Abbreviations CDW cell dry weight EI electron impact EPA eicosapentanoic acid FA fatty acid FAME fatty acid methyl ester GC gas chromatography IS internal standard MS mass spectrometry MUFA monounsaturated fatty acids PUFA polyunsaturated fatty acids RT retention time SFA saturated fatty acids SIM selected ion monitoring 1 INTRODUCTION

Microalgae are a group of photosynthetic microorganisms of high diversity (Metting, 1996). While adapting to various environments, plenty of bioactive substances have evolved (Metting, 1996; Pulz & Gross, 2004). Today, various microalgal substances e.g., antioxidants, carotenoids, as well as proteins, are of high importance in various sectors, such as pharmacy, cosmetic, and food industry (Pulz & Gross, 2004; Vanthoor-Koopmans et al., 2013). Due to the high fatty acid (FA) content, several microalgae species are also considered an interesting platform for a targeted FA production in the biofuel and food industry (Abomohra et al., 2013; Adarme-Vega et al., 2012; El-Sheekh et al., 2013). A high content of saturated fatty acids (SFA) is required for biofuel production, whereas a high content of polyunsaturated fatty acids (PUFA) is suitable for applications in the food industry (Piligaev et al., 2015; Riediger et al., 2009; Ruxton et al., 2004). Omega-3 FA such as linolenic acid (18:3), and the omega-6 FA, linoleic acid (18:2), are the basis for longer-chain PUFA, such as eicosapentanoic acid (EPA) and can be used as supplements for human diet (Brenna, 2002). Besides the degree of unsaturation, the ratio of these two FA is essential for healthy nutrition as well. Several diseases are caused by a high ratio of omega-6/omega-3 FA (Simopoulos, 2004). Another cause of adverse health effects are trans FA (Gebauer et al., 2007; Mozaffarian et al., 2006). Transisomers of unsaturated FA are naturally present in several microbial food products and in industrially processed vegetable oils, such as margarine (Dhaka et al., 2011; Kuhnt et al., 2011; Sommerfeld, 1983). Hence, to produce a suitable FA composition in microalgae for applications in the food or biofuel industry, a regulation of the FA metabolism during microalgae cultivation is of high importance.

It is well known that the FA metabolism in microalgae can be influenced by cultivation with selected parameters (Breuer et al., 2012; Mandotra et al., 2016). For instance, the total lipid content in microalgae can be raised by nitrogen-limiting conditions in the cultivation media. However, this results in reduced growth, which counterbalances the total lipid yield (El-Sheekh et al., 2013). More recent studies showed an elevation of the lipid content without compromising the microalgae growth (Abomohra et al., 2018, 2019, 2020; Abomohra & Almutairi, 2020; Almarashi et al., 2020; Esakkimuthu et al., 2020). For example, the utilization of phytohormones and the integration of seaweeds anaerobic digestate were shown to increase both growth and lipid production (Abomohra & Almutairi, 2020; Esakkimuthu et al., 2020). In a very novel approach, it was also shown, that a pretreatment of microalgae with low-dose cold atmospheric plasma (CAAP) resulted in an enhancement of growth and lipid content in Chlorella vulgaris (Almarashi et al., 2020). Variation of the cultivation temperature is another factor to influence the FA metabolism in microalgae. Beside the lipid content, also the degree of saturation and the FA composition can be influenced by the microalgae cultivation conditions. One of the underlying mechanisms of temperature adaptation in plants, microorganisms, and green algae is the modification of the degree of FA saturation to regulate the cell membrane fluidity (Alfonso et al., 2001; Collados et al., 2006; de Mendoza & Cronan Jr, 1983; Degraeve-Guilbault et al., 2021; Patterson, 1970).

