Combination of Light Emitting Diodes (LEDs) for photostimulation of carotenoids and chlorophylls synthesis in Tetradesmus sp.

Algal Research 43 (2019) 101649

Contents lists available at ScienceDirect

Algal Research journal homepage: www.elsevier.com/locate/algal

Combination of Light Emitting Diodes (LEDs) for photostimulation of carotenoids and chlorophylls synthesis in Tetradesmus sp.

T

⁎

Vanessa Daneluz Gonçalvesa, , Márcia Regina Fagundes-Klenb, Daniela Estelita Goes Triguerosb, Adilson Ricken Schuelterb, Alexander Dimitrov Kroumovc, Aparecido Nivaldo Módenesb a

Department of Environment, State University of Maringá, UEM, Av. Ângelo Moreira da Fonseca 1800, 87506-370 Umuarama, PR, Brazil Department of Chemical Engineering Postgraduate Program, State University of West Paraná, UNIOESTE, Rua da Faculdade 645, Jd. Santa Maria, 85903-000 Toledo, PR, Brazil c The “Stephan Angeloff” Institute of Microbiology-Bulgarian Academy of Sciences, Acad. G. Bonchev str., Bl. 26, Sofia 1113, Bulgaria b

A R T I C LE I N FO

A B S T R A C T

Keywords: LEDs Biopigments Tetradesmus sp.

In this work, Tetradesmus sp. was investigated regarding biomass and pigment production. The cultivation of Tetradesmus sp. was carried out in a BG-11 medium in addition to a vertical tubular photobioreactor (PBR) irradiated with different spectral combinations and intensities, within a 24:00 h photoperiod. The LEDs emitting light with white; red; yellow; green and blue spectrum were used alone and/or combined, with intensities varying from 13 to 190 μmol m−2 s−1. ANOVA suggested significant effects of the light combination, intensity and interaction between the factors on biomass, carotenoid and chlorophyll responses. The blue light accelerated the growth of Tetradesmus sp. in all three experiments. The combinations white:green (95 μmol m−2 s−1), red:blue (50 μmol m−2 s−1) and blue (25 μmol m−2 s−1) favored biomass growth; while white:green (190 μmol m−2 s−1), red:green (50 μmol m−2 s−1) and red (25 μmol m−2 s−1) favored the synthesis of carotenoids and chlorophylls. Culture reproduction under the spectral regions favoring pigment synthesis indicated that Tetradesmus sp. was photostimulated to synthesize both pigments, especially with red:green (50,50%) at 50 μmol m−2 s−1, where the highest production of carotenoids (6.09 ± 0.29 mg (g biomass)−1) and chlorophyll a (10.08 ± 0.75 mg (g biomass)−1) was obtained.

1. Introduction The industrial cultivation of microalgae for the production of biomass and plastidial pigments [1], as well as other bioproducts, was promoted by the growing demand for compounds of natural origin and clean energy by the food, pharmaceutical, cosmetic and bioenergy industries [2–4]. Global biomass production rose from 5000 tons in 2004 to 15,000 tons in 2013 [5]. Each species of microalgae shows anabolic and catabolic specificities, varying in carbon assimilation processes, which makes it difficult to define operational conditions that optimize biomass growth associated with bioproducts of interest [6–8]. The definition of optimal culture conditions for each species requires investigation of the factors that interfere with the rate values of biomass growth and plastidial or cytoplasmic pigment synthesis [9]. These factors are most likely connected to the combination of the visible electromagnetic spectrum [10,11], light intensity [11–17], interaction with the components of the culture medium [18] and understanding of the CO2 fixation process

⁎

[19]. First and foremost, the kind of relationship between biomass growth and pigment synthesis must be defined for each case, as a function of microalgal physiology and operational conditions. This information is crucially important for the overall research strategy in order to minimize the experiments required to achieve the desired goals. Abreu et al. [20] significantly increased biomass production of Chlorella vulgaris in a mixotrophic culture with a medium enriched with hydrolyzed whey. Bouarab, Dauta and Loudiki [21] found the highest growth rate (0.865 d−1) of Micractinium pusillum in a medium containing glucose as a carbon source, and the lowest rate (0.2 d−1) with an inorganic carbon source. Smith et al. [22] cultivated Micractinium inermum in an autotrophic system using air injected with 5% CO2, and estimated a maximum productivity 10.4 times higher than the productivity determined in the cultivation of the species with atmospheric air, which contains 0.04% CO2. Přibyl et al. [23] reached 12 g L−1 biomass, a specific maximum growth rate of 2.695 ± 0.014 d−1 and 19.01 ± 1.45 mg L−1 d−1 carotenoids in the cultivation of Scenedesmus sp. in a closed indoor

Corresponding author. E-mail address: [email protected] (V.D. Gonçalves).

https://doi.org/10.1016/j.algal.2019.101649 Received 27 March 2019; Received in revised form 1 September 2019; Accepted 2 September 2019 2211-9264/ © 2019 Elsevier B.V. All rights reserved.

Algal Research 43 (2019) 101649

V.D. Gonçalves, et al.

tubular photobioreactor (PBR) irradiated with 500 μmol m−2 s−1 (Photosynthetically Active Radiation - PAR) and enriched with 2% CO2. Kim et al. [24] investigated the cultivation of Gracilaria tikvahiae under different energy sources, and determined maximum production of carotenoids (~0.25 mg g−1) and chlorophyll (~1.75 mg g−1), and minimum specific growth rate (~6.2% d−1), in the spectral combination of the red:blue region (50:50%) with 100 μmol m−2 s−1. Despite the various analyses, the use of Light Emitting Diode (LED) as a light source for the cultivation and stimulation of microalgae for the synthesis of high value bioproducts is still insufficient [7,16,25], since a great part of the studies available to date evaluate the cultivations, especially in the red and blue spectral region [14,26,27]. In view of the particularities of each species in terms of luminous intensity and spectral combinations, evaluations of a broader spectrum are required, with combinations of single and multiple wavelengths revealing possible interactions between the different pigment absorption responses in microalgae cells [27]. In addition, there are numerous unexplored species with potential for synthesis of bioproducts [28] with high-value bioactive properties, such as the microalgae Tetradesmus sp., formerly Acutodesmus sp., belonging to Chlorophycophyta group [29]. The few studies with Tetradesmus sp. demonstrate their ability to remove drugs in aqueous media [30]. Hence, the information about the biopotential of this attractive alga for industrial application is very scarce and needs further research. The objective of this research was to investigate the effects of spectral conditions of energy and luminous intensity that induced high photostimulation of Tetradesmus sp. for the synthesis of carotenoids and chlorophylls a and b, by cultivation in a vertical tubular PBR injected with CO2-enriched air. 2. Material and methods

Fig. 1. Illustration of the photobioreactor. (i) graduated pipette (2 mL) for the injection of CO2-enriched air; (ii) access for addition of culture medium or collection of material; (iii) air outlet; (iv) styrofoam interleaved with plastic film seal; (v) test tube; (vii) PVC tube of 150 mm diameter and 350 mm height; (vii) spiral LED strip in the inner portion of the PVC pipe.

2.1. Microalga species and maintaining

2.3. Cultivation in photobioreactor

The microalga Tetradesmus sp., with 97% similarity to T. acuminatus (LC192133.1), was isolated from a sample obtained by Hinterholz et al. [18] and Schuelter et al. [31] in the municipality of Matelândia (25°14′27″S; 53°59′47″W), Paraná, located in southern Brazil. It was maintained in a solid BG-11 medium in Petri dishes and placed under continuous fluorescent lighting (28 μmol m−2 s−1) at 20 °C in a Biochemical Oxygen Demand (BOD) incubator. Prior to the cultures, the microalgae was acclimated in a PBR for 120 h in 100 mL BG-11 medium using 250 mL Erlenmeyer flasks sealed with plastic film, which were kept in a horizontal shaker at 65 rpm, room temperature, continuous fluorescent lighting (56 μmol m−2 s−1) and pH 7.0 ± 0.5.

The cultivation was carried out in a glass beaker (500 mL), with its upper portion sealed by two layers of plastic film intercalated with a styrofoam plate, containing three accesses: (i) connected to a 2 mL pipette for the downstream air with CO2; (ii) for the addition of culture medium and sample collection; and (iii) for the air outlet. Each beaker was placed inside a polyvinyl chloride (PVC) tube of 150 mm in diameter and 350 mm high, internally filled with a spiral-round LED strip (4,8 W m−1, 12 V, 3528 60LED/M, Maxtel) (Fig. 1). Aeration enriched with 7.9 ± 1.5% CO2 was kept constant (30 L h−1). The cultures were performed with a 24:00 h (light:dark) photoperiod, at room temperature (25 ± 3 °C) and pH of 7.6 ± 0.3. Fig. 2 shows the respective LEDs emission spectra, obtained using the spectrofluorimeter (model LS 55, Perkin Elmer), in the bands of the visible (white), red, yellow, green and blue applied in this work. The intensity of the energy irradiated in each experiment was set up utilizing a dimmer.

