Impact of green and blue‐green light on the growth, pigment concentration, and fatty acid unsaturation in the microalga Monoraphidium braunii

The spectral composition of light is an important factor for the metabolism of photosynthetic organisms. Several blue light‐regulated metabolic processes have already been identified in the industrially relevant microalga Monoraphidium braunii. However, little is known about the spectral impact on this species' growth, fatty acid (FA), and pigment composition. In this study, M. braunii was cultivated under different light spectra (white light: 400–700 nm, blue light: 400–550 nm, green light: 450–600 nm, and red light: 580–700 nm) at 25°C for 96 h. The growth was monitored daily. Additionally, the FA composition, and pigment concentration was analyzed after 96 h. The highest biomass production was observed upon white light and red light irradiation. However, green light also led to comparably high biomass production, fueling the scientific debate about the contribution of weakly absorbed light wavelengths to microalgal biomass production. All light spectra (white, blue, and green) that comprised blue‐green light (450–550 nm) led to a higher degree of FA unsaturation and a greater concentration of all identified pigments than red light. These results further contribute to the growing understanding that blue‐green light is an essential trigger for maximized pigment concentration and FA unsaturation in green microalgae.


INTRODUCTION
Light is a vital parameter for the metabolism of photosynthetic organisms such as microalgae.The light intensity and light regime are crucial for growth and photosynthetic activity. 1 Additionally, the spectral composition of light is highly relevant for microalgal metabolism.][4][5] Recent studies have reported a high biomass production upon green and yellow light under high light conditions as well as high culture densities.][10][11] For instance, it is known that blue light is required for the cell division of the microalga Chlamydomonas reinhardtii. 12In the green microalga Monoraphidium braunii, many well-known blue light-induced processes have been identified.4][15] Moreover, it is also an activation signal for enzymes involved in the nitrogen metabolism of this organism. 16Activation of the enzyme nitrate reductase by blue light has also been reported in other algae species and is presumably widespread among green microalgae. 9,17Furthermore, blue and green light can influence the composition of photosynthetic pigments and fatty acids (FA).Recently, we have reported that blue-green light between 450 and 550 nm is required for a maximized FA unsaturation and a maximized pigment concentration in the green microalga Acutodesmus obliquus. 180][21] Many of such light-triggered processes in organisms are highly conserved in the evolution of organisms. 22,23Therefore, investigating the impact of blue-green light on the FA and pigment composition in photosynthetic organisms is of great interest, both phylogenetically and biochemically.
Besides this foundational knowledge, investigating these spectrally triggered metabolic processes in microalgae may have practical applications.The detailed data can be harnessed for the targeted production of valuable compounds, such as FA, for the food, feed, and biofuel industries.][34] Polyunsaturated fatty acids (PUFA), such as alpha-linolenic acid (ALA, 18:3n-3), are produced by different microalgae species. 35,36The value of ALA for human nutrition is very high, due to its key function as a precursor for higher chained PUFA, which are in turn important for different physiological reactions in the human body. 37Therefore, the food industry aims to produce products enriched in ALA. 38But the n-3 /n-6 FA ratio is also vital for the nutritional value of food products.In particular, the ratio of the n-3 FA ALA toward the n-6 FA linoleic acid (LA, 18:2n-6) can have a strong impact on human health.0][41] However, the supply of these PUFA from traditional sources, such as aquaculture and the fishery industry, is insufficient to satisfy global demand. 42Moreover, these sources are associated with adverse environmental effects. 43,44In contrast, microalgae are a renewable production platform for crop production and other agricultural demands. 45Additionally, they have several advantages over land plants, such as no requirement for arable land, a lower water quality requirement, and higher growth rates. 46,47n this study, a combined investigation of the impact of different broad light spectra on the growth, FA, and pigment composition of M. braunii was conducted.It provides new insights into the light-related metabolic processes of green microalgae.The species was chosen due to the many already known blue light-triggered metabolic processes and its high industrial relevance. 5,48,49Moreover, it has a close phylogenetic relation to A. obliquus, for which the impact of blue-green light on the FA composition and pigment concentration has already been described in detail Ref. [5,50].

