Influence of light quality on Chlorella growth, photosynthetic pigments and high-valued products accumulation in coastal saline-alkali leachate

Soil salinization is a widespread environmental problem worldwide, and there are approximately 954 million hectares of saline-alkali land in the world (Yue et al. 2020). Coastal saline-alkali soils, have a high content of exchangeable sodium, which is easy to cause soil consolidation, and the high salinity in the soil prevents plant roots from absorbing water and obtaining nutrients (Tang et al. 2020). In addition, as saline-alkali land is an important reserve land resource for food production, the restoration of saline-alkali land is essential to global food demand. Nowadays, the improvement of saline-alkali land often adopts various methods such as physical restoration, chemical modification and bioremediation, and the common practiced method in the world is the combination of irrigation and drainage, which removes the salt in the soil through leaching (Wang et al. 2018; Meena et al. 2019). Although this method can improve the soil quality, it also produces a large amount of wastewater with high salinity. The wastewater contains inorganic salts, which is an abundant source of energy to be recovered and reused, but if it is directly discharged, it may cause groundwater pollution and deterioration of water quality (Fang et al. 2019). At present, the global water consumption is gradually increasing, and a large part of the population still lives in water-scarce areas (Zhu & Dou 2018; Archer et al. 2019). Given the above, the resource utilization of saline-alkali leachate is essential to alleviate the shortage of water resources and maintain the sustainable development of the social economy. Our previous studies have shown that the coastal saline-alkali leachate can be used to cultivate three oleaginous microalgae at low cost to realize the conversion of wastewater resources (Liu et al. 2020).

As a global emerging industry, microalgae cultivation has been extensively researched and marketed in recent years due to its enormous economic and social potential (Mata et al. 2010). Microalgae are highly efficient photosynthetic cell factories, which contain high amount of high-valued substances, such as proteins, pigments, carbohydrates and lipids. Among the possible applications of the microalgae, they can be used in the food, feed, life sciences and renewable fuels, for example carbohydrates and fatty acids can be converted into alcohol and biodiesel (Chew et al. 2017; Chandra et al. 2019). However, the cost of large-scale production of microalgae and related biological products is still high, which has brought challenges to the development of microalgae biotechnology. Current researches are aimed at increasing the biomass of microalgae, improving the contents of lipid and high-valued substances, or reducing the cost of microalgae cultivation (Chen et al. 2009). How to improve the cultivation efficiency of microalgae in saline-alkali leachate and the content of high-valued substances is of great significance for reducing the production cost of microalgae-based biological products.

It is known that environmental factors such as light, nutrients, temperature and CO₂ concentration can significantly affect the algal growth and lipid accumulation including content and composition (Ananthi et al. 2021). Light, as the energy source of microalgae photosynthesis, is one of the important factors affecting the growth of algae and the accumulation of active substances. In indoor microalgae cultivation, artificial light sources such as fluorescent light are commonly used as energy sources, which undoubtedly increase the power consumption and may exacerbate the greenhouse phenomenon (Sun et al. 2017). Light emitting diode (LED) has been widely used in microalgae cultivation due to its advantages of narrowing specific wavelengths with low power consumption, small chip size, and long duration (Kim et al. 2014). It has been reported that the microalgal photosystem II and photosystem I can be enhanced or induced by red and blue light wavelengths, respectively (Ravelonandro et al. 2008). In addition, since pigments such as chlorophyll a and chlorophyll b are sensitive to light of different wavelengths, it would be pertinent to select LEDs to regulate the distribution of various products in microalgae (Schulze et al. 2014). Shu et al. (2012) found that light quality has a significant effect on algae cell growth and product formation, and this effect is dependent on the algae species. Abiusi et al. (2014) investigated the influence of light quality on algae cell size, growth, productivity, photosynthetic efficiency of Tetraselmis suecica F&M-M33, and found that red light can be profitably used for the production of this marine microalga for aquaculture.

Therefore, in order to further improve the cultivation efficiency of Chlorella in the coastal saline-alkali leachate and the content of high-valued substances, the effects of five different light sources (including monochromatic blue, red, white LED light and blue-red mixed light, blue-white mixed light) on the growth of Chlorella and the accumulation of lipid, pigments, protein, total sugar and starch were explored. The findings can provide a theoretical guidance on how to accurately use light quality to make Chlorella in coastal saline-alkali leachate obtain higher content of high-valued substances. This will not only provide a resource utilization method for the coastal saline-alkali leachate, but also reduce the production cost of microalgae-based biological products.