2.2. Cultivation medium preparation The BG-11 medium was composed of NaNO3 (17.60 mM), K2HPO4 (0.23 mM), MgSO4·7H2O (0.30 mM), CaCl2·2H2O (0.24 mM), citric acid·H2O (0.031 mM), iron and ammonium citrate (0.021 mM), Na2EDTA·2H2O (0.0027 mM), Na2CO3 (0.19 mM), H3BO3 (46.00 mM), MnCl2·4H2O (9.00 mM), ZnSO4·7H2O (0.77 mM), Na2MoO4·2H2O (1.60 mM), CuSO4·5H2O (0.30 mM), and Co(NO3)2·6H2O (0.17 mM). The final solution was sterilized in a vertical autoclave (model CS150, Prismatec, Brazil) at 1 atm for 20 min and stored for further use under refrigeration. For the solidification of the BG-11 medium, a solution of agar (15 g L−1) was prepared and autoclaved (1 atm for 20 min). A sterile solution of sodium thiosulfate pentahydrate (248 g L−1, 1.00 mM) was added to the agar solution. Inside a laminar flow hood, the solution was mixed with the liquid BG-11 medium, both at a temperature range of 45 to 50 °C. The mixture was partitioned into Petri dishes, and after their solidification, the dishes were closed, sealed with plastic film and stored under refrigeration.

2.4. Spectral combinations and luminous intensity Three experiments of Tetradesmus sp. photostimulation were performed without replicates, as well as an additional experiment aiming to reproduce the best conditions found. The cultures of Tetradesmus sp. were monitored visually using a microscope. Biomass production and pigment synthesis were determined in triplicate (N = 3), except in the reproduction of the experiment with the visible:green combination and 50 μmol m−2 s−1 light intensity (N = 2). Experiment 1 was carried out with an initial biomass concentration of 0.03 g L−1, during a period of 12 days with the cultures irradiated by white LED light and a second LED with a different spectrum in an 86.7%:13.3% ratio, respectively. The following LED combinations were performed: white:red; white:yellow; white:green; white:blue. The light 2

Algal Research 43 (2019) 101649

V.D. Gonçalves, et al.

4,0

[35]:

3,5

Chla = 12.25 A663.2 − 2.79 A646.8

(1)

Chlb = 21.50 A646.8 − 5.10 A663.2

(2)

3,0

W cm-2

2,5

White Red Yellow Green Blue

TC =

(1000 A 470 − 1.82 Chla − 85.02 Chlb) 198 −1

(3) −1

where, Chla: chlorophyll a [μg mL ]; Chlb: chlorophyll b [μg mL ]; TC: total carotenoids; A646.8: absorbance in 646.8 nm; A663.2: absorbance in 663.2 nm; and A470: absorbance in 470 nm. The quantitative determination of chlorophylls and carotenoids by UV–VIS spectroscopy is complicated by the choice of sample, solvents and spectrophotometer used. Pigments like chlorophyll b and carotenoids can absorb light in overlapping spectral regions. In order to have a precision spectroscopic measurement, absorbance should be measured between 0.3 and 0.85, otherwise, there are several interfering factors [35].

2,0 1,5 1,0 0,5 0,0 250 300 350 400 450 500 550 600 650 700 750 800 Wavelength (nm)

2.7. Statistical evaluation

Fig. 2. Emission spectra of white, red, yellow, green and blue LEDs. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

The factorial ANOVA was used to verify if there is interaction between the i-combinations of the spectrum and the j-light intensities, as well as if there is a significant difference (p < 0.05) between the means of these factors used in the k-type culture. When a statistical difference was observed, Tukey test was performed (p < 0.05). All statistical analyses were achieved using Statistica software.

intensities for each LED combination were 90 and 190 μmol m−2 s−1, respectively. Control cells were exposed to white light alone (90 and 190 μmol m−2 s−1). Experiment 2 was accomplished with an initial biomass concentration of 0.03 g L−1, during a period of 12 days with the cultures irradiated by red LED light and a second LED with a different spectrum in a 50%:50% ratio, respectively. The following LED combinations were performed: red:yellow; red:green; red:blue. The light intensities for each LED combination were 25 and 50 μmol m−2 s−1, respectively. Control cells were exposed to red light alone (were 25 and 50 μmol m−2 s−1). Experiment 3 was achieved with an initial biomass concentration of 0.89 g L−1, during a period of 6 days with the cultures irradiated by white, red, yellow, green and blue LEDs, at light intensities of 13 and 25 μmol m−2 s−1. The reproduction of the spectral combinations that stimulated the highest carotenoid synthesis in each of the previous experiments were performed by fixing the following conditions: Tetradesmus sp. with 0.03 g L−1 of initial concentration was cultivated for 12 days with an intensity of 50 μmol m−2 s−1: red, white:green and red:green.

3. Results and discussion 3.1. Biomass growth The blue spectrum significantly accelerated the growth of Tetradesmus sp. in all three experiments (Fig. 3). The culture irradiated in the combination white:blue at 95 μmol m−2 s−1 (Fig. 3a) developed a reduced lag phase with exponential growth after the first day. This culture was overtaken in biomass production (3.45 g L−1) on the 9th day, by cells irradiated with white:green at 95 μmol m−2 s−1 (3.59 g L−1). As observed, the culture irradiated with the visible spectrum (white) was the only one that showed an enhanced productivity and specific growth rate with an increase in the light intensity, in this case from 13 to 25 μmol m−2 s−1, as well as from 25 to 95 μmol m−2 s−1 (see Table 1). With an increase to 190 μmol m−2 s−1 (Experiment 1), the kinetic parameters quantifying the growth decreased due to incident light with intensity above the cellular absorption capacity, a phenomenon that causes photoinhibition [36]. Photoinhibition has been reported by You and Barnett [37], who showed that Porphyridium cruentum grew well at the light intensity of 70 μmol m−2 s−1 and presented inhibition effects with 90 μmol m−2 s−1. Khalili et al. [32] also detected an increase in the growth of Chlorella vulgaris by increasing light intensity from 50 μmol m−2 s−1 to 80 μmol m−2 s−1, and photoinhibition of growth under the intensity of 110 μmol m−2 s−1. On the other hand, Atta et al. [38] cultivated C. vulgaris with light energy in the blue spectral region, under a photoperiod of 16 h:08 h (light:dark); reaching 2.3 g L−1 of biomass with 200 μmol m−2 s−1, and 0.817 g L−1 with 300 μmol m−2 s−1. Ravelonandro et al. [39] did not witness photoinhibition of Spirulina sp. by a cultivation with energy in the green spectral region, emitting at about 25; 50; 64 and 76 μmol m−2 s−1. Through the analysis of variance, the light spectrum and luminous intensity, in addition to the interaction between them, promote significant effects on the responses of final biomass concentration for the three experiments with Tetradesmus sp. with low variability (Table 2). Fig. 4 presents the response of the final biomass in the different spectral combinations and levels of light intensity. The increase from 95 to 190 μmol m−2 s−1 promoted a significant

2.5. Biomass and kinetic parameters quantification The biomass concentration was determined by the gravimetric method and correlated to optical density at a wavelength of 683 nm (OD683) for diluted samples, measured in a Spectrophotometer UV1800 (Shimadzu) [17,32]. The estimate of the biomass concentration from the calibration curve (OD683 = 1998 g L−1) was realized regularly after refilling evaporated water from PBRs [23] with distilled and deionized water. The specific growth rate (μ) (d−1) and productivity (P) (g L−1 d−1) were estimated according to Setyoningrum and Nur [33]. 2.6. Quantification of chlorophylls and carotenoids The method adapted from Wagner et al. [17] and León-Saiki [34] was applied, where centrifugation of 5 mL of microalgae culture (1008 ×g, 8 min), supernatant discard and sediment freezing (−15 °C for 24 h) were performed. Subsequently, 5 mL of acetone solution (80%) was added, maintaining the sample in ultrasonic bath for 20 min with subsequent centrifugation (1008 ×g, 8 min). The supernatant absorbance was measured at wavelengths of 470; 646.8 and 663.2 nm against a zero consisting of acetone (80%). The concentration of pigment [μg (mL extract)−1] was calculated using Eqs. (1), (2) and (3) 3

Algal Research 43 (2019) 101649

V.D. Gonçalves, et al.

