Evaluation of Scenedesmus dimorphus under Different Photoperiods with Eutrophicated Lagoon Water

Abstract

Given the need to improve bioenergy production processes, it is necessary to focus on low-cost culture media and environmental conditions of radiation and temperature. The Scenedesmus dimorphus species was cultured in eutrophicated lagoon water and Bayfolan 0.3% as culture media under four photoperiods with the objective of evaluating the biomass productivity, bioremediation capacity and influence of illumination on the composition and lipid content. It is concluded that the increase of light hours in the culture with eutrophicated lagoon water produces a decrease in the biomass productivity and COD removal percentage. The highest biomass productivity was obtained in photoperiod F1 (10.5:13.5) hours L:O, 0.053 ± 0.0015 g/L day and a removal of 95.6%. Bayfolan 0.3% with F2 (11.5:12.5) and F3 (12.5:11.5) did not show significant differences in the biomass productivity and COD removal. The increase in light hours in the photoperiod induced an increase of 1.01% and 2.84% of saturated fatty acids and 0.8% and 2.14% of monounsaturated fatty acids, as well as a decrease of 3.85% and 2.88% of polyunsaturated fatty acids in eutrophicated lagoon water and Bayfolan 0.3%, respectively.

1. Introduction

Microalgae are unicellular organisms containing carbohydrates, lipids and pigments. Their cultivation has been studied for the production of food, fuels and pharmaceuticals [1]. Renewable fuels, such as biodiesel and bioethanol, are promising and environmentally friendly alternatives [2]. They are considered third generation when produced from microalgal biomass [3]. For the exploitation of microalgal metabolites, it is necessary to decrease energy inputs, improve energy balances and reduce greenhouse gas emissions in the conversion processes to obtain more efficient production of biofuels [4], in addition to improving microalgal biomass production yields with low-cost culture media and decreased energy consumption during the process.

The factors that influence microalgal growth are nutrient availability and the C:N ratio in the culture medium, light penetration, carbon source, pH, salinity and temperature [5]. The light:dark cycles in microalgal culture influence the synthesis of organic compounds and nutrient metabolism [6].

Various artificial culture media and wastewater from different sources have been used for the cultivation of different species of microalgae, which are summarized in Table 1. Research has focused on biomass productivity under different metabolisms, finding higher productivities in mixotrophic than phototrophic cultures [7,8,9], in addition to the use of artificial culture media and urban and industrial wastewater, under a fixed photoperiod; however, the use of eutrophicated natural water as a microalgal culture medium has not been investigated so far.

Little is known about the influence of the photoperiod on biomass productivity. Spirulina platensis shows the optimal biomass yield when using a photoperiod of 12.5:11.5 light:dark and drastically decreases its biomass when increasing the illumination to more than 12.5 [10]. Using artificial light in microalgae cultivation increases the cost of biomass production, so it is necessary to investigate microalgal growth under natural photoperiods.

Table 1. Biomass produced under different growing conditions.

Microalgae	Culture Medium	Metabolism	Photoperiod L:D	Culture Conditions	Biomass Obtained	Reference
Chlorella vulgaris	Water, raw sewage	Mixotrophic	12:12	37,000 lux, 25 °C, 16 days	69.8 mg/Ld	[11]
	Water contaminated with 10 g/L of oil	Mixotrophic	12:12	2000 lux, 25 °C	0.41 g dry biomass	[12]
	Water contaminated with 20 g/L of oil				0.33 g dry biomass
	Biological reactor wastewater	Mixotrophic	12:12	180 µm/m2/d, 24 ± 1 °C, Air 0.5 vvm	20.3 mg/Ld	[13]
Chlorella pyrenoidosa	Dairy wastewater	Mixotrophic	12:12	10 W/m2, 25 °C	Not reported	[14]
Scenedesmus acutus	Raw wastewater	Mixotrophic	12:12	37,000 lux, 25 °C, 16 days	61.5 mg/Ld	[11]
Scenedesmus sp.	Domestic wastewater	Mixotrophic	14:10	60 µm/m2/d, 25 ± 2 °C, 7 days	61.4 mg/Ld	[15]
	Leachate from sanitary landfill, 20%	Mixotrophic	12:12	80 µm/m2/d, air 4.5 L/min, 25 ± 2 °C	3.9 mg/Ld	[9]
	Leachate from sanitary landfill, 60%	Mixotrophic			319.9 mg/Ld
	Leachate from sanitary landfill, 80%	Mixotrophic			421.9 mg/Ld
	Leachate from sanitary landfill, 100%	Mixotrophic			163 mg/Ld
	CHU	Phototrophic			34.6 mg/Ld
Scenedesmus dimorphus and Scenedesmus minutum	Municipal wastewater	Mixotrophic	16:8	150 µm/m2/d, 22 ± 2 °C	16 mg/L	[16]
Scenedesmus dimorphus	Wastewater with lactic acid	Mixotrophic	14:10	2500 lux, 25 °C, 10 days	2.5 g/L	[17]
	Wastewater with lactic acid + 0.8 g/L de NaNO3, 4 mg/L K2HPO4-3H2O	Mixotrophic	14:10		4.5 g/L
	BG11	Phototrophic	12:12	CO2 atmospheric 11 L/min, 25 °C, 20 days	96.5 mg/Ld	[7]
	BG11 + Apple pomace hydrolyzate 2% w/v	Mixotrophic	12:12		140.3 mg/Ld
	BBM	Phototrophic	16:8	120 µm/m2/s 11 L/min air	96.4 mg/Ld	[8]
	BBM + Hydrolyzed sugar cane bagasse 10 g/L	Mixotrophic	16:8		105.9 mg/Ld
	BBM + Hydrolyzed sugar cane bagasse 5 g/L	Mixotrophic	16:8		119.2 mg/Ld

