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Article

Research article

J. Microbiol. Biotechnol. 2020; 30(6): 868-877

Published online June 28, 2020 https://doi.org/10.4014/jmb.1910.10035

Copyright © The Korean Society for Microbiology and Biotechnology.

β-Carotene Production from Dunaliella salina Cultivated with Bicarbonate as Carbon Source

Yimei Xi , Jinghan Wang , Song Xue and Zhanyou Chi *

School of Bioengineering, Dalian University of Technology, Dalian 116024, P. R. China

Correspondence to:Zhanyou  Chi
chizhy@dlut.edu.cn

Received: October 16, 2019; Accepted: March 10, 2020

Abstract

Bicarbonate has been considered as a better approach for supplying CO2 to microalgae cells microenvironments than gas bubbling owing to cost-effectiveness and easy operation. However, the β-carotene production was too low in Dunaliella salina cultivated with bicarbonate in previous studies. Also, the difference in photosynthetic efficiency between these two carbon sources (bicarbonate and CO2) has seldom been discussed. In this study, the culture conditions, including NaHCO3, Ca2+, Mg2+ and microelement concentrations, were optimized when bicarbonate was used as carbon source. Under optimized condition, a maximum biomass concentration of 0.71 g/l-1 and corresponding β-carotene content of 4.76% were obtained, with β-carotene yield of 32.0 mg/l-1, much higher than previous studies with NaHCO3. Finally, these optimized conditions with bicarbonate were compared with CO2 bubbling by online monitoring. There was a notable difference in Fv/Fm value between cultivations with bicarbonate and CO2, but there was no difference in the Fv/Fm periodic changing patterns. This indicates that the high concentration of NaHCO3 used in this study served as a stress factor for β- carotene accumulation, although high productivity of biomass was still obtained.

Keywords: 6Dunaliella salina, &beta,-carotene, bicarbonate, microelement, carbon source

Introduction

Dunaliella salina is a halo-tolerant microalgae species that accumulates high levels of β-carotene [1, 2]. Although it has been used in commercial production of carotene for several decades, its production cost is still very high, limiting its application to only high-value nutraceuticals [3]. For products of lower value but much larger market size, such as animal and aquaculture feed additives, the current production cost of Dunaliella is much too high to be economically feasible, and needs to be significantly reduced.

Carbon accounts for about 50% of microalgae’s dry weight, and its supply is very important in the development of cost-effective microalgae cultivation processes. In conventional cultivation systems, inorganic carbon is usually supplied as gaseous CO2, which has caused serious technological difficulties in photobioreactor (PBR) design and operation [4,5,6], ultimately resulting in high production cost. Recently, bicarbonate has been suggested as a better approach to supplying carbon, with the advantages of easy transportation, handling, and storage. In such conditions, an aeration-free PBR can be used as a low-cost cultivation system [7-10].

There have been some reports on employing sodium bicarbonate to stimulate β-carotene accumulation in D. salina [8, 9, 11, 12]. However, β-carotene production reached in these studies was much lower than the conventional cultivation with CO2, amounting to only 8.25 ± 0.01 mg/l [12], 7.10 ± 0.08 mg/l [9], and 20.43 ± 2.84 mg/l [8], respectively. These levels are too low to be used as routine practice in large-scale production. As a matter of fact, when bicarbonate is used as carbon source, the optimal culture condition for β-carotene accumulation may be different from that with CO2. In this situation, it is necessary to disclose the effects of different factors on cell growth and β-carotene accumulation with bicarbonate as carbon source.

Also, when bicarbonate is used in the culture, pH drift is inevitable, and HCO3- converts to CO32- at high pH. Seawater is usually used as culture medium for Dunaliella sp., and the high concentration of Ca2+ and Mg2+ in it may precipitate with CO32-, since CaCO3 precipitation usually occurs when pH exceeds 8.5, and Mg(OH)2 precipitation forms when pH exceeds 10. Although pH adjustment with acid can avoid this precipitation, it is not practical for outdoor large-scale cultivation since additional chemicals and sophisticated equipment are required. An alternative method to avoid this precipitation is reducing the concentrations of Ca2+ and Mg2+, but this reduction to a certain level may limit the production of carotene. Thus, the effects of Ca2+ and Mg2+ concentrations need to be carefully investigated.

