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 Energy Centre, Maulana Azad National Institute of Technology, Bhopal (M.P), India
2
4 Department of Mechanical Engineering, Maulana Azad National Institute of Technology,
5 Bhopal (M.P), India
3,
6 Faculty of Mechanical Engineering, Universiti Malaysia Pahang, 26600 Pahang, Malaysia

PT
7 E mail: rumaarora14@gmail.com ;sudhakar.i@manit.ac.in

8 Abstract

RI
9 Background

SC
10 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

U
12 water, can be harvested and processed easily. It has very high content of macro and
AN
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
M

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
D

18 source of nutraceutical and pharmaceuticals.

TE

19 Scope and Approach

20 The primary objective of this paper is to review the utilization of Spirulina as a dietary
EP

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.
C

23 Key findings and conclusion

AC

24 The important growth parameters have been studied to enhance Spirulina biomass
25 productivity qualitatively and quantitatively. This review provides useful information on
26 commercially viable technology for Spirulina cultivation. Mass cultivation and Innovative
27 formulations are further needed to fortify conventional foods with Spirulina based protein
28 system.

29 Keywords: Spirulina; Pharmaceutical; nutritional use; dietary supplement; open pond; PBR

30

Page 1 of 42
 ACCEPTED MANUSCRIPT
31 Nomenclature

32 (PX) Cell productivity

33 (TC) Cultivation time
34 Γ Productivity of the system
35 µ Specific growth rate
36 x Biomass concentration

PT
37 (Xm- Xi) Cell concentration
38 N0 Initial population size

RI
39 t Amount of time that has past
40 Nt Population size at time

SC
41 G Generation time
42 N.S – Not Specified

U
43 1. Introduction
AN
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
M

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
D

48 specificity (Becker.2007). Sometimes cyanobacteria are also included in microalgae.

49 Microalgae classification includes prokaryotic and eukaryotic unicellular and multicellular.
TE

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
EP

52 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
C

54 biotechnology are biofuels, agricultural biostimulants for crop plants, waste water treatment
55 etc. Microalgal biotechnologies refer to the production of different products as phycocyanin,
AC

56 carotenoids, fatty acids and lipids for application in health food, cosmetics, food supplements,
57 pharmaceuticals and fuel production. Microalgal groups of major importance are chlorophyte,
58 bacillariophytes, while macroalgae are harvested from natural habitats. Algae that is currently
59 cultivated for its maximum protein content is the cyanobacterium species Athrospira, which
60 is commonly known as Spirulina.
61
62

Page 2 of 42
 ACCEPTED MANUSCRIPT
63 Spirulina was first discovered by Spanish Scientist Hernando Cortez and Conquistadors in
64 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.
67 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

PT
69 plant, Sosa Texcoco, was set up in 1969 by the French.
70 Spirulina is the most nutritious, concentrated food that is known to mankind containing

RI
71 antioxidants, phytonutrients, probiotics, and nutraceuticals.Spirulina is fast emerging as a
72 complete answer to the varied demands due to its imposing nutrient composition which can

SC
73 be used for therapeutic uses. The United Nations world at food conference declared that
74 Spirulina as the best food for future, and it is gaining popularity nowadays (Pulz and Gross
75 2004). World Health Organization has described spirulina as Mankind’s best health product.
76

U
According to UNESCO, spirulina is most ideal food for tomorrow. According to NASA and
AN
77 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”.
M

79 Intergovernmental institution permitted for the use of Micro-algae Spirulina against

80 Malnutrition (IIMSAM).
D

81 The two most important species of Spirulina are Spirulina maxima and Spirulina platensis. It
82 has a considerable high content of micro and macronutrients.Its dry weight chemical
TE

83 composition includes 60-70% proteins, carbohydrates, vitamins like provitamin A, vitamin C,

84 vitamin E, minerals such as iron, calcium, chromium, copper, magnesium, manganese,
EP

85 phosphorus, potassium, sodium and zinc. Essential fatty acids γ-linolenic acid (GLA),
86 pigments like chlorophyll a, phycocyanin and carotenes are also present. Spirulina is also
C

87 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
AC

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;
92 Sudhakar and Premlatha 2015).
93 Spirulina is a planktonic photosynthetic cyanobacterium that forms huge populations in
94 tropical as well as subtropical bodies of water which contain a high amount of salts such as
95 carbonate and bicarbonate with alkaline pH 9.5(Sjors 2010; Habib et al 2008). Generally,
96 microalgae have higher growth rates, higher CO2 fixation efficiency and larger quantities of

Page 3 of 42
 ACCEPTED MANUSCRIPT
97 high-value products, such as dietary supplements for human along with animals (Zeng et al
98 2011; Anupama 2000).Cost effectiveness and composition of cultivation media along with
99 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).

PT
103 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

RI
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

SC
107 for nutritional products. The article starts with the illustration of spirulina growth chain
108 from identifying suitable strain to the final product.
109 The present study focuses on growth rate, productivity, growth parameters, different
110

U
cultivation systems (outdoor and indoor systems), harvesting and drying techniques of
AN
111 Spirulina. This review focuses on following aspects:
112 • Strain selection and cultivation of Spirulina.
•
M

113 Optimum parameters for growth of Spirulina.

114 • Harvesting and drying techniques of Spirulina.
D

115 • Commercial applications of Spirulina as pharmaceutical and nutraceuticals product.

116
TE

117 2. Review of growth system

118 Cultivation of algae can be done in open systems like ponds, lakes or lagoons or in a closed
EP

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
C

121 approaches are used commercially to produce high-value products.

122 2.1 Open pond system
AC

123 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
125 categorized into natural waters as lakes, lagoons, ponds and artificial ponds or containers.
126 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

Page 4 of 42
 ACCEPTED MANUSCRIPT
130 requirement of large acres of land. Also, due to inefficient aeration in open cultivation
131 systems, their mass transfer rates are very poor resulting in less biomass productivity. The
132 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

PT
136 Sharma 2012).
137 Table 1 summarizes the advantages and limitations of open ponds, photobioreactors and

RI
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

SC
140 protect them from foreign particles in the air. The optimally designed algae greenhouse and
141 controlled environment systems can increase productivity 10 fold compared to outdoor
142 growth. Construction of greenhouse includes design and optimizing for improved biomass
143

U
yield. Controlled environment algae facilities are gaining momentum due to improved yields
AN
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
M

146 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
D

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
TE

150 complete protection against undesirable weather conditions, while the full vertical vent
151 promotes optimum light and air movement.
EP

152
153
C

154
155
AC

156
157
158
159
160
161
162
163

Page 5 of 42
 ACCEPTED MANUSCRIPT
164 Species Selection
Phase- I

165
166
167 Microalgae Macroalgae Cyanobacteria
168 (a)
169

PT
Growth System of Spirulina
170
171
Phase- II

RI
172
173 Open Raceway Photo Hybrid System
Ponds bioreactors

SC
174
175 (b)
176

U
Growth Parameters of Spirulina
177
AN
Phase- III

178
179
Mother Media Mixing & Temp. & pH Light Growth
M

180 culture Selection Aeration Intensity Rate&

Productivity
181
D

182 (c)
183 Harvesting System of Spirulina
TE
Phase- IV

184
185
Normal Filter
EP

Centrifugation
186
187 (d)
C

188
Drying
189
AC
Phase- V

190 Grinding/Powdering
191
Pellets/ Capsules
192
193 (e)
Figure 1 : Different phases of Spirulina cultivation system (a) strain selection (b) Growth systems
194
(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
199 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

PT
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,

RI
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

SC
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

U
2002). In order to overcome the problems with open ponds, much attention is now focused on
AN
212 the development of suitable closed systems such as flat-plate, tubular, vertical column and
213 internally-illuminated photobioreactor. Generally, laboratory-scale photobioreactors are
M

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
D

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
TE

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
EP

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
C

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
AC

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.