Cultivation device and test conditions
Only a brief description of the cultivation experiments and methodological differences is provided here since they have already been described in detail in Helamieh et al. 5 .
Microalgae cultivation was carried out in column reactors as a batch culture for 96 h, with the cultivation temperature set at 25 ± 0.5°C to promote high growth and a high FA unsaturation.Two Philips MSR HR CT, 575 W metal halide lamps with a sun-like light spectrum provided the light.The metal halide lamps generated the white light spectrum, which was further referred to as "white light" (380-700 nm).The white light was additionally attenuated with a shading net (polyethylene, aperture size 2 × 10 mm, Hermann Meyer KG, Rellingen, Germany).Other light spectra were obtained using the optical filter foils "light red" "dark green," and "dark blue" (LEE-Filters, England) in combination with the metal halide lamps.These light spectra were named "red light" (580-700 nm), "green light" (450-600 nm), and "blue light" (400-550 nm).All microalgae experiments were conducted at a photon flux density of 210 μmol photons m −2 s −1 for all tested light spectra (shown in the Appendix S1).
The cultivation experiments started at an optical density of 0.5 and were measured with a UV/VIS spectrometer at 750 nm (Pharmacia LKB Ultrospec III). 5,51The cell dry weight (CDW) was calculated using a linear correlation between optical density at 750 nm (OD 750 ) and CDW, previously determined in measurements: Samples were taken daily and subsequently stored at −80°C for the FA and pigment analysis.The CDW was determined using the linear correlation by measuring the OD 750 immediately after daily sampling.

Fatty acid analysis
The analytical method was performed according to Helamieh et al. 5 In contrast, a volume equivalent to 0.015 g CDW of the thawed samples was used for the FA extractions and heneicosanoic acid (Sigma Aldrich, Taufkirchen, Germany) 20 μmol dissolved in hexane was employed as an internal standard (IS) in this study.The extraction and transesterification of all FA-containing lipids into fatty acid methyl ester (FAME) were carried out according to Ref. [5,52-54].
The retention times (RT) in a GC-MS chromatogram with the identified FA can be found in the Appedix S1.The ratio of the identified FAME was set into relation with the respective area of the IS, and the relative percentage of the total FAME was calculated.

Pigment analysis
The pigment analysis was performed according to Helamieh et al. 18

Data evaluation and statistical analyses
The cultivations were performed in triplicates for each tested condition in two independent experiments, and the means ± standard errors were calculated from the values obtained.All statistical tests were done according to Helamieh et al. 18

Growth
In the growth experiments, M. braunii was cultivated for 96 h at 25°C, with equal photon flux densities under white, red, green, and blue light conditions.Under white light treatment, the CDW reached 1.94 ± 0.04 g L −1 .With red light, a maximum of 1.62 ± 0.04 g L −1 CDW was reached.Green light and blue light treatment led to a maximum CDW of 1.37 ± 0.04 and 1.06 ± 0.01 g L −1 , respectively (Figure 1).The differences in biomass production after 96 h cultivation are significant (p ≤ 0.05) for all spectra, except for the green light cultivation compared to the red light cultivation (Figure 1).

Fatty acids
All identified FA of M. braunii are shown in Table 1, and a chromatogram of the GC-EI/MS analysis is shown in the supplementary data (see Appendix S1).The FA 18:1, 16:1, and 16:3 were observed in different, not specifically characterized isomeric forms.Under white light, the dominant PUFA were 18:2 (18.2% ± 0.6%) and 18:3 (15.7% ± 0.6%), with a total PUFA percentage of 57.2% ± 0.6%.The rare PUFA 16:4 and 18:4 are typical for microalgae and were also identified in M. braunii (Table 1). 55he light spectrum had a strong effect on the FA composition.Higher proportions of the FA 18:3 were found in the white, green, and blue light cultivation compared to CDW = 0.436 × OD 750 − 0.099 red light conditions (Figure 2).A similar pattern was also observed for the PUFA 16:4, even though these differences were not significant (see Figure S2).
Contrary to the aforementioned effect on the FA 18:3, a reverse tendency was observed for the lower desaturated n-C18 FA (18:1 and 18:2).Upon red light cultivation, a higher percentage of the 18:1 and 18:2 was detected, compared to the other spectral cultivations.Taken together, upon white, green, and blue light cultivation, a higher proportion of 18:3 toward 18:2 and 18:1 was identified, in comparison to red light cultivation (Figure 2).Additionally, a higher percentage of the summarized PUFA was also observed under white, green, and blue light, compared to red light cultivation (Figure 3).Therefore, the white, green, and blue light treatment led to a higher total degree of FA unsaturation than monochromatic red light.
The light spectrum had an influence on the concentration of the identified photosynthetic pigments.Pigment concentrations for all identified pigments reached a higher value in the white, green, and blue light cultivations, compared to the red light cultivation (Table 2; Figure 4).For example, only 69.0% of lutein was found upon red light treatment compared to white light cultivation.Likewise, the values for the red light-treated samples reached 72.6% for neoxanthin, 78.5% for violaxanthin, 63.6% for alpha-carotene, and 80.0% and 87.9% for chlorophyll a and b, in comparison to white light.A similar relation of pigments can also be observed if red light treatment is compared to the green or blue light-cultivated samples (Table 2; Figure 4).