The microalga Chlorella sp. HQ (No. GCMCC7601) was used in the experiment, which was isolated in our previous study and has been kept in China General Microbiological Culture Collection Center. The strain was routinely cultivated in an axenic Selenite Enrichment (SE) medium, which contained 250 mg·L⁻¹ of NaNO₃, 75 mg·L⁻¹ of K₂HPO₄·3H₂O, 75 mg·L⁻¹ of MgSO₄·7H₂O, 25 mg·L⁻¹ of CaCl₂·2H₂O, 175 mg·L⁻¹ of KH₂PO₄, 25 mg·L⁻¹ of NaCl, 5 mg·L⁻¹ of FeCl₃·6H₂O, 0.81 mg·L⁻¹ of FeCl₃, 10 mg·L⁻¹ of Na₂EDTA, 2.86 mg·L⁻¹ of H₃BO₃, 1.81 mg·L⁻¹ of MnCl₂·4H₂O, 0.22 mg·L⁻¹ of ZnSO₄·H₂O, 0.079 mg·L⁻¹ of CuSO₄·5H₂O, and 0.039 mg·L⁻¹ of (NH₄)₆Mo₇O₂₄·4H₂O. All chemicals were of analytical grade. Prior to the experiments, cultures were maintained in Erlenmeyer flask at 25 ± 1 °C, 4800 lux incident light intensity in a 14:10 h light/dark cycle and the flasks were shaken for three times a day.

The coastal saline-alkali soil samples used in this study were taken from Dongying, Shandong Province, China. The detailed preparation method of the coastal saline-alkali leachate was described by Liu et al. (2020). The obtained leachate was filtered through 0.45-μm filtration membrane and sterilized at 121 °C for 30 min, and was stored after cooling to room temperature for later use.

The experiments were performed in a climate chamber equipped with LEDs as the light source. Chlorella sp. HQ was cultivated in 250 mL Erlenmeyer flasks with 180 mL sterile coastal saline-alkali leachate and initial cell density of 2 × 10⁵cells·mL⁻¹, and the temperature was maintained at 25 ± 1 °C. The illumination was supplied at an average light intensity of 4500 lux and a photoperiod of 14:10 h light/dark. Based on light intensity, two mixed LED light treatments (red:blue = 1:2; blue:white = 1:2) and three monochrome LED light treatments (monochrome red; blue; and white LED) were set up in this study. In order to avoid the influence of the light source on the other experimental groups, each treatment group was separated by aluminum foil.

In the experiment, the algal density was measured every other day, and the biomass, lipids, TAGs, total sugar, starch, protein, and photosynthetic pigment content of the microalgae were measured at the end of cultivation. The Erlenmeyer flask was manually shaken three times a day to ensure that the algae cells and the culture medium were thoroughly mixed, and to reduce experimental errors.

Algal density was determined using a Neubauer hemocytometer. To determine the yield of biomass, a 20 mL algae solution sample was filtered through a pre-weighed 0.45-μm membrane. The membrane was washed twice with distilled water, dried at 110 °C for 24 h until completely dehydrated and weighed again, then the biomass yield was calculated.

In the above formula, N_t (cells·mL⁻¹) is cell density at cultivation time t (d). K, a and r represent the maximum cell density during the whole cultivation period, the relative position from the origin and algae cell intrinsic growth rate, respectively. We determined the parameters (K, a, r) of each light treatment group through non-linear regression, and R_max (maximum population growth rate, in which case N equals to half of K) were calculated (Equation (3)).

The lipid content was determined based on the method of modified chloroform-methanol extraction (Bligh & Dyer 1959). Using a high-speed refrigerated centrifuge (CR22G, HITACHI, Japan), 40 mL of algae liquid was centrifuged at 12,000 rpm, 4 °C for 10 min, and concentrated to 0.8 mL. For extraction, 2 mL of chloroform, 2 mL of methanol, and 1 mL of distilled water were added to the above concentrated algae solution in this order, and mixed well. After extraction, centrifuged again for 10 min (4,000 rpm, 4 °C), then transferred the bottom chloroform layer to the pre-weighed dry glass tubes, blown it to a constant weight with a nitrogen blower. Finally, the microalgal lipid was calculated by gravimetric method. In addition, after the measurement of lipid was completed, 0.4 mL of isopropyl alcohol was added to the glass tube, and the triacylglycerols (TAGs) was determined by the enzyme colorimetric kit method (A110-1 GPO-PAP M Enzyme Method Single Reagent Type) (Li et al. 2010).