Table 1. Biomass produced under different growing conditions.

Microalgae	Culture Medium	Metabolism	Photoperiod L:D	Culture Conditions	Biomass Obtained	Reference
Chlorella vulgaris	Water, raw sewage	Mixotrophic	12:12	37,000 lux, 25 °C, 16 days	69.8 mg/Ld	[11]
	Water contaminated with 10 g/L of oil	Mixotrophic	12:12	2000 lux, 25 °C	0.41 g dry biomass	[12]
	Water contaminated with 20 g/L of oil				0.33 g dry biomass
	Biological reactor wastewater	Mixotrophic	12:12	180 µm/m2/d, 24 ± 1 °C, Air 0.5 vvm	20.3 mg/Ld	[13]
Chlorella pyrenoidosa	Dairy wastewater	Mixotrophic	12:12	10 W/m2, 25 °C	Not reported	[14]
Scenedesmus acutus	Raw wastewater	Mixotrophic	12:12	37,000 lux, 25 °C, 16 days	61.5 mg/Ld	[11]
Scenedesmus sp.	Domestic wastewater	Mixotrophic	14:10	60 µm/m2/d, 25 ± 2 °C, 7 days	61.4 mg/Ld	[15]
	Leachate from sanitary landfill, 20%	Mixotrophic	12:12	80 µm/m2/d, air 4.5 L/min, 25 ± 2 °C	3.9 mg/Ld	[9]
	Leachate from sanitary landfill, 60%	Mixotrophic			319.9 mg/Ld
	Leachate from sanitary landfill, 80%	Mixotrophic			421.9 mg/Ld
	Leachate from sanitary landfill, 100%	Mixotrophic			163 mg/Ld
	CHU	Phototrophic			34.6 mg/Ld
Scenedesmus dimorphus and Scenedesmus minutum	Municipal wastewater	Mixotrophic	16:8	150 µm/m2/d, 22 ± 2 °C	16 mg/L	[16]
Scenedesmus dimorphus	Wastewater with lactic acid	Mixotrophic	14:10	2500 lux, 25 °C, 10 days	2.5 g/L	[17]
	Wastewater with lactic acid + 0.8 g/L de NaNO3, 4 mg/L K2HPO4-3H2O	Mixotrophic	14:10		4.5 g/L
	BG11	Phototrophic	12:12	CO2 atmospheric 11 L/min, 25 °C, 20 days	96.5 mg/Ld	[7]
	BG11 + Apple pomace hydrolyzate 2% w/v	Mixotrophic	12:12		140.3 mg/Ld
	BBM	Phototrophic	16:8	120 µm/m2/s 11 L/min air	96.4 mg/Ld	[8]
	BBM + Hydrolyzed sugar cane bagasse 10 g/L	Mixotrophic	16:8		105.9 mg/Ld
	BBM + Hydrolyzed sugar cane bagasse 5 g/L	Mixotrophic	16:8		119.2 mg/Ld

The chemical composition of the biomass is determinant for its industrial application; biomass rich in lipids is useful in the production of biodiesel, while that rich in carbohydrates is useful in the production of bioethanol. An effective tool for the determination of the biochemical composition of microalgae is Fourier transform spectroscopy (FT-IR), as it dissects functional chemical groups in different absorbance regions, lipids and proteins, and carbohydrates have characteristic absorbance in different frequency regions, which allows an elucidation of the biochemical composition of the biomass [18].

As described above, it is vital to investigate biomass production with low energy inputs and low-cost culture media. In this study, the influence of four different photoperiods on the biomass productivity of Scenedesmus dimorphus was evaluated; the light:dark cycles were selected because the geographical area where this research was conducted presents such photoperiods under natural conditions. The use of eutrophicated lagoon water and Bayfolan as the culture media has the dual purpose of evaluating the nutrient removal capacity and the biochemical composition of the microalgae.