(b) 6 Wh95 WhRe95 WhY e95 WhGr95 WhBl95

5 4 3 2 1 0 0

1

2

3

4

5

6

7

8

Wh190 WhRe190 WhY e190 WhGr190 WhBl190

5

Biomass (g L-1)

Biomass (g L-1)

(a) 6

4 3 2 1 0

9 10 11 12

0

1

2

3

4

Time (d)

7

8

9 10 11 12

(d) 6

5

Re25 ReY e25 ReGr25 ReBl25

4 3 2 1 0 0

1

2

3

4

5

6

7

8

5

Biomass (g L-1)

Biomass (g L-1)

6

Time (d)

(c) 6

Re50 ReY e50 ReGr50 ReBl50

4 3 2 1 0

9 10 11 12

0

1

2

3

4

Time (d)

5

6

7

8

9 10 11 12

Time (d)

(e) 6

(f) 6 Wh13 Re13 Y e13 Gr13 Bl13

5 4 3 2 1

Wh25 Re25 Y e25 Gr25 Bl25

5

Biomas (g L-1)

Biomass (g L-1)

5

4 3 2 1

0

0 0

1

2

3

4

5

6

0

1

2

Time (d)

3

4

5

6

Time (d)

Fig. 3. Growth of Tetradesmus sp. biomass over time in the cultures from Experiment 1: (a) 95 μmol m−2 s−1; (b) 190 μmol m−2 s−1; Experiment 2: (c) 25 μmol m−2 s−1; (d) 50 μmol m−2 s−1; and Experiment 3 (e) 13 μmol m−2 s−1; (f) 25 μmol m−2 s−1. Visible (Wh); red (Re); yellow (Ye); green (Gr); blue (Bl); intensity in μmol m−2 s−1 light. Biomass (X0). Experiment 1 and 2: X0 = 0.03 g L−1; 12 days. Experiment 3: X0 = 0.89 g L−1; 6 days. Symbol: mean; box: mean ± standard error; whisker: mean ± standard deviation. (N = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

similarity may be a consequence of the high contribution of the visible spectrum, white (86.7%), which met the microalgae metabolic demand for energy and made the addition of the red, yellow, green, and blue spectral bands (13.3%) insufficient. In Experiment 2, light sources in the red and blue regions favored the production of biomass (Fig. 3c, d), with the highest value of 3.42 ± 0.10 g L−1 observed with the red:blue-50 combination, followed by 3.39 ± 0.01 g L−1 with red:blue-25 and 3.02 ± 0.01 g L−1 with red-50 (Table 3); all with kinetic similarities of productivity and specific growth rate (Table 1). These spectral regions comprise the absorption bands of chlorophyll a and b [40], responsible for triggering

biomass decrease (p-value = 0.0002) in the cultures with white:green and white:red (Table 2, Fig. 4a), attributed to light intensity irradiation (190 μmol m−2 s−1) above the initial cell (0.03 g L−1) absorption ability, promoting an interaction effect between the spectral combination and light intensity (p-value = 0.0013). This reaction of Tetradesmus sp. suggests sensitivity to exposure in the green and red bands. Further, the white:green and white:red spectral combinations, both with 95 μmol m−2 s−1, resulted in equal values of productivity and specific growth rate (Table 1). These cultures reached 4.06 ± 0.8 g L−1 biomass on the 12th day, statistically equal to white:blue-190, white:blue95, white-95; white-190 and white:yellow-95 (Table 3). This statistical

Table 1 Kinetic parameters of productivity (P) (g L−1 d−1) and specific growth rate (μ) (d−1) of Tetradesmus sp. in PBRs irradiated by LED lights. Experiment 1

Experiment 2

Spectrum

Intensity (μmol m

86.7:13.3%

95

Wh:Re Wh:Ye Wh:Gr Wh:Bl Wha

−2 −1

s

)

190

P

μ

P

μ

0.34 0.23 0.34 0.30 0.27

0.42 0.39 0.42 0.41 0.40

0.20 0.20 0.18 0.31 0.27

0.37 0.37 0.36 0.40 0.39

Experiment 3 −2 −1

Spectrum

Intensity (μmol m

50:50%

25

Re:Ye Re:Gr Re:Bl Rea

s

)

50

P

μ

P

μ

0.08 0.09 0.28 0.15

0.31 0.31 0.41 0.36

0.08 0.06 0.28 0.25

0.29 0.27 0.39 0.38

Spectrum

Intensity (μmol m−2 s−1)

100%

13

Re Ye Gr Bl Wha

25

P

μ

P

μ

0.09 0.04 0.04 0.27 0.07

0.08 0.04 0.04 0.17 0.06

0.15 0.06 0.06 0.35 0.15

0.12 0.05 0.06 0.20 0.11

a Control treatment composed by 100% LED. Wh:Re: composed by the visible:red LED light, Wh:Ye: composed by the visible:yellow LED light, Wh:Gr: composed by the visible:green LED light, Wh:Bl: composed by the visible:blue LED light, Re:Ye: composed by the red:yellow LED light, Re:Gr: composed by the red:green LED light, Re:Bl: composed by the red:blue LED light, Wh: visible LED light, Re: red LED light, Ye: yellow LED light, Gr: green LED light, Bl: blue LED light. Initial biomass (X0). Experiments 1 and 2: X0 = 0.03 g L−1; 12 days. Experiment 3: X0 = 0.89 g L−1; time duration of 6 days.

4

Algal Research 43 (2019) 101649

V.D. Gonçalves, et al.

Table 2 ANOVA (α = 0.05) of the spectrum combination and light intensity for the response variable final biomass of the Tetradesmus sp. in PBRs irradiated by LED lights. Factor

Experiment 1 (86.7%:13.3%)

X1 X2 X1 : X2 Error Total

Experiment 2 (50%:50%)

Experiment 3 (100%)

d.f.

SS

MS

Fcalc

p-Value

d.f.

SS

MS

Fcalc

p-Value

d.f.

SS

MS

Fcalc

p-Value

4 1 4 20 29

3.3 4.2 5.2 3.9 16.6

0.8 4.2 1.3 0.2 0.6

4.2 21.4 6.8

0.0118 0.0002 0.0013

3 1 3 16 23

25.8 0.3 1.9 0.5 28.5

8.6 0.3 0.6 0.03 1.2

280 9,1 20,4

< 0.0001 0.0081 < 0.0001

4 1 4 20 29

9.8 0.6 0.2 0.01 10.6

2.5 0.6 0.04 5.3E−4 0.4

4655 1172 84

< 0.0001 < 0.0001 < 0.0001

X1: spectral combination; X2: light intensity; d.f.: degrees of freedom; SS: Square Sum; MS: Mean Square Sum. (N = 3). Initial biomass (X0). Experiment 1 and 2: X0 = 0.03 g L−1; time duration of 12 days. Experiment 3: X0 = 0.89 g L−1; time duration of 6 days.

In Experiment 3, the highest concentration of biomass was detected with blue spectrum irradiation (25 μmol m−2 s−1), which resulted in 2.97 ± 0.03 g L−1 at the end of the 6th day of cultivation (Table 3). Ravelonandro et al. [39] cultivated the prokaryotic microorganism Spirulina sp. for 14 to 18 days with a light intensity of 64 μmol m−2 s−1 under different spectra, and found the lowest biomass and productivity with blue light (1.73 g L−1, 0.10 g L−1 d−1), followed by red (1.87 g L−1, 0.11 g L−1 d−1), visible (2.28 g L−1, 0.14 g L−1 d−1) and green (2.44 g L−1, 0.17 g L−1 d−1). In a study with visible, red, yellow, green and blue spectral bands, Chen et al. [13] also estimated the lowest concentration of biomass in Spirulina sp. cultures with blue light (< 0.1 g L−1) regardless of the intensity (750–3000 μmol m−2 s−1) and the highest biomass concentration (0.45 g L−1) in red light cultivation at maximum intensity. Khalili et al. [32] verified a higher concentration of biomass (~1.25 g L−1) with red light, when compared to blue (~1.0 g L−1), in Chlorella vulgaris cultures irradiated with 80 μmol m−2 s−1. Severes et al. [41] found increased absorbance (600 nm) of Chlorella sp. with red irradiation intensity when it was increased from 20 lx (0.29 ± 0.01), to 60 lx (0.33 ± 0.02) and 220 lx (0.46 ± 0.02). An elevation in biomass with the elevation of the luminous intensity from 50 μmol m−2 s−1 to 150 μmol m−2 s−1 was also observed in the cultivation of Nannochloropsis sp. BR2 [11], Chlorella fusca LEB 111 and Synechococcus nidulans LEB 115 [14]. Cell growth under a light source is specific to the microalgae species and culture conditions [27,32]. In this work, the cultivation of Tetradesmus sp. with visible spectrum resulted in the maximum biomass production of 3.32 g L−1 under the intensity of 95 μmol m−2 s−1 (Table 3), while Přibyl et al. [23] estimated 12 g L−1 of biomass and 2.695 ± 0.014 d−1 specific maximum growth rate when cultivating Scenedesmus sp. with 500 μmol m−2 s−1 (Photosynthetically Active Radiation) 24 h a day.