Page 7 of 42
 ACCEPTED MANUSCRIPT
232 • 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

PT
238 (Roberto 2015).

Open systems Closed systems Hybrid system

Factor

RI
(raceway ponds) (photobioreactors) (Open Pond + PBR)

Space required High Low High

SC
Area/volume ratio Low (5–10 m−1) High (20–200 m−1) Variable

U
Evaporation High No evaporation Minimized
AN
Water loss Very high Low Less

CO2-loss High Low Minimizes

M

Temperature Highly variable Required cooling Controlled

D

Weather dependence High Low Low

TE

Process control Difficult Easy Difficult

Cleaning Easy Required Difficult

EP

Biomass quality Variable Reproducible Better

C

Population density Medium High Medium

AC

Harvesting
Medium High High
efficiency

Harvesting cost High Lower High

Light utilization
Poor Good Better
efficiency
Most costly Oxygen and
Mixing Temp control
parameters temperature control

Page 8 of 42
 ACCEPTED MANUSCRIPT
Open systems Closed systems Hybrid system
Factor
(raceway ponds) (photobioreactors) (Open Pond + PBR)
Contamination
Difficult Easy Easy
control

Capital investments Low High Low

PT
3–5 times more 5-7 times more
Productivity Low
productive productive

RI
Hydrodynamic stress
Very low Low–high Low
on Spirulina

SC
239
240 2.2.1 Vertical-column PBR
241 Vertical-column photobioreactors are easy to operate compact and low-cost (Miron et al
242
U
2002). Various designs and scales of vertical column photobioreactors have been reported for
AN
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
M

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
D

247 2002). Some bubble column photobioreactors are equipped with either draft tubes or
248 constructed as split cylinders.
TE

249 2.2.2 Flat plate PBR

250 For cultivation of photosynthetic microorganisms flat-plate photobioreactors have received
EP

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
C

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
AC

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. Accumulation of dissolved oxygen concentrations in horizontal tubular
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

Page 9 of 42
 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
Reactor Location Light Photosynt Productivity Reference

PT
system path hetic (ton ha-1yr-
1
Efficiency )

RI
Raceway La 0.1- 1.5% 43.1 ( Olguin et al 2003)
pond Mancha, 0.25

SC
Mexico
Raceway Florence, 0.035 1.5% 20.0 (Tredici and Materassi
pond Italy 1992)

U
AN
Raceway Malaga, 0.30 1.5% 23.6-30.0 (Jimenez et al 2003)
pond Spain
M

Raceway Australia 0.30 1.5% 91.0 (Borowitzka 1999)

pond
D

Horizontal Florence, 0.06 1.8-3% 30.0 (Tredici and Materassi

TE

Tubular Italy 1992)

Flat Panel US 0.10 3.8% 22.1 (Richmond and Zhang,

EP

2001)

267 2.2.3 Tubular PBR

C

268 A tubular photobioreactor is the most suitable types of bioreactors for outdoor mass
AC

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

Page 10 of 42
 ACCEPTED MANUSCRIPT
277 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

PT
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

RI
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

SC
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

U
be minimized. A continuous supply of light to the photobioreactor can be maintained both
AN
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
M

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
D

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
TE

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
EP

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
C

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
AC

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).

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)
Culture Dimensions Specific Prospects Limitations Images

PT
systems growth

RI
rate
Open Variable 0.30day-1 Relatively economical, easy to Little control of culture conditions,

SC
systems clean up after cultivation, difficulty in growing algal cultures
good for mass cultivation of for long periods,

U
algae poor productivity,

AN
occupy large land mass,

M
limited to few strains of algae,
cultures are easily contaminated

D
Vertical 0.2 m 0.015 ± High mass transfer, Small illumination surface area,

TE
Column diameter 0.002 good mixing with low shear construction require sophisticated
−1
PBR and 4 m h ) stress, materials,
EP
column low energy consumption, shear stress to algal cultures,
height[ high potentials for scalability, the decrease of illumination surface
C

easy to sterilize, area upon scale-up

AC

good for immobilization of

algae,
reduced photoinhibition and

Page 12 of 42
 ACCEPTED MANUSCRIPT

photo-oxidation
Flat plate 0.07 m Large illumination surface Scale-up require many
PBR wide, area, compartments and support

PT
1.5 m height suitable for outdoor cultures, materials,

RI
, 2.5 m good for immobilization of difficulty in controlling culture
length algae, temperature,

SC
Volume good light path, good biomass some degree of wall growth,
250lts productivities, the possibility of hydrodynamic

U
Productivity relatively cheap, stress to some algal strains

AN
- 1.0 g/L easy to clean up,
day readily tempered,

M
low oxygen buildup

D
Tubular D = 3-10cm 0.055 h−1 Large illumination surface Gradients of pH,

TE
PBR area, dissolved oxygen and CO2 along
suitable for outdoor cultures, the tubes, fouling,
EP
fairly good biomass some degree of wall growth,
productivities, requires large land space
C

relatively cheap
AC

Page 13 of 42
 ACCEPTED MANUSCRIPT

Internally Not Large illumination surface Outdoor mass cultivation of algae

Illuminated Specified area, require some technical efforts.
PBR can utilize both solar and

PT
artificial light system,

RI
contamination can be
minimized in this system

SC
Hybrid Not Minimize microbial Requires large areas of land and
System Specified contamination, maximize some technical efforts

U
biomass and product yield,

AN
Maximize CO2 supply

M
Poly Bags D <30cm 0.20day-1 site flexibility, Polyethylene bag cultures have a

D
Low-cost materials, relatively short life because the

TE
easy scalability, internal surface attracts culture
optimal light exposure, debris and bacteria, which
EP
isolation of the crop from collectively reduce light penetration
predators, and are a source of contamination
C

very high biomass

AC

concentration,
low energy consumption
effective weather protection

Page 14 of 42
 ACCEPTED MANUSCRIPT
1 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

PT
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

RI
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

SC
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

U
widened and inflated to approximate an open pond; this design is mainly aimed at reducing
AN
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.
M

17 Polybags

18 The cultivation of algae using natural ponds is easy, but turning it into a viable feedstock is
D

19 very difficult. So to enable higher production levels, least investments and operating costs,
TE

20 greater biomass density, better climatic controlled conditions, and industrial scalability, this
21 technique can be implemented. Thin, floating, flexible, multi-compartment photobioreactors
EP

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
C

25 productivity results have indicated that growing algae in floating bags can be much more
AC

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).