Another important parameter to influence FA production in microalgae is light. It is known that light regulates the activity and triggers the expression of various FA-desaturases (Berestovoy et al., 2020; Collados et al., 2006; Kis et al., 1998). Most studies on microalgae had focused on the effect of the light intensity on biomass production and FA composition. However, the light spectrum can influence various metabolic processes in microalgae as well. Additional night illumination with colored light-emitting diodes was also shown to influence the fatty acid composition (Abomohra et al., 2019). Especially, blue light can trigger several enzymatic reactions in microalgae (Aparicio et al., 1994; Giráldez et al., 1998). It was also shown that exposure to green light increases the percentage of PUFA in Chlorella vulgaris (Hultberg et al., 2014) and the expression level of omega-3 desaturases in Chlorella sp. (Osman et al., 2018). Therefore, the influence of green, blue, and red light on the FA composition is subject to further investigation. Moreover, there is only little knowledge about the combined effects of temperature and light spectrum on the FA composition in microalgae.

This study aims to investigate the combined impact of the light spectrum and temperature on the FA composition in Acutodesmus obliquus. The green microalga A. obliquus was chosen due to its beneficial FA profile and high growth rate (Abomohra et al., 2013; El-Sheekh et al., 2013; Hindersin et al., 2013). In order to evaluate the growth and FA profile, A. obliquus was cultivated at 20, 30, and 35°C in three successive experiments. In all experiments, the cultivation tubes were irradiated with red light, blue light, and green light. Produced biomass of A. obliquus was assessed and the FA profile was analyzed by gas chromatography coupled with electron impact ionization mass spectrometry (GC-EI/MS). It could be shown that temperature and light spectrum have a major impact on the FA composition in A. obliquus.

2 MATERIALS AND METHODS 2.1 Chemicals

The cultivation medium for A. obliquus was composed of Flory Basis Fertilizer 1 (Euflor, Germany) and KNO3 (Fisher Scientific, Germany) and kept at a pH of 7.0 ± 0.5 with the usage of HCl (Fisher Scientific, Germany) and NaOH (Fisher Scientific, Germany). Culture medium was prepared in distilled water for all cultivation experiments. The internal standard (IS), heptadecanoic acid (17:0) was purchased from Sigma Aldrich (Taufkirchen, Germany). Hydrochloric acid, chloroform, methanol, and n-hexane for the FA extractions were purchased from Carl Roth (Karlsruhe, Germany) in GC ultragrade.

2.2 Microalgae preparation

The microalgae strain A. obliquus (No. U169) from the Microalgae and Zygnematophyceae Collection Hamburg (MZCH, previously SVCK) microalgae collection of the University of Hamburg was used. Microalgae precultivations were done in Schott flasks at 25°C and a constant photon flux density of 150 μmol m−2 s−1 emitted by a Sylvania T9 circline, 32 W fluorescent tube with a white light spectrum. The precultures were aerated with CO2-enriched air (5% v/v) and stirred by using a magnetic stirrer. The cultivation medium was composed of 2 g L−1 Flory Basis I (Euroflor, Germany) and 3.22 g L −1 KNO3 and kept at a pH of 7 ± 0.5 daily by manual adjustment. Cell dry weight (CDW) was determined gravimetrically, and a correlation of CDW and optical density at 750 nm (OD750) was set up for each experiment from different dilution steps of the culture of each respective experiment (Chen et al., 2012; Girard et al., 2014; Reymann et al., 2020). The defined volume was filtered, subsequently dried at 80°C for 24 h and the measured CDW was set into relation with the respective OD750. It was previously tested that the filters kept constant weight after 3–8 h, depending on the applied biomass.

2.3 Cultivation device

Cultivation experiments were performed in glass tubes of a length of 490 mm and a diameter of 40 mm each holding a volume of 350 ml. Up to 12 glass tubes were submerged into a transparent acrylic glass water bath and kept in a vertical position in black acrylic brackets. These brackets prevented ambient light from reaching the tubes from the back and the sides. All tubes were irradiated from the front side of the water bath with metal halide lamps (Philips MSR HR CT, 575 W), which emit a sun-like light spectrum (Figure 1a). Different light spectra were generated by optical filter foils: light red, dark green, and dark blue (LEE-Filters, England) which were fixed to the outer front side of the water bath. The resulting spectra of the metal halide lamps, and the LEE filters were measured and adjusted to an equal photon flux density, respectively (Figure 1b–d, Table 1). Absolute photon fluxes were determined by a UV–Vis spectrometer (BLACK-Comet, StellarNET, Tempa, USA) within a range of λ = 400–700 nm.