Biomass (g L -1)

(a) 5 4 3 2 Intensity 95 mol m -2 s-1 Intensity 190 mol m -2 s-1

1 0 Wh

Biomass (g L-1)

(b) 5

Wh:Re

Wh:Ye

Wh:Gr

Wh:Bl

Intensity 25 mol m -2 s-1 Intensity 50 mol m -2 s-1

4 3 2 1 0 Re

Re:Ye

Re:Gr

Re:Bl

Biomass (g L-1)

(c) 5 Intensity 13 mol m -2 s-1 Intensity 25 mol m -2 s-1

4 3 2 1 0 Wh

Re

Ye

Gr

Bl

Fig. 4. Mean ± 95% confidence interval for the final biomass concentration of Tetradesmus sp. cultivated in PBR irradiated with LED lights. (a) Experiment 1, bichromatic 86.7%:13.3% and Wh 100% as the control; (b) Experiment 2, bichromatic 50%:50% with Re 100% as the control; and (c) Experiment 3, monochromatic with Wh 100% as the control. Visible (Wh); red (Re); yellow (Ye); green (Gr); blue (Bl). Biomass (X0). Experiment 1 and 2: X0 = 0.03 g L−1; 12 days. Experiment 3: X0 = 0.89 g L−1; time duration of 6 days. Symbol: mean; whisker: mean ± least square means. (N = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.2. Total carotenoids The synthesis of total carotenoids by Tetradesmus sp. was significantly influenced by spectral combination, light intensity, and the interaction between them (Table 4). In spite of the predominance of an augmentation of carotenoid concentration by increasing light intensity, the highest intensity (190 μmol m−2 s−1) did not necessarily result in the highest total carotenoids (TC) concentrations, possibly due to the significant effect of spectral combination and photoinhibition (Fig. 5). Microalgae exposed to electromagnetic radiation, can produce carotenoids as a response to light stress [15,23]. Increased carotenoid production with elevated light intensity was noted in other studies, as well. After six days of cultivation, Duarte and Costa [14] observed a higher carotenoid production of Chlorella fusca LEB 111 and Synechococcus nidulans LEB 115 by approximately 79% by increasing the intensity from 50 μmol m−2 s−1 to 150 μmol m−2 s−1 of blue irradiation. With six days of visible LED cultivation, Grama et al. [42] discerned a higher synthesis of canthaxanthin by Acutodesmus sp. by increasing the energy from 100 μmol m−2 s−1 −1 −2 −1 (0.94 ± 0.04 mg L ) to 900 μmol m s (1.44 ± 0.03 mg L−1).

the photochemical reactions of energy production in the form of ATP and NADPH, with consequent CO2 fixation, carbohydrate synthesis and cell growth [36]. The increase in light intensity from 25 to 50 μmol m−2 s−1 stimulated microalgae growth (p-value = 0.0081) (Fig. 4b). However, when performing the analysis of variance excluding the control treatment, red-25 and red-50, an absence of interaction between the spectrum combination and intensity (p-value = 0.3083) and the intensity effect on the biomass response (p-value = 0.3271) was observed. While the red:blue light combination, regardless of the intensity, increased the biomass growth (p-value < 0.00001).

5

Algal Research 43 (2019) 101649

V.D. Gonçalves, et al.

Table 3 Mean final biomass concentration (X) (g L−1) ± standard deviation (SD) and Tukey test (α = 0.05) relative to the Experiments 1, 2 and 3 with Tetradesmus sp. in PBR supplied with LED lights. Experiment 1 (86.7%:13.3%)

Experiment 2 (50%:50%)

Spectrum

X ± SD

Wh:Gr-190 Wh:Ye-190 Wh:Re-190 Wh:Ye-95 Wh-190 Wh-95 Wh:Bl-95 Wh:Bl-190 Wh:Re-95 Wh:Gr-95

2.14 2.48 2.48 2.80 3.31 3.32 3.60 3.71 4.05 4.06

± ± ± ± ± ± ± ± ± ±

0.01c 0.01b,c 0.02b,c 0.02a,b,c 0.01a,b,c 0.01a,b,c 0.03a,b 0.04a,b 0.47a 1.31a

Experiment 3 (100%)

Spectrum

X ± SD

Re:Gr-50 Re:Ye-50 Re:Ye-25 Re:Gr-25 Re-25 Re-50 Re:Bl-25 Re:Bl-50 – –

0.76 1.02 1.03 1.08 1.87 3.02 3.39 3.42 – –

± ± ± ± ± ± ± ±

0,01c 0,01c 0,35c 0,33c 0,01b 0,01a 0,01a 0,10a

Spectrum

X ± SD

Ye-13 Gr-13 Ye-25 Gr-25 Wh-13 Re-13 Wh-25 Re-25 Bl-13 Bl-25

1.14 1.15 1.22 1.28 1.31 1.44 1.77 1.79 2.54 2.97

± ± ± ± ± ± ± ± ± ±

0.02g 0.01g 0.01f 0.03e,f 0.02e 0.01d 0.03c 0.03c 0.01b 0.03a

Wh:Gr-190: composed by the visible:green LED light with 190 μmol m−2 s−1 light intensity, etc. Initial biomass (X0). (N = 3). Experiments 1 and 2: X0 = 0.03 g L−1; 12 days. Experiment 3: X0 = 0.89 g L−1; time duration of 6 days; means followed by the same letters do not differ statistically (Tukey-test, α = 0,05).

Similarly, an increase from 100 μmol m−2 s−1 to 1000 μmol m−2 s−1 of visible light promoted higher production of β-carotene by D. salina [15]. In the case of Arthrospira platensis cultivated with light energy promoted by fluorescent lamps, a decrease of carotenoids (2.90 ± 0.01 mg g−1 to 2.65 ± 0.12 mg g−1) was observed, with an escalation in the mean intensity from 159 to 343 μmol m−2 s−1 [12]. Chlamydomonas reinhardtii reduced the synthesis of total carotenoids when the intensity increased from 25 μmol m−2 s−1 (17.05 mg g−1) to 150 μmol m−2 s−1 (8.91 mg g−1) and to 200 μmol m−2 s−1 (5.77 mg g−1) of visible light [17]. Acutodesmus sp. also exhibited a lutein concentration decrease by increasing light intensity [42]. Nannochloropsis sp. showed carotenoid productivity and maximum yields about two times higher in low light intensity (50 μmol m−2 s−1) when compared to those obtained at high intensity (150 μmol m−2 s−1), using a photoperiod of 16:8 h (light:dark) [11]. In Experiment 1, the highest TC concentration (3.31 ± 0.26 mg g−1) determined in the white:green-190 condition was the same as those measured under white:yellow-190 (2.74 ± 0.37 mg g−1) and white:red-190 (2.65 ± 0.19 mg g−1) (Table 5). The cultures carried out with 13 μmol m−2 s−1 (Experiment 3) presented the same production of carotenoids, regardless of the LED light combination. This low energy supply was sufficient to promote biomass growth (Fig. 4c), but was not sufficient to increase carotenoid biosynthesis (Fig. 5c). Table 5 indicates that the highest carotenoid synthesis (3.47 ± 0.30 mg g−1), observed with red-25, was statistically equal to that obtained with blue-25 (2.94 ± 0.08 mg g−1). The blue color, which comprises the wavelength range from 440 to 485 nm, corresponding to the absorption of chlorophylls and carotenoids, such as lutein and β-carotene [40], may have stimulated carotenoid synthesis. Wagner et al. [17] noted the highest concentration of TC under green (19.87 mg g−1), followed by blue (18.95 mg g−1), visible

(17.05 mg g−1) and red (15.02 mg g−1) light, in Chlamydomonas reinhardtii cultures with 25 μmol m−2 s−1. With the red:green combination at the light intensity of 50 μmol m−2 s−1, Tetradesmus sp. synthesized 6.09 ± 0.29 mg g−1 of carotenoids, the highest production recorded among Experiments 1, 2 and 3. In contrast, a Gracilaria tikvahiae culture was investigated in the following light sources (100 μmol m−2 s−1): fluorescent; red; green; blue; red:green (50:50%); red:blue (50:50%); green:blue (50,50%); and red:green:blue in proportions of 40:40:20%; 40:20:40%; and 20:40:40%, resulting in a maximum production of carotenoids (~0.25 mg g−1) and chlorophyll (~1.75 mg g−1), and a minimum specific growth rate (~6.2% d−1) with 50% red light and 50% blue light [24]. The lowest carotenoid production (1.07 ± 0.12 mg g−1) measured in Experiment 2 was obtained with the spectral combination red:blue25. The response to photostimulation is specific to the microalgal metabolism, Wagner et al. [17] estimated 19.52 mg g−1 of carotenoids in a culture of Chlamydomonas reinhardtii (25 μmol m−2 s−1) under red:blue light in a 50:50% ratio. With the visible spectrum, the highest carotenoid value (2.77 ± 0.18 mg g−1) was detected at a light intensity of 25 μmol m−2 s−1 in Experiment 3 (Table 5). Chlorella zofingiensis cultivated in a PBR with 245 μmol m−2 s−1 of visible light and under conditions of nitrogen depletion produced from 0.8 up to 4.5 mg g−1 of total carotenoids [43]. Chlorella vulgaris and Scenedesmus obliquus synthesized 2.0 mg g−1 and 2.7 mg g−1 TC, respectively, when cultivated with 150 μmol m−2 s−1 provided by fluorescent lamps [28]. A Scenedesmus sp. strain cultivated in visible light with high intensity, 500 μmol m−2 s−1, showed a production of 19.01 ± 1.45 mg L−1 d−1 of total carotenoids or 2.08 ± 0.03% in dry weight after 14 days of cultivation [23]. While Acutodesmus sp. cultivated under different light intensities, 100–900 μmol m−2 s−1 (PAR), presented concentrations of the carotenoid canthaxanthin varying between 0.65 and 0.85 mg g−1 of