Page 15 of 42
 ACCEPTED MANUSCRIPT
32 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

PT
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

RI
40 (Algae Industry Magazine, 2012).

41 3. REVIEW OF GROWTH PARAMETERS

SC
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

U
44 (Sudhakar et al 2011)
AN
45 3.1 Climatic factors

46 Temperature is an important climatic factor influencing the rate of growth of Spirulina.

M

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
D

49 but direct sunlight is not recommended, 30% of full sunlight is actually better, except that
TE

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
EP

53 synthesis of proteins and respiration.

54 3.2 Media
C
AC

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.

Page 16 of 42
 ACCEPTED MANUSCRIPT
63 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.

PT
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)

RI
Rao’s CFTRI OFERR Conventi
Zarrouk’ Reduced
Media(g Media(g Media(g George’s onal

SC
s Cost
Ingredient ms/l) ms/l) ms/l) Media(g growth
Media(g Media(g
ms/l) Media(g

U
ms/l) ms/l)
ms/l)
AN
NaHCO3 16.80 15 4.5 8.0 - 16 16.8

K2HPO4 0.50 0.50 0.5 - 0.02 - 0.235

M

NaNO3 2.50 2.50 1.5 - - - -

D

K2SO4 1.00 0.60 1.0 0.5 - 0.5 0.353

TE

NaCl 1.00 0.20 1.0 5.0 - 1.00 0.471

MgSO4·7 0.04 1.2 0.16

0.20 0.02 0.1 -
EP

H2O

EDTA 0.08 - - - - - 0.353

C

CaCl2·2H2 0.008 0.04 -

AC

0.04 - 0.1 0.176

O

FeSO4·2H2 - 0.01 0.05

0.01 - - 0.265
O

H3BO3 2.86 - - 0.052ml - - 2.86

MnCl2·4H - - -
1.180 - - 1.81
2O

Page 17 of 42
 ACCEPTED MANUSCRIPT
ZnSO4·7H - - -
0.222 - - 0.222
2O

Na2MoO3· 0.015 - - - - - 0.0177

CuSO4·5H - - -
0.074 - - 0.079
2O

PT
NH4VO3 22.9 - - - - - -

NiSO4·7H2 - - -

RI
47.8 - - -
O

SC
NaWO2 17.9 - - - - - -

Ti2(SO4)3· - - -
4.4 - - -

U
6H2O AN
Co(NO3)2· - - -
4.4 - - -
6H2O
M

Ferric - - -
- 0.035 - -
citrate
D

Peptone - - - - 1.00 - -
TE

KNO3 - - - - - 2.00 -

(NH4)2HP - - -
EP

- - 0.1 -
O4

- - - 2
C

squeezes
AC

Chelated
- - (¼ -
Iron
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

72 3.3 Mother culture

73 For Inoculums preparation and culture maintenance fully grown concentrated Spirulina

PT
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

RI
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

SC
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

U
80 the area of the culture exposed to light, it is recommended to maximize this area at all times.
AN
81 It is reported that minimum cell population is necessary to initiate and sustain Spirulina
82 cultures.
M

83 3.4 Mixing and aeration

84 Agitation of the culture is necessary to homogenize and ensure a good distribution of lighting
D

85 among all the filaments of Spirulina. Mixing plays an important role in the productivity of
TE

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
EP

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
C

91 illumination. It also helps to distribute carbon dioxide concentration uniformly and removes
AC

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

Page 19 of 42
 ACCEPTED MANUSCRIPT
100 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

PT
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
Spirulina can grow at 200C- 370C. The best temperature for Spirulina growth is between 290C

RI
108
109 - 350C. During night growth of Spirulina is least or almost zero. It is reported that the effect

SC
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
113 •
U
Available carbon alteration, which may interfere with photosynthesis.
AN
114 • Through the disruption of cell membrane processes.
115 This may have a direct impact on the accumulation of antioxidants (Matsunaga et al 1991).
M

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
D

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
TE

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
EP

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
C

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
AC

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
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
133 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

PT
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

RI
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

SC
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

U
exposed to two rhythms of the dark and light regime. These cycles impose a unique
AN
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
M

149 decrease of the growth rate of Spirulina.

150 The attenuation coefficient was observed to scale linearly with microorganism density.
D

151 The irradiance attenuation coefficient at wavelength λ, αλ, is calculated according to,
TE

152 Gλ(z)/Gλ(0) = e −αλz Eq.1

153 The Spectral Irradiances at different depths z is calculated according to above equation
EP

154 knowing the value of αλ,

155 The spectral attenuation cross section, Aλ, is defined as, Aλ = αλ/X Eq.2
C

156 Using the irradiance attenuation coefficients for each culture, αλ,
AC

157 , Gλ(z) = Gλ(0)e −αλz Eq.3

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

Page 21 of 42
 ACCEPTED MANUSCRIPT
163 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

PT
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

RI
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,

SC
173 when cell concentration reached 5800 mg L−1, the growth stopped likely due to photo
174 saturation or shadowing.

U
175
176 3.7 Growth rate & productivity
AN
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
M

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,
D

181 under normal growth conditions the specific growth rate is described by the following
TE

182 equation:

t dx
183 µ= Eq. 4
x dt
EP

184 Calculation of specific growth rate has been described in many ways. Most commonly used
C

185 formula is
ln x2 − ln x1
AC

186 µ= Eq. 5
t 2 − t1
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:
ln 2 0.693
190 g= == = d .t Eq. 6
µ µ
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
 ACCEPTED MANUSCRIPT
193 According to Grobbelaar (Miron et al 1999], one of the most important factors to obtain high
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

PT
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

RI
201 in the range of usual open raceway performance (Bharathiraja et al 2015).
202 The productivity of the system γ is defined as

SC
203 γ =µx Eq. 7
204 Where µ is the specific growth rate in units of reciprocal of time and x is the biomass
205 concentration.
206

U
The cell productivity (PX) is calculated as the ratio of the variation in cell concentration (Xm-
AN
207 Xi) to the cultivation time (TC)
208 PX = (Xm – Xi ) / TC Eq. 8
M

209 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.
D

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
TE

213 relatively high value in open pond cultivation system.

214 Toyoshima et al 2015 reported the maximum biomass productivities of Spirulina platensis in
EP

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
closed bioreactor. The maximum specific growth rate of 0.141 was found at 320C for
C

217
218 Spirulina platensis and that of 0.144 was found at 37 0 C for Spirulina fusiformis.Maximum
AC

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 -1
and chlorophyll - a
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

Page 23 of 42
 ACCEPTED MANUSCRIPT
227 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
232 • Percentage of proteins in the Spirulina is highest in the morning.

PT
233 • Cool temperature makes the work easier.
234 • More sunshine hours will be available to dry the product.

