Accepted Manuscript

Spirulina cultivated under different light emitting diodes: enhanced cell growth
and phycocyanin production

Denise da Fontoura Prates, Elisângela Martha Radmann, Jessica Hartwig

Duarte, Michele Greque de Morais, Jorge Alberto Vieira Costa

PII: S0960-8524(18)30144-5
DOI: https://doi.org/10.1016/j.biortech.2018.01.122
Reference: BITE 19479

To appear in: Bioresource Technology

Received Date: 30 November 2017

Revised Date: 24 January 2018
Accepted Date: 25 January 2018

Please cite this article as: Fontoura Prates, D.d., Radmann, E.M., Hartwig Duarte, J., de Morais, M.G., Costa, J.A.V.,
Spirulina cultivated under different light emitting diodes: enhanced cell growth and phycocyanin production,
Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech.2018.01.122

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Spirulina cultivated under different light emitting diodes: enhanced cell growth

and phycocyanin production

Denise da Fontoura Prates1, Elisângela Martha Radmann1, Jessica Hartwig Duarte1,

Michele Greque de Morais1, Jorge Alberto Vieira Costa1*

1
Laboratory of Biochemical Engineering, College of Chemistry and Food Engineering,

Federal University of Rio Grande, Rio Grande, Brazil

*Corresponding Author: Prof. Dr. Jorge Alberto Vieira Costa - Laboratory of

Biochemical Engineering - College of Chemistry and Food Engineering - Federal

University of Rio Grande - P.O. Box 474, 96203-900 - Av. Itália, km 8 - Rio Grande,

RS, Brazil. Phone: +55 53 32336908. Fax: +55 53 32336968. E-mail:

jorgealbertovc@terra.com.br

ABSTRACT

This study evaluated light emitting diodes (LEDs) as a light source in Spirulina sp. LEB

18 cultures in terms of growth parameters and biomass composition. Different

photoperiods (partial and integral) and colors (blue, green, red and white) were

assessed. Blue, green, red and white LEDs increased biomass productivity and

maximum specific growth rate of such cultivations. The maximum biomass

concentration (1.77 ± 0.02 g L-1) was obtained when red LEDs in integral light

photoperiod were applied to cultivations. The biomass composition showed around 12.8

% carbohydrates (w w-1), 57.4 % proteins (w w-1) and 12.7 % lipids (w w-1). The major

fatty acids produced during cultivations were palmitic, linoleic and γ-linolenic. Green

LEDs in partial light photoperiod promoted a higher concentration of phycocyanin

(126.39 mg gbiomass-1). The potential of LEDs as an energy source in Spirulina sp. LEB

18 cultures was demonstrated by the biomass and bioproducts photostimulation.

1
Keywords: biomass composition, cyanobacteria, fatty acids, LEDs, pigment.

1. Introduction

The rising world population, coupled with the global warming impacts and the fossil

fuels depletion, threaten the natural resources production such as food and energy

(Wishkerman and Wishkerman, 2017). Microalgae and cyanobacteria cultivation are an

alternative to overcome these problems. These microorganisms perform the atmospheric

CO2 mitigation and can reduce the impacts caused by global warming (Duarte et al.,

2017). Some species produce high content of essential amino acids and polyunsaturated

fatty acids. In addition, some groups of microalgae, such as cyanobacteria, contain

phycocyanin, a photosynthetic pigment with recognized nutraceutical properties, which

can be applied to human food, helping health promotion (Vanthoor-Koopmansa et al.,

2013; Lee et al., 2017). Besides, microalgae and cyanobacteria carbohydrates and lipids

can even be extracted from the biomass and converted into biofuels, such as bioethanol

and biodiesel (Wang et al., 2014; Singh and Olsen, 2011).

Spirulina is a cyanobacteria that is characterized by its filamentous and spiral form

(Tomaselli, 1997). These microorganisms have been extensively studied and marketed

for different purposes. Due to its composition, which is rich in proteins (50-70%),

composed of essential amino acids, polyunsaturated fatty acids, vitamins (including

B12), pigments and phenolic compounds, Spirulina has been marketed as a food

supplement (Thajuddin and Subramanian, 2005). In addition, studies demonstrate the

Spirulina potential to atmospheric CO2 mitigation, biofuels production and phycocyanin

obtaining (Duarte et al., 2017; Rosa et al., 2015; Kim et al., 2013; Chen et al., 2013).

Light is one of the most important factors during microalgae and cyanobacteria

cultivation, so that this energy is captured by the pigments to carry out the

2
photosynthesis process (Atta el al., 2013). On a large scale, most cultivations use sun

light as energy source. However, for the high added value biocompounds production,

such as phycocyanin and essential fatty acids, artificial light is more commonly used in

the cultivations, with efficient and standardized control of photosynthetic photon flux

density, resulting in high productivities (Blanken et al., 2013). Fluorescent lamps are the

most commonly artificial energy sources used in microalgae cultures. Light emitting

diodes (LEDs) are considered an ecologically correct energy source and an economical

technology (Atta et al., 2013; Zhao et al., 2013; Teo et al., 2014). The main advantages

of LEDs are: (1) high efficiency of electricity conversion; (2) reduced energy

consumption; (3) smaller material mass and volume; (4) non-polluting and durable; (5)

lower heat dissipation and (6) single wavelength (Atta et al., 2013; Carvalho et al.,

2011).