The light spectrum of the MSR 575 HR CT metal halide lamp. Unfiltered (a); in combination with the optical foils: light red (b); dark green (c), and dark blue (d) (Lee filters, England). The relative emission spectra (Jλ,rel in counts) were determined by a UV–vis spectrometer (BLACK-comet, StellarNET, Tempa, USA) within a range of λ = 300 to 800 nm (integration time = 10 ms)

TABLE 1. Test conditions during the different experiments Experiment Conditions Red light Green light Blue light 1 Temperature (°C) 20 ± 0.5 20 ± 0.5 20 ± 0.5 Photon flux (μmol m−2 s −1) 469 ± 51 487 ± 27 482 ± 7 2 Temperature (°C) 30 ± 0.5 30 ± 0.5 30 ± 0.5 Photon flux (μmol m−2 s−1) 469 ± 51 487 ± 27 482 ± 7 3 Temperature (°C) 35 ± 0.5 35 ± 0.5 35 ± 0.5 Photon flux (μmol m−2 s−1) 469 ± 51 487 ± 27 482 ± 7 Note: The temperatures and the estimated accuracies of measurement. Values for the photon fluxes represent means ± standard deviation of triplicates. 2.4 Experimental conditions Pre-cultures were diluted to an OD750 of 0.2 for each experiment, which was determined by a UV/VIS spectrometer (Pharmacia LKB Ultrospec III). The cultivation was started with a volume of 350 ml of microalgae suspension. Microalgal growth was monitored via the OD750, which was measured directly after sampling. The CDW was calculated from the OD750 by using the coefficient determined from the linear correlation between OD750 and CDW:

After inoculation, batch cultivation started in triplicates for a duration of 96 h. The microalgae were irradiated constantly with a photon flux density of 480 μmol m-2 s–1 ± 51 μmol m−2 s−1 at all experiments of different wavebands (Figure 1b–d and Table 1). The microalgae suspension was mixed by aeration with humidified and CO2-enriched air (5% v/v) and an airflow of 0.2 L min−1. The temperature in the water bath was kept at 20°C ± 0.5°C, 30°C ± 0.5°C, and 35°C ± 0.5°C by a chiller (AD15R-30, VWR European) in three successive experiments (Table 1). The pH was kept at pH 7 ± 0.5 manually and adjusted daily by the addition of 1 M HCl or 1 M NaOH. Samples for the FA analysis were taken from the preculture. After the cultivation was started, samples were taken after 1, 3, 6, 24, 48, 72, and 96 h of cultivation. For the GC–MS measurements, 3–20 ml samples were taken, depending on the biomass concentration, and subsequently stored at −80°C prior to analysis.

2.5 Sample preparation and FA analysis

Samples were thawed, homogenized, and the volume of microalgae suspension, containing 0.0025 g CDW, determined via OD750 -CDW-correlation, was used for the FA-extraction. Heptadecanoic acid was used as IS and a stock solution of 1.0 mg ml−1 was prepared. The samples were centrifuged at 5137g for 20 s (Rotanta 460R, Hettich Zentrifugen, Tuttlingen, Germany), the supernatant was discarded, and 20 μmol of the IS stock solution dissolved in hexane were added to the pellet. A modified Folch extraction (Reich et al., 2012, 2013), in which the pellet was resuspended in a CHCl3 /MeOH mixture (2:1, v/v), was applied. Upon full resuspension, the samples were shaken at 200 rpm (IKA HS 501 digital, Jahnke and Kunkel and Co IKA Labortechnik, Staufen, Germany) for 1 h. Afterward, they were centrifuged for 20 s at 5137g (Rotanta 460R, Hettich Zentrifugen, Tuttlingen, Germany), and the supernatant containing the extracted lipids was collected in a glass tube. This procedure was repeated twice with shaking times of 3 and 12 h. Previous test extractions showed highest FA yields with three extraction steps. In a further extraction, less than 0.1% of extracted FA of the first three extractions were found. The supernatants, containing virtually all extractable lipids were all transferred and collected in one glass tube, and the solvents were evaporated under a constant and gentle stream of nitrogen. The transesterification was performed according to the method of Ichiara and Fukubayashi (Ichihara & Fukubayashi, 2010). The dried lipid extracts were resuspended in 0.2 ml of chloroform, 2 ml of methanol were added, and acidified with 0.1 ml of concentrated hydrochloric acid (35% w/w). This solution was transferred into a screw-capped glass tube, overlaid with nitrogen, and the tube was tightly closed. Upon vortexing, the tube was heated to 100°C for 1 h and subsequently cooled down at room temperature for 10 min. In order to extract the fatty acid methyl esters (FAME), 2 ml of hexane and 2 ml of water were added, the tube was vortexed, and the hexane phase was collected after phase separation. This solution was diluted 1:10 with hexane, of which 1 μl was injected for GC–MS analysis.