Table 4 ANOVA (α = 0.05) of the spectrum combination and light intensity for the response variable total carotenoids of the Tetradesmus sp. cultivation in PBR supplied with LED lights. Factor

X1 X2 X1 : X2 Error Total

Experiment 1 (86.7%:13.3%)

Experiment 2 (50%:50%)

Experiment 3 (100%)

d.f.

SS

MS

Fcalc

p-Value

d.f.

SS

MS

Fcalc

p-Value

d.f.

SS

MS

Fcalc

p-Value

4 1 4 20 29

5.4 9.1 4.3 1.2 20

1.3 9.1 1.1 0.06 0.7

23.1 156.5 18.7

< 0.0001 < 0.0001 < 0.0001

3 1 3 16 23

35.9 6.2 5.0 2.7 49.8

12.0 6.2 1.7 0.17 2.2

70.8 36.5 9.9

< 0.0001 < 0.0001 0.0006

4 1 4 20 29

1.7 19.4 2.6 1.1 24.8

0.4 19.4 0.6 0.06 0.9

7.6 340.3 11.2

0.0007 < 0.0001 < 0.0001

X1: spectral combination; X2: light intensity; d.f.: degrees of freedom; SS: square sum; MS: mean square sum. (N = 3). Initial biomass (X0). Experiment 1 and 2: X0 = 0.03 g L−1; 12 days. Experiment 3: X0 = 0.89 g L−1; time duration of 6 days. 6

Algal Research 43 (2019) 101649

V.D. Gonçalves, et al.

mg TC (g biomass)-1

(a)

7 6 5 4 3 2 1 0

dry biomass [42]. Intensity 95 mol m-2 s -1 Intensity 190 mol m-2 s -1

Wh

mg TC (g biomass)-1

(b)

7 6 5 4 3 2 1 0

Wh:Re

mg TC (g biomass)-1

7 6 5 4 3 2 1 0

Wh:Gr

The spectral combination, light intensity and their interaction promoted significant effects on chlorophyll a and chlorophyll b concentration in Experiments 1, 2 and 3 (Table 6). The upsurge in light intensity stimulated an increase in chlorophyll a and b concentrations in Experiments 1 and 2; and a decrease in Experiment 3 (Fig. 6). The low light intensity (13 μmol m−2 s−1) may have stimulated microalgae to multiply the number of energy capture centers (chlorophylls) to meet their metabolic demand. A decrease of chlorophyll production due to the elevation in light intensity from 150 μmol m−2 s−1 to 650 μmol m−2 s−1 was observed in the cultivation of Dunaliella salina [15]. Acutodesmus sp. also presented a decrease in the chlorophyll a (Chla) and chlorophyll b (Chlb) concentration with an increase in light intensity from 100 μmol m−2 s−1 to 900 μmol m−2 s−1 [42]. Chlamydomonas reinhardtii, cultivated in visible light, revealed a decrease in chlorophyll concentration by raising the intensity from 25 μmol m−2 s−1 (53.02 mgChla g−1; 18.02 mgChlb g−1) to −2 −1 −1 150 μmol m s (14.75 mgChla g , 7.0 mgChlb g−1), and a slight increase in 200 μmol m−2 s−1 [17]. Spirulina sp. increased chlorophylls a and b concentration from 10.2 mg g−1 to 13.2 mg g−1 by changing the intensity from 175 to 343 μmol m−2 s−1 [12]. Chlorella fusca LEB 111 and Synechococcus nidulans LEB 115 also increased chlorophyll synthesis by boosting the light irradiation from 50 μmol m−2 s−1 to 150 μmol m−2 s−1 of blue irradiation [14]. Table 7 shows that the highest production of Chla (10.08 ± 0.75 g L−1) and Chlb (2.04 ± 0.66 g L−1), which was obtained with the combination red:green with 50 μmol m−2 s−1 illumination (Experiment 2), the same condition that resulted in higher TC concentration (Table 5). In Experiment 1, the highest values of chlorophyll a and b were recorded with white:green-190 (5.16 ± 0.42 mg Chla g−1; 0.98 ± 0.03 mg Chlb g−1) and white-190 (4.34 ± 0.41 mg Chla g−1; 0.89 ± 0.05 mg Chlb g−1). In Experiment 3, the highest concentrations of Chla were obtained under the following conditions: white-25 (2.89 ± 0.14 mg Chla g−1) and red-25 (2.35 ± 0.52 mg Chla g−1), which also provided the highest value of Chlb (1.35 ± 0.12 mg Chlb g−1). However, this value was statistically equal to 5 other essays. The chlorophyll synthesis is dependent on the microalgal species, for example Spirulina sp. (prokaryotic) cultivated in different light sources, presented a higher concentration of chlorophylls, 8 mg L−1, when cultivated in red light with an intensity of 3000 μmol m−2 s−1 [13]. In this case, red light also promoted the highest total chlorophyll concentration result, 14.86 mg L−1 in the red-50, referring to Experiment 2. Cultivation of the microalgae Chlamydomonas reinhardtii at the

Wh:Bl

Intensity 25 mol m-2 s -1 Intensity 50 mol m-2 s -1

Re

(c)

Wh:Ye

3.3. Chlorophylls

Re:Ye

Re:Gr

Re:Bl

Intensity 13 mol m-2 s -1 Intensity 25 mol m-2 s -1

Wh

Re

Ye

Gr

Bl

Fig. 5. Mean ± 95% confidence interval for the final total carotenoids of Tetradesmus sp. cultivated in photobioreactor irradiated with LED lights. (a) Experiment 1, bichromatic 86.7%:13.3% and Wh 100% as the control; (b) Experiment 2, bichromatic 50%:50% with Re 100% as the control; and (c) Experiment 3, monochromatic with Wh 100% as the control. Visible (Wh); red (Re); yellow (Ye); green (Gr); blue (Bl). Biomass (X0). Experiment 1 and 2: X0 = 0.03 g L−1; 12 days. Experiment 3: X0 = 0.89 g L−1; time duration of 6 days. Symbol: mean; whisker: mean ± least square means. (N = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 5 Final values of total carotenoids (TC) (mg TC (g biomass−1)), standard deviation (SD) and Tukey test (α = 0.05) relative to the Experiments 1, 2 and 3 with Tetradesmus sp. in PBRs irradiated by LED lights. Experiment 1 (86.7%:13.3%) Spectrum Wh:Bl-95 Wh:Re-95 Wh:Gr-95 Wh:Bl-190 Wh:Ye-95 Wh-190 Wh-95 Wh:Re-190 Wh:Ye-190 Wh:Gr-190

Experiment 2 (50%:50%) TC ± SD 0.80 0.80 1.35 1.68 1.80 2.18 2.31 2.65 2.74 3.31

± ± ± ± ± ± ± ± ± ±

Spectrum e

0.06 0.10e 0.11d,e 0.28c,d 0.07c,d 0.33b,c 0.35b,c 0.19a,b 0.37a,b 0.26a

Experiment 3 (100%) TC ± SD

Re:Bl-025 Re-25 Re:Bl-50 Re-50 Re:Ye-50 Re:Ye-25 Re:Gr-25 Re:Gr-50 – –

1.07 2.01 2.07 2.66 3.14 3.19 3.63 6.09 – –

± ± ± ± ± ± ± ±

e

0.12 0.43d,e 0.21c,d,e 0.50b,c,d 0.83b,c,d 0.13b,c 0.27b 0.29a

Spectrum

TC ± SD

Re-13 Ye-13 Wh-13 Bl-13 Gr-13 Ye-25 Gr-25 Wh-25 Bl-25 Re-25

0.88 1.09 1.20 1.24 1.33 1.88 2.72 2.77 2.94 3.47

± ± ± ± ± ± ± ± ± ±

0.38d 0.24d 0.13c,d 0.18c,d 0.19c,d 0.32c 0.20b 0.18b 0.08a,b 0.30a

Wh:Gr-190: composed by the visible:green LED light with 190 μmol m−2 s−1 light intensity, etc. Initial biomass (X0). (N = 3). Experiments 1 and 2: X0 = 0.03 g L−1; 12 days. Experiment 3: X0 = 0.89 g L−1; time duration of 6 days; means followed by the same letters do not differ statistically (Tukey-test, α = 0,05). 7

Algal Research 43 (2019) 101649

V.D. Gonçalves, et al.

