism. Spirulina also requires a specific range of intensity for its
139
growth (Sudhakar et al 2012) Zarrouks did the first detailed study on the response of
140
Spirulina maxima to light (zarrouk 1966). The optical density of the culture is directly
141
proportional to the light intensity. Higher the optical density higher is the requirement of light
142
and lower is the optical density, lower is the requirement of light (Samuel et al 2010). The
143
light intensity is an important variable in cyanobacteria cultivation. High values of light
144
intensity promote growth parameters such as maximum specific growth rate, whereas low
145
values result in a biomass that is rich in pigments and proteins.Outdoor algal cultures are
146
exposed to two rhythms of the dark and light regime. These cycles impose a unique
147
physiological regime on the adjustment or acclimatization of outdoor algal cells to light.
148
Increasing the cell concentration of culture, increases the self-shading and results in a
149
decrease of the growth rate of Spirulina.
150
The attenuation coefficient was observed to scale linearly with microorganism density.
151
The irradiance attenuation coefficient at wavelength λ, αλ, is calculated according to,
152
Gλ(z)/Gλ(0) = e −αλz
153
The Spectral Irradiances at different depths z is calculated according to above equation
154
knowing the value of αλ,
155
The spectral attenuation cross section, Aλ, is defined as, Aλ = αλ/X
156
Using the irradiance attenuation coefficients for each culture, αλ,
157
, Gλ(z) = Gλ(0)e −αλz
158
Where Gλ(z) = spectral irradiance at depth z
159
Gλ(0)= Incident spectral irradiance just below the culture surface.
160
X = microorganism density in grams of dry biomass per liter (g/l).
161
It has been observed that decreasing the depth of a pond from 20 cm to 10 cm achieve the
162
targeted biomass density of 0.19 grams dry biomass per liter (g/l).The shallower ponds
Eq.1
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Eq.2
Eq.3
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164
harvesting the resultant biomass.
165
Bezerra et al , 2011 reported that the maximum cell concentration (Xm) increased from 5200
166
to 5800 mg L−1 when the light intensity was increased from 36 to 72 µmol photons m−2 s−1,
167
highlighting growth limitation by light intensity within this irradiance level. On the other
168
hand, an additional increase in light intensity up to 108 µmol photons m−2 s−1 led only to a
169
reduction in the cultivation time from 8 to 6 days. Similar results were obtained by Danesi et
170
al. (2004) using urea as a nitrogen source in the light intensity range of 2–5 klux. This
171
behavior suggests that, at a relatively high light intensity (108 µmol photons m−2 s−1), cell
172
growth was accelerated by the faster photosynthetic production of ATP and NADPH; but,
173
when cell concentration reached 5800 mg L−1, the growth stopped likely due to photo
174
saturation or shadowing.
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3.7 Growth rate & productivity
177
Salinity or nutrient concentration affects the growth rate of algae. Specific growth rates of
178
Spirulina were reported to be lower in increased salinity concentrations. The highest growth
179
was achieved at the lowest salinity ratio for studies performed with various concentrations of
180
NaHCO3 and NaCl salts. The growth rate of Spirulina undergoes simple cell division. Thus,
181
under normal growth conditions the specific growth rate is described by the following
182
equation:
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µ=
t dx x dt
Eq. 4
Calculation of specific growth rate has been described in many ways. Most commonly used
185
formula is
186
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µ=
ln x2 − ln x1 t 2 − t1
Eq. 5
187
Where x1 and x2 are biomass concentration at time interval t1 and t2 The simple equation that
188
combines the specific growth are (µ) and the doubling time or the generation time (g) of the
189
culture is:
190
g=
ln 2
µ
==
0.693
µ
= d .t
Eq. 6
191
Cell productivity (PX) is a function of the independent variable, which is described as the
192
lowest difference in the cultivation time (TC). Page 22 of 42
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194
biomass productivity is the nutritional content of the culture medium. The use of certain
195
nutrients can alter production costs and affect growth or biomass composition (Grobbelaar
196
2007; Sassano et al 2007). Annual biomass production of Spirulina in PBRs is 3000 tonnes
197
which are maximum when compared to other microalgae species (Jayati et al 2015,
198
Bharathiraja et al 2015) Productivity is a measure of how much algal biomass is produced
199
per area per unit of time. Production up to 127,000 kg ha-1 yr-1 can be achieved in high-rate
200
raceway ponds. Productivity rates between 20 and 30 gm-2day-1 (73–109,000kg ha-1yr-1) are
201
in the range of usual open raceway performance (Bharathiraja et al 2015).