Therefore, this work contributes to the scientific community by identifying whether the culture photoperiod influences the biomass productivity, nutrient removal capacity and biochemical composition of Scenedesmus dimorphus obtained by culturing it in eutrophicated lagoon water and Bayfolan.

2. Materials and Methods

2.1. Characteristics of the Culture Medium

The eutrophicated water was taken from a lagoon called “El Conejo”, located at coordinates latitude: 22.41881 and longitude: −97.87649, in the municipality of Altamira, Tamaulipas, Mexico. The water was subjected to autoclave heat treatment at 121 °C and 15 psi for 10 min. In addition, a 0.3% Bayfolan solution was used as a control medium. The species Scenedesmus dimorphus was provided by the Phycology Department of the Institute of Biology, UNAM.

2.2. Culture Conditions

The microalgae were cultured in 1.5 L PET containers disinfected for 24 h with 0.14 mL/L of 5% sodium hypochlorite, followed by neutralization with 0.1 mL/L of 24.81% sodium thiosulfate (Fermont) and washing with sterile distilled water [19]. A culture volume of 1.25 L with 20% inoculum was used under the illumination of two 18 W Led lamps (lux) at room temperature.

Table 2. Experimental factorial design of eight treatments for two culture media and four photoperiods.

Treatment	Culture Medium	Photoperiod (Light:Dark)
1	Eutrophicated lagoon water	F1 (10.5:13.5)
2	Eutrophicated lagoon water	F2 (11.5:12.5)
3	Eutrophicated lagoon water	F3 (12.5:11.5)
4	Eutrophicated lagoon water	F4 (13.5:10.5)
5	Bayfolan 0.3%	F1 (10.5:13.5)
6	Bayfolan 0.3%	F2 (11.5:12.5)
7	Bayfolan 0.3%	F3 (12.5:11.5)
8	Bayfolan 0.3%	F4 (13.5:10.5)

describes the full factorial design of experiments: the first factor is the different culture mediums (lagoon water and Bayfolan); the second factor is the four different photoperiods.

This design allows for examination of the effect of each factor and their interaction on biomass production. The growth curve was performed by optical density at 685 nm in a UV/Vis spectrophotometer, model Cintra 303 (GBC, Regents Park, NSW, Australia). All the experiments were performed in triplicate.

2.3. Biomass Harvesting

The biomass was left to sediment for 24 h and then centrifuged at 6000 rpm for 15 min in a centrifuge, model EBA 21 (Hettich, Saint-Laurent, QC, Canada), and the supernatant was eliminated. The samples were then lyophilized by freezing in 50 mL falcon tubes covered with aluminum foil with 5 holes. The process was carried out at −80 °C for 2 days, and then the samples were placed in a 2.5 L FreeZone lyophilizer benchtop freeze dryer (Labconco, Kansas, MO, USA).

2.4. Lipid Extraction

Ultrasound-assisted extraction was performed at a 20 mL:g ratio, 20% amplitude, for 50 min, at room temperature in a UP200Ht ultrasonic processor (Hielscher, Teltow, Germany). The process was repeated three times, followed by filtration and evaporation of the solvent [12,20,21,22]. The solvent evaporation was performed in a Rotary evaporator, model R-134 (Buchi, Mumbai, India).

For the purification of the microalgae oil, 3 mL of H₂SO₄ 0.5M [6] was added to the sample to remove the chlorophyll, followed by centrifugation. The supernatant containing the chlorophyll was decanted, then the sample was washed twice with 5 mL of hexane and heated at 90 °C for 15 min; once cooled, it was centrifuged. The solvent was allowed to evaporate from the supernatant to obtain the lipid [11].

The solvents chloroform and methanol at 99.8% purity (Fermont, Playa del Carmen, México) were used.

2.5. Characterization

The infrared spectra (FTIR) were obtained in a Fourier transform spectrometer, model spectrum 100 (PerkinElmer, Waltham, MA, USA). The measurements were performed in the mid-infrared in the range between 450 and 4000 cm⁻¹ for a total of 12 scans.

The lipid content was identified by gas chromatography using the AOAC 996.06 2001 methodology.

3. Results

The following tables present the results obtained under the four photoperiods studied: F1 (10.5:13.5 L:O); F2 (11.5:12.5 L:O); F3 (12.5:11.5 L:O); F4 (13.5:10.5 L:O). The data were analyzed using an analysis of variance (ANOVA) with α = 0.05. In all cases in which the F test was significant, Tukey HSD analyses with α = 0.05 were performed with the Excel statistical tool (Microsoft Office).

Table 3. Biomass productivity in both culture media under the different photoperiods analyzed.