Recently, a number of researchers have reported the application of LEDs as light source

in microalgae and cyanobacteria cultivations. The blue and red LEDs increased the

biomass productivity in the Nannochloropsis sp. and Botryococcus braunii cultivation,

respectively (Das et al., 2011; Baba et al., 2012). The lipid productivity was increased in

the Tetraselmis sp., Nannochloropsis sp. and Chlorella vulgaris biomass when blue

LEDs were used as an energy source in the cultivations (Teo et al., 2014; Atta et al.,

2013). Relatively few studies have evaluated the use of LEDs in Spirulina cultures. In

this context, the objective of this study was to evaluate the application of LEDs as a

light source in Spirulina sp. LEB 18 cultivation, analyzing the cell growth parameters

and the biomass composition.

2. Materials and Methods

2.1 Microorganism and culture medium

3
The microorganism used was Spirulina sp. LEB 18 from the Culture Collection of the

Laboratory of Biochemical Engineering at Federal University of Rio Grande (FURG).

This strain was isolated from Mangueira Lagoon (southern Brazil, latitude 32º32'05" S

and longitude 33º31'57" W). The culture medium used in the experiments was Zarrouk

(as described by Costa et al., 2004).

2.2 Cultivation conditions

The experiments were performed in triplicate in a closed acrylic vertical tubular

photobioreactor, with 600 mm of length and 75 mm of diameter (Morais and Costa,

2007). Cultures were carried out for 10 d, with an initial cell concentration of 0.2 g L-1,

at 30 °C in a growth chamber. Stirring was effected by compressed air injection at 0.05

vvm (flow regulated from flowmeters – COLE PARMER – Illinois – USA) through a

porous stone sparger fastened to the base of the photobioreactor. The pumped air was

filtered by glass wool filters in order to avoid contamination. The evaporated water

during the experiments was replenished daily prior to sampling by the addition of sterile

water in the cultivations. The illumination of cultivations was provided by 40 W white

fluorescent tubular lamps and by different wavelengths of LEDs ribbons and

photoperiods (Table 1). The lamps and LEDs ribbons were fixed on a support 10 cm

beside the photobioreactor. The control experiment was performed using fluorescent

light as energy source under 12 h light/dark photoperiod.

2.3 Biomass concentration and pH determination

The Spirulina sp. LEB 18 biomass concentration in the cultivations was monitored daily

by optical density at a wavelength of 670 nm using a digital spectrophotometer

(QUIMIS Q798DRM, Diadema – SP – Brazil). A microalga standard growth curve was

generated previously to correlate the optical density with the dry weight biomass (Costa

4
et al., 2002). The pH measurements were performed on the cultures using a digital pH

meter (QUIMIS Q400RS). For these analysis, 5 mL of sample was collected for each

experiment.

2.4 Growth parameters

The biomass productivity was obtained from the equation PX = (Xt - X0)/(t - t0), where

Xt is the biomass concentration (g L-1) at time t (d) and X0 is the biomass concentration

(g L-1) at time t0 (d). The maximum specific growth rate was obtained via linear

regression applied to the logarithmic growth rate of each assay obtained from a plot of

ln X (g L-1) versus t (d).

2.5 Biomass harvesting and biomass characterization

The biomass from the experiments was recovered from the liquid medium by

centrifugation (Hitachi Himac CR-GIII, Tokyo – Japan) at 15,000 g for 12 min, at 20

ºC. Afterwards, the biomass was resuspended in distilled water and centrifuged again

under the same conditions to improve nutrient removal. The biomass was frozen at -80

°C and lyophilized for characterization.

2.5.1 Carbohydrates

The carbohydrates concentration was determined via the Dubois phenol–sulfuric

method using a standard glucose curve (Dubois et al., 1956).

2.5.2 Proteins

The proteins were determined via colorimetric method (Lowry et al., 1951), from

thermal and alkaline pretreatment of the microalgal biomass, using bovine serum

albumin as a standard.

2.5.3 Lipids

The lipids concentration was determined by the Folch method (Folch et al., 1957).

5
2.5.4 Fatty acid profile

The derivatization of fatty acids in methyl esters (FAMEs) was performed using the

methodology proposed by Joseph and Ackman (1992). Separation and identification of

FAMEs were performed in a gas chromatograph (GC-FID 7890A - Agilent, Germany)

equipped with automatic injector, operating in the split mode (1:25), with a OV225

column (60 m×0.25 mm×0.25 μm). The equipment was operated with an injector at

250°C, detector at 280°C, using H2 as carrier gas at 1 mL min-1 and flow rate of the gas

in the flame ionization detector (FID) of 30:30:300 mL min-1 (H2:N2:synthetic air).