Total sugar, starch and protein were determined using modified standard methods previously described by Liu et al. (2019).

All experiments were carried out in triplicates, and the data were the mean values ± standard deviation from three independent experiments. All statistical analyses were conducted using SPSS software. One-way ANOVA analysis and Duncan's multiple range test were performed to assess the significant differences among the light source treatments (p < 0.05).

In order to improve the growth efficiency of Chlorella in coastal saline-alkali leachate, LED lights with different light qualities were used as the light source for the growth of Chlorella. The growth curves of Chlorella sp. HQ in the coastal saline-alkali leachate under different light sources are shown in Figure 1. The five treatments exposed to blue, red, white, blue-red and blue-white mixed light, Chlorella sp. HQ maintained high speed growth, and the cell density exceeded 1.00 × 10⁷ cells·mL⁻¹ after 5 days of cultivation. After the end of cultivation, it was found that the algal density under blue light was the highest, reaching 8.71 ± 0.15 × 10⁷ cells·mL⁻¹, which was about 2–3 times that of the other treatment groups. In addition, Chlorella reached the log phase earlier under blue light than the other treatment groups, which indicated that the growth of Chlorella was significantly affected by light quality. Meanwhile, the biomass yield of Chlorella in the coastal saline-alkali leachate was determined at the end of cultivation under five different light sources (Figure 2). The results showed that biomass yield of Chlorella grown under monochromatic blue and white light were significantly higher than that of other groups (p < 0.05). And, the algal biomass obtained by Chlorella using white light in coastal saline-alkali leachate was 1.76 times higher than that using red light, which indicated that choosing a light source with a specific light quality can significantly increase the biomass of microalgae. At present, there are currently studies on increasing the biomass of microalgae by adjusting other parameters. Gao et al. (2019) achieved the purpose of increasing the biomass of Chlorella sp. G-9 by adjusting the initial organic carbon-nitrogen ratio of simulated wastewater, and the results showed the algal biomass productivities obtained in wastewater with TOC/TN ratio of 1 and 3 were 1.2 and 1.5 times higher than the value in photoautotrophic condition (TOC/TN = 0).

Based on the the experimental data of microalgae cultivation exhibited in Figure 1, a Logistic model was used to describe the light sources effects on the growth of Chlorella sp. HQ. Through the model fitting, the fitting parameters of K, r and R_max are listed in Table 1. The results showed that using red light as the light source, the specific growth rate of Chlorella sp. HQ in coastal saline-alkali leachate was the highest, reaching 0.60 d⁻¹. Maaitah et al. (2020) used olive oil washing wastewater to provide a low-cost substrate for algal biomass production and found that the maximum specific growth rate of C. pyrenoidosa was 0.4872 d⁻¹, which was lower than the maximum specific growth rate obtained in this experiment. Compared with r and K, R_max is more suitable for predicting the growth potential of microalgae (Campos et al. 2014). In this study, the R_max values of Chlorella in the monochromatic blue, white light, and blue-white mixed light groups were higher, which was consistent with the law of algal density at the end of cultivation. In contrast, the blue-red mixed light group gave the lowest growth performances in view of the R_max. This showed that Chlorella sp. HQ was more suitable to use blue light as a light source to obtain higher algal biomass in the coastal saline-alkali leachate.

Several studies have shown that the growth ability of microalgae under monochromatic blue or red light is similar or enhanced compared to white light (Mouget et al. 2004; Abiusi et al. 2014). Herein, monochromatic blue light was the optimal light source for Chlorella sp. HQ growth because it achieved the highest cell density and the highest value of R_max. Similarly, Atta et al. (2013) found that Chlorella vulgaris grew faster under blue light compared to white light with the light intensity of 200 μmol photons m⁻²·s⁻¹. In another study, Nannochloropsis sp. and Tetraselmis sp. were cultured for 14 d under blue, red, red-blue LED light and white fluorescent light, based on the results of optical density measurements, it was found that microalgae exhibited better growth curve under blue light (Teo et al. 2014). Researchers have reported that blue light not only participates in photosynthesis and energy activation, but also helps regulate gene transcription and enzyme activation (Ruyters 1984).

However, many studies have obtained different results, that is, red light is more suitable for the growth of microalgae. This may be due to the different responses of different species to light, which is reflected in product formation and cell growth of microalgae (Shu et al. 2012). Wang et al. (2007) found that the biomass yield and specific growth rate of Spirulina platensis under red light were higher than those of blue light. Xu et al. (2013) found that under different C/N ratios, the amount of algal dry weight exposed by the red, white, and yellow light groups was similar, much higher than that of the purple and blue light groups.