Factor B	Factor A—Biomass Productivity (g/L/Day)
Growing Medium	F1 (10.5:13.5)	F2 (11.5:12.5)	F3 (12.5:11.5)	F4 (13.5:10.5)
Eutrophicated lagoon water	0.055	0.043	0.040	0.023
	0.054	0.050	0.033	0.025
	0.052	0.041	0.035	0.023
Bayfolan at 0.3%	0.038	0.036	0.033	0.023
	0.035	0.039	0.032	0.023
	0.036	0.036	0.033	0.026

shows the biomass productivities obtained in g/Lday, while

Table 4. Results of ANOVA analysis of biomass productivities in both culture media under different photoperiods.

Origin of Variances	Sum of Squares	Degrees of Freedom	Mean Squares	F	Probability	Critical Value for F
Sample	0.000170	1	0.000170	27.48	8.04 × 10−5	4.49
Columns	0.001514	3	0.000504	81.30	6.69 × 10−10	3.23
Interaction	0.000385	3	0.000128	20.67	9.44 × 10−6	3.23
In-group	9.93 × 10−5	16	6.20 × 10−6
Total	0.002169	23

shows the results of the analysis of variance (ANOVA) of these productivities.

A two-factor ANOVA was performed with a significance level of 0.05. It was established that the H₀:biomass productivities were equal in all the photoperiods (Factor A), the H₀:biomass productivities were equal in both culture media (Factor B) and in H₁, there are no interactions between the two factors. Based on the results obtained, H₀ is rejected, that is to say, there is a statistically significant difference between the means of biomass productivity among the four photoperiods and the two culture media. H₁ is accepted, as there is interaction between both factors.

To identify the difference between the photoperiods, a one-factor ANOVA and Tukey’s test were performed for each culture medium.

The results of the single factor ANOVA for the results of the biomass productivities in the eutrophicated lagoon water are shown in

Table 5. Results of ANOVA analysis of biomass productivities obtained for eutrophicated lagoon water.

Origin of Variances	Sum of Squares	Degrees of Freedom	Mean Squares	F	Probability	Critical Value for F
Between groups	0.001471	3	0.00049033	50.29	1.55 × 10−5	4.06
Within groups	0.000078	8	0.00000975
Total		11

. Based on the results, the H₀ is rejected, i.e., there is a significant difference in the average biomass productivity among the four photoperiods evaluated. The results of Tukey’s HSD test with a significance level of 0.05 are shown in

Table 6. Tukey HSD test of biomass productivities for eutrophicated lagoon water in the analyzed photoperiods.

	F1	F2	F3	F4
F1	-	0.0090	0.0177	0.0300
F2	-	-	0.0087	0.0210
F3	-	-	-	0.0123
F4	-	-	-	-

(HSD = 0.00816 and probability of 1.55 × 10⁻⁵). Variation was observed between photoperiods F1 and F2, F1 and F3, and F1 and F4. In addition, in F2 and F3, F2 and F4, and F3 and F4, the greatest difference in the means was observed between photoperiods F1 and F4.

A single factor ANOVA was performed for the results of the biomass productivities in Bayfolan 0.3%. As shown in

Table 7. Results of ANOVA analysis of biomass productivities obtained in Bayfolan 0.3%.

Origin of Variances	Sum of Squares	Degrees of Freedom	Mean Squares	F	Probability	Critical Value for F
Between groups	0.000428	3	0.000142	53.54	1.22 × 10−5	4.06
Within groups	2.13 × 10−5	8	2.66 × 10−6
Total		11

, based on the results, the H₀ is rejected, that is, there is a significant difference in the average biomass productivity among the four photoperiods evaluated. The results of the Tukey HSD test with a significance level of 0.05 are shown in

Table 8. Tukey HSD test of biomass productivities in Bayfolan 0.3%.

	F1	F2	F3	F4
F1	-	−0.0006	−0.0030	0.0123
F2	-	-	−0.0023	0.0130
F3	-	-	-	0.0153
F4	-	-	-	-

(HSD = 0.00427 and probability of 1.22 × 10⁻⁵). Variation was observed between photoperiods F1 and F4, F2 and F4, and F3 and F4.

As shown in Figure 1, the biomass productivity in crops grown with eutrophicated lagoon water decreased as light hours increased. On the other hand, in the crops grown with 0.3% Bayfolan, a slight increase in productivity was observed, reaching a maximum of 0.032 g/L ± 0.0005 in photoperiod three; however, as the light hours increased in photoperiod four, the productivity dropped drastically to 0.024 ± 0.0017 g/L/día.

A calibration curve of absorbance versus the dry weight concentration of the Scenedesmus dimorphus biomass was performed. In Figure 2, the growth curves of Scenedesmus dimorphus in eutrophicated lagoon water are presented, and Figure 3 illustrates the growth curves in Bayfolan at 0.3%. Figure 2 shows a decrease in the biomass concentration in dry weight as the photoperiod light hours increased; the maximum concentration was 1.23 g/L in F1 and the minimum was 0.86 g/L in F4. The growth curves did not reach the stationary phase.