The determination of FAMEs was performed according to the conditions adapted from

the method described by David et al. (2005). Quantification was assessed by area

normalization, calculating the relative percentage of FAMEs´ area, identified in relation

to the sum of the areas of all methyl esters of the chromatogram (total area). Peak

identification was performed by comparing the retention time of the 37 FAMEs patterns

(Supelco, USA) with those of the peaks observed in the samples analyzed under the

same conditions. Confirmation of the compounds was done using an Agilent 7890 GC-

MS coupled with a quadrupole mass analyzer (Agilent 5975c), using EI as the

ionization source. The analysis was realized in Scan mode (50-550m/z) and

identification was done using the NIST11 mass spectra library, observing the agreement

of at least 80 % of the spectra obtained with the library spectra.

2.5.5 Phycocyanin

In order to perform phycocyanin biomass extraction, lyophilized samples (0.08 g) were

immersed in 1 mL of 0.1 M sodium phosphate buffer (pH 6.9) and kept in the ultrasonic

bath (40 Hz) for 15 min. Soon after, the samples were centrifuged at 6000 rpm for 10

min, and the supernatant collected for further analysis. The procedure described above

6
was repeated 3 times. The phycocyanin extraction was validated following International

Union of Pure and Applied Chemistry (Thompson et al., 2002). The validation

characteristics were: precision and accuracy. The repeatability of the extraction was

evaluated with 7 repetitions of the extraction on the same day, and the intermediate

precision was determined by performing the extraction on 3 consecutive days (n=3). For

the accuracy test, the standard addition recovery test was performed at 3 levels of

concentration. The recovery percentage of phycocyanin was 91% and the coefficient of

variation was <10% for each sample, indicating that the intermediate accuracy and

repeatability were acceptable. The phycocyanin concentration was determined from the

optical densities at 652 and 620 nm, according to equation proposed by Bennett and

Bogorad (1973).

2.6 Statistical analysis

The responses were assessed using an analysis of variance followed by Tukey’s test at a

95.0 % confidence level.

3. Results and discussion

3.1 Growth parameters

The highest (p < 0.05) biomass concentration was obtained in the experiment with red

LEDs in integral light photoperiod (Table 2). This result was about 2 times greater than

control experiment, which used only fluorescent light as energy source. Wang et al.

(2007) and Chen et al. (2010) also obtained high Spirulina biomass concentrations when

red LEDs were used as energy source in the cultivations. The main photosynthetic

pigments present in cyanobacteria absorb energy at wavelengths in the 430 and 680 nm

(chlorophyll a) and 550 and 620 nm (phycobiliproteins) bands. The red LEDs cover the

pigments light absorption spectrum (620-645 nm) (Markou, 2014), which can lead to a

7
greater energy utilization and, consequently, higher biomass production by the cells, as

observed from the results obtained for Spirulina sp. LEB 18. All LEDs experiments

showed biomass concentration, productivity and maximum specific growth rate results

higher (p < 0.05) than control experiment with only fluorescent light. Thus, the longer

LEDs light exposure times in the cultures favored the microalga cell growth. The

wavelengths of the fluorescence light emission spectrum have low photosynthetic

activity for certain microalgae and cyanobacteria species (Carvalho et al., 2011). On the

other hand, LEDs have specific wavelengths bands, which can cause a greater energetic

use by these microorganisms and, consequently, a greater cell photosynthetic activity

(Schulze et al., 2014). The cultures using as energy source green, red and white LEDs

with integral light photoperiod showed higher (p < 0.05) biomass concentrations than

the partial light photoperiod. However, the biomass productivity and the specific growth

rate in cultures with blue, green and white LEDs with integral light photoperiod did not

show any significant difference (p > 0.05) for the partial light photoperiod. Thus, in

order to obtain high growth parameters in cultures under these conditions, the partial

illumination can be used, thus increasing the LEDs useful life and reducing the

expenses with energy source.

The cultures did not show a physiological adaptation phase (Figure 1) in any of the

LEDs evaluated conditions. The LEDs wavelengths used comprise the fluorescent light

spectral range used in the Spirulina sp. LEB 18 maintenance, which may justify the cell

adaptation phase absence. Green and red LEDs with integral light photoperiod showed a

more pronounced cell growth than the other conditions. Thus, Spirulina sp. LEB 18

may have used more energy from these wavelength ranges and, therefore, were finalized

still in the cell growth exponential phase. The rest of the experiments presented a

8
tendency to stationary phase in 10 d of culture. The pH of the cultures varied from 9 to

10.45, prevailing the ideal condition for the cyanobacteria cell growth, as Spirulina

(Tomaselli, 1997).

3.2 Biomass biochemical composition

3.2.1 Carbohydrates, proteins and lipids

The carbohydrate concentrations in the Spirulina sp. LEB 18 biomass, cultured with

different colors of LEDs and photoperiods, were the same (p > 0.05) compared to the

control experiment with fluorescent light (Table 3). The carbohydrates concentrations

are in accordance with Rosa et al. (2015), when the same cyanobacteria strain was

cultivated under standard conditions (14.4±1.4 %). Carbohydrates from Spirulina sp.