2.6 Instrumental conditions (GC)

GC/EI-MS was performed with a Thermo Scientific™ ISQ™ 7000 Single Quadrupole GC–MS system. The samples (1 μl) were injected with an autosampler, and the injector was operated in splitless mode and kept at 260°C. For the separation of the target compounds, a TRACE™ TR FAME fused silica capillary column (0.25 mm, 0,25 μm × 30 m) with helium as carrier gas was used with a constant pressure of 100 kPa and a flow of 1,5 ml min−1. The oven temperature was set to start at 60°C for 1 min, followed by a ramp rate of 6.5°C min−1 until the final temperature of 260°C was reached and then held for 8 min. The electron energy was 70 eV, the ion source was set to 270°C, and the mass range of m/z 60–400 was recorded in the full scan mode. Fragment ions included m/z 74, m/z 79, m/z 81, and m/z 87 for the FAME that were detected during the measurement in the GC/EI-MS selected ion monitoring (SIM) mode (Reich et al., 2013). A chromatogram with retention times (RT) of the identified FA is shown in Figure 2.

GC/MS chromatogram of Acutodesmus obliquus acquired in SIM mode. The sample was taken after 5 days of cultivation with 150 μmol m−2 s −1 white light at 25°C

2.7 Data evaluation

The mass spectra and the RT were used for the qualitative analysis of the separated FAME. The peak area ratio of the identified FAME was set into relation with the respective area of the IS, and the share of each FA (in %) was calculated. All samples were taken and measured in triplicates, and the mean values ± standard deviation were calculated.

Additionally, to the in-depth descriptive analysis and visualization of the data, analysis of variance with repeated measures were conducted to examine statistical effects of the light spectrum on the CDW, the percentage of the FA 16:4, 18:3, 18:4, and the isomers of the FA 16:1.

As the samples were taken in triplicates, degrees of freedom for statistical analyses were limited. Missing values were replaced with means of existent values. The individual samples were randomly grouped to test the effect of light spectrum on the dependent variables in a within-subjects design. As such sufficient data were obtained to test assumptions for a repeated-measures ANOVA and the analysis was conducted respectively. In the interpretation of results, the focus lies on the spectral effects on dependent variables, and not on conclusions on the growth. For statistical inference, conservative measures of Greenhouse–Geisser corrected values due to the small sample size are reported. Furthermore, measures included in the ANOVA concerned the measurement timepoints after 24, 48, and 96 h of cultivation.

3 RESULTS 3.1 Microalgal growth

The biomass concentration was determined daily for the whole cultivation period (Figure 3a–c). The light spectrum strongly influenced the growth patterns of A. obliquus. In all experiments, the red light regime resulted in the highest increase of biomass after 96 h of cultivation. A maximum of 2.42 g L−1 CDW was observed after 96 h cultivation at 30°C and under red light (Figure 3b).

Biomass production of Acutodesmus obliquus exposed to 480 μmol m−2 s−1 red light (circle; 580–720 nm), green light (triangle; 450–600 nm), and blue light (diamonds; 380–540 nm) at 20°C (a), 30°C (b) and 35°C (c). The cell dry weight (CDW) was determined by a correlation with the optical density at 750 nm. Values represent means ± standard deviation of triplicates

Blue light treatment resulted in the lowest amount of produced CDW at all tested temperatures. Irradiation with blue light resulted in a 34.3%–36.8% reduced biomass production in 96 h compared to red light, in all experiments (Figure 3a–c). Biomass production under green light was higher than under blue light, but still 11.6%–16.8% lower than under red light (Figure 3a–c). Nevertheless, the green light regime resulted in a relatively high amount of produced biomass. The highest CDW was measured for all spectra at a temperature of 30°C (Figure 3b). In comparison, the maximum CDW were decreased by 21.1% at 20°C and 12.4% at 35°C, after 96 h cultivation (Figure 3a,c).