Figure 3 shows a maximum concentration of 1.38 g/L in F1 and a minimum concentration of 1.08 g/L in F4 at the end of the culture; however, in this culture medium, the stationary growth phase is not observed for the F1, F2 and F3 photoperiods. It is only observed in F4: on day 22, a concentration of 1.12 g/L is observed. This implies that the longer the light exposure time in the photoperiod, the faster the growth is, reaching its stationary growth phase followed by the death phase.

Table 9. Percentage of COD removal in both culture media under the different photoperiods analyzed.

Factor B	Factor A—% Removal of Chemical Oxygen Demand
	F1 (10.5:13.5)	F2 (11.5:12.5)	F3 (12.5:11.5)	F4 (13.5:10.5)
Eutrophicated lagoon water	95.1	93.1	87.8	59.9
	97.1	91.9	82.5	61.0
	94.5	91.0	80.5	60.5
Bayfolan at 0.3%	59.5	89.0	87.2	43.6
	60.1	87.6	89.6	40.8
	61.2	89.1	89.7	45.1

shows the COD removal percentages in the photoperiods and culture media studied, while

Table 10. Results of the ANOVA analysis of the percentage of COD removal in both culture media under different photoperiods.

Origin of Variances	Sum of Squares	Degrees of Freedom	Mean Squares	F	Probability	Critical Value for F
Sample	868.806666	1	868.806666	247.25	3.75 × 10−11	4.49
Columns	4986.05833	3	1662.01944	473.00	7.93 × 10−16	3.23
Interaction	1380.29333	3	460.097777	130.94	1.80 × 10−11	3.23
In-group	56.22	16	3.51375
Total	7291.37833	23

shows the results of the analysis of variance (ANOVA) of these productivities. This was performed with a significance level of 0.05. It is established that the H₀:COD removal percentages are equal in all the photoperiods (Factor A), the H₀:COD removal percentages are equal in both culture media (Factor B) and in H₁, there are no interactions between the two factors. Based on the results obtained, H₀ is rejected, meaning there is a statistically significant difference between the removal percentages among the four photoperiods and the two culture media. H₁ is accepted, as there is interaction between the two factors.

The single factor ANOVA for the results of COD removal in eutrophicated lagoon water is shown in

Table 11. Results of ANOVA analysis of COD percentage removal obtained for eutrophicated lagoon water.

Origin of Variances	Sum of Squares	Degrees of Freedom	Mean Squares	F	Probability	Critical Value for F
Between groups	2240.995833	3	746.998611	170.77	1.36 × 10−7	4.06
Within groups	34.99333333	8	4.37416666
Total	2275.989167	11

. Based on the results, H₀ is rejected because there is a significant difference in COD removal. The results of the Tukey HSD test with a significance level of 0.05 are shown in

Table 12. Tukey HSD test of percentage of COD removal for eutrophicated lagoon water in the analyzed photoperiods.

	F1	F2	F3	F4
F1	-	3.5666	11.9666	35.1000
F2	-	-	8.4000	31.5333
F3	-	-	-	23.1333
F4	-	-	-	-

(HSD = 5.46 and probability of 1.366 × 10⁻⁷). Variation was observed between photoperiods F1 and F3 and F1 and F4, in addition to F2 and F3, F2 and F4, and F3 and F4. The greatest difference in means was observed between photoperiods F1 and F4.

The single factor ANOVA for the results of COD removal in Bayfolan 0.3% water is shown in

Table 13. Results of the ANOVA analysis of the percentage COD removal in Bayfolan 0.3%.

Origin of Variances	Sum of Squares	Degrees of Freedom	Mean Squares	F	Probability	Critical Value for F
Between groups	4125.35583	3	1375.11861	518.26	1.68 × 10−9	4.06
Within groups	21.2266667	8	2.6533333
Total		11

. Based on the results, H₀ is rejected because there is a significant difference in COD removal among the four photoperiods. The results of the Tukey HSD test with a significance level of 0.05 are shown in

Table 14. Tukey HSD test for % COD removal in Bayfolan 0.3%.

	F1	F2	F3	F4
F1	-	−28.3000	−28.5600	14.4300
F2	-	-	−0.2600	42.7300
F3	-	-	-	43.0000
F4	-	-	-	-

(HSD = 4.26 and probability of 1.686 × 10⁻⁹). Variation was observed between photoperiods F1 and F2, F1 and F3, F1 and F4, F2 and F4, and F3 and F4. The greatest difference in means was observed between photoperiods F3 and F4.

Since the biomass showed significant differences in productivity under F1 and F4 photoperiods in both culture media, characterization with FT-IR spectrophotometry of the biomass and gas chromatography of the extracted lipids was performed. The samples were identified considering the culture medium and photoperiod. ALF1 and ALF4 refer to the biomass and lipid obtained from the culture in eutrophicated lagoon water under F1(10.5:13.5) and F4(13.5:10.5) photoperiods. BF1 and BF4 refer to the biomass and lipid obtained from the culture in 0.3% Bayfolan under F1(10.5:13.5) and F4(13.5:10.5) photoperiods.