The generation of photosynthetic pigments is related to the growth of microalgae. Generally, the percentage of pigments in the algal biomass that is finally harvested from the culture medium is constant (Maaitah et al. 2020). In this study, the effect of five different light sources on pigment contents of Chlorella sp. HQ in the coastal saline-alkali leachate are presented in Figure 3. The total chlorophylls content and total carotenoids content of Chlorella sp. HQ in the coastal saline-alkali leachate were 0.81%–1.70% and 0.08%–0.25%, respectively. It has been reported that the relative proportion of photosynthetic pigments changes with the spectral quality of light (Rochet et al. 1986). The photosynthetic pigments content of Chlorella in the coastal saline-alkali leachate under the blue-red mixed light was significantly higher than that of monochromatic blue and red light (p < 0.05). The chlorophyll a content of Chlorella in the blue-white mixed light group was 1.38 times and 2.27 times that of the monochromatic blue and white light groups, respectively. Similarly, the content of chlorophyll b and carotenoids in the blue-white mixed light group was also higher than that in the monochromatic blue and white light groups. The above results showed that compared with the monochromatic light group, the mixed light group was more conducive to the accumulation of pigments of Chlorella sp. HQ in the coastal saline-alkali leachate. Pereiraa & Oterob (2019) reported that blue light can increase the total carotenoid content in Dunaliella salina, and when it was mixed with red light, due to the synergistic effect, the carotenoid content was significantly higher than the other two monochromatic light experimental groups. The explanation on the higher production of chlorophyll and carotenoid under mixed LED light treatments (blue-red and blue-white mixed light) can be related to complementary chromatic adaptation. According to Emerson and Brody's theory, complementary chromatic adaption is the process of adjusting relative content of major light harvesting pigments which have similar absorption spectra patterns to that of the incident light. And complementary chromatics adaptation can improve the algae cells photosynthetic ability (Rochet et al. 1986; Mouget et al. 2004).

In addition, the chlorophyll b and carotenoids content in the blue light group were higher than those in the red light group, while the content of chlorophyll a was not significantly different (p < 0.05). The reason may be that chlorophyll a in microalgae can absorb blue and red light, while other auxiliary pigments (such as carotenoids) absorb blue light (Kandilian et al. 2013). Similarly, Vadiveloo et al. (2015) found that the content of chlorophyll a in Nannochloropsis sp. exposed the blue light was higher than that of red light. Tamburic et al. (2014) found that the microalgae had a higher photosynthetic rate when exposed to monochromatic blue light than to monochromatic red light, which due to that blue light was more easily absorbed by microalgae than red light and was more efficiently transmitted to photosystem II under light-saturated illumination. Therefore, the photosynthetic efficiency of Chlorella under blue light was higher than red light, and higher biomass productivity can be obtained. In this work, we obtained similar results, that is, the biomass yield of Chlorella in the coastal saline-alkali leachate under blue light was higher than that of red light (Figure 2).

Figure 4 demonstrates the total lipid per algal biomass and TAGs content per total lipids yield of Chlorella sp. HQ in the coastal saline-alkali leachate with different light sources at the end of cultivation. The results showed that the lipid content of Chlorella in the coastal saline-alkali leachate was 29.31%–62.95%, and the value under red light was the highest, which was significantly higher than the blue light group and the blue-red mixed light group (p < 0.05). Maaitah et al. (2020) found that using different dilutions of olive oil washing wastewater as the culture medium of Chlorella pyrenoidosa, the lipid content of Chlorella under undiluted conditions was the highest, reaching 51.5%, which was lower than the highest lipid content of Chlorella sp. HQ in the coastal saline-alkali leachate.

The TAGs content per lipid yield of Chlorella was the highest under blue-white mixed light, reaching 7.10 ± 0.35%, which were 2.73 and 1.48 times that of monochromatic blue and white light, respectively. The accumulation of lipids in algal biomass is often associated with a decrease in biomass productivity (Duarte et al. 2019). In this experiment, the total lipid content per unit biomass under red light was significantly higher than the blue light and blue-red mixed light, while the biomass under red light was the lowest (Figures 2 and 4). This indicated that red light was conductive to lipid accumulation but was not good to the growth of biomass of Chlorella in coastal saline-alkali leachate.