Results of the main ANOVA further show that the main effect of light spectrum on the CDW was highly significant with large effect sizes across all temperature conditions. The effect of the light spectra on CDW was highest at 35°C at F(1.1; 2.1) = 68.8 at p = 0.01 with a large effect of partial eta2 = 0.98. Within-subject contrasts show that CDW is significantly different under blue light condition than under red light condition under all temperature conditions. The difference is also significant for green and red light conditions at 35°C.

3.2 Fatty acids

In this study, a total of 14 FA were identified in A. obliquus, which are shown in Figure 2 and Table 2. The FA 14:0, 15:0, 22:0, and 24:0 were always found in low share (<1%) and were therefore dismissed for further study. The cis/trans (c/t) isomers of unsaturated FA were summed up for the comparison of the relative FA relations. Strong variations of 16:1 (c/t) isomers were found, and the relative changes toward different test conditions are separately shown in Section 3.5.

TABLE 2. Fatty acid (FA) profile of Acutodesmus obliquus FA % FA % 14:0 ≤1 18:1Δ9 7.8 ± 0.1 15:0 ≤1 18:2Δ9,12 10.2 ± 0.1 16:0 38.2 ± 0.9 18:3Δ9,12,15 21.9 ± 0.5 16:1Δ9 -trans 1.4 ± 0.1 18:4Δ6,9,12,15 2.4 ± 0.1 16:1Δ9 -cis 1.1 ± 0.1 22:0 ≤1 16:2Δ7,10 1.0 ± 0.1 24:0 ≤1 16:3Δ7,10,13 5.2 ± 0.1 SFA 41.4 16:4Δ4,7,10,13 7.5 ± 0.2 MUFA 10.3 18:0 2.1 ± 0.2 PUFA 48.3 Note: The samples were taken after 5 days of cultivation with 150 μmol m−2 s−1 white light at 25°C. Values represent means ± standard deviation of triplicates. 3.3 Effect of light spectrum on the degree of saturation

The light spectrum had a strong impact on the FA composition. In all experiments, red light caused a lower degree of desaturation, in comparison to blue and green light. The relative proportions of the 16:4, 18:3, and 18:4 FA decreased by up to 64% under a red light regime compared to green- and blue-light treatments (Figure 4a–c). Accordingly, the percentage of the lower desaturated FA increased (Figure 5b–d). These differences in the degree of desaturation were already detectable after 1 and 3 h of cultivation (data not shown). Nevertheless, it became evident in all experiments after 24 h of cultivation (Figures 4a–c, 5b–d, 6a–c and 7a–c). The SFA were not affected by the light spectrum (Figures 5a and 7d).

Relative proportions (%) of the fatty acids 16:4 (a), 18:3 (b) and 18:4 (c) in Acutodesmus obliquus, cultivated at a photon flux density of 480 μmol m−2 s −1 red light (black columns; 580–720 nm), green light (light gray colums, 450–600 nm), blue light (dark gray columns; 380–540 nm), and a temperature of 30°C. values represent means ± standard deviation of triplicates

Relative proportions (%) of the fatty acids 16:0 (a), 16:1 (b), 16:2 (c), and 16:3 (d) in Acutodesmus obliquus, cultivated at a photon flux density of 480 μmol m−2 s −1 red light (black columns; 580–720 nm), green light (light gray columns; 450–600 nm), blue light (dark gray columns; 380–540 nm), and a temperature of 30°C. values represent means ± standard deviation of triplicates

Relative proportions (%) of the fatty acids 16:4 (a), 18:3 (b), and 18:4 (c) in Acutodesmus obliquus, cultivated at a photon flux density of 480 μmol m−2 s −1 red light (black columns; 580–720 nm), green light (light gray columns; 450–600 nm), blue light (dark gray columns; 380–540 nm), and a temperature of 20°C. values represent means ± standard deviation of triplicates