Figure 4A shows the spectrum of the freeze-dried biomass that was cultured in eutrophicated lagoon water, while Figure 4B shows the spectrum of the one cultured in Bayfolan 0.3%.

Table 15. Signals obtained in FTIR spectrophotometer.

ALF1	ALF4	BF1	BF4	Wavenumber Range cm−1	Functional Group	References
950–1200 Carbohydrate Band						[18]
1019	1021			980–1072	C-O-C polysaccharides	[23,24]
		1036
			1053
		1036		1030–1099	P=O nucleic acids	[25]
			1053
1075	1074	1074
1075	1074	1074		1070–1140	C-O-C	[26]
1151	1149			1134–1174	C-O-C polysaccharides	[23]
			1219	1210–1240	P=O polysaccharides	[27]
		1240
		1240		1230–1244	P=O polysaccharides	[25]
		1240	1231	1230–1310	C-N secondary amide	[26]
		1262	1264
		1350		1191–1356	P=O polysaccharides, phosphodiester	[23]
	1379			1370–1398	CH3, CH2, C-O proteins, and carboxyl groups	[25]
		1385	1381
	1413	1401	1400	1390–1430	C-N amide stretching	[26]
			1405
			1411
	1413	1401	1400	1392–1460	C-O carboxyl groups	[25]
	1444		1405
			1411
1450–1720 Amino acid band						[28]
1490–1710 Protein band						[18]
	1452	1454	1454	1450–1456	CH2, CH3 Lipids and proteins	[23,25]
1539	1536	1538	1533	1515–1570	N-H secondary amide	[26]
1546			1537
1639	1630	1631	1634	1630–1680	C=O secondary amide	[26]
2800–3000 Lipid band						[18]
2852	2851	2852	2851	2850–2960	CH2 symmetrical nucleic acids	[29]
2874	2873	2871	2873	2960–2975	CH3 asymmetric Lipids	[25]
2922	2919	2921	2919	2916–2936	CH2 asymmetric Lipids	[29]
		2955	2960	2952–2972	CH3 symmetrical Lipids	[29]
3280	3281	3280	3281	3170–3370	N-H secondary amide	[26]
3285

shows the details of the signals obtained in the FTIR analysis of each of the samples grouped by wavelength range and their respective functional group.

The identification and lipid content calculation were performed by gas chromatography using the AOAC 996.06 2001 methodology. The results are shown in

Table 16. Fatty acid profile of Scenedesmus dimorphus biomass grown in eutrophicated lagoon water and Bayfolan 0.3%, under F1 (10.5:13.5) and F4 (13.5:10.5) photoperiods.

	BF1	BF4	ALF1	ALF4
Saturated Fatty Acids (%)	60.52	62.66	53.02	55.86
Caprylic acid (C8:0)	2.5	3.01	3.9	4.37
Capric acid (C10:0)	2.15	2.61	3.4	3.81
Lauric acid (C12:0)	2.40	2.73	3.57	4.08
Tridecanoic acid (C13:0)	1.33	1.5	1.86	2.03
Myristic acid (C14:0)	2.82	4.02	4.16	4.65
Pentadecanoic acid (C15:0)	1.26	1.65	1.81	2.01
Palmitic acid (C16:0)	18.17	18.71	11.59	13.09
Margaric acid (C17:0)	4.11	2.01	3.03	2.78
Stearic acid (C18:0)	9.66	8.35	11.24	8.79
Arachidic acid (C20:0)	2.73	3.44	4.37	5.13
Heneicosanoic acid (C21:0)	1.52	0	0	0
Behenic acid (C22:0)	7.87	9.71	0	5.12
Tricosanoic acid (C23:0)	1.21	1.46	4.09	0
Lignoceric acid (C24:0)	2.79	3.42	0	0
Monounsaturated fatty acids	17.60	18.40	23.96	24.97
Myristoleic acid (C14:1 cis 9)	1.39	1.67	2.18	2.28
Pentadecanoic acid (C15:1 cis 10)	1.52	1.94	2.44	0
Hexadecenoic acid (C16:1 cis 9)	1.81	2.09	2.39	6.5
Margaroleic acid (C17:1 cis 10)	1.56	1.91	2.46	2.64
Oleic acid (C18:1 cis 9)	9.57	9.2	12.41	11.31
Eicosenoic acid (C20:1 cis 11)	1.75	1.59	2.08	2.24
Polyunsaturated fatty acids	21.87	18.99	23.02	19.17
Linoleic acid (C18:2 cis 9, 12)	12.05	9.81	11.55	10.36
Gamma-linoleic acid (C18:3 cis 6,9, 12)	1.59	1.97	2.13	2.45
Alpha-linolenic acid (C18:3 cis 9, 12, 15)	4.14	5.78	5.42	6.36
Eicosadienoic acid (C20:2 cis 11, 14)	1.23	0	0	0
Eicosatrienoic acid (C20:3 cis 8, 11, 14)	1.53	0	0	0
Arachidonic acid (C20:4 cis 5, 8, 11, 14)	1.33	1.43	1.89	0
Docosahexaenoic acid (C22:6 cis 4, 7, 10, 13, 16, 19)	0	0	2.03	0
Trans fatty acids	0	0	0	0

.