LEB 18 can be used, for example, for bioethanol production (Salla et al., 2016). The

main advantage of obtaining this type of biofuel from microalgae biomass is the ease in

carbohydrates hydrolysis when compared to other biomass sources (Singh and Olsen,

2011).

The highest proteins concentrations were obtained in the experiments in which the

highest biomass concentrations were found (blue, green and red LEDs with integral

light photoperiod). Under optimum cultivation conditions, microalgae and

cyanobacteria tend to produce more proteins in order to maintain cell multiplication and

biomass production (Solomon et al., 1999). With the exception of the white LED with

integral light photoperiod experiment, the remaining cultures had higher protein

concentrations (p < 0.05) than the control experiment, with only fluorescent light as

energy source. Coward et al. (2016) cultivated Porphyridium purpureum with blue and

green LEDs and also obtained increased amounts of proteins in microalgae biomass.

9
Spirulina biomasses with high protein content can be applied in the human and animal

nutrition, helping to promote health (Santos et al., 2016).

The use of green, red and white LEDs as energy source in Spirulina sp. LEB 18 cultures

photostimulated the lipids production by the cells, showing this biomolecule

concentration higher (p < 0.05) than the control experiment. The conditions used to

increase the lipid content in microalgae and cyanobacteria biomasses generally lead to a

decrease in the cultivation biomass productivity. However, from the results obtained, it

was verified that the LEDs application to the Spirulina sp. LEB 18 cultures, besides

increasing the lipid content, did not influence the cultures biomass productivity,

obtaining superior results (p < 0.05) than the control experiment. Chlorella vulgaris,

Tetraselmis sp. and Nannochloropsis sp. cultivations using LEDs as an energy source

also showed high lipid and biomass productivities simultaneously (Atta et al., 2013, Teo

et al., 2014). The lipids concentrations in the Spirulina sp. LEB 18 biomass were not

influenced (p > 0.05) by the different photoperiods evaluated in the experiments. Thus,

in cultures where the objective is to accumulate lipids in the Spirulina sp. LEB 18

biomass, the partial light photoperiod can be used in order to increase the LEDs lamps

operating time and reduce cultivation costs.

3.2.2 Fatty acid profile

In relation to the fatty acid profile (Table 4), it was observed that 40 % were composed

of saturated and 60 %, unsaturated. In addition, among the unsaturated fatty acid, 44.4

% were identified as polyunsaturated (PUFAs). The fatty acids present in the Spirulina

sp. LEB 18 biomass in greater proportions were palmitic (C16: 0), linoleic (C18: 2 Cys)

and γ-linolenic (C18: 3 n-6). The proportion of these fatty acids in the biomass did not

differ (p > 0.05) between the different LEDs and photoperiods evaluated and the control

10
experiment. The proportions obtained from these fatty acids in the Spirulina sp. LEB 18

biomass were similar to the results obtained by Rahman et al. (2017) with the Spirulina

maxima (40.20 % palmitic acid, 17.87 % linoleic acid and 17.34 % γ-linolenic acid).

Linoleic and γ-linolenic acids are very important essential fatty acids for human

nutrition and health (Fan and Chapkin, 1998). The high concentrations of saturated and

long chains fatty acids in the Spirulina sp. LEB 18 biomass are desirable characteristics

for biodiesel production. The main advantages of the presence of these fatty acids in the

biomass are the increased calorific value, cetane number and oxidation stability, thus

guaranteeing the biofuel resistance to oxidation (Kondamudi et al., 2009). Besides, the

low fatty acid concentration with carbon chains with more than 19 carbons obtained in

the study (approximately 0.23 %) is other advantage for biodiesel production with low

viscosity (Teo et al., 2014).

3.2.3 Phycocyanin

The LEDs application as an energy source to Spirulina sp. LEB 18 cultivation

photostimulated the phycocyanin production by the cells, so that almost all the

experiments showed, at the end of the cultivations, this pigment concentration superior

(p < 0.05) to the control experiment. LEDs are characterized by specific wavelengths,

which may, in some cases, hinder the energy absorption by chlorophyll molecules in

microalgae and cyanobacteria cells. Thus, there may be increased production of other

pigments by cells, such as phycocyanin, in order to increase energy efficiency uptake.

The phycocyanin molecules are responsible for energy absortion in the visible light

spectrum that are poorly utilized by chlorophyll and convey the energy to

photosynthetic reaction center to be used by chlorophyll a (Hemlata and Fatma, 2009).

11
The highest (p < 0.05) phycocyanin concentration was obtained in the experiment using

green LEDs with partial light photoperiod. This value being about 2.7 times greater than

the control experiment. This result is in agreement with Chen et al. (2010), who

obtained a similar phycocyanin concentration (149 mg g biomass-1) from Spirulina

platensis, cultivated with green LEDs.

For all LEDs colors evaluated, the phycocyanin concentration was higher (p < 0.05) in

the partial light photoperiod than in the integral light photoperiod experiments.