RI
235 Harvesting is carried out in two steps:
236 • Filtration - to obtain a biomass containing about 10 % dry matter and 50 % residual

SC
237 culture medium,
238 • Removals of the residual culture medium to obtain the fresh Spirulina biomass,
239 containing about 20 % dry matter.
240 Different harvesting techniques used are
U
AN
241 • filtration,
242 • flotation,
M

243 • centrifugation,
244 • precipitation,
D

245 • ion exchange,

•
TE

246 Electrolytic and

247 • Ultrasonic vibration.
248 Harvesting of microalgae Spirulina is done using a filter or mesh cloth of at least 50 microns
EP

249 to efficiently collect Spirulina from its medium..

250 4.1 Centrifugation
C

251 Centrifugation is a method to separate Spirulina algae from the media. Centrifugation and
AC

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.

Page 24 of 42
 ACCEPTED MANUSCRIPT
260 4.2 Filtration
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

PT
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.

RI
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

SC
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

U
device for harvesting. Next step is the washing of excess salts from the biomass. The washed
AN
274 cake is frequently homogenized before being dried.
275
M

276 4.3 Drying

277 Though Spirulina can be consumed fresh, it has to be used after slight drying (Ankita et al
D

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
TE

280 greenhouses or in a solar drier.

281 Spirulina is relatively easily digestible in its fresh form (Richmond and Vonshak 1978).
EP

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 (
C

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
AC

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. In
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

Page 25 of 42
 ACCEPTED MANUSCRIPT
294 rods as spaghetti laid on a perforated tray. In the former case the air flows horizontally over
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

PT
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

RI
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

SC
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
U
As far as drying treatment is concerned, significant amounts of energy are needed to
AN
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
M

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
D

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
TE

314 amount of cultivated and harvested biomass that it is needed to acquire 1 kg of dried
315 material differs. (Papadaki et al 2017)
EP

316
317 4.4 Grinding/powdering
C

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,
AC

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
Page 26 of 42
 ACCEPTED MANUSCRIPT
328 available Spirulina are powder and tablets. It is also an ingredient in some protein and
329 energy-boosting powder mixes.

Sirulina

PT
flakes

RI
330 Powdered Spirulina

331 (a) (b)

332 Figure 2 :Dried Spirulina (a) Spirulina flakes (b) Powdered form of Spirulina

SC
333
334 4.5 Pellets/ capsules

U
335 Spirulina powder is pressed together into a tablet or granule shape ( Ogbonda et al 2007) for
AN
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.
M

339 Advantages of Spirulina pellets are as follows

340 Excellent water stability
D

341 Easily consumable

TE

342 Contains extra levels of preservative and antioxidants

343 Longer shelf life.
EP

344
345 4.6 Spirulina products
346 Spirulina fights against aging, oxidative stress, diabetes, cardiovascular diseases,
C

347 hypertension, arthritis, infertility and cancer. Spirulina is considered as a superfood as it is

AC

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)

Page 27 of 42
 ACCEPTED MANUSCRIPT
356 Spiruline de Burkina (Burkina Faso) (spirulineburkina.org)
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)

PT
362 Sanatur Spirulina (Germany) (sanatur.de)
363 Marcus Rohrer Spirulina (Netherlands) (spirulina.nl)

RI
364 Taiwan Chlorella (Taiwan) (taiwanchlorella.com)
365 RBC Life Sciences (USA) (rbclifesciences.com)

SC
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

U
spirulina bar, spirulina green tea, Spirulina personal care products, Spirulina chocolates,
AN
370 spirulina drinks, spirulina honey etc.

371 5. Spirulina benefits

M

372 5.1 Nutritional composition of Spirulina

373 Spirulina is a microalga that has been consumed for decades due to its high nutritional value
D

374 and reported health benefits. Today Spirulina is endorsed as a secret, potent superfood, also
TE

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
EP

378 elements, and essential fatty acids are present. Unlike other algae, Spirulina is easier to
379 consume
C

380
AC

381 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

Page 28 of 42
 ACCEPTED MANUSCRIPT
388 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
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

PT
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.

RI
396
397 Minerals

SC
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

U
Balasubramani, R et al 2016). Spirulina is very advantageous for athletes, vegetarians,
AN
402 pregnant women, and teenagers. Average nutritional analysis of Spirulina per 100gm is
403 shown in Table 5.
M

404 Table:5: Average nutritional analysis of Spirulina per 100g (Roberto 2015)

Components Nutritional Value(in Components Nutritional Value(in

D

mgs) mgs)
TE

Plant Protein 63000 Calcium 1000

EP

Carbohydrates 22000 Phosphorus 800

C

Fat 2200 Magnesium 400

AC

Minerals 8000 Iron 58

Dietary Fibre 7000 Zinc 3

Vitamin A 212 Copper 1.2

Chlorophyll 600 Manganese 0.5

Page 29 of 42
 ACCEPTED MANUSCRIPT
Vitamin E 10 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

PT
Vitamin B5 0.2 Vitamin B9 0.05

RI
Vitamin B6 6 Vitamin B12 0.35

SC
405
406

U
407 5.2 Pharmaceuticals and nutraceutical applications
408 Spirulina is the best complete nutritional food source of protein, beta carotene, GLA, B
AN
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
M

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
D

413 products which are commercially available with researched and approved health benefits. The
TE

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
EP

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–
C

419 cancer, hypocholesterolemic and health improvement agent is getting attention as a

AC

420 nutraceutical and pharmaceutical.

421 Spirulina has the following health benefits
422 • Helps athletes with long lasting energy and vitality
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

Page 30 of 42
 ACCEPTED MANUSCRIPT
428 • 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
431 effects
432 • 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

PT
434 • Functions as an anti-inflammatory agent
435 • Reduce the inflammation characteristic of arthritis

RI
436 • Govern the appetite and helps to stimulate the metabolism
437 Hailed by health obsessives as the superfood to conquer viruses, prevent aging and even ward

SC
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

U
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
AN
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.
M

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
D

446 from 1000 – 4500INR /Kg. Spirulina capsules range from 250- 900INR / 60 capsules.
TE

447 Spirulina face packs vary from 360 – 900INR / 100gm.

448 6. Future outlook

EP

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
C

451 cultivation system, growth efficient techniques and use of organic fertilizer may be adopted
452 to maximize the protein content of Spirulina.
AC

453 • The food processing technique including drying of Spirulina biomass is an

454 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
457 liter -1 day-1 has been reported.
458 • Open pond cultivation system has many drawbacks such as improper light
459 intensity, contamination and requires large acres of land.

Page 31 of 42
 ACCEPTED MANUSCRIPT
460 • 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
464 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.

PT
466 • 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

RI
468 ton ha-1yr-1.
469 • The daily production system according to the work reported is greater with

SC
470 PBR and lesser with the open raceway ponds, So, qualitatively as well as
471 quantitatively when measured, PBR systems are more efficient.

U
472 • Hybrid reactors are fortunate outcomes of groundbreaking strategies and
AN
473 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
M

476 can be a good option if economically tested.

•
D

477 Climatic factors play an important role in Spirulina cultivation where the
478 optimum temperature is very important. So, in very hot or cold or less humid
TE

479 conditions greenhouse can be used for Spirulina cultivation. Different

480 greenhouse designs have been studied to design for Spirulina.
EP

481 • Various harvesting system has been reported among them centrifugation may
482 not be suggested for Spirulina as it is expensive and it may also break
C

483 Spirulina cells on separation.

484 • Normal filter or mesh cloth of 30µ - 450µ is highly used and it may separate
AC

485 Spirulina very easily and efficiently.

486 • 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 • Dried powder may be transformed to easy and consumable form than pellets,
490 powder and capsules which are already available in the market.
491

Page 32 of 42
 ACCEPTED MANUSCRIPT
492 7. Summary

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.

