Microalga strain, chemicals and culture medium
The green microalga strain of M. reisseri FM1, isolated from pipe-type photobioreactor in a local microalgae cultivation company was used in the present study (Liu et al. 2021b). The algal strain was purified by streak plate method to satisfy the requirements of pure cultivation. The purified algal strain was cultured in liquid medium to obtain seed liquid. NaAc, NaNO3, K2HPO4, MgSO4•7H2O, CaCl2•2H2O, KH2PO4, NaCl, FeCl3•6H2O, EDTA-Na, NaOH, HCl, ethanol and other reagents were all analytical reagent and purchased from domestic chemical reagent company. The modified soil extract (SE) medium referenced from Freshwater Algae Culture Collection of the Institute of Hydrobiology, China (http://algae.ihb.ac.cn/MeSearch.aspx) was adopted to cultivate the M. reisseri FM1, in which the soil extract (40 mL) was removed compared with normal SE. The medium consisted of (g L-1): 0.25 g NaNO3, 0.75 g K2HPO4, 0.75 g MgSO4∙7H2O, 0.25 g CaCl2∙2H2O, 0.175 g KH2PO4, 0.25 g NaCl, 0.05 g FeCl3∙6H2O, EDTA-Fe, 1 mL of A5 solution [0.22 g ZnSO4∙7H2O, 1.86 g MnCl4∙4H2O, 2.86 g H3BO3, 0.08 g CuSO4∙5H2O, 0.05 g Na2Mo7O24∙7H2O, 0.08g Co (NO3)2∙6H2O].
Purified M. reisseri FM1 colony from the SE agar plate was transferred into sterilized SE liquid medium in 250-mL flasks to prepare the inoculum. M. reisseri FM1 cells of seed cultures in the exponential phase were used for subsequent experiments. To investigate the effects of NaAc on the growth, cellular components (pigments, proteins, carbohydrates), lipid content and fatty acid composition of M. reisseri FM1, different concentrations and feeding modes (batch and fed-batch) of NaAc were added into SE medium as carbon source. In batch culture, different concentrations (1, 2, 4, 6, 8 and 10 g L-1) of NaAc were supplemented into 250 mL flask containing 100 mL sterile SE medium, photoautotrophic group without adding NaAc was used as the control. In the batch culture process, 4 g L-1 and 8 g L-1 of NaAc were directly added into SE medium. Fed-batch culture was designed that NaAc was intermittently fed three times at 1, 2 g L-1 concentration during cultivation at 2, 4 and 6 days respectively, which ensured that the final concentration was 4 and 8 g L-1. Initial NaAc concentrations of 4 g L-1 and 8 g L-1 under batch cultures were used as the control.
All the culture medium were sterilized at 121 °C and 0.1 MPa for 20 min, the purified inoculum of M. reisseri FM1 was inoculated with a volume of 5% (v/v). All the cultures were cultivated in a shaker incubator at 150 rpm and 25±1 ℃ under 5000 lux (around 95 μmol m-2 s-1), 12: 12 h light-dark period (HNY-2102C, Honour, China).
Determination of growth curve and kinetic parameters
The growth profile of M. reisseri FM1 was measured at 680 nm spectrophotometrically (V-5000, Shanghai, China) after every 24 h for all test groups to plot growth curve (Li et al. 2015). The dry weight concentration of algal cells was measured by gravimetric method. The algae liquid was centrifuged at 3052 g for 10 min, and the supernatant was discarded. The algae pellet was collected after being washed twice with deionized water, then dried until constant weight. The relationship between optical density and DCW was established by linear regression. The specific growth rate (μ, day−1) and biomass productivity (PB, mg L-1 d-1) were calculated using equation (1) and (2):
μ = ln (W2/W1) / (t2-t1) (1)
PB = (W2-W1) / (t2-t1) (2)
where, W2 and W1 are the values of biomass concentration (g L-1) on the days t2 and t1, respectively. Biomass productivity (PB, g L-1 d-1) was obtained according to the following calculation (Song and Pei 2018).
