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

Using saline-alkali leachate to cultivate microalgae is an effective way to realize the utilization of wastewater and alleviate the shortage of water resources. Light source is usually used as an optimized parameter to further improve the cultivation ef ﬁ ciency of microalgae. In this work, the in ﬂ uence of light qualities on the growth and high-valued substances accumulation of Chlorella sp. HQ in coastal saline-alkali leachate were investigated. The speci ﬁ c growth rate of Chlorella in coastal saline-alkali leachate was 0.27 – 0.60 d (cid:1) 1 . At the end of cultivation, the algal density under blue light reached 8.71 ± 0.15 × 10 7 cells·mL (cid:1) 1 , which was signi ﬁ cantly higher than the other light groups. The lipid content in the biomass was 29.31 – 62.95%, and the highest lipid content and TAGs content were obtained under red light and blue-white mixed light, respectively. Percentages of total chlorophylls (0.81 – 1.70%) and carotenoids (0.08 – 0.25%) were obtained in the ﬁ nal biomass of the coastal saline-alkali leachate. In addition, the contents of photosynthetic pigments and three high-valued products under mixed light were higher than those of monochromatic light, and the protein, total sugar and starch content under blue-red mixed light was 1.52 – 3.76 times, 1.54 – 3.68 times and 1.06 – 3.35 times of monochromatic blue light and red light, respectively.


GRAPHICAL ABSTRACT INTRODUCTION
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. ). 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. ). 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. ; Meena et al. ). 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 it 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. ). At present, the global water consumption is gradually increasing, and a large part of the population still lives in waterscarce areas (Zhu & Dou ; Archer et al. ). 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.
And our previous studies have shown that the coastal salinealkali leachate can be used to cultivate three oleaginous microalgae at low cost to realize the conversion of wastewater resources (Liu et al. ).
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. ). 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. ; Chandra et al. ). 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. ). 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 CO 2 concentration can significantly affect the algal growth and lipid accumulation including content and composition (Ananthi et al. ).
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. ).
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. ). 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. ). 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. ). Shu et al. () 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. () 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.

Microorganism
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 con- 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.

Cultivation setup
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. (). 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 × 10 5 cells·mL À1 , 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 ¼ 1:2; blue:white ¼ 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.

Microalgal growth and mathematic model analysis
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. A generalized Logistic model was fitted to the measured data (cell density) with Origin software.
In the above formula, N t (cells·mL À1 ) 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)).

Lipid and triacylglycerols determination
The lipid content was determined based on the method of modified chloroform-methanol extraction (Bligh & Dyer ). 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 trans-

Photosynthetic pigments determination
The photosynthetic pigments analysis followed the modified procedure as used from Kirk & Allen (). A 5 mL sample was centrifuged at 8,000 rpm for 5 min. The pellets were re-suspended in 5 mL of 80% acetone solution at 4 C for 24 h in darkness. After centrifugation at 4 C, 4,000 rpm for 15 min, the obtained supernatant was measured for absorbance at 480 nm, 645 nm, and 663 nm, respectively. The 80% acetone solution was used as the blank. The photosynthetic pigments content was calculated as shown below: Chlorophyll a (mgÁL À1 ) ¼ 12:7 × OD 663 À 2:69 × OD 645 ð1Þ where OD λ is optical density at wavelength λ (nm).

Protein, total sugar and starch determination
Total sugar, starch and protein were determined using modified standard methods previously described by Liu et al.

Statistics
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 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. ).

RESULTS AND DISCUSSION
Similarly, Vadiveloo et al. () found that the content of chlorophyll a in Nannochloropsis sp. exposed the blue light was higher than that of red light. Tamburic et al. () 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).   (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.

Comparison of high-valued products accumulation of
In addition, in the previous study, the fluorescent lamp was used as light source, the protein, total sugar and starch content   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. ).
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. ).
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.

CONCLUSION
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 highvalued products. Therefore, if obtaining the more biomass and high-valued substances is the desired goal, then a twostage control of light quality can be considered, such as using blue light first and then using blue-red mixed light as light source, so as to maximize the benefits of saline-alkali leachate resources utilization.