202
The productivity of the system γ is defined as
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Eq. 7
Where µ is the specific growth rate in units of reciprocal of time and x is the biomass
205
concentration.
206
The cell productivity (PX) is calculated as the ratio of the variation in cell concentration (Xm-
207
Xi) to the cultivation time (TC)
208
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PX = (Xm – Xi ) / TC
Eq. 8
As demonstrated in the earlier work (Miron et al 1999; Neils et al 2011), there is an optimal
210
biomass concentration which corresponds to the highest productivity.
211
Masojı´dek et al. (2003) applied a peristaltic pump as circulation apparatus to cultivate
212
Spirulina platensis and obtained a cell productivity of 0.5 g/ L/day, which was considered a
213
relatively high value in open pond cultivation system.
214
Toyoshima et al 2015 reported the maximum biomass productivities of Spirulina platensis in
215
the warm temperature habitat. 9 g dry biomass m−2 day−1 in summer and in the subtropical
216
habitat 10 g dry biomass m−2 day−1 in autumn and. 6 g dry biomass m−2 day−1 in winter in the
217
closed bioreactor. The maximum specific growth rate of 0.141 was found at 320C for
218
Spirulina platensis and that of 0.144 was found at 37 0 C for Spirulina fusiformis.Maximum
219
biomass production of 2.4 g l-1 and chlorophyll a production of 16.6 mg l -1 were observed at
220
320C for Spirulina platensis. Maximum biomass production of 2.3 g l
221
production of 14.2 mg l -1 were observed at 370C for Spirulina fusiformis ( Rafiqul Islam, M
222
et al 2003, Allen, K. A 2016). Spirulina, (Arthrospira platensis) is normally cultivated in
223
high salinity (>100 g/L) media or in high bicarbonate (16 g/L alkalinity) waters to allow
224
stable growth and reduce the harmful bacteria and fungi invasions. The maximum
225
productivity of biomass Spirulina is in the range of 21 -13.2 g m2 /d (Vonshak A
226
1997).Maximum biomass yield of Spirulina reported in the large open pond is lower than
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and chlorophyll - a
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other species.Spirulina biomass yield of 35 tonnes/ hectare/yr has been reported in a
228
commercial open mass cultivation pond at Siam Algae, Bangkok (Habib et al 2008)
229 230
4. Review of harvesting system
231
The best time for harvesting is early morning for following reasons •
Percentage of proteins in the Spirulina is highest in the morning.
233
•
Cool temperature makes the work easier.
234
•
More sunshine hours will be available to dry the product.
•
culture medium,
237 238
•
Removals of the residual culture medium to obtain the fresh Spirulina biomass, containing about 20 % dry matter.
239 240
Filtration - to obtain a biomass containing about 10 % dry matter and 50 % residual
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Harvesting is carried out in two steps:
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Different harvesting techniques used are •
filtration,
242
•
flotation,
243
•
centrifugation,
244
•
precipitation,
245
•
ion exchange,
246
•
Electrolytic and
247
•
Ultrasonic vibration.
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Harvesting of microalgae Spirulina is done using a filter or mesh cloth of at least 50 microns
249
to efficiently collect Spirulina from its medium..
250
4.1 Centrifugation
251
Centrifugation is a method to separate Spirulina algae from the media. Centrifugation and
252
chemical precipitation are economically feasible, where centrifugation being in appreciably
253
better A centrifuge is an equipment, driven by a motor, that puts an object in rotation around
254
a fixed axis, applying a force perpendicular to the axis. This method is reasonably efficient,
255
but sensitive algal cells may be damaged by pelleting against the rotor wall. Centrifugation
256
and drying are currently considered too expensive for personal use, though viable on a
257
commercial and industrial scale. The centrifuge works using the sedimentation principle,
258
where the centripetal acceleration is used to evenly distribute substances of greater and lesser
259
density.
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During commercial production processes filtration devices are used for harvesting. These are
262
of two types, i.e. inclined or vibrating screens. Inclined Screens are 380-500 mesh with a
263
filtration area of 2-4 m2 per unit and are capable of harvesting nearly about 10-18 m3 of
264
Spirulina culture per hour (Ogbonna et al 1999). Efficiencies of biomass harvesting are very
265
high which nearly 95%. Inclined, stationary screen is considered as a better solution for
266
harvesting Spirulina. Vibrating screens filter the same volume per unit time as the inclined
267
screens, but require one-third of the area. Their harvesting efficiencies are often very high.