Phycocyanin is photosensitive (Jespersen et al., 2005), thus high light intensity may

result in a decrease in the number of chromophore proteins, reducing the amount of

phycobilisomes per cell. In this way, continuous cultures illumination with LEDs may

have caused this pigment degradation in the Spirulina cells, decreasing their

concentration in the final biomass.

The main challenge in microalgae and cyanobacteria cultures to obtain bioproducts is to

ally high productivities of the compound of interest and biomass. Generally, ideal

cultivation conditions for high phycocyanin productivities are not the same for

obtaining high biomass productivities. However, in the present study, it is observed that

all LEDs experiments showed high phycocyanin and biomass productivities

simultaneously, obtaining results superior (p < 0.05) to the control experiment using

only fluorescent light.

4. Conclusion

The application of LEDs as an energy source in Spirulina sp. LEB 18 cultures

photostimulated biomass and bioproducts production. Red LEDs in integral light

photoperiod provided the best growth parameters of cultures. Green, red and white

LEDs as energy source in Spirulina sp. LEB 18 cultures photostimulated the lipids

12
production. The major fatty acids methyl esters obtained in the biomasses were

palmitic, linoleic and γ-linolenic. The highest phycocyanin concentration (green LEDs

in partial light photoperiod) was about 2.8 times greater than the control experiment

with fluorescent light (126.39 mg gbiomass-1).

5. Acknowledgements

The authors acknowledge CAPES (Coordination for the Improvement of Higher

Education Personnel), CNPq (National Council of Technological and Scientific

Development), MCTIC (Ministry of Science Technology, Innovation and

Communication) and the researchers Milene Teixeira Barcia, Wellington Oliveira,

Cristiano Ballus and Helena Godoy.

6. References

1. Atta, M., Idris, A., Bukhari, A., Wahidin, S. Intensity of blue light: A potential

stimulus for biomass and lipid content in fresh water microalgae Chlorella vulgaris.

2013. Bioresour. Technol. 148, 373-378.

2. Baba, M., Kikuta, F., Suzuki, I., Watanabe, M.M., Shiraiwa, Y., 2012. Wavelength

specificity of growth, photosynthesis, and hydrocarbon production in the oil producing

green alga Botryococcus braunii. Bioresour. Technol. 109, 266-270.

3. Bennett, A., Bogorad, L. 1973. Complimentary chromatic adaptation in a filamentous

blue-green alga. J. Cell Biol., 58, 419-435.

4. Blanken, W., Cuaresma, M., Wijffels, R.H., Janssen, M. 2013. Cultivation of

microalgae on artificial light comes at a cost. Algal Res. 2, 333-340.

5. Carvalho, A.P., Silva, S.O., Baptista, J.M., Malcata, F.X., 2011. Light requirements

in microalgae photobioreactors: an overview of biophotonic aspects. Appl. Microbiol.

Biotechnol. 89, 1275-1288.

13
6. Chen, C.Y., Kao, P.C., Tsai, C.J., Lee, D.J., Chang, J.S. 2013. Engineering strategies

for simultaneous enhancement of C-phycocyanin production and CO2 fixation with

Spirulina platensis. Bioresour. Technol. 145, 307-312.

7. Chen, H.B., Wu, J.Y., Wang, C.F., Fu, C.C., Shieh, C.J., Chen, C.I., Wang, C.Y., Liu,

Y.C. 2010. Modeling on chlorophyll a and phycocyanin production by Spirulina

platensis under various light-emitting diodes. Biochem. Eng. J. 53, 52-56.

8. Costa, J.A.V., Colla, L.M. Filho, P.D., 2004. Improving Spirulina platensis biomass

yield using a fed-batch process. Bioresour. Technol. 92, 237-241.

9. Costa, J.A.V., Colla, L.M., Duarte Filho, P., Kabke, K., Weber, A., 2002. Modelling

of Spirulina platensis growth in fresh water using response surface methodology. World

J. Microb. Biot. 18, 603-607.

10. Coward, T., Grunewald, C.F., Silkina, A., Radcliffe, D.L.O., Llewellyn, G., Lovitt,

R.W. 2016. Utilising light-emitting diodes of specific narrow wavelengths for the

optimization and co-production of multiple high-value compounds in Porphyridium

purpureum. Bioresour. Technol. 221, 607-615.

11. Das, P., Lei, W., Aziz, S.S., Obbard, J.P. 2011. Enhanced algae growth in both

phototrophic and mixotrophic culture under blue light. Bioresour. Technol. 102, 3883-

3887.

12. David, F., Sandra, P., Vickers, A.K. 2005. Column Selection for the Analysis of

Fatty Acid Methyl Esters - Application. Agilent Technologies. Available at

http://www.chem.agilent.com/Library/applications/5989-3760EN.pdf. Accessed on

January 20, 2017.

14
13. Duarte, J.H., Morais, E.G., Radmann, E.M., Costa, J.A.V. 2017. Biological CO2

mitigation from coal power plant by Chlorella fusca and Spirulina sp. Bioresour.

Technol. 234, 472-475.

14. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956.

Colorimetric method for determination of sugars and related substances. Anal. Chem.

28, 350-356.

15. Fan, Y., Chapkin, R.S., 1998. Importance of dietary γ-linolenic acid in human health

and nutrition. J. Nutr. 128, 1411-1414.

16. Folch, J., Lees, M., Stanley, G.H.S., 1957. A simple method for the isolation and

purification of total lipides from animal tissues. J. Biol. Chem. 226, 497-509.

17. Hemlata, Fatma, T. 2009. Screening of cyanobacteria for phycobiliproteins and

effect of different environmental stress on its yield. Bull Envirom. Contam. Toxicol. 83,

509-515.

18. Thompson, M., Ellison, S.L.R., Wood, R. 2002. IUPAC – International Union of

Pure and Applied Chemistry. Harmonized guidelines for single-laboratory validation of

methods of analysis. Pure Appl. Chem. 74, 835-855.

19. Jespersen, L., Strømdahl, L.D., Olsen, K., Skibsted, L.H. 2005. Heat and light

stability of three natural blue colorants for use in confectionery and beverages. Eur.

Food Res. Technol. 220, 261-266.

20. Joseph, J.D., Ackman, R.G. 1992. Capillary column gas chromatographic method

for analysis of encapsulated fish oils and fish oil ethyl esters: Collaborative study. J.

AOAC Int. 75, p. 488-506.

15
21. Kim, K., Hoh, D., Ji, Y., Do, H., Lee, B., Holzapfel, W. Impact of light intensity,

CO2 concentration and bubble size on growth and fatty acid composition of Arthrospira

(Spirulina) platensis KMMCC CY-007. 2013. Biomass Bioenerg., 49, 181-187.

22. Kondamudi, N., Strull, J., Misra, M., Mohapatra, S., 2009. A green process foe

producing biodiesel from feater meal. J. Agric. Food Chem. 57, 6163-6166.

23. Lee, N.K., Oh, H.M., Kim, H.S., Ahn, C.Y. 2017. Higher production of C-

phycocyanin by nitrogen-free (diazotrophic) cultivation of Nostoc sp. NK and

simplified extraction by dark-cold shock. Bioresour. Technol. 227, 164-170.

24. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein

measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275.

25. Markou, G. 2014. Effect of various colors of light-emitting diodes (LEDs) on the

biomass composition of Arthrospira platensis cultivated in semi-continuous mode.

Appl. Biochem. Biotechnol. 172, 2758-2768.

26. Morais, M.G., Costa, J.A.V., 2007. Carbon dioxide mitigation with Chlorella

kessleri, Chlorella vulgaris, Scenedesmus obliquus and Spirulina sp. cultivated in flasks

and vertical tubular photobioreactors. Biotechnol. Lett. 29, 1349-1352.

27. Rahman, M.A., Aziz, M.A., Al-khulaidi, R.A., Sakib, N., Islam, M. 2017. Biodiesel

production from microalgae Spirulina maxima by two step process: Optimization of

process variable. Radiat. Res. Appl. Sci. 10, 140-147.

28. Rosa, G.M., Moraes, L., Cardias, B.B., Souza, M.R., Costa, J.A.V., 2015. Chemical

absorption and CO2 biofixation via the cultivation of Spirulina in semicontinuous mode

with nutrient recycle. Bioresour. Technol. 192, 321-327.

29. Salla, A.C.V., Margarites, A.C., Seibel, F.I., Holz, L.C., Brião, V.B., Bertolin, T.E.,

Colla, L.M., Costa, J.A.V. 2016. Increase in the carbohydrate content of the microalgae

16
Spirulina in culture by nutrient starvation and the addition of residues of whey protein

concentrate. Bioresour. Technol. 29, 133-141.

30. Santos, R.R., Araújo, O.Q.F., Medeiros, J.L., Chaloub, R.M. 2016. Cultivation of

Spirulina maxima in medium supplemented with sugarcane vinasse. Bioresour.

Technol. 204, 38-48.

31. Schulze, P.S.C., Barreira, L.A., Pereira, H.G.C., Perales, J.A., Varela, J.C.S. 2014.

Light emitting diodes (LEDs) applied to microalgal production. Trends Biotechnol. 32,

422-430.

32. Singh, A., Olsen, S.I., 2011. A critical review of biochemical conversion,

sustainability and life cycle assessment of algal biofuels. Appl. Energ. 88, 3548-3555.

33. Solomon, E.P., Berg, L.R., Martin, D.W., 1999. Biology, 5th ed. Saunders College

Publishing, Fort Worth.

34. Teo, C.L., Atta, M., Bukhari, A., Taisir, M., Yusuf, A.M., Idris, A. 2014. Enhancing

growth and lipid production of marine microalgae for biodiesel production via the use

different LED wavelegengths. Bioresour. Technol. 162, 38-44.

35. Thajuddin, N., Subramanian, G. 2005. Cyanobacterial biodiversity and potential

applications in biotechnology. Curr. Sci. India, 89, 47-57.