PT
497 The following conclusions are drawn from the study.

498 • Spirulina is mainly reported to be grown in open raceway ponds for commercial or

RI
499 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

SC
501 productivity.

502 • Commercial Industrial scale cultivation of spirulina uses inorganic chemicals which

U
503 are expensive and requires optimization of media concentration to avoid intrusion of
AN
504 prevention of facultative pathogens.

505 • Climatic factors, light intensity and aeration are very important in Spirulina growth
M

506 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
D

508 increased by bringing variations in used growth media.

•
TE

509 There has been a significant change in functional properties of Spirulina under
510 stressed conditions. Environmental stresses like high pH, light, salinity and
511 temperature affect growth and nutrient productivity.
EP

512 • Spirulina is contended to have several health benefits as it contains essential proteins,
513 carbohydrates, essential fatty acids, vitamins, minerals, carotenes, chlorophyll a and
C

514 phycocyanin to fight against malnutrition. So it can be used in nutraceuticals and

AC

515 pharmaceuticals applications.

516 • 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 • Development of various Spirulina fortified foods is required to create nutritional

520 awareness and increase the acceptance level in developing countries.

Page 33 of 42
 ACCEPTED MANUSCRIPT
521 • 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.

524 Acknowledgement

525 We are very thankful to the Honourable Ex-Director, Dr. K.K.Appukuttan, Maulana Azad

PT
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
agencies in the public, commercial, or not-for-profit sectors.

RI
528

529 References

SC
530 Allen, K. A. T. H. L. E. E. N. (2016). Evaluating Spirulina as a protein source in Nile Tilapia
531 (Oreochromis niloticus) grow-out diets.

U
532 Aloni, M.N., Lukusa, A.K., Matondo, F.K., Nkuadiolandu, A.B., & Takaisi, K. (2016).
AN
533 Spirulina Supplements Improved the Nutritional Status of Undernourished Children Quickly
534 and Significantly: Experience from Kisantu, the Democratic Republic of the Congo.
535 International journal of pediatrics.
M

536 Ankita, Juneja, Ruben Michael Ceballos, and Murthy GantiS. "Effects Of Environmental
537 Factors And Nutrient Availability On The Biochemical Composition Of Algae For Biofuels
D

538 Production: A Review." Energies (19961073) 6.9 (2013)

TE

539 Anupama, P.R., 2000. Value-added food: single cell protein. Biotechnology Advances. 18:
540 459–479.
541 AsterioSa´nchezMiro´n, Antonio Contreras Go´mez, Francisco Garcı´a Camacho, Emilio
EP

542 Molina Grima, Yusuf Chisti,Comparative evaluation of compact photobioreactors for large-
543 scale monoculture of microalgae, Journal of Biotechnology 70 (1999) 249–270
C

544 Balasubramani, R., Gupta, S. K., Cho, W., Kim, J., Lee, S., Jeong, K., ... & Choi, H. (2016).
AC

545 Microalgae potential and multiple roles-current progress and future prospects-an overview.
546 Sustainability, 8(12).
547 Bezerra, R. P., Montoya, E. Y. O., Sato, S., Perego, P., de Carvalho, J. C. M., & Converti, A.
548 (2011). Effects of light intensity and dilution rate on the semicontinuous cultivation of
549 Arthrospira (Spirulina) platensis. A kinetic Monod-type approach. Bioresource technology,
550 102(3), 3215-3219.
551 Bharathiraja B., Chakravarthy M., Ranjith Kumar R., Yogendran D., Yuvaraj D.,
552 Jayamuthunagai J., PraveenKumar R , Palani S., Aquatic biomass(algae) as a future feedstock

Page 34 of 42
 ACCEPTED MANUSCRIPT
553 for bio-refineries:Areviewoncultivation,processingandproducts,Renewable and Sustainable
554 Energy Reviews47(2015)634–653
555 Camacho Rubio, F., Acie´nFerna´ndez, F.G., Sa´nchezPe´rez, J.A., Garcıa Camacho, F.,
556 Molina Grima, E., 1999. Prediction of dissolved oxygen and carbon dioxide concentration
557 profiles in tubular photobioreactors for microalgal culture. Biotechnology and
558 Bioengineering 62, 71–86.

PT
559 Chaumont, D., Thepenier, C., Gudin, C., 1988. Scaling up a tubular photobioreactor for
560 continuous culture of Porphyridiumcruentum – from laboratory to pilot plant. In: Stadler, T.,

RI
561 Morillon, J., Verdus, M.S., Karamanos, W., Morvan, H., Christiaen, D. (Eds.), Algal
562 Biotechnology. Elsevier Applied Science, London, pp. 199–208.

SC
563 Chen, C. Y., Yeh, K. L., Aisyah, R., Lee, D. J., & Chang, J. S. (2011). Cultivation,
564 photobioreactor design and harvesting of microalgae for biodiesel production: a critical
565 review. Bioresource technology, 102(1), 71-81.
566

U
Chetsumon, A., Umeda, F., Maeda, I., Yagi, K., Mizoguchi, T., Miura, Y., 1998. Broad
AN
567 spectrum and mode of action of an antibiotic produced by Scytonema sp. TISTR 8208 in a
568 seaweed-type bioreactor. Applied Biochemistry and Biotechnology. 70–72, 249–256
M

569 ChiniZittelli, G., Rodolfi, L., Tredici, M.R., 2003. Mass cultivation of Nannochloropsis sp. in
570 annular reactors.Journal of Applied Phycology. 15, 107–114.
D

571 Chisti, Y. "Large-scale production of algal biomass: raceway ponds." Algae Biotechnology.
572 Springer International Publishing, 2016. 21-40.
TE

573 Chojnacka, K., & Noworyta, A. (2004). Evaluation of Spirulina sp. growth in
574 photoautotrophic, heterotrophic and mixotrophic cultures. Enzyme and Microbial
575 Technology, 34(5), 461-465.
EP

576 CuaresmaM, JanseenM, Vilchez, Wijffels RH. “Horizontal or Vertical photobioreactrs?How

577 to improve microalgae photosynthetic efficiency,” Bioresource Technology,102, 2011,
C

578 5129-37.
AC

579 Degen, J., Uebele, A., Retze, A., Schmidt-Staigar, U., Trosch, W.A., 2001.A novel airlift
580 photobioreactor with baffles fro improved light utilization through flashing light effect.
581 Journal of Biotechnology. 92, 89–94.
582 Dubey RC (2006). A textbook of Biotechnology. Fourth revised and enlarged edition, S.
583 hand and Company Limited, pp. 419-421.
584 Famelart M, Kobilinsky A, Bouillamnne C, Desmazeaud MJ (1987).Influence of
585 temperature, pH and dissolved oxygen on growth of Brevibacterium linen in a fermentor.
586 Applied Microbiology Biotechnology 25: 442-448.