Relative proportions (%) of the fatty acids 16:4 (a), 18:3 (b), 18:4 (c) and 16:0 (d) in Acutodesmus obliquus, cultivated at a photon flux density of 480 μmol m−2 s −1 red light (black columns; 580–720 nm), green light (light gray columns; 450–600 nm), blue light (dark gray columns; 380–540 nm), and a temperature of 35°C. values represent means ± standard deviation of triplicates

3.4 Impact of temperature and light spectrum on the degree of saturation

In this study, a strong impact of the temperature on the degree of FA saturation of A. obliquus was observed. These temperature-triggered FA changes interacted with the aforementioned light related FA changes. The precultures of all experiments were cultivated at 25°C (see Section 2.2). The cultivation at 20°C resulted in a maximum increase of the PUFA 16:4, 18:3, and 18:4 by 37.9%, 32.8%, and 23.1% of the relative amounts, respectively (Figure 6a–c). However, this increase was significantly less expressed during cultivation under red light regime, compared to the blue- and green light groups (Figure 6a–c).

The cultivation at 30°C and red light resulted in a reduction of the PUFA 16:4, 18:3, and 18:4 after 96 h of cultivation by up to 74.97%, 41.55%, and 43.47% (Figure 4a–c), respectively. This decrease was significantly less expressed in the green and blue light groups (Figure 4a–c). Cultivation at 35°C resulted in a maximum decrease of 16:4, 18:3, and 18:4 by 75.57%, 35.51%, and 64.76%, respectively, after 96 h of cultivation with red light (Figure 7a–c). However, this temperature-triggered decrease was also less expressed under green as well as blue light at 35°C (Figure 7a–c). Still, this was not significant for all measured timepoints.

In summary, results show that exposure to red light and higher temperatures (30 and 35°C) resulted in lower relative shares of the PUFA 16:4, 18:3, and 18:4, whereas exposure to blue or green light and low temperatures (20°C) gave rise to elevated shares (Figures 4a–c, 6a–c and 7a–c). No impact of the light spectrum was found on the relative proportions of SFA. In contrast, high temperatures 35 and 30°C resulted in an elevation of the relative amounts of the FA 16:0 by up to 23.2% (35°C) and 11.7% (30°C) in the course of the experiment (Figures 5a and 7d). At a cultivation temperature of 20°C, the relative amount of the FA 16:0 was maintained at a level of the preculture (data not shown).

Repeated-measures ANOVA exposed a significant effect of the light spectrum on the FA 16:4, 18:3, and 18:4 across all temperature conditions. This effect was the largest at a temperature of 30°C, at F (1.4; 2.8) = 308.4 at p = 0.001 with a large effect size of partial eta2 = 0.99. Furthermore, the differences in FA composition are significant for green light against red light conditions across all temperatures in the experiment. The FA composition under blue light significantly differed from that under red light under 20 and 30°C; however, there are no significant differences regarding the FA composition between red and blue light under 35°C.

3.5 cis-trans isomerism of the fatty acid 16:1

Both cis and trans isomers of the FA 16:1 were identified in A. obliquus. The light spectrum and temperature had a strong impact on the 16:1 c/t ratio. A significant difference between samples irradiated with red light and the ones irradiated with green light was found (Figures 8 and 9). In contrast, no strong differences between the green light- and blue light-treated samples were observed, which is why the data of the blue light cultivations are not shown and discussed together with the green light cultivation data.

Relative amounts of the 16:1 trans (black columns) and 16:1 cis (gray columns) isomers in Acutodesmus obliquus after cultivation at a photon flux density of 480 μmol m−2 s −1 red light (580–720 nm) (a), green light (450–600 nm) (b), and a temperature of 35°C. values represent means ± standard deviation of triplicates

Relative amounts of the 16:1 trans (black columns) and 16:1 cis (gray columns) isomers in Acutodesmus obliquus upon cultivation at a photon flux density of 480 μmol m−2 s −1 red light (580–720 nm) (a), green light (450–600 nm) (b), and a temperature of 20°C. values represent means ± standard deviation of triplicates (a)

Results of the repeated-measures ANOVA show significant effects of the light spectrum on cis-trans isomerism of the FA 16:1 across all temperature conditions. Here, the effect was largest at 20°C with F(1;2) = 4629 at p = 0.001 with a larger effect size of partial eta2 = 1. Within-subject contrasts show that these differences are significant for green light compared to red light conditions, and for blue light compared to red light conditions, across all temperatures in the experimental setup.