36. Tomaselli, L., 1997. Morphology, ultrastructure and taxonomy of Arthrospira

(Spirulina). In: Vonshak, A. (Ed.), Spirulina platensis (Arthrospira) Physiology, cell-

biology and biotechnology. Taylor & Francis, London, pp. 01-16.

37. Vanthoor-Koopmansa, M., Wijffelsa, R.H., Barbosac, M.J., Eppinka, M.H.M. 2013.

Biorefinery of microalgae for food and fuel. Bioresour. Technol. 135, 142-149.

38. Wang, C.Y., Fu, C.C., Liu, Y.C. 2007. Effects of using light-emitting diodes on the

cultivation of Spirulina platensis. Biochem. Eng. J. 37, 21-25.

17
39. Wang, H., Ji, S., Bi, S., Zhou, P., Chen, L., Liu, T. 2014. Joint production of

biodiesel and bioethanol from filamentous oleaginous microalgae Tribonema sp.

Bioresour. Technol. 172, 169-173.

40. Wishkerman, A., Wishkerman, E. 2017. Application note: A novel low-cost open-

source LED system for microalgae cultivation. Comput. Electron. Agric. 132, 56-62.

41. Zhao, Y., Wang, J., Zhang, H., Yan, C., Zhang, Y. 2013. Effects of various LED

light wavelengths and intensities on microalgae-based simultaneous biogas upgrading

and digestate nutrient reduction process. Bioresour. Technol. 136, 461-468.

Figure captions

Figure 1: Spirulina sp. LEB 18 cell growth curves: (+) Control experiment; () Blue

LEDs and partial light photoperiod; () Blue LEDs and integral light photoperiod; (∆)

Green LEDs and partial light photoperiod; (▲) Green LEDs and integral light

photoperiod; (□) Red LEDs and partial light photoperiod; (■) Red LEDs and integral

light photoperiod; () White LEDs and partial light photoperiod; () White LEDs and

integral light photoperiod.

Figure 2: Phycocyanin concentrations in the Spirulina sp. LEB 18 biomass cultivated

with different LEDs colors and photoperiods.

Tables and Figures:

Table 1: Experiments with different LEDs colors and photoperiods.

18
 Experiment Colors Ligh period Photoperiod (h)

Control White Partial 12F:12D

2 Blue Partial 12F: 06L: 06D

3 Blue Integral 12F: 12L: 00D

4 Green Partial 12F: 06L: 06D

5 Green Integral 12F: 12L: 00D

6 Red Partial 12F: 06L: 06D

7 Red Integral 12F: 12L: 00D

8 White Partial 12F: 06L: 06D

9 White Integral 12F: 12L: 00D

F: White fluorescent light source (3200 µmol m-2s-1); L: Light emitting diodes (500 µmol m-
2 -1
s ); D: Dark (without illumination).

Table 2: Maximum cell growth (Xmax, g L-1), biomass productivity (P max, g L-1 d-1) and

specific growth rate (μmax, d-1) under different conditions.

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 Xmax Pmax µmax
Experiment LEDs Photoperiod
(g L-1) (g L-1 d-1) (d-1)

1 Control Partial 0.87±<0.01g 0.10±<0.01d 0.08±<0.01c

2 Blue Partial 1.13±<0.01de 0.12±<0.01cd 0.14±<0.01ab

3 Blue Integral 1.16±<0.01d 0.12±<0.01bcd 0.13±<0.01b

4 Green Partial 1.33±<0.01c 0.15±<0.01ab 0.15±<0.01ab

5 Green Integral 1.44±0.03b 0.14±<0.01bc 0.13±<0.01ab

6 Red Partial 1.34±<0.01c 0.16±<0.01a 0.11±<0.01b

7 Red Integral 1.77±0.02a 0.17±<0.01a 0.16±<0.01a

8 White Partial 1.08±<0.01f 0.12±<0.01bcd 0.11±<0.01b

9 White Integral 1.15±<0.01d 0.14±<0.01abc 0.13±<0.01b

Means ± standard deviations. The same letters in the same column indicate that no significant difference

between values at a 95 % confidence level (p > 0.05).

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Table 3: Carbohydrates (%, w w-1), proteins (%, w w-1) and lipids (%, w w-1)

concentrations in the Spirulina sp. LEB 18 biomass under different conditions.

Photoperiod Blue LEDs Green LEDs Red LEDs White LEDs

Carbohydrates (%, w w-1)

Partial 12.64±0.39aA 12.70±0.67aA 12.34±0.12aA 14.01±1.96aA

Integral 12.27±0.12aA 13.50±0.43aA 12.91±0.29aA 12.23±0.09aA

Control 11.72±0.44A 11.72±0.44A 11.72±0.44A 11.72 ±0.44A

Proteins (%, w w-1)

Partial 58.06±0.60aA 56.03±0.02aB 54.17±1.48aB 54.53±0.50aA

Integral 60.61±1.28aA 61.79±1.90aA 61.10±0.30aA 52.96±0.36bA

Control 51.98±1.13B 51.98±1.13C 51.98±1.13B 51.98±1.13A

Lipids (%, w w-1)

Partial 11.63±0.95aA 13.25±0.12aA 12.17±0.17aA 13.75±0.16aA

Integral 11.92±1.14bA 13.30±0.19aA 12.19±0.81abA 13.24±0.17abA

Control 10.45±0.40A 10.45±0.40B 10.45±0.40B 10.45±0.40B

Means ± standard deviations. The same letters in the same line indicate that no significant difference

between the different LEDs colors at a 95 % confidence level (p > 0.05). The same letters in the same

column indicate that no significant difference between the photoperiods and control experiments at a 95

% confidence level (p > 0.05).