β-carotene accumulation is usually triggered by environmental stress such as light, temperature, salinity, or nitrogen depletion [13, 14]. Fv/Fm is the photosystem II (PS II) maximum photochemical quantum yield. It is sensitive to stress conditions for microalgae, and has been established as a quantitative indicator that shows certain stress levels. For example, the value of Fv/Fm can reflect fatty acid accumulation in response to nitrogen depletion [15]. Fv/Fm was also used as an indicator in Dunaliella sp. in previous study to disclose the mechanism of β-carotene accumulation in response to different stresses [12]. Thus, the Fv/Fm was monitored in this study, to compare the stress the cells experienced in cultivation with either bicarbonate or CO2, which finally affects the β-carotene accumulation.

The present study, therefore, investigated the effects of various factors on D. salina growth and β-carotene production when sodium bicarbonate is used as carbon source. Different concentrations of sodium bicarbonate, as well as Ca2+ and Mg2+ were investigated for D. salina growth and β-carotene accumulation. Central composite design experiments were carried out for studying the effect of microelements on biomass and β-carotene content of D. salina. Finally, maximal PS II quantum yield and β-carotene content were analyzed and compared between bicarbonate and CO2-based cultivation.

Materials and Methods

Strain and Medium

The microalgae D. salina CCAP 19/18 was purchased from Culture Collection of Algae and Protozoa agencies (UK), and it was maintained in Artificial Sea Water (ASW). The nutrient medium was 1.5 M NaCl, 5 mM KNO3, 4.5 mM MgCl2·6H2O, 0.5 mM MgSO4·7H2O, 3 mM CaCl2·2H2O, 0.13 mM K2HPO4, 0.02 mM FeCl3, 0.02 mM EDTA, 25 mM NaHCO3, 1 ml of trace elements stock with 50 mM H3BO3, 10 mM MnCl2·4H2O, 0.8 mM ZnSO4·7H2O, 1.0 mM CuSO4·5H2O, 2 mM NaMoO4·2H2O, 1.5 mM NaVO3, 0.8 mM CoCl2 ·6H2O, and the pH was adjusted to 7.5 by addition 40mM of Tris-buffer [16]. Before inoculation, microalgae were cultivated in batch mode to promote fast growth in 500 mL conical flasks with light intensity of 40 μmol/m-2/s-1 and alternating 12 h/ 12 h light/dark cycles. After inoculation, the initial cell concentration in each horizontal photobioreactor (PBR) was about 0.2 ×106 cells/ml-1. The horizontal PBRs were polystyrene boxes of 12 cm × 12 cm, with a working volume of 250 ml and a light path of 20 mm. Light was provided by white LEDs, with intensity on the top surface of the PBR controlled at 200 μmol/m-2/s-1 and under 12 h/12 h light/dark cycles. Cultivation temperature was controlled at 25 ± 0.5°C in the illumination incubator. Each experiment below was carried out in triplicate.

Growth Analysis

Cell numbers were counted daily using a hematocyto-meter. Dry weight (DW, g/l) was measured by using pre-weighed Whatman GF/C filters [17, 18]. Ten milliliter cultures were filtered and washed three times with 2 ml 0.5 M ammonium bicarbonate and then were dried below 60°C for over 16 h until the weight was constant. The DW of the microalgae cells was calculated according to the final weight and volume of the filtered sample.

Analysis of microalgal β-carotene was based on methods described in Mojaat [19]. For β-carotene content measurement, 10 mg of dried biomass was extracted with 1 ml acetone and vortexed for 20 s. Then, it was centrifuged at 10,000 ×g for 10 min. This extraction was repeated twice. The extracts were filtered by 0.45-μm pore size (PTFE) membrane syringe filters (1.7 cm2). All extracts were treated using amber glass vials with screw caps to protect carotenoids from degradation under light. The β-carotene analysis was carried out by High-Performance Liquid Chromatography (HPLC, Agilent Technologies 1100, USA). The mobile phase was 10% acetonitrile and 90% methanol. The flow rate was 1 ml/min-1, and the detection wavelength was 452 nm. The standard sample of β-carotene was purchased from Sigma (Sigma-Aldrich, USA).