In addition, in the previous study, the fluorescent lamp was used as light source, the protein, total sugar and starch content of Chlorella sp. HQ in coastal saline-alkali leachate were higher than those of SE medium, indicating that the accumulation of high-valued substances can be improved under salinity stress (Liu et al. 2020). In this study, under blue light conditions where Chlorella sp. HQ can obtain higher biomass in saline-alkali leachate, the protein and total sugar content of Chlorella was 50.09% and 24.73%, respectively. Rashid et al. (2019) found that Ettlia sp. and Chlorella sp. HS-2 was co-cultured in BG11, and its protein and carbohydrate content were 44 and 33%, respectively. This indicated that the coastal saline-alkali leachate can obtain a higher content of high-value substances under the adjustment of suitable light quality. As shown in Figures 5 and 6, the accumulation characteristics of high-value substances of Chlorella under different light qualities have been further explored. The results showed that at the end of experiment, the protein, total sugar and starch contents in blue-red mixed light cultures were higher than those in monochromatic red and blue light, which were 64.71 ± 1.63 × 10⁻⁷ μg·cell⁻¹, 46.92 ± 1.05 × 10⁻⁷ μg·cell⁻¹ and 10.20 ± 0.23 × 10⁻⁷ μg·cell⁻¹, respectively. In addition, statistically significant differences were observed among blue-white mixed light, monochromatic blue, and white light groups, the protein, total sugar and starch content in blue-white mixed light cultures remaining higher than the monochromatic blue and white light groups (p < 0.05). This indicated that mixed light treatments (blue-red and blue-white) were more appropriate for microalgal protein, total sugar and starch accumulation than monochromatic LED light groups (monochromatic red, blue, and white) in the coastal saline-alkali leachate.

The total sugar and starch content of Chlorella in the coastal saline-alkali leachate under blue light lower than those of other light groups, which may be due to the fact that blue light can promote the production and activity of respiratory enzymes in microalgae. The increase in the concentration and activity of respiratory enzymes accelerates the rate of decomposition of carbohydrates, resulting in a reduction in carbohydrate content (Kowallik 1982; Ruyters 1984). Similarly, Vadiveloo et al. (2015) found that the carbohydrate content of Nannochloropsis sp. under blue light was lower than that of other light treatments. However, several studies have shown that under different light qualities, the light-harvesting pigments and membranes of microalgae are changed due to various types of algae, resulting in different aspects responses of microalgae to light spectra (Saavedra & Voltolina 1994).

Besides, the effect of light quality on the microalgae growth is a complex process, which is not only related to photosynthesis pathways, but also stimulates photoreceptors, affects microalgae cell division, enzyme activation, gene transcription, and so on (Oldenhof et al. 2006; Abiusi et al. 2014). For example, in this paper, the total lipid content of Chlorella sp. HQ in the coastal saline-alkali leachate under red light was significantly higher than that of monochromatic blue and white light, while another study found that Nannochloropsis sp. had significantly higher lipid content under blue light than that of red light and white light (Vadiveloo et al. 2015). The reason may be that the activities of carbonic anhydrase and ribulose biphosphate carboxylase/oxygenase which play a vital role in the regulation of the microalgal carbon cycle are enhanced under blue light (Eskins et al. 1991).

In general, by optimizing the light quality of the light source required for culturing Chlorella in saline-alkali leachate, the light conditions can be controlled more accurately, and the high yield of algal biomass and the maximum utilization of coastal saline-alkali leachate resources can be achieved.

Based on the experimental results obtained in this study, optimizing light quality can harvest higher algal biomass from the coastal saline-alkali leachate. Compared with monochromatic lights, blue-red mixed light and blue-white mixed light were more conducive to the accumulation of photosynthetic pigment of Chlorella, and complementary chromatics adaptation can improve the algae cells photosynthetic ability. While the highest total lipid content per unit biomass was achieved under monochromatic red light. In addition, mixed LED light (blue-red light and blue-white light) can be used to improve the production of high-valued products. Therefore, if obtaining more biomass and high-valued substances is the desired goal, then a two-stage control of light quality can be considered, such as using blue light first and then using blue-red mixed light as the light source, so as to maximize the benefits of saline-alkali leachate resources utilization.

The authors would like to thank Prof. Hu Hong-Ying for the experimental facilities. This study was supported by National Natural Science Foundation (No. 52071030) and the Fundamental Research Funds for the Central Universities (No. 2017ZY43).

All relevant data are included in the paper or its Supplementary Information.