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 Table 4: Fatty acid concentrations (wt, %) in the Spirulina sp. LEB 18 biomass under different conditions.

Experiments 1 2 3 4 5 6 7 8 9
Fatty acid
Capric (C10:0) 4.86±2.15a 4.26±0.11a 4.84±0.84a 6.27±0.06a 6.05±1.11a 5.35±0.50a 4.70±0.23a 5.94±1.08a 5.73±0.02a
Docecanoic (C12:0) 0.59±<0.01a 0.35±0.02ab 0.33±<0.01b 0.52±0.11ab 0.37±0.02ab 0.42±0.05ab 0.51±0.01ab 0.42±0.04ab 0.52±0.01ab
Myristic (C14:0) 0.36±0.02a 0.32±<0.01a 0.37±0.04a 0.22±<0.01a 0.28±0.05a 0.32±0.03a 0.27±0.06a 0.28±0.03a 0.20±0.01a
Myristoleic (C14:1) 0.25±0.01bc 0.34±0.09abc 0.43±0.05ab 0.41±0.04abc 0.59±0.02a 0.56±0.01a 0.17±0.02c 0.43±0.06ab 0.45±0.01ab
Pentanoic (C15:0) *N.D 0.08±0.01a *N.D *N.D 0.03±0.03b *N.D *N.D 0.07±0.01ab 0.05±0.01ab
Monopentanoic (C15:1) 0.80±0.04a 0.92±0.02a 1.27±0.70a 0.85±0.21a 0.54±0.03a * 0.43±0.09a 1.26±0.18a 0.61±0.01a
Palmitic (C16:0) 41.29±1.30a 46.65±0.35a 44.53±0.08a 43.40±0.26a 43.27±1.90a 43.37±2.00a 42.95±2.03a 44.68±0.05a 45.28±0.01a
Palmitoleic (C16:1) 4.94±0.31a 3.89±0.16a 4.62±0.37a 4.67±0.15a 6.32±1.20a 5.04±0.32a 5.31±0.46a 4.40±0.43a 4.43±0.01a
Stearic (C18:0) 1.64±0.55a 2.10±0.15a 1.76±0.47a 1.61±0.14a 1.20±<0.01a 2.37±0.32a 1.09±0.08a 1.95±0.02a 1.40±0.01a
Oleic (C18:1 Cis) 2.80±0.05b 3.63±0.15ab 2.92±0.14ab 6.32±1.25ab 3.87±0.27a 4.45±1.21ab 3.74±0.07ab 3.18±0.55ab 3.73±0.01ab
Linoleic (C18:2 Cis) 15.61±0.04a 14.07±0.08a 14.16±0.26a 13.13±0.18a 13.61±0.50a 16.24±1.70a 14.42±0.12a 13.76±0.21a 14.09±0.01a
γ-linolenic (C18:3 n-6) 26.87±1.79a 23.08±0.19a 24.77±0.44a 22.21±1.94a 23.63±1.40a 21,67±1,4a 26,15±1,75a 23.26±0.08a 23.04±0.01a
gadoleic (C20: ln-9) *N.D 0.08±0.01a *N.D *N.D 0.06±<0.01a 0,07±0,01a *N.D 0.06±0.01a 0.05±0.01a
Eicosadienoic (C20:2) *N.D 0.11±0.02b *N.D 0.21±0.04a 0.17±<0.01ab 0,12±0,01b 0,14±0,0ab 0.17±0.01ab 0.21±0.01a
Means ± standard deviations. The same letters in the same line indicate that no significant difference between the different LEDs colors and photoperiods at a 95 % confidence level (p > 0.05).

*N.D: Not detected.

22
Figure 1:

Figure 2:

140
126,39
120
Phycocyanin (mg g biomass -1)

105,09
100
81,96 83,72
77,69 76,5
80
58,56
60 54,27
46,36
40

20

0
Control Blue Blue Green Green Red (Partial) Red White White
(Partial) (Integral) (Partial) (Integral) (Integral) (Partial) (Integral)
LED color (photoperiod)

23
24
25
26
Highlights

 Blue, green, red and white LEDs increased the growth parameters of cultivations.

 LEDs increased the lipids amount on the Spirulina sp. LEB 18 biomass.

 Major fatty acids obtained were palmitic, linoleic and γ-linolenic.

 Green LEDs photostimulated phycocyanin production by the cells up to 2.8 times.

27