The relative percentage of the 16:1c increased from 44.4% in the preculture to 76.4% after 96 h of cultivation at 20°C in the blue and green light group (Figure 9b). However, no such increase was observed in the red-light group at the same temperature (Figure 9a). Upon cultivation with 35°C, the relative percentage of the 16:1c decreased from 42.8% in the preculture to 30.8%–33.2% for all tested spectra (Figure 8a,b). Over the time course of all experiments, the relative percentage of the 16:1c was significantly reduced in the red-light group, with respect to the green light- and blue light-treated samples (Figures 8a,b and 9a,b). Independent of the influences of temperature and light spectrum, a third effect was observed. In all experiments, a strong increase of the 16:1 c was evident in the first 24 h of cultivation (Figures 8a,b and 9a,b). In the first hours of cultivation, biomass concentration was always under 1 g L−1 (Figure 3a–c). In the following 72 h, the CDW in all experimental approaches increased to higher values, combined with a concomitant reduction of the relative percentage of the 16:1c isomers (Figures 3a-c, 8a,b and 9a,b). This leads to the conclusion that the 16:1c/t ratio is also influenced by the biomass concentration.

Presumably, the isomeric ratio changes are also influenced by temperature. While the maximum percentage of the 16:1 c was reached after 24–48 h of cultivation time with 20°C (Figure 9a,b), the maximum value of the 30°C cultivation was reached after 3–6 h (data not shown) and 1–3 h in the case of 35°C (Figure 8a,b). In general, lower temperatures, blue-green light as well as a low biomass concentration resulted in a high relative percentage of 16:1c. In contrast, red light, higher temperatures, and higher biomass concentrations gave rise to a lower 16:1c/t ratio.

4 DISCUSSION

In general, a maximum biomass of 2.42 g L−1 CDW was reached with a cultivation temperature of 30°C and red light (Figure 3b). This temperature is close to the optimum growth temperature for A. obliquus (Hindersin et al., 2013). The maximum of produced biomass was reduced by 21.1% and 12.4% at cultivation temperatures of 20 and 35°C, compared to the 30°C cultivation (Figure 3a–c). Among all tested temperatures, the maximum of produced biomass for A. obliquus was observed under red-light conditions (Figure 3a–c). The red light spectrum, (wavelength between 600 and 700 nm, Figure 1b), is effectively absorbed by the main photosynthesis pigments in microalgae (Sandmann, 1991). Therefore, the growth results might be related to the high absorption of these wavebands by chlorophyll a. The utilized blue light has a high overlap with the chlorophyll a absorption maximum at 430 nm (Figure 1d). Nevertheless, blue light regime caused the lowest amounts of produced biomass of all tested light spectra at all tested temperatures (Figure 3a–c). Beside chlorophyll a, carotenoids and xanthophylls also absorb light at this waveband in A. obliquus (Niyogi et al., 1997). These pigments mainly convert light energy into heat and, therefore, may reduce the contribution of this waveband to the biomass production (Wilhelm et al., 1985). This can provide an explanation for the low performance under blue light regime. Contrary to common assumptions, green light treatment resulted in a relatively high amount of produced biomass at all tested temperatures. A comparably high biomass production was reached with green compared to red light (Figure 3a–c). Due to the low absorption of this waveband, many studies on microalgae postulate that green light only has a low contribution to biomass production in microalgae (Kim et al., 2013, 2014). However, more recent studies have shown that light spectra that are weakly absorbed by microalgae can outperform all other light spectra in biomass productivity (Mattos et al., 2015; de Mooij et al., 2016; Ooms et al., 2017). In another study on the green microalga Scenedesmus bijuga, green light treatment resulted in the highest biomass production of all tested light spectra (Mattos et al., 2015). Due to the close phylogenetical relation of A. obliquus and Scenedesmus bijuga, it might be reasonable to compare the growth results of this species with the ones received for A. obliq