4. Discussion

The genus Scenedesmus has the necessary characteristics to combine CO₂ fixation, lipid synthesis and water treatment [30]. The species Scenedesmus dimorphus was isolated from a wastewater effluent of the petrochemical industry of the city of Altamira, Tamaulipas, the study area of this research [31], which indicates its ability to develop under environmental conditions. Its growth potential has been investigated under different metabolisms in diverse culture media, which were summarized in Table 1, in which it can be observed that the investigations with Scenedesmus dimorphus have not varied based on the photoperiod, but by the addition of nutrients to change the phototrophic metabolism to mixotrophic, maintaining the same operating conditions. The biomass productivity of Scenedesmus dimorphus with residual water containing lactic acid is 2.5 g/L and is increased by adding 0.8 g/L NaNO₃ + 4 mg/L K₂HPO₄-3H₂O up to 4.5 g/L biomass using the same conditions of 1500 lux and a 14:10 photoperiod [17]. When cultivated in the artificial culture medium BG11, it has a productivity of 96.5 mg/Ld and increases with the addition of apple pomace hydrolysate at 2% w/v up to 140.3 mg/Ld when cultivated with a 12:12 photoperiod and atmospheric CO₂ at a rate of 11 L/min [7]. Similarly, when cultivated in BBM, 96.4 mg/Ld are obtained. The production increases with the addition of 5 g/L of hydrolyzed sugarcane bagasse to 105.9 mg/Ld; however, the addition of 10 g/L of this nutrient decreases production to 105.9 mg/Ld because the turbidity of the substrate decreases the passage of illumination. These experiments were carried out under illumination of 120 µmol/m²/s, a photoperiod of 16:8, and air supplementation [8].

Biomass productivity is a key factor for industrial applications of microalgae because they are photosynthetic organisms. Carbon and nitrogen elements are the most important in the metabolic pathway of microalgae [32]. The biomass productivities in eutrophicated lagoon water and 0.3% Bayfolan solution are different because eutrophicated lagoon water contains higher levels of nutrients and organic carbon; therefore, its metabolism is mixotrophic, and growth in 0.3% Bayfolan solution results in phototrophic metabolism.

The influence of the photoperiod on the growth of Scenedesmus dimorphus has not been evaluated previously; however, it was found that it has been cultivated in 12:12, 14:10 and 16:8 light:dark photoperiods. In this research study, it was found that the biomass productivity was different in the four photoperiods of study in lagoon water: the productivity was 0.053 g/L in photoperiod F1 (10.5:13.5), while 0.023 g/L was obtained in photoperiod F4 (13.5:10.5). In the Bayfolan 0.3% solution, no significant differences in productivity were observed among photoperiods F1, F2 and F3. Differences were only observed between photoperiods F1 and F4, where the productivities were 0.036 and 0.024 g/L, respectively.

The percentage of COD removal showed significant differences among the four photoperiods of study. The greatest difference was observed between F1 and F4, being 95.56 and 60.46%, that is, in photoperiod F1 (10.5:13.5), the greatest removal was obtained. On the other hand, in the Bayfolan 0.3% solution cultures, significant differences were also shown in the removal means. The greatest difference was observed between the F3 and F4 photoperiods: the removals were 88.83 and 43.16% respectively, that is, a greater removal was obtained in the F3 photoperiod (12.5:11.5). The results obtained in this research are related to those obtained in reported investigations, where the reported removal was 95.6% of COD by Scenedesmus dimorphus in wastewater with lactic acid [17]. Another study reported a removal of 95.9% of COD in domestic wastewater by Scenedesmus sp. [15], while Chlorella vulgaris was able to achieve 100% removal of COD in wastewater from a biological reactor [13].

The presence of the functional groups that form carbohydrates, amino acids, proteins and lipids was identified due to the fact that microalgae are organisms that are composed of these compounds [33]. Proteins are a sequence of amino acids, called the primary structure, where secondary amide bonds link the repeated units in a protein [26]. In the infrared spectrum, 9 amide bands can be observed, named A, B and I–VII. Among them, amide I is the most intense one, representing the C=O bond occurring in the 1600–1700 region coupled to the N-H and C-N bending [28]. Amide I is observed in the 1585–1724 cm⁻¹ range, while amide II is in the 1490–1585 cm⁻¹ range [18].