Table 6 ANOVA (α = 0.05) of the spectrum combination and light intensity for the response variable chlorophylls of the Tetradesmus sp. in PBRs irradiated by LED lights. Factor

Experiment 1 (86.7%:13.3%)

Experiment 2 (50%:50%)

Experiment 3 (100%)

SS

MS

Fcalc

p-Value

d.f.

SS

MS

Fcalc

p-Value

d.f.

SS

MS

Fcalc

p-Value

Chlorophyll a 4 X1 X2 1 X1 : X2 4 Error 20

14.5 22.5 19.6 3.5

3.6 22.5 4.9 0.17

20.8 129.2 28.1

< 0.0001 < 0.0001 < 0.0001

3 1 3 16

116.7 16.8 30.6 8.8

38.9 16.8 10.2 0.6

71.2 30.8 18.7

< 0.0001 < 0.0001 < 0.0001

4 1 4 20

4.8 2.5 6.6 2.0

1.2 2.5 1.7 0.10

12.2 25.8 16.8

< 0.0001 < 0.0001 < 0.0001

Chlorophyll b X1 4 X2 1 X1 : X2 4 Error 20

0.2 1.0 0.9 0.1

0.05 1.0 0.2 0.01

8.0 159.3 33.1

0.0005 < 0.0001 < 0.0001

3 1 3 16

3.7 2.0 1.8 0.9

21.1 34.8 10.4

< 0.0001 < 0.0001 0.0005

4 1 4 20

2.0 0.3 0.3 0.5

0.5 0.3 0.1 0.03

20.0 12.5 3.4

< 0.0001 0.0021 0.0298

d.f.

1.2 2.0 0.6 0.06

X1: spectral combination; X2: light intensity; d.f.: degrees of freedom; SS: Square Sum; MS: Mean Square Sum. (N = 3). Initial biomass (X0). Experiment 1 and 2: X0 = 0.03 g L−1; time duration of 12 days. Experiment 3: X0 = 0.89 g L−1; time duration of 6 days.

(b)

12 10 8

Intensity 95 mol m -2 s -1 Intensity 190 mol m -2 s -1

mg Chlb (g biomass)-1

mg Chla (g biomass)-1

(a)

6 4 2 0 Wh:Ye

Wh:Gr

(d) Intensity 25 mol m -2 s -1 Intensity 50 mol m -2 s -1

10 8 6 4 2

Wh:Re

Wh:Ye

Wh:Gr

Wh:Bl

2.5 2.0

Intensity 25 mol m -2 s -1 Intensity 50 mol m -2 s -1

1.5 1.0 0.5

Re:Ye

Re:Gr

Re:Bl

Re

12 Intensity 13 mol m -2 s -1 Intensity 25 mol m -2 s -1

6 4 2 0

(f)

2.5

mg Chlb (g biomass)-1

mg Chla (g biomass)-1

0.5

0.0 Re

8

1.0

Wh

12

10

1.5

Wh:Bl

mg Chlb (g biomass)-1

mg Chla (g biomass)-1

Wh:Re

0

(e)

Intensity 95 mol m -2 s -1 Intensity 190 mol m -2 s -1

2.0

0.0 Wh

(c)

2.5

2.0

Re:Ye

Re:Gr

Re:Bl

Intensity 13 mol m -2 s -1 Intensity 25 mol m -2 s -1

1.5 1.0 0.5 0.0

Wh

Re

Ye

Gr

Bl

Wh

Re

Ye

Gr

Bl

Fig. 6. Mean ± 95% confidence interval for the final total chlorophylls of Tetradesmus sp. cultivated in PBR irradiated with LED lights. Experiment 1, bichromatic 86.7%:13.3% and Wh 100% as the control: (a) chlorophyll a; (b) chlorophyll b. Experiment 2, bichromatic 50%:50% with Re 100% as the control: (c) chlorophyll a (d) chlorophyll b; and Experiment 3, monochromatic with Wh 100% as the control: Experiment 3: (e) chlorophyll a (f) chlorophyll b. Visible (Wh); red (Re); yellow (Ye); green (Gr); Blue (Bl). Biomass (X0). Experiment 1 and 2: X0 = 0.03 g L−1; 12 days. Experiment 3: X0 = 0.89 g L−1; time duration of 6 days. Symbol: mean; whisker: mean ± least square means. (N = 3).

light intensity of 25 μmol m−2 s−1, promoted high Chla and Chlb concentration in the red-light reactors (40.82 mg Chla g−1, −1 10.03 mg Chlb g ) and red:blue in the ratio of 50:50% (59.57 mg Chla g−1; 20.18 mg Chlb g−1) [17].

(mg g−1) synthesis was higher in the first week of cultivation, period of higher radiation incidence per cell, and reduced as the biomass increased in the PBR (Fig. S1). The combinations red:green and white:green boosted the production of TC and chlorophylls, especially on the sixth day of cultivation. On the ninth day, the combination red:green resulted in higher chlorophyll accumulation compared to the other spectral arrangements (Table 8). However, the evaluation of the results of carotenoids and chlorophylls in μg mL−1 suggested the predominance of a higher pigment concentration with the time of cultivation (12 d). Duarte and Costa [14] also discovered a similar tendency of increase in these pigments (μg mL−1) in Chlorella fusca LEB 111 and

3.4. Tetradesmus sp. cultivation with the white:green, red:green and red combinations When cultivating Tetradesmus sp. for 12 days on PBRs with red; white:green and red:green at the light intensity of 50 μmol m−2 s−1, it was possible to verify that the total carotenoids and chlorophylls 8

Algal Research 43 (2019) 101649

V.D. Gonçalves, et al.

Table 7 Final values of Chla and Chlb (mg Chl (g biomass)−1), standard deviation (SD) and Tukey test (α = 0.05) relative to the Experiments 1, 2 and 3 with Tetradesmus sp. in PBRs irradiated by LED lights. Experiment 1 (86.7%:13.3%) Spectrum

Chla ± SD1

Wh:Re-95 Wh:Bl-95 Wh:Gr-95 Wh:Ye-95 Wh-190 Wh:Bl-190 Wh-95 Wh:Ye-190 Wh:Re-190 Wh:Gr-190

0.68 0.90 1.75 2.16 2.64 2.87 3.54 3.63 4.34 5.16

± ± ± ± ± ± ± ± ± ±

Experiment 2 (50%:50%) Chlb ± SD

0.06e 0.08e 0.11d,e 0.65d 0.15c,d 0.68c,d 0.60b,c 0.33b,c 0.41a,b 0.42a

0.13 0.26 0.26 0.48 0.54 0.55 0.70 0.72 0.89 0.98

± ± ± ± ± ± ± ± ± ±

0.01f 0.03e,f 0.02e,f 0.06d,e 0.13c,d 0.15c,d 0.12b,c,d 0.03b,c 0.05a,b 0.03a

Experiment 3 (100%)

Spectrum

Chla ± SD

Chlb ± SD

Re:Bl-25 Re:Bl-50 Re-25 Re:Ye-50 Re-50 Re:Ye-25 Re:Gr-25 Re:Gr-50 – –

0.74 ± 0.06d 1.84 ± 0.76c,d 2.90 ± 0.56b,c 3.24 ± 1.07b,c 4.17 ± 1.22b 4.20 ± 0.11b 4.80 ± 0.53b 10.08 ± 0.75a – –

0.11 0.39 0.30 0.53 0.74 0.44 0.54 2.04 – –

± ± ± ± ± ± ± ±

0.01b 0.13b 0.05b 0.03b 0.07b 0.03b 0.05b 0.66a

Spectrum

Chla ± SD

Bl-25 Ye-25 Re-25 Gr-25 Ye-13 Wh-13 Bl-13 Gr-13 Re-13 Wh-25

0.55 0.83 1.01 1.10 1.54 1.66 1.85 1.89 2.35 2.89

± ± ± ± ± ± ± ± ± ±

0.06e 0.25d,e 0.22c,d,e 0.15c,d,e 0.51b,c,d 0.05b,c,d 0.45b,c 0.28b,c 0.52a,b 0.14a