Page 35 of 42
 ACCEPTED MANUSCRIPT
587 FedekarFadelMadkour, Abd El-WahabKamil, HodaShafik Nasr, Production and nutritive
588 value of Spirulina platensis in reduced cost media,Egyptian Journal of Aquatic Research
589 (2012) 38, 51–57
590 Grobbelaar J.U., Photosynthetic characteristics of Spirulinaplatensis grown in commercial-
591 scale open outdoor raceway ponds: what do the organisms tell us? Journal of Applied
592 Phycology, 19, 2007, 591–598.

PT
593 Habib, M.A.B., Parvin, M., Huntington, T.C. &Hasan, R.M. (2008). A Review on Culture,
594 Production and Use of Spirulina as Food for Humans and Feeds for Domestic Animals and

RI
595 Fish, FAO Fisheries and Aquaculture Circular. No. 1034, Rome-Italy, ISBN 978-92- 5-
596 1061060, pp. 1-41

SC
597 Hall, D.O., Fernandez, F.G.A., Guerrero, E.C., Rao, K.K., Grima, E.M.,2003. Outdoor helical
598 tubular photobioreactors for microalgalproduction:modeling of fluid-dynamics and mass
599 transfer and assessmentof biomass productivity. Biotechnology and Bioengineering. 82, 62–
600 73.
U
AN
601 Harker, M., Tsavalos, A.J., Young, A.J., 1996. Autotrophic growth and carotenoid production
602 of Haematococcuspluvialis in a 30 liter airlift photobioreactorJournal of Fermation and
M

603 Bioengineering. 82, 113–118.

604 HilalKarginYilmaz, DenizAyas and HarunYilmaz (2010). The effect of different salinity
D

605 ratios on reproductive rates and proximate composition of Spirulinaplatensis in a helical.

606 Journal of Animal and Veterinary Advances 9: 2427-2431.
TE

607 Hoekema, S., Bijmans, M., Janssen, M., Tramper, J., Wijffels, R.H., 2002.A pneumatically
608 agitated flat-panel photobioreactor with gas recirculation:anaerobic photoheterotrophic
EP

609 cultivation of a purple nonsulfur bacterium. Intenational Journal of Hydrogen Energy. 27,
610 1331–1338.
C

611 Hu, Q., Guterman, H., Richmond, A., 1996. A flat inclined modular photobioreactor for
612 outdoor mass cultivation of phototrophs. Biotechnology and Bioengineering. 51, 51–60.
AC

613 Jayati Trivedi, Mounika Aila, D.P. Bangwal, Savita Kaul, M.O. Garg, Algae based
614 biorefinery-How to make sense?, Renewable and Sustainable Energy Reviews
615 47(2015)295-307
616 Jiménez C, Cossío BR, Niell FX. Relationship between physicochemical variables and
617 productivity in open ponds for the production of Spirulina: A predictive model of algal yield.
618 Aquaculture. 2003;221:331-45.

Page 36 of 42
 ACCEPTED MANUSCRIPT
619 Kaewpintong, K., Shotipruk, A., Powtongsook, S., Pavasant, P., 2007. Photoautotrophic
620 high-density cultivation of vegetative cells of Haematococcuspluvialis in airlift bioreactor.
621 Bioresource Technology. 98, 288–295.
622 Koru, E. (2009).Earth Food Spirulina(Arthrospira): Production and Quality Standards,
623 Turkey Journal of Agriculture, - May-June 2008, Issue:11, Year:3, pp:133-134,
624 Lee, Y.K., Low, C.S., 1991. Effect of photobioreactor inclination on the biomass productivity

PT
625 of an outdoor algal culture. Biotechnology and Bioengineering. 38, 995–1000.
626 Licamele, J.D. & White, C.L. (2011). V-trough photobioreactor system and method of use.

RI
627 U.S. Patent Application US2011/0258920 A1 (27 October 2011).
628 Low-Cost Algae Production—Is It Finally With Us? May 13, 2012

SC
629 http://www.algaeindustrymagazine.com/low-cost-algae-production-is-it-finally-with-us/
630 Lucie Novoveskáa, Anastasia K.M. Zapataa, Jeffrey B. Zabolotneya, Matthew C. Atwood,
631 Eric R. Sundstrom,Optimizing microalgae cultivation and wastewater treatment in large-scale
632

U
offshore photobioreactors, Algal Research 18 (2016) 86–94.
AN
633 Madhu, G. M., Satyanarayana, S. V., Kalpana, P., & Bindiya, P. (2015). Equilibrium and
634 kinetic studies of lead biosorption by three Spirulina (Arthrospira) species in open raceway
M

635 ponds. Journal of Biochemical Technology, 6(1), 894-909.

636 Matsunaga T. , Takeyama H., Sudo H., Oyama N, Ariura S., Takano H., Hirano M., Burgess
D

637 J.G., Sode K., Nakamura N. , Glutamate production from CO2 by marine cyanobacterium
638 Synechococcus sp. using a novel biosolar reactor employing light diffusing optical fibers
TE

639 Applied Biochemistry and Biotechnology. (28/29) (1991), pp. 157–167

640 Molina, E., Ferna´ndez, J., Acie´n, F.G., Chisti, Y., 2001. Tubular photobioreactor design for
EP

641 algal cultures. Journal of Biotechnology. 92, 113–131

642 Mori, K., Photoautotrophic bioreactor using visible solar rays condensed by fresnel lenses
C

643 and transmitted through optical fibers Biotechnology and bioengineering symposium
644 Journal., 15 (1985), pp. 331–345
AC

645 Newsted J.L. Effect of light, temperature, and pH on the accumulation of phenol by
646 Selenastrumcapricornutum, a green alga. Ecotoxicology and Environmental Safety.
647 2004;59:237–243. [PubMed]
648 Niels-Henrik Norsker,Maria J. Barbosa , Marian H. Vermuë , René H. Wijffels ,Microalgal
649 production — A close look at the economics, Biotechnology Advances 29 (2011) 24–27
650 Ogbonda K.H., Aminigo R.E., Abu G.O. Influence of temperature and pH on biomass
651 production and protein biosynthesis in a putative Spirulina sp. Bioresource Technology.
652 2007;98:2207–2211. [PubMed]

Page 37 of 42
 ACCEPTED MANUSCRIPT
653 Ogbonna, J.C., Ichige, E., Tanaka, H., 2002. Interactions between photoautotrophic and
654 heterotrophic metabolism in photoheterotrophic cultures of Euglena gracilis. Applied
655 Microbiology Biotechnology. 58, 532–538.
656 Ogbonna, J.C., Soejima, T., Tanaka, H., 1999. An integrated solar and artificial light system
657 for internal illumination of photobioreactorsJournal of Biotechnology. 70, 289–297
658 Olguín E, Galicia S, Mercado G, Pérez T. Annual productivity of Spirulina (Arthrospira) and

PT
659 nutrient removal in a pig wastewater recycling process under tropical conditions.
660 2003;15:249-57.