The ALF1 sample does not present signals between 1191 and 1244 cm⁻¹ corresponding to the presence of nucleic acids. On the other hand, the confirmation of secondary amides is given by the presence of two C-N bonds and one N-H bond. Secondary amides are present in proteins, and despite having 4 signals in the N-H bonds, this does not present the confirmatory signals in C-N of a secondary amide; however, it presents the signals 1639, 3280 and 3285 cm⁻¹ of the C=O group and two N-H signals, confirming the presence of a primary amide [26].

Nitrogen is an essential component in the synthesis of proteins and nucleic acids in microalgae and is related to their growth [9] due to the fact that the culture in the lagoon water with F1 presented a higher concentration in mg/L in comparison with the one cultivated in F4. It is possible that the concentration of nitrogen in the culture was being depleted during the cultivation.

The biomass obtained from ALF4, BF1 and BF4 present signals corresponding to the presence of amide I [28] and amide II [18]; the presence of amides I and II is characteristic of protein chains [27]. Only sample BF4 presents three signals in the C-N range stretching amide and two signals in the N-H range of a secondary amide together with the C=O group of a secondary amide. Considering that secondary amides link the repeated units of the protein and two signals in C-N, one in N-H and the C=O should be present [26], it can be affirmed that only sample BF4 presents two protein-forming secondary amides. With respect to the 950–1200 cm⁻¹ band that characterizes the absorption of polysaccharides, it can be deduced that the microalgae cultivated in Bayfolan have a higher carbohydrate content. The use of Bayfolan favors the increase of pigments and proteins in the microalgae [13].

Regarding the representative bands of a lipid profile, the similarity of the signals among the samples was observed. The lipids have characteristic absorption bands, the absorption band of the C=O stretching of the ester and the vibration of the C-H stretching in the acyl chains around 2800–3000 cm⁻¹. This last one can characterize the lipid content [18]. The asymmetric and symmetric CH₃ and CH₂ groups in the 2800–3000 band are lipid hydrocarbons; these signals are representative of lipid accumulation in microalgae [27]. These results indicate that the samples have the same lipid profile.

The lipid profile showed the presence of saturated, monounsaturated and polyunsaturated fatty acids. The lipids obtained from the biomass cultured in Bayfolan 0.3% and in the eutrophicated lagoon water with photoperiod F4 (13.5:10.5) showed an increase in the percentage of saturated and monounsaturated fatty acids, in addition to a decrease in polyunsaturated fatty acids with respect to the lipid percentages obtained from the biomass cultured in photoperiod F1 (10.5:13.5). The increase in light hours in the photoperiod of cultivation influenced the fatty acid profile: caprylic, capric, capric, lauric, tridecanoic, arachidic, behenic and myristoleic fatty acids presented an increase, while stearic, oleic and linolenic acids presented a decrease. On the other hand, the palmitic acid content was constant in the % lipids obtained from the biomass grown in Bayfolan in both photoperiods. As for the lipid percentages obtained from the biomass grown in eutrophicated lagoon water under photoperiods F1 and F4, a higher palmitic acid content was observed in photoperiod F4 with respect to photoperiod F1. The fatty acid profile obtained from Scenedesmus dimorphus agrees with those obtained in related research. Among the lipids that have been obtained from this species are mainly C16 to C18 fatty acids, although C20 to C22 can also be obtained depending on the culture conditions [7,16,17].

The percentage of lipids found in the microalgae Scenedesmus dimorphus grown in Bayfolan 0.3% and eutrophicated lagoon water were 18.71 and 13.09% palmitic acid, 8.3 and 8.79% stearic acid, 9.2 and 11.31% oleic acid and 9.81 and 10. 36% of linoleic acid, respectively, which are lower than those reported by other studies, where in BG11 medium, wastewater with lactic acid and municipal wastewater had 24.23, 25.01 and 21.6% palmitic acid, 0, 53.24 and 23.3% oleic acid, and 44.01, 0 and 24.01% linolenic acid, respectively [16,17].

5. Conclusions

By increasing the number of hours of light, the cultures in the eutrophicated lagoon water produced a decrease in the biomass productivity and percentage of COD removal. The highest biomass productivity was obtained in photoperiod F1 (10.5:13.5) hours L:O, 0.053 ± 0.0015 g/Lday and a removal of 95.6%, while in the cultures grown in Bayfolan 0.3% under photoperiods F2 (11.5:12.5) and F3 (12.5:11.5) hours L:O, the biomass productivities and COD removal percentages were obtained without significant differences.

The same functional groups C=O, CH, CH₃ and CH₂ belonging to fatty acid formation were identified in the biomass of Scenedesmus dimorphus grown in both culture media under F1 and F4 photoperiods, by FTIR spectroscopy.

The main fatty acids identified by the gas chromatography technique were palmitic stearic, oleic and linolenic acids in both culture media. The increase in light hours in the photoperiod induced the increase of saturated and monounsaturated fatty acids, as well as the decrease of polyunsaturated fatty acids.