Chlb ± SD 0.89 0.25 1.35 0.71 0.78 0.90 0.94 0.93 1.27 0.58

± ± ± ± ± ± ± ± ± ±

0.07a,b 0.04c 0.12a 0.13b,c 0.26b 0.07a,b 0.23a,b 0.14a,b 0.26a 0.04b,c

Wh:Gr-190: composed by the visible:green LED light with 190 μmol m−2 s−1 light intensity, etc. Initial biomass (X0). (N = 3). Experiments 1 and 2: X0 = 0.03 g L−1; 12 days. Experiment 3: X0 = 0.89 g L−1; time duration of 6 days; means followed by the same letters do not differ statistically (Tukey-test, α = 0.05). Table 8 Final values of Chla, Chlb and total carotenoids (TC) (mg pigment (g biomass)−1), standard deviation (SD) and Tukey test (α = 0.05) regarding the Tetradesmus sp. cultivation in spectral combinations of higher carotenoid production in the previous experiments. Spectrum

6 day

9 day

Chla WhGr50 (86,7:13,3%) ReGr50 (50:50%) Re50 (100%)

Chlb

TC

12 day

Chla

Chlb

a

5.1 ± 0.9ª

2.0 ± 0.2ª

2.0 ± 0.2

8.2 ± 1.2ª

6.2 ± 1.1ª

2.2 ± 0.04ª

2.7 ± 0.1ª

1.9 ± 0.4b

1.2 ± 0.3b

1.1 ± 0.1b

1.8 ± 0.05b

7.0 ± 1.2

b

TC

Chla

Chlb

TC

1.0 ± 0.2ª

3.3 ± 0.3ª

3.3 ± 0.4ª

0.8 ± 0.02ª

1.8 ± 0.01ª

1.2 ± 0.1ª

3.3 ± 0.1ª

3.1 ± 0.2ª

0.8 ± 0.03ª

1.2 ± 0.03c

0.7 ± 0.03b

2.3 ± 0.3b

2.1 ± 0.3b

0.6 ± 0.05b

1.6 ± 0.004

b

Wh:Gr: composed by the visible:green LED light, etc. Experiments 4: Initial biomass, X0 = 0.03 g L−1; 12 days. Light intensity: 50 μmol m−2 s−1. Wh - visible; Gr green; Re - red; means followed by the same letters do not differ statistically (Tukey-test, α = 0,05). (WhGr50: N = 2; ReGr50 and Re50: N = 3).

Synechococcus nidulans LEB 115, when cultivated for 18 days with light energy in the blue spectral region. The highest carotenoid production (2.22 mg g−1) observed with the combination red:green was statistically the same as that measured with the white:green combination (2.01 mg g−1). The red LED stimulated the cell growth of the microalgae. It must be noted that the combination with the green LED (red:green) most likely triggered/unlocked the pigment biosynthesis.

Authors' contributions

4. Conclusions

Statement of informed consent, human/animal rights

Vanessa Daneluz Gonçalves planned and conducted the experiments, analyzed the data and drafted the article. Márcia Regina Fagundes Klen and Daniela Estelita Goes Trigueros analyzed the data and revised the article. Aparecido Nivaldo Módenes and Alexander Dimitrov Kroumov revised the article and approved the final version of the article. Adilson Ricken Schuelter supplied study material and revised the article.

No conflicts, informed consent, human or animal rights applicable.

The color LED combination and intensity significantly affected the biomass, carotenoids and chlorophyll a and b production of the microalga Tetradesmus sp., which presented high growth rate when cultivated under the highest energy blue light range. Surprisingly, the highest concentration of biomass (4.06 ± 1.31 g L−1) at the end of the cultivation was observed with the combination of visible light with an increase in green light at the light intensity of 95 μmol m−2 s−1. In terms of biopigments, the red:green light combination, in intensity of 50 μmol m−2 s−1, promoted the highest concentrations of total carotenoids and chlorophylls (6.09 ± 0.29 mgTC (g biomass−1); 10.08 ± 0.75 mg Chla (g biomass−1); 2.04 ± 0.66 mg Chlb (g biomass−1)).

References [1] A. Ben-Amotz, New mode of Dunaliella biotechnology - 2-phase growth for betacarotene production, J. Appl. Phycol. 7 (1995) 65–68, https://doi.org/10.1007/ bf00003552. [2] D. Gangl, J.A.Z. Zedler, P.D. Rajakumar, E.M.R. Martinez, A. Riseley, A. Włodarczyk, S. Purton, Y. Sakuragi, C.J. Howe, P.E. Jensen, C. Robinson, Biotechnological exploitation of microalgae, J. Exp. Bot. 66 (2015) 6975–6990, https://doi.org/10.1093/jxb/erv426. [3] T. Suganya, M. Varman, H.H. Masjuki, S. Renganathan, Macroalgae and microalgae as a potential source for commercial applications along with biofuels production: a biorefinery approach, Renew. Sust. Energ. Rev. 55 (2016) 909–941, https://doi. org/10.1016/j.rser.2015.11.026. [4] R.A. Andersen, The microalgal cell, Handbook of Microalgal Culture: Applied Phycology and Biotechnology, 1st ed., 2013. [5] B. dos S.A.F. Brasil, L.C. Garcia, Microalgas: alternativas promissoras para a indústria, Agroenergia em revista: microalgas, Agroenergia e Rev, 10 2016, pp. 6–11. [6] M. Janssen, P. Slenders, J. Tramper, L.R. Mur, R.H. Wijffels, Photosynthetic efficiency of Dunaliella tertiolecta under short light/dark cycles, Enzym. Microb. Technol. 29 (2001) 298–305, https://doi.org/10.1016/S0141-0229(01)00387-8. [7] G. Markou, E. Nerantzis, Microalgae for high value compounds and biofuels production: a review with focus on cultivation under stress conditions, Biotechnol.

Acknowledgements We would like to thank the National Council for Scientific and Technological Development (CNPq) for the financial support (Project CNPq 400771/2014-4).

9

Algal Research 43 (2019) 101649

V.D. Gonçalves, et al.

Adv. 31 (2013) 1532–1542, https://doi.org/10.1016/j.biotechadv.2013.07.011. [8] M.A. Gomaa, L. Al-Haj, R.M.M. Abed, Metabolic engineering of cyanobacteria and microalgae for enhanced production of biofuels and high-value products, J. Appl. Microbiol. 121 (2016) 919–931, https://doi.org/10.1111/jam.13232. [9] F.B. Scheufele, C.L. Hinterholz, M.M. Zaharieva, H.M. Najdenski, A.N. Módenes, D.E.G. Trigueros, C.E. Borba, F.R. Espinoza-Quiñones, A.D. Kroumov, Complex mathematical analysis of photobioreactor system, Eng. Life Sci. (2018) 1–14, https://doi.org/10.1002/elsc.201800044. [10] S. Baer, M. Heining, P. Schwerna, R. Buchholz, H. Hübner, Optimization of spectral light quality for growth and product formation in different microalgae using a continuous photobioreactor, Algal Res. 14 (2016) 109–115, https://doi.org/10. 1016/j.algal.2016.01.011. [11] R. Ma, S.R. Thomas-Hall, E.T. Chua, E. Eltanahy, M.E. Netzel, G. Netzel, Y. Lu, P.M. Schenk, LED power efficiency of biomass, fatty acid, and carotenoid production in Nannochloropsis microalgae, Bioresour. Technol. 252 (2018) 118–126, https://doi.org/10.1016/j.biortech.2017.12.096. [12] K.V. Ajayan, M. Selvaraju, K. Thirugnanamoorthy, Enrichment of chlorophyll and phycobiliproteins in Spirulina platensis by the use of reflector light and nitrogen sources: an in-vitro study, Biomass Bioenergy 47 (2012) 436–441, https://doi.org/ 10.1016/j.biombioe.2012.09.012. [13] H.B. Chen, J.Y. Wu, C.F. Wang, C.C. Fu, C.J. Shieh, C.I. Chen, C.Y. Wang, Y.C. Liu, Modeling on chlorophyll a and phycocyanin production by Spirulina platensis under various light-emitting diodes, Biochem. Eng. J. 53 (2010) 52–56, https://doi. org/10.1016/j.bej.2010.09.004. [14] J.H. Duarte, J.A.V. Costa, Blue light emitting diodes (LEDs) as an energy source in Chlorella fusca and Synechococcus nidulans cultures, Bioresour. Technol. 247 (2018) 1242–1245, https://doi.org/10.1016/j.biortech.2017.09.143. [15] P.P. Lamers, C.C.W. Van De Laak, P.S. Kaasenbrood, J. Lorier, M. Janssen, R.C.H. De Vos, R.J. Bino, R.H. Wijffels, Carotenoid and fatty acid metabolism in light-stressed Dunaliella salina, Biotechnol. Bioeng. 106 (2010) 638–648, https://doi.org/10. 1002/bit.22725. [16] L. Ramanna, I. Rawat, F. Bux, Light enhancement strategies improve microalgal biomass productivity, Renew. Sust. Energ. Rev. 80 (2017) 765–773, https://doi. org/10.1016/j.rser.2017.05.202. [17] I. Wagner, C. Steinweg, C. Posten, Mono- and dichromatic LED illumination leads to enhanced growth and energy conversion for high-efficiency cultivation of microalgae for application in space, Biotechnol. J. 11 (2016) 1060–1071, https://doi.org/ 10.1002/biot.201500357. [18] C.L. Hinterholz, A.R. Schuelter, A.N. Módenes, D.E.G. Trigueros, C.E. Borba, A. Espinoza-Quiñones, F.R. Kroumov, Microalgae flat plate-photobioreactor (FPPBR) system development: computational tools to improve experimental results, Acta Microbiol. Bulg. 33 (2017) 119–124. [19] A.D. Kroumov, A.N. Módenes, D.E.G. Trigueros, F.R. Espinoza-Quiñones, C.E. Borba, F.B. Scheufele, C.L. Hinterholz, A systems approach for CO2 fixation from flue gas by microalgae—theory review, Process Biochem. 51 (2016) 1817–1832, https://doi.org/10.1016/j.procbio.2016.05.019. [20] A.P. Abreu, B. Fernandes, A.A. Vicente, J. Teixeira, G. Dragone, Mixotrophic cultivation of Chlorella vulgaris using industrial dairy waste as organic carbon source, Bioresour. Technol. 118 (2012) 61–66, https://doi.org/10.1016/j.biortech.2012. 05.055. [21] L. Bouarab, A. Dauta, M. Loudiki, Heterotrophic and mixotrophic growth of Micractinium pusillum Fresenius in the presence of acetate and glucose:effect of light and acetate gradient concentration, Water Res. 38 (2004) 2706–2712, https:// doi.org/10.1016/j.watres.2004.03.021. [22] R.T. Smith, K. Bangert, S.J. Wilkinson, D.J. Gilmour, Synergistic carbon metabolism in a fast growing mixotrophic freshwater microalgal species Micractinium inermum, Biomass Bioenergy 82 (2015) 73–86, https://doi.org/10.1016/j.biombioe. 2015.04.023. [23] P. Přibyl, V. Cepák, P. Kaštánek, V. Zachleder, Elevated production of carotenoids by a new isolate of Scenedesmus sp, Algal Res. 11 (2015) 22–27, https://doi.org/ 10.1016/j.algal.2015.05.020. [24] J.K. Kim, Y. Mao, G. Kraemer, C. Yarish, Growth and pigment content of Gracilaria tikvahiae McLachlan under fluorescent and LED lighting, Aquaculture 436 (2015) 52–57, https://doi.org/10.1016/j.aquaculture.2014.10.037. [25] P.S.C. Schulze, L.A. Barreira, H.G.C. Pereira, J.A. Perales, J.C.S. Varela, Light emitting diodes (LEDs) applied to microalgal production, Trends Biotechnol. 32 (2014) 422–430, https://doi.org/10.1016/j.tibtech.2014.06.001. [26] Y.K. Choi, R.S. Kumaran, H.J. Jeon, H.J. Song, Y.H. Yang, S.H. Lee, K.G. Song,