DISCUSSION
In the cultivation experiments, the biomass production of M. braunii was strongly affected by the light spectrum.This underlines the importance of the parameter light spectrum for the biomass production of microalgae.A comparably low growth upon blue light was also observed in recent studies on microalgae. 5,56Even though the absorption spectrum of the primary photosynthetic pigment, chlorophyll a, has a high overlap with the blue light spectrum, this waveband is inefficiently used for the biomass production of M. braunii (Figure 1).Besides chlorophyll a and b, carotenoids and xanthophylls in M. braunii can also absorb blue light. 57Xanthophylls and carotenoids mainly convert light energy into heat as a photoprotective mechanism, which might compromise biomass production. 58,59his is one possible explanation for the low growth performance under blue light.
In contrast, the green light treatment led to higher biomass production than blue light (Figure 1).In classical plant physiology, the prevailing assumption of a low contribution of green light to photosynthetic production dominates. 604][5]56 Due to the low absorptance, green light has a lower contribution to the photosynthetic production in plants and microalgae compared to red, and blue light at low photon flux densities.However, green light might efficiently contribute to biomass production under high and medium photon flux densities and high biomass concentrations.In this case, the low absorptance of green light is compensated by a more uniform absorption and more efficient use of light energy. 4Green light can penetrate deeper layers, enabling better light dilution in the leaf tissue or microalgae solution.In contrast, red and blue light are strongly absorbed by the first layers of chloroplasts in leaf layers or microalgae solutions, whereas deeper layers remain unilluminated.This results in a lower photon quantum yield due to unequal illumination, a higher heat dissipation, and perhaps, even a higher degree of photoinhibition of the photosystems. 2,4,5,56Very efficient use of red light and green light under medium photon flux densities was also observed in our study on A. obliquus. 5evertheless, a combination of blue and red light was found to cause higher photosynthetic efficiencies compared to monochromatic red light in the same algae strain. 61This was also observed in our recent study on the microalgae A. obliquus and might explain the high biomass production under white light, which contains red light and blue light (Figure 1).The FA composition of M. braunii has a high content of PUFA, and therein a comparably high percentage of the FA 18:3 and 18:2 (Table 1).These are essential FA for human nutrition. 62The FA composition is in accordance with other studies on the genus Monoraphidium. 49,55A strong impact of the light spectrum on the FA composition of M. braunii was observed.The degree of FA unsaturation was higher upon white, green, and blue light treatment, compared to red light conditions.In our recent study on the green microalga A. obliquus, we observed a similar spectral effect.We hypothesized that blue-green light between 450 and 550 nm is required for a maximum FA unsaturation in A. obliquus. 5The waveband between 450 and 550 nm was contained in all tested light spectra except red light.Therefore, the aforementioned FA desaturation effect is apparently also present in M. braunii and might explain the higher degree of unsaturation upon white, blue, and green light compared to red light in this study (Figures 2 and 3).This spectral waveband might be required for the maximum activity of FA-desaturase enzymes in green microalgae.Some of these FA-desaturases in photosynthetic organisms are regulated via different environmental cues, such as temperature and light. 63,64owever, it is not yet known which part of the light spectrum is required for the light regulation of FA-desaturases.
The changes in the ratio of the n-C18 FA indicate that the different light spectra impact the FA desaturation process (Figure 2).In particular, the desaturation of the FA 18:2, which is the last biochemical step in the biosynthesis of 18:3, seems to require blue-green light in M. braunii.It is well-known that the FA are desaturated step by step via FA-desaturases that are specific for each reaction. 21,64,65oreover, the activation of desaturases by light is already recognized as widespread in different phylogenetically distanced organisms. 21,63,64Therefore, we assume that blue-green light (450-550 nm) is also required for a maximized degree of unsaturation in M. braunii, presumably by activating FA-desaturases.
In the pigment analysis, six photosynthetic pigments were analyzed (Figure 4a,b; Table 2).The pigment composition is in accordance with previous studies on this species. 66Furthermore, a spectral influence on the pigment composition of M. braunii was observed in this study.It is already known that blue light triggers the production of xanthophylls and photosynthetic pigments. 34,67,68This was also observed in this study for M. braunii.Similar to the higher FA unsaturation, the concentrations of all pigments were also higher upon white, green, and blue light treatment compared to the red light cultivation (Figure 4a,b; Table 2).Both, the different pigment concentrations and the different degrees of FA saturation, might be related to rearrangement processes of thylakoid membranes of chloroplasts. 5,19,200][21] Therefore, the application of blue-green light might trigger an elevation of the thylakoid membrane system in M. braunii.This potentially explains the higher values of photosynthetic pigments and PUFA upon blue-green light-containing spectra.
Many light-regulated processes are highly conserved in the evolution of organisms. 22,23Therefore, further investigation of the impact of blue-green light on the PUFA and pigments in different phylogenetically distant organisms is required.This investigation might reveal the extend to which the light wavelength dependent effect on the FA and pigment composition is spread among photosynthetic organisms.To validate the hypothesized mechanism of thylakoid membrane rearrangements, additional research is necessary.For example, electron microscopic images of microalgae samples cultivated under specific light spectra might visualize blue-green light-triggered rearrangement processes in the chloroplasts of microalgae.To uncover the potential role of FA-desaturases, a detailed investigation of these enzymes is required.A transcriptome analysis of genes encoding FA-desaturases or enzyme assays involving FA-desaturases can provide insights into the role of these enzymes in blue-green light-triggered FA unsaturation.A detailed analysis can contribute to a deeper understanding of the light-regulated mechanisms in microalgae and plants.5][26][27][28] Further practical applications of this knowledge are photovoltaic-photosynthesis hybrid systems with comprehensive spectral use of the sunlight for the production of electricity and biomass in one system. 61