Extraction and determination of cellular components
Pigments Photosynthetic pigments (chlorophylls and carotenoids) were analyzed according to previous work with minor modifications (Basu et al. 2013). 5 mL of culture from each sample were collected and centrifuged at 3052 g for 10 min. The algal pellet was extracted with 95 % ethanol, mixed well and incubated at 4 °C in dark overnight. Afterwards, mixture was centrifuged at 3052 g and the supernatant was used for pigment estimation by measuring the absorbencies at 470, 649 and 665 nm, Total chlorophyll (Ct) and carotenoid (Cc) concentrations were calculated using Eq. (3) to (7) as follows (Kong et al. 2020):
Ca = 13.95 × A665 - 6.88 × A649 (3)
Cb =24.96 × A649 - 7.32 × A665 (4)
Ct = Ca + Cb (5)
Cc = (1000 × A470 - 2.05 × Ca - 114.8 × Cb) / 254 (6)
C (mg g-1) = [Ct (or Cc) × Vt × D] / W (7)
where C: pigment content (mg g−1), Ct: total chlorophylls content (mg mL−1), Cc: carotenoids content (mg L−1), Vt: total volume of extract (L), D: dilution factor, and W: dry weight of algal cells (g).
Lipids Total lipids in the algal biomass were extracted and analyzed using the sulfo-phospho-vanillin assay described by Mishra et al (2014). Briefly, 25 mg of the lyophilized algal biomass was resuspended in 5 mL of distilled water and mixed by a vortex mixer completely. 100 μL of algal liquid, 1 mL of deionized water and 2 mL of concentrated sulfuric acid were added to fresh tube successively. The mixture was heated for 10 min in boiling water, then cooled for 5 min in ice bath. 5 mL of freshly prepared phospho-vanillin reagent were added into each tube and measured the optical density at 530 nm after incubation for 15 min at room temperature. Refined olive oil was used as a standard. Total lipid content was confirmed using Eq. (8) as follows:
Total lipid content (mg mL-1) = 2.1585 × A530 - 0.0159 (8)
Carbohydrates The carbohydrate content was estimated by phenol-sulphuric acid method as described by Vijay et al (2021). 50 mg of disrupted and ground algal biomass was resuspended in 25 mL distilled water and kept in water bath at 80℃ for 1 h for the extraction of soluble carbohydrates, and the process was repeated once again. The mixture was then centrifuged at 3052 g for 15 min, and the supernatant was collected and merged. Finally, the constant volume of the extract was set to 50 mL with distilled water. 1 mL of supernatant was mixed with 1 mL distilled water, 1 mL of 6% phenol and 98% sulphuric acid, shaken well and incubated for 20 min. The mixture was used to measure the absorbance at 490 nm. Total carbohydrate content was quantified from the calibration curve prepared using glucose as standard and expressed as percentage of dry cell weight (%, Eq. 9).
Carbohydrate content (%) = (C × VT × N) * 100 / (W × VS × 106) (9)
where C: glucose content (μg), VT: total volume of extraction liquid (mL), N: Diluted multiples, W: the sample quality (g), VS: the volume of the sample taken for determination, and W: dry weight of algal biomass (g).
Proteins The proteins content was determined by the micro-Kjeldahl method using 100 mg of the lyophilized and ground algal powder and calculated the proteins content according to the following Eq. (10) and (11) (Aziz and Mitu 2019).
Sample Nitrogen content (%) = [C ×(V1-V2) ×0.014] × 100 / W (10)
Protein content (%) = Nitrogen content (%) × 6.25 (11)
where C: average quantity of standard solution of hydrochloric acid when titrated sample (mol L-1), V1: average quantity of standard solution of hydrochloric acid when titrated control (mL), V2: and W: dry weight of algal biomass (g).
Determination of fatty acid compositions by GC-MS
Lyophilized and disrupted algal biomass was blended with chloroform/methanol (2:1, v/v). The mixture was agitated and extracted for 3 h. The chloroform phases were collected and evaporated by rotary evaporator (RE-2000E, Yarong, China) to determine the fatty acid methyl esters (FAMEs). Extracted lipids were subjected to methyl esterification reaction via KOH-methanol method according to GB/T 17376-2008. Samples were then assayed by GC-MS (Agilent, 7890B-5977A, USA). Optimized detecting conditions of GC-MS were described in our previous study (Kong et al. 2020).
All experiments were carried out in triplicates. The data shown in table and figure were expressed as the Mean ± SD (standard deviation). The statistical differences between experimental groups were determined by analysis of variance (ANOVA) using the SPSS Statistics (18.0, USA).