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36] [37]

[38]

[39]

[40]

[41]

[42]

[43]

10

K.J. Kim, V. Singh, H.J. Kim, LED light stress induced biomass and fatty acid production in microalgal biosystem, Acutodesmus obliquus, Spectrochim. Acta - Part A Mol. Biomol. Spectrosc. 145 (2015) 245–253, https://doi.org/10.1016/j.saa.2015. 03.035. M. Glemser, M. Heining, J. Schmidt, A. Becker, D. Garbe, R. Buchholz, T. Brück, Application of light-emitting diodes (LEDs) in cultivation of phototrophic microalgae: current state and perspectives, Appl. Microbiol. Biotechnol. 100 (2016) 1077–1088, https://doi.org/10.1007/s00253-015-7144-6. L.D. Patias, A.S. Fernandes, F.C. Petry, A.Z. Mercadante, E. Jacob-lopes, L.Q. Zepka, Carotenoid pro fi le of three microalgae/cyanobacteria species with peroxyl radical scavenger capacity, Food Res. Int. 100 (2017) 260–266, https://doi.org/10.1016/j. foodres.2017.06.069. M.J. Wynne, J.K. Hallan, Reinstatement of Tetradesmus G. M. Smith (Sphaeropleales, Chlorophyta), Feddes Repert 126 (2015) 83–86, https://doi.org/ 10.1002/fedr.201500021. C. Escapa, R.N. Coimbra, S. Paniagua, A.I. García, M. Otero, Comparison of the culture and harvesting of Chlorella vulgaris and Tetradesmus obliquus for the removal of pharmaceuticals from water, J. Appl. Phycol. 29 (2017) 1179–1193, https://doi.org/10.1007/s10811-016-1010-5. A.R. Schuelter, A.D. Kroumov, C.L. Hinterholz, A. Fiorini, D.E.G. Trigueros, E.G. Vendruscolo, M.M. Zaharieva, A.N. Módenes, Isolation and identification of new microalgae strains with antibacterial activity on food-borne pathogens. Engineering approach to optimize synthesis of desired metabolites, Biochem. Eng. J. 144 (2019) 28–39, https://doi.org/10.1016/J.BEJ.2019.01.007. A. Khalili, G.D. Najafpour, G. Amini, F. Samkhaniyani, Influence of nutrients and LED light intensities on biomass production of microalgae Chlorella vulgaris, Biotechnol. Bioprocess Eng. 20 (2015) 284–290, https://doi.org/10.1007/s12257013-0845-8. T.M. Setyoningrum, M.M.A. Nur, Optimization of C-phycocyanin production from S. platensis cultivated on mixotrophic condition by using response surface methodology, Biocatal. Agric. Biotechnol. 4 (2015) 603–607, https://doi.org/10.1016/j. bcab.2015.09.008. G.M. León-Saiki, N. Ferrer Ledo, D. Lao-Martil, D. van der Veen, R.H. Wijffels, D.E. Martens, Metabolic modelling and energy parameter estimation of Tetradesmus obliquus, Algal Res. 35 (2018) 378–387, https://doi.org/10.1016/j. algal.2018.09.008. H.K. Lichtenthaler, C. Buschmann, Chlorophylls and carotenoids: measurement and characterization by UV–VIS spectroscopy, Handb. Food Anal. Chem. 2 (2) (2001) 171–178, https://doi.org/10.1002/0471709085.ch21. J. Masojídek, G. Torzillo, M. Koblíıžek, Photosynthesis in microalgae, Handbook of Microalgal Culture, 2004, https://doi.org/10.1002/9780470995280. T. You, S.M. Barnett, Effect of light quality on production of extracellular polysaccharides and growth rate of Porphyridium cruentum, Biochem. Eng. J. 19 (2004) 251–258, https://doi.org/10.1016/j.bej.2004.02.004. M. Atta, A. Idris, A. Bukhari, S. Wahidin, Intensity of blue LED light: a potential stimulus for biomass and lipid content in fresh water microalgae Chlorella vulgaris, Bioresour. Technol. 148 (2013) 373–378, https://doi.org/10.1016/j.biortech.2013. 08.162. P.H. Ravelonandro, D.H. Ratianarivo, C. Joannis-Cassan, A. Isambert, M. Raherimandimby, Influence of light quality and intensity in the cultivation of Spirulina platensis from Toliara (Madagascar) in a closed system, J. Chem. Technol. Biotechnol. 83 (2008) 842–848, https://doi.org/10.1002/jctb.1878. D.L. Nelson, M.M. Cox, Princípios de Bioquímica de Lehninger, https://www. amazon.com.br/Princípios-Bioquímica-Lehninger-David-Nelson/dp/8582715331? tag=goog0ef-20&smid=A1ZZFT5FULY4LN&ascsubtag=go_729680143_ 34002717090_172477348789_pla-590479558470_c_, (2014) , Accessed date: 8 February 2019. A. Severes, S. Hegde, L. D'Souza, S. Hegde, Use of light emitting diodes (LEDs) for enhanced lipid production in micro-algae based biofuels, 170 (2017) 235–240, https://doi.org/10.1016/j.jphotobiol.2017.04.023. B.S. Grama, S. Chader, D. Khelifi, B. Stenuit, C. Jeffryes, S.N. Agathos, Characterization of fatty acid and carotenoid production in an Acutodesmus microalga isolated from the Algerian Sahara, Biomass Bioenergy 69 (2014) 265–275, https://doi.org/10.1016/j.biombioe.2014.07.023. K.J.M. Mulders, J.H. Janssen, D.E. Martens, R.H. Wijffels, P.P. Lamers, Effect of biomass concentration on secondary carotenoids and triacylglycerol (TAG) accumulation in nitrogen-depleted Chlorella zofingiensis, Algal Res. 6 (2014) 8–16, https://doi.org/10.1016/j.algal.2014.08.006.