268
The combination of both inclined filter and a vibrating screen is used. In the process of
269
pumping the algal culture, the Spirulina filaments may be damaged physically. Repeated
270
harvesting leads to the increasing enrichment of the culture with unicellular microalgae or
271
short filaments of Spirulina, which can pass through the screen easily. According to the work
272
reported in large-scale production of Spirulina the vibrating screen may not be the optimum
273
device for harvesting. Next step is the washing of excess salts from the biomass. The washed
274
cake is frequently homogenized before being dried.
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4.3 Drying
277
Though Spirulina can be consumed fresh, it has to be used after slight drying (Ankita et al
278
2013). Spirulina should be consumed within 6 hours of its harvest although it can be
279
preserved for later consumption for a period of up to one or more year by sun drying or in
280
greenhouses or in a solar drier.
281
Spirulina is relatively easily digestible in its fresh form (Richmond and Vonshak 1978).
282
Health and nutrition companies have tried to minimize the nutrients lost during drying and
283
maximizing the pure microalgae biomass recovered, while still keeping cost effective (
284
Sierra et al 2008). Different drying methods include sun drying, freeze drying, spray drying,
285
drum drying and cooking. Since Spirulina has a thin, fragile cell wall so, sun drying is
286
sufficient to sterilize the algae and make it consumable. Sun drying is the most popular
287
drying method, but requires a few precautions. Direct sun drying must be very quick,
288
otherwise the chlorophyll will be destroyed and the dry product will appear blue.
289
industries spray drier is used for Spirulina which flash dries fine droplets at very high
290
temperature and yields an extremely fine powder of low apparent density. Although freeze
291
drying considered as the best way of drying but far too expensive and complicated. The
292
biomass to be dried must be thin enough to dry before it starts fermenting. Fundamentally
293
two types of shapes are used, thin layers of rather fluid biomass laid on a plastic film, and
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295
the film, while in the later case it flows vertically through the tray. The rod shape is
296
theoretically better as evaporation can take place all around; rods are obtained by extrusion to
297
a diameter of 1 to 2 mm. But rods must be sturdy enough to maintain their shape, so this type
298
of drying is restricted to biomass that can be dewatered by pressing. The total duration of the
299
drying should not be less than 2 hours. Drying temperature should be limited to 68°C and
300
drying time is limited to 7 hours. For better preservation under storage, moisture should not
301
exceed 3-4%. During the drying process as well as afterward the product must be protected
302
against contaminations from dust and insects and should not be touched by hands. Incipient
303
fermentation during drying can be detected by smelling during the drying process as well as
304
afterward. For long time storage of Spirulina, it is vacuum dried and packed air‐tight where it
305
sustains its nutritional qualities for at least five years. The best storage is in heat sealed,
306
aluminized plastic bags.
307
As far as drying treatment is concerned, significant amounts of energy are needed to
308
evaporate water from the high moisture containing biomass. The evaporation energy
309
for 1 kg of water is 2.257 kJ, while depending on the drying equipment the efficiency of
310
the process varies. In the case of solar drying the efficiency is considered to be around
311
50 % since the material is exposed to open air, while for vacuum drying the efficiency
312
can rise up to 80 %. Taking into consideration the final moisture content that can be
313
achieved, specifically 4 % and 2.5 % for solar and vacuum drying, respectively, the
314
amount of cultivated and harvested biomass that it is needed to acquire 1 kg of dried
315
material differs. (Papadaki et al 2017)
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4.4 Grinding/powdering
318
The dry chips or rods are usually converted to powder by grinding to increase their apparent
319
density. Spirulina is used as a whole food/ dietary supplement which is available in tablet,
320
flake and powder form (Figure 2). Spirulina can be directly ground to ultra fine powder
321
form. It is also used as a feed supplement in the aquaculture, aquarium and poultry industries.
322
Commercial Spirulina is most often sold as a deep green-coloured powder or a tablet. It is
323
used as an ingredient in packaged health food snacks and drinks. The strained Spirulina algae
324
paste is laid out and triple-washed with potable water for salt removal before it goes into a
325
drying vessel that converts it into powder form. The dried Spirulina flakes are crushed using
326
high impact ultrafine grinding mill. Grinding is continued for about 6 to 10 hours, till the
327
average powder size reaches 200 ~ 800nm. The two most common forms of commercially
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available Spirulina are powder and tablets. It is also an ingredient in some protein and
329
energy-boosting powder mixes.