CONCLUSION AND IMPLICATIONS
Monochromatic red light and green light led to higher biomass production than monochromatic blue light.This underlines the high relevance of red and green light for microalgal growth.However, the highest biomass production was observed upon white light treatment.In addition, the strong impact of blue-green light on the FA and pigment composition of M. braunii was observed.Light spectra that contained blue-green light (450-550 nm) led to a maximized FA unsaturation and concentration of photosynthetic pigments.These results contribute to a growing understanding that blue-green light is a crucial environmental trigger for the lipid metabolism of photosynthetic organisms.The knowledge generated in this study may potentially be used to influence the metabolism of microalgae for industrial applications.It might pave the way for a targeted production of specific FA by light wavelength management for the biofuel and food industry.A lower degree of FA unsaturation is suitable for the production of biofuels, whereas a high content of specific PUFA is associated with beneficial nutritional effects.The degree of FA saturation in microalgae can be influenced by light wavelength management during microalgae cultivation, particularly by choosing a light spectrum that includes or excludes the waveband between 450 and 550 nm.

F I G U R E 1 1
Biomass production of Monoraphidium braunii exposed to 210 μmol photons m −2 s −1 white light (400-700 nm), red light (580-700 nm), green light (450-600 nm), and blue light (400-550 nm) at 25°C.The cell dry weight (CDW) was determined by a correlation with the optical density at 750 nm.Values represent means ± standard errors of two experiments.Different superscript letters (a-c) mean significant differences (p ≤ 0.05) between the groups.Fatty acid (FA) profile of Monoraphidium braunii.

T A B L E 2
Results of the HPLC-DAD pigment analysis in Monoraphidium braunii.

F I G U R E 4
Results of the HPLC-DAD analysis of Monoraphidium braunii.Chl a: Chlorophyll a; Chl b: Chlorophyll b (A).Alpha, Alpha-Carotene; Neo, Neoxanthin; Vio, Violaxanthin (B).CDW, cell dry weight.The analysis was repeated two times.Values represent means ± standard error.Different superscript letters (A, B) mean significant differences (p ≤ 0.05) between the groups.