RI
661 Ouassim Zeriouh, José Vicente Reinoso-Moreno, Lorenzo López-Rosales, María del Carmen
662 Cerón-García, Asterio Sánchez-Mirón, Francisco García-Camacho, and Emilio Molina-
663 GrimaCritical Reviews in Biotechnology Vol. 0 , Iss. 0,0

SC
664 Pal, R., Gupta, A., & Tripathi, A. (2011). Impact of environmental factors on the biomass
665 production of Spirulina in different conditions. Vegetos-An International Journal of Plant
666 Research, 24(2), 142-148.
667
U
Pandey J.P., Tiwari A. Optimization of biomass production of Spirulina maxima. Journal of
AN
668 Algal Biomass Utilization. 2010;1:20–32.
669 Pandey, J. P., Tiwari, A., & Mishra, R. M. (2010). Evaluation of biomass production of
M

670 Spirulina maxima on different reported media. J. Algal Biomass Utln, 1(3), 70-81.
671 Papadaki, S., Kyriakopoulou, K., Stramarkou, M., Tzovenis, I., & Krokida, M. (2017).
D

672 Environmental Assessment of Industrially Applied Drying Technologies for the Treatment of
673 Spirulina Platensis. IOSR Journal of Environmental Science, Toxicology and Food
TE

674 Technology, 11(1), 41-46.

675 Pirt, S.J., Lee, Y.K., Walach, M.R., Pirt, M.W., Balyuzi, H.H.M., Bazin,M.J., 1983. A tubular
EP

676 photobioreactor for photosynthetic production of biomass from carbon dioxide: design and
677 performance. Journal of Chemical Technology and Biotechnology. 33B, 35–38.
C

678 Pohl, P., Kohlhase, M., Martin, M., 1988. Photobioreactors for the axenic mass cultivation of
679 microalgae. In: Stadler, T., Mollion, J., Verdus, M.C., Karamanos, Y., Morvan, H.,
AC

680 Christiaen, D. (Eds.), Algal Biotechnology. Elsevier Applied Science, London and New
681 York, pp. 209–217.
682 Powls SB (1985). Photo-inhibition of photosynthesis induced by visible light. Ann. Rev.
683 Plant Physiology 35: 15-44.
684 Pragya, Namita, Krishan K. Pandey, and P.K. Sahoo. "A Review On Harvesting, Oil
685 Extraction And Biofuels Production Technologies From Microalgae." Renewable &

Page 38 of 42
 ACCEPTED MANUSCRIPT
686 Sustainable Energy Reviews 24.(2013): 159-171. Academic Search Premier. Web. 10 Dec.
687 2015
688 Pruvost, J., Pottier, L., Legrand, J., 2006. Numerical investigation of hydrodynamic and
689 mixing conditions in a torus photobioreactorChemical Engineering Science 61, 4476–4489
690 Pulz, M.O., Gross, W., 2004. Valuable products from biotechnology of microalgae. Applied
691 Microbiology Biotechnology 6, 635-648

PT
692 R. M. Atlas and L. C. Parks, Handbook of Microbiological Media, CRC. Press, Boca Raton,
693 Fla, USA, 2nd edition, 1997

RI
694 Rafiqul Islam, M., Hassan, A, Sulebele, G., Orosco C and Roustaian, P. 2003. Influence of
695 Temperature on Growth and Biochemical Composition of Spirulina platensis and Spirulina

SC
696 fusiformis. Iranian International Journal of Sciences, 4(2): 97-106.
697 Ramos de Ortega, A., Roux, J.C., 1986. Production of Chlorella biomass in different types of
698 flat bioreactors in temperate zones. Biomass 10, 141–156.
699

U
Raoof, B., Kaushika, B.D. &Prasanna, R. 2006. Formulation of a low-cost medium for mass
AN
700 production of Spirulina. Division of Microbiology,
701 Richmond A, Vonshak A (1978). Spirulina culture in Israel. Arch. Hydrobiol. Bein. Ergebn.
702 Limnology 11: 274-280.
M

703 Richmond A, Zhang C-W. Optimization of a flat plate glass reactor for mass production of
704 Nannochloropsis sp. outdoors. Journal of Biotechnology. 2001;85:259-69
D

705 Richmond, A., Boussiba, S., Vonshak, A., Kopel, R., 1993. A new tubular reactor for mass
TE

706 production of microalgae outdoorsJournal of Applied Phycology. 5, 327–332.

707 Roberto Parra-Saldivar,Photosynthetic bioenergy utilizing CO2: an approach on flue gases
708 utilization for third generation biofuels,Journal of Cleaner Production 98 (2015) 53-65
EP

709 Saeid, A., & Chojnacka, K. (2015). Toward production of microalgae in photobioreactors
710 under temperate climate. Chemical Engineering Research and Design, 93, 377-391.
C

711 Samson, R., Leduy, A., 1985. Multistage continuous cultivation of bluegreen alga Spirulina
AC

712 maxima in the flat tank photobioreactors.Chemical Engineering Journal. 63, 105–112.
713 Samuel, G. S., Sofi, M. Y. and Masih, S. 2010. Potential of different light intensities on the
714 productivity of Spirulinaplatensis under agra conditions. Research Journal of Agriculture
715 Science. 1(4): 468-469
716 Sassano C.E.N., Gioielli L.A., Almeida K.A., Sato S., Perego P., A. Converti, J.C.M.
717 Carvalho, Cultivation of Spirulinaplatensis by continuous process using ammonium chloride
718 as nitrogen source, Biomass and Bioenergy, 31, 2007, 593–598.

Page 39 of 42
 ACCEPTED MANUSCRIPT
719 Sierra E., Acien F.G., Fernandez J.M., Garcıa,C J.L.. Gonzalez, Molina E.,Characterization
720 of a flat plate photobioreactor for the production of microalgae, Chemical Engineering
721 Journal 138 (2008) 136–147
722 Singh R.N., Shaishav Sharma, “Development of suitable photobioreactor for algae production
723 – A review,” Renewable and Sustainable Energy Reviews,16 ,(2012), 2347– 2353
724 SjorsV.I.,Alessandro F. Algae based biofuels, Applications and coproducts. Environment and

PT
725 natural resources management working paper. Environment climatechange.Bioenergy
726 monitoring and assessment;2010.