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Powdered Spirulina
330
(a)
331
Figure 2 :Dried Spirulina (a) Spirulina flakes (b) Powdered form of Spirulina
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332
(b)
333
4.5 Pellets/ capsules
335
Spirulina powder is pressed together into a tablet or granule shape ( Ogbonda et al 2007) for
336
improved acceptance and performance. It is formulated as a completely balanced diet which
337
provides optimum growth and health (Slade et al 2013). It contains proteinated trace
338
minerals for higher stability, biological availability and overall human health.
339
Advantages of Spirulina pellets are as follows
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Excellent water stability
341
Easily consumable
342
Contains extra levels of preservative and antioxidants
343
Longer shelf life.
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4.6 Spirulina products
346
Spirulina fights against aging, oxidative stress, diabetes, cardiovascular diseases,
347
hypertension, arthritis, infertility and cancer. Spirulina is considered as a superfood as it is
348
the best food supplement. Different healthcare industries make Spirulina products. Major
349
companies which are involved in cultivating spirulina globally are :
350
Earthrise Nutritionals (USA California) (earthrise.com)
351
DIC Lifetec Spirulina (Japan) (dlt-spl.co.jp/business/en/spirulina/)
352
Cyanotech Spirulina (USA Hawaii) (cyanotech.com)
353
Boonsom Spirulina Farm (Thailand) (boonsomfarm.com)
354
FEBICO (Far East Bio-Tec Co.) (Taiwan) (febico.com)
355
Spirulinea (France/Laos) (spirulinea.com)
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357
Green Valley (Germany) (greenvalley.de)
358
Natesis Spirulina (France) (natesis.com)
359
Spirulina.PL (Poland) (spirulina.pl)
360
All Seasons Health (United Kingdom) (allseasonshealth.com)
361
NaturKraftWerke Spirulina (Switzerland) (naturkraftwerke.com)
362
Sanatur Spirulina (Germany) (sanatur.de)
363
Marcus Rohrer Spirulina (Netherlands) (spirulina.nl)
364
Taiwan Chlorella (Taiwan) (taiwanchlorella.com)
365
RBC Life Sciences (USA) (rbclifesciences.com)
366
Gerophyta Nutraceuticals Company in Tamil Nadu, India offers a wide range of products,
367
as Spirulina Powder, Spirulina Capsules, Spirulina tablets, Spiruvita-C, Dr. Spirulina Diavita-
368
C, Spirulina herbal face pack. Other companies also offers a wide range of products as
369
spirulina bar, spirulina green tea, Spirulina personal care products, Spirulina chocolates,
370
spirulina drinks, spirulina honey etc.
371
5. Spirulina benefits
372
5.1 Nutritional composition of Spirulina
373
Spirulina is a microalga that has been consumed for decades due to its high nutritional value
374
and reported health benefits. Today Spirulina is endorsed as a secret, potent superfood, also
375
considered as the miracle that grows naturally in oceans and salty lakes in subtropical
376
climates. Spirulina contains practically all the components found in the ideal complete food.
377
A considerable proportion of proteins, vitamins, mineral salts, carbohydrates, pigments, trace
378
elements, and essential fatty acids are present. Unlike other algae, Spirulina is easier to
379
consume
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Protein
382
Spirulina is the richest source of proteins.Spirulina is abundant in plant protein, which makes
383
up 60%-70% of its weight (Balasubramani, R et al 2016). Soya flour, contains about 35%
384
protein. Qualitatively, Spirulina provides complete proteins as it contains the full range of
385
essential amino acids which is 47% of total protein weight.
386 387
Vitamins
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389
content is unusually high which is about 30 times higher than found in a carrot. Spirulina is
390
also exceptionally rich in vitamin B12 cobalamin. This vitamin is, most difficult to get from a
391
vegetarian diet because no fruit, vegetable, grain, or legume contains it. Spirulina has four
392
times as much vitamin B12 than raw liver, which was considered to be the best source of this
393
nutrient. Spirulina is also recognized as an excellent source of vitamin E comparable to those
394
found in wheat gram(Yin, C et al 2017). The primary antioxidant vitamins contained in
395
Spirulina are ß-carotene, carotenoids, and vitamin E.
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Minerals
398
Spirulina contains mineral such as iron, magnesium, calcium, and phosphorus. Spirulina is a
399
splendid source of iron which contains 20 times more iron than wheat gram. Iron is a mineral
400
that is mainly present in foods from animals, such as meat, and fish (Roberto 2015;
401
Balasubramani, R et al 2016). Spirulina is very advantageous for athletes, vegetarians,
402
pregnant women, and teenagers. Average nutritional analysis of Spirulina per 100gm is
403
shown in Table 5.
404
Table:5: Average nutritional analysis of Spirulina per 100g (Roberto 2015) Nutritional Value(in
mgs)
mgs) Calcium
1000
Phosphorus
800
2200
Magnesium
400
Minerals
8000
Iron
58
Dietary Fibre
7000
Zinc
3
Vitamin A
212
Copper
1.2
Chlorophyll
600
Manganese
0.5
Fat
22000
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63000
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Chromium
0.03
Vitamin B1
3.5
Potassium
1.4
Vitamin B2
0.4
Gamma-linoleic acid
1
Vitamin B3
1.3
Vitamin B8
0.005
Vitamin B5
0.2
Vitamin B9
Vitamin B6
6
Vitamin B12
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0.05
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408
Spirulina is the best complete nutritional food source of protein, beta carotene, GLA, B
409
Vitamins, minerals, chlorophyll, sulfolipids, glycolipids, superoxide dismutase, phycocyanin,
410
enzymes, RNA, DNA, and supplies many nutrients that are lacking in most of the people's
411
diets. Nutraceutical food products supplement the diet as well as facilitate the prevention or
412
treatment of a disease or disorder. There are many Nutraceutical and Functional food
413
products which are commercially available with researched and approved health benefits. The
414
current estimated global market size for nutraceuticals products is approximately 30 to 60
415
billion dollars, which is primarily in the United States, Japan, and Europe. Spirulina products
416
have a potential short-term growth market demand of over 197 billion dollars. As the demand
417
for nutraceuticals and food supplements is increasing, organisms that can rapidly produce
418
nutritional compounds are in demand. The ability of Spirulina as a potent for anti-viral, anti–
419
cancer, hypocholesterolemic and health improvement agent is getting attention as a
420
nutraceutical and pharmaceutical.
421
Spirulina has the following health benefits
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•
423
•
Nourishes people with digestion, assimilation & elimination
424
•
Prevents diabetes
425
•
Aids in reducing stress
426
•
Prevents depression
427
•
Concentrated impressive nutrients to weight loss
Helps athletes with long lasting energy and vitality
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•
Improves memory and mental clarity
429
•
Stimulates immune system to destroy invading disease organisms and carcinogens
430
•
Enhance the immune system with its antiviral, anti-tumor and interferon inducing effects
431
•
Promotes tissue repair in wounds and burns and also has the anti-infectious properties
433
•
Decreases cholesterol levels and helps to lower the risk of cardiovascular disease
434
•
Functions as an anti-inflammatory agent
435
•
Reduce the inflammation characteristic of arthritis
436
•
Govern the appetite and helps to stimulate the metabolism
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Hailed by health obsessives as the superfood to conquer viruses, prevent aging and even ward
438
off cancer, Spirulina may be able to play another, much more significant role as a way to
439
combat malnutrition in developing countries.In light of Spirulina’s gamut of nutritional
440
goodness, a number of individuals and organizations are developing Spirulina programmes to
441
address malnutrition. Aloni et al 2016 reported that the administration of Spirulina at a dose
442
of 10 g per day seemed to significantly and quickly improve the nutritional status of
443
undernourished children in the intervention group when compared to the control group.
444
Indeed, the rate of global acute malnutrition decreased from 30% before the Spirulina
445
supplements to 20% at day 30. According to The Hindu Survey Spirulina powder ranges
446
from 1000 – 4500INR /Kg. Spirulina capsules range from 250- 900INR / 60 capsules.
447
Spirulina face packs vary from 360 – 900INR / 100gm.
448
6. Future outlook
449
Spirulina is a promising food source with protein content about 65-70%.However the
450
maximum protein content reported in literature till date is 59%. Proper designing of
451
cultivation system, growth efficient techniques and use of organic fertilizer may be adopted
452
to maximize the protein content of Spirulina.
454
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The food processing technique including drying of Spirulina biomass is an
important step to retain the nutrition and active compound.
455
•
Further efforts should be made to increase protein content and biomass yield.
456
•
Open raceway pond is economical but the annual production of only 0.8gms liter -1 day-1 has been reported.