RI
727 Slade, Raphael, and AusilioBauen. "Micro-Algae Cultivation For Biofuels: Cost, Energy
728 Balance, Environmental Impacts And Future Prospects." Biomass & Bioenergy 53.(2013):

SC
729 29-38
730 Sudhakar , Suresh S, Premalatha M., An Overview Of Co2 Mitigation Using Algae
731 Cultivation Technology, International Journal of Chemical Research ISSN: 0975-3699 &
732

U
E-ISSN: 0975–9131, Vol. 3, Issue 3, 2011, pp-110-117
AN
733 Sudhakar K. and Premalatha M., Theoretical Assessment of Algal Biomass Potential for
734 Carbon Mitigation and Biofuel Production, Iranica Journal of Energy & Environment 3
M

735 (3): 232-240, 2012 ISSN 2079-2115

736 Sudhakar K., Rajesh M. & Premalatha M. A Mathematical Model to Assess the Potential of
D

737 Algal Bio-fuels in India, Energy Sources, Part A: Recovery, Utilization, and Environmental
738 Effects, (2012): , 34:12, 1114-1120
TE

739 Sudhakar K.and Premalatha M. (2015) Characterization of Micro Algal Biomass Through
740 FTIR/TGA /CHN Analysis: Application to Scenedesmus sp., Energy Sources, Part A:
EP

741 Recovery, Utilization, and Environmental Effects, 37:21, 2330-2337

742 Torzillo, G., Pushparaj, B., Bocci, F., Balloni, W., Materassi, R., Florenzano, G., 1986.
C

743 Production of Spirulina biomass in closed photobioreactors. Biomass 11, 61–74.

744 Toyoshima, M., Aikawa, S., Yamagishi, T., Kondo, A., & Kawai, H. (2015). A pilot-scale
AC

745 floating closed culture system for the multicellular cyanobacterium Arthrospira platensis
746 NIES-39. Journal of Applied Phycology, 27(6), 2191–2202. http://doi.org/10.1007/s10811-
747 014-0484-2
748 Tredici, M.R., ChiniZittelli, G., 1998. Efficiency of sunlight utilization: tubular versus flat
749 photobioreactors. Biotechnology and Bioengineering. 57, 187–197.
750 Tredici, M.R., Materassi, R., 1992. From open ponds to vertical alveolar panels: the Italian
751 experience in the development of reactors for the mass cultivation of photoautotrophic
752 microorganisms. Journal of Applied Phycology. 4, 221–231

Page 40 of 42
 ACCEPTED MANUSCRIPT
753 Tredici, Mario R. "Mass production of microalgae: photobioreactors." Handbook of
754 microalgal culture: Biotechnology and applied phycology 1 (2004): 178-214.
755 Tsoglin LN, Gabel BV, Fal’kovich TN, Semenenko VE. Closed photobioreactors for
756 microalgal production. Russ, Journal of Plant Physiology1996;43(1):131–6.
757 Ugwu CU, Aoyagi H, Uchiyama H. “Photobiorectors for mass cultivation of algae,”
758 Bioresource Technology, 99, 2008, 4021-8.
759 Ugwu, C.U., Ogbonna, J.C., Tanaka, H., 2002. Improvement of mass transfer characteristics

PT
760 and productivities of inclined tubular photobioreactors by installation of internal static
761 mixers. Applied Microbiology Biotechnology. 58, 600–607.

RI
762 Ugwu, C.U., Ogbonna, J.C., Tanaka, H., 2003. Design of static mixers for inclined tubular
763 photobioreactorsJournal of Applied Phycology. 15, 217–223.

SC
764 Ugwu, C.U., Ogbonna, J.C., Tanaka, H., 2005a. Light/dark cyclic movement of algal cells in
765 inclined tubular photobioreactors withwith internal static mixers for efficient production of

U
766 biomass. Biotechnology Letters. 27, 75–78.
767 Vardaka, E., Kormas, K. A., Katsiapi, M., Genitsaris, S., & Moustaka-Gouni, M. (2016).
AN
768 Molecular diversity of bacteria in commercially available “Spirulina” food supplements.
769 PeerJ, 4, e1610.
M

770 Vega-Estrada, J., Montes-Horcasitas, M.C., Dominı ´gues-Bocanegra, A.R., Can˜ izares-
771 Villanueva, R.O., 2005. Haematococcuspluvialis cultivation in split-cylinder internal-loop
D

772 airlift photobioreactor under aeration conditions avoiding cell damage. Applied
TE

773 Microbiology and Biotechnology. 68, 31–35

774 Venkataraman, L. V., Bhagyalakshmi, N. and Ravishankar, G. A. 1995. Commercial
775 production of micro and macro algae problems and potentials. Indian Journal of
EP

776 Microbiology. 35, 1–19.

777 Vonshak A, Guy R (1987). Photo-inhibition as a limiting factor in outdoor cultivation of
C

778 Spirulinaplatensis. In Stadler T (Ed), Algal Biotechnology. Elsevier Applied Science

AC

779 Publishers pp. 365-370

780 Vonshak A. Spirulina: growth, physiology and biochemistry Spirulina platensis (Arthrospira).
781 In: Vonhask A, editor. Physiology cell-biology and biotechnology. London: Taylor and
782 Francis; 1997. p. 43–65.
783 Vree, J. H., Bosma, R., Janssen, M., Barbosa, M. J., & Wijffels, R. H. (2015). Comparison of
784 four outdoor pilot-scale photobioreactors. Biotechnology for biofuels, 8(1), 215.
785 Vymazal J. Uptake of heavy metals by Cladophoraglomerata. ActaHydrochimHydrbiol.
786 1990;18:657–665

Page 41 of 42
 ACCEPTED MANUSCRIPT
787 Watanabe, Y., Saiki, H., 1997. Development of photobioreactor incorporating Chlorella sp.
788 for removal of CO2 in stack gas. Energy Conversion Management 38, 499–503
789 Yin, C., Daoust, K., Young, A., Tebbs, E. J., & Harper, D. M. (2017). Tackling community
790 undernutrition at Lake Bogoria, Kenya: The potential of spirulina (Arthrospira fusiformis) As
791 a food supplement. African Journal of Food, Agriculture, Nutrition and Development, 17(1),
792 11603-11615.

PT
793 Zarrouk, C. 1966. Contribution à l'étuded'unecyanophycéeinfluencée de divers facteurs
794 physiques etchimiquessur la croissanceet la photosynthèse de Spirulina maxima (Setch. et

RI
795 Gardner) Geitler.University of Paris, Paris, France. (PhD Thesis).
796 Zeng, X.-H., Danquah, M.K., Chen, X.D., Lu, Y.-H., 2011. Microalgae bioengineering:

SC
797 Autotrophic cultivation from CO2 fixation to biofuel production. Renewable and
798 Sustainable Energy Reviews. 15 (6), 3252–3260.
799 Zhang, K., Kurano, N., Miyachi, S., 2002. Optimized aeration by carbon dioxide gas for
800

U
microalgal production and mass transfer characterization in a vertical flat-plate
AN
801 photobioreactor. Bioprocess and Biosystem Engineering. 25, 97–101.
802 Zhang, L., Chen, L., Wang, J., Chen, Y., Gao, X., Zhang, Z., & Liu, T. (2015). Attached
M

803 cultivation for improving the biomass productivity of Spirulina platensis. Bioresource
804 technology, 181, 136-142.
D

805 Zittelli, G.C., Biondi, N., Rodolfi, L., &Tredici, M.R. (2013). Photobioreactors for mass
806 production of microalgae. In: Richmond, A. & Hu, Q., editors. Handbook of microalgal
TE

807 culture: applied phycology and biotechnology. Oxford: Blackwell Publishing, p 225-266.
808
EP

809
C
AC

Page 42 of 42
 ACCEPTED MANUSCRIPT

Highlights

• Various Methods of spirulina cultivation are appraised.

• Growth conditions and Spirulina yield are reviewed.

PT
Spirulina growth for nutritional use is highlighted.

• Recommendations of spirulina as super food is suggested

RI
U SC
AN
M
D
TE
C EP
AC