457 458 459
•
Open pond cultivation system has many drawbacks such as improper light intensity, contamination and requires large acres of land. Page 31 of 42
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•
Aeration is very necessary for getting good quality and better yields of
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Spirulina species. Aeration should be done every 3-4 hrs, to avoid clump
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formation.
463
•
It has been reported on a dry weight basis for Spirulina, productivity and total biomass production at the end of a production cycle in PBR were 30 mg L-1
465
day -1 and 0.9 g.L-1.
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Highest productivity with Spirulina plantesis was reported in raceway pond at
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Australia with a photosynthetic efficiency of 1.5% and areal productivity of 91
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ton ha-1yr-1. •
The daily production system according to the work reported is greater with
SC
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PBR and lesser with the open raceway ponds, So, qualitatively as well as
471
quantitatively when measured, PBR systems are more efficient.
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Hybrid reactors are fortunate outcomes of groundbreaking strategies and technology advancements of Spirulina cultivation. Productivity is expected to
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increase in the case of the hybrid system.The hybrid system proves to be a
475
better solution to overcome the drawbacks of open pond and PBR. Poly bags
476
can be a good option if economically tested.
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Climatic factors play an important role in Spirulina cultivation where the optimum temperature is very important. So, in very hot or cold or less humid
479
conditions greenhouse can be used for Spirulina cultivation. Different
480
greenhouse designs have been studied to design for Spirulina.
481
•
485 486
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Various harvesting system has been reported among them centrifugation may not be suggested for Spirulina as it is expensive and it may also break
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Spirulina cells on separation.
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Normal filter or mesh cloth of 30µ - 450µ is highly used and it may separate
Spirulina very easily and efficiently.
•
Greenhouse-based solar drying is preferred over open drying in order to
487
maintain the nutritional quality. Microwave or oven drying method can also be
488
used as an alternate method.
489 490
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Dried powder may be transformed to easy and consumable form than pellets, powder and capsules which are already available in the market.
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ACCEPTED MANUSCRIPT 7. Summary
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The main purpose of this review is to call attention for different cultivation systems, growth
494
parameters, and productivity of Spirulina in various climatic conditions. The present review
495
had revealed that significant studies have been carried out on various growth techniques to
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increase the protein productivity of Spirulina.
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The following conclusions are drawn from the study.
498
•
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Spirulina is mainly reported to be grown in open raceway ponds for commercial or industrial purposes. New hybrid techniques such as poly bags and greenhouse can
500
also be implemented to increase cost economics and the annual spirulina biomass
501
productivity. •
Commercial Industrial scale cultivation of spirulina uses inorganic chemicals which
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499
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are expensive and requires optimization of media concentration to avoid intrusion of
504
prevention of facultative pathogens.
505
•
Climatic factors, light intensity and aeration are very important in Spirulina growth system. The culture conditions also influence the growth phases of Spirulina
507
platensis, causing changes in its composition. Growth rate and doubling time can be
508
increased by bringing variations in used growth media.
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There has been a significant change in functional properties of Spirulina under stressed conditions. Environmental stresses like high pH, light, salinity and
511
temperature affect growth and nutrient productivity. •
carbohydrates, essential fatty acids, vitamins, minerals, carotenes, chlorophyll a and
513
phycocyanin to fight against malnutrition. So it can be used in nutraceuticals and
514
pharmaceuticals applications.
515 516
Spirulina is contended to have several health benefits as it contains essential proteins,
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Innovative formulations are required to fortify conventional foods with Spirulina and
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more scientific, clinical and toxicological research has to be carried out for extensive
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usage of Spirulina in food and pharma industry.
519 520
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Development of various Spirulina fortified foods is required to create nutritional awareness and increase the acceptance level in developing countries.
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Despite their evident use as a nutritional product, Industrial production of Spirulina
522
are still more or less confined to the limited natural areas. Mass cultivation of
523
spirulina has to be encouraged globally to avoid food shortages in near future. Acknowledgement
525
We are very thankful to the Honourable Ex-Director, Dr. K.K.Appukuttan, Maulana Azad
526
National Institute of Technology Bhopal, India for his continued support and guidance to
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complete this research work. This research did not receive any specific grant from funding
528
agencies in the public, commercial, or not-for-profit sectors.
529
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Highlights Various Methods of spirulina cultivation are appraised.
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Growth conditions and Spirulina yield are reviewed.
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Spirulina growth for nutritional use is highlighted.
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Recommendations of spirulina as super food is suggested
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