Experimental Design and Data Analysis

Influence of sodium bicarbonate concentrations on D. salina growth. To test the effect of NaHCO3 concentrations on D. salina CCAP 19/18, six concentration gradients were selected, i.e., 25, 50, 100, 200, 300, and 500 mM. The concentrations of NaCl, Ca2+, and Mg2+ in the culture medium were 1.5 M, 3.0 mM, and 5.0 mM, respectively.

Influence of Ca2+ and Mg2+concentrations on D. salina growth. When optimizing the concentrations of Ca2+ and Mg2+ for D. salina growth, gradients of Ca2+ and Mg2+ concentrations were from 0.3 to 3.0 mM, and from 0.5 to 5.0 mM, respectively, as listed in Table 1. Optimized NaHCO3 concentration (200 mM) was adopted for all cultures with various Ca2+ and Mg2+ concentrations.

Table 1 . Different concentrations of Ca2+, Mg2+ investigated in this study..

No.Ca2+ (mM)Mg2+ (mM)
A0.30.5
B0.61.0
C1.22.0
D1.83.0
E2.44.0
F3.05.0


Central Composite Design

With a Plackett-Burman (PB) design, three significant microelements listed in Table 2 were screened from FeCl3·6H2O, H3BO3, ZnSO4·7H2O, CoCl2·6H2O, CuSO4·5H2O, MnCl2·4H2O, NaMoO4·2H2O, and NaVO3, as displayed in Table S1. A central composite design was used to investigate their effects on dry cell weight and D. salina β-carotene yield. The design matrix was a 24 full factor design combined with five central points, and eight axial points where one variable was set at an extreme level while other variables were set at their central point levels. The coded and real values of each parameter are shown in Table 2. Based on Table 3, the responses (cell dry weight and β-carotene yield) were correlated as a function of variables by a second-order polynomial equation, i.e.,

Table 2 . The coded and real values of the independent variables in the central composite design..

VariablesUnit-2-1012
FeCl3•6H2OμM1.855.929.9914.118.1
CoCl2•6H2OμM0.340.971.602.232.86
NaVO3μM0.330.91.482.052.62

Table 3 . The central composite design of the significant (in coded level) with DW and β-carotene yield as responses..

RunFeCl3•6H2OCoCl2•6H2ONaVO3DW (g/l)β-carotene (%)
111-10.6454.35
21-110.6374.27
3-1110.6854.76
4-1-1-10.6644.27
5-2000.714.28
62000.6294.59
70-200.6914.16
80200.6654.55
900-20.6714.21
100020.7083.85
110000.6953.97
120000.6963.94
130000.7073.86
140000.694.02
150000.7093.86


Y = β0 ΣβiXi++ΣβiXi2 Σ+βijXiXj (1)

where Y is the predicted response, β is the coefficient of the equation, and xi and xj are the coded levels of variables i and j, respectively. The software Design-Expert (Stat-Ease Inc., USA) was adopted for this correlation through non-linear regression. An F-test was used to evaluate the significance of the models.

NaHCO3 vs. CO2 Cultivation with Online Monitoring

To confirm the experimental results obtained from the NaHCO3, D. salina was cultivated with a multi-device-equipped flat-plate photobioreactor (PBR)-Algal station [15] in both NaHCO3-based (optimal NaHCO3 concentration) and CO2-based (2%) cultivations. Light was provided by white LEDs, with intensity on the top surface of the PBR controlled at 200 μmol/m-2/s-1 and under 12 h/12 h light/dark cycles. Light path of the PBR was 20 mm and total cultivation volume was 1 L. The cultivation temperature was automatically controlled at 25±0.5°C. Cultures in the PBR were agitated with 0.2-μm membrane-filtered air at 200 ml/min (NaHCO3-based culture) or with 2% CO2 (CO2-based culture). Maximal PS II quantum yield (Fv/Fm), OD, and pH value were recorded online.

Results

Effect of NaHCO3 Concentration on Cell Growth and β-Carotene Accumulation

Growth curves for D. salina cultivated under a series of NaHCO3 concentrations are shown in Fig.1A, indicating D. salina could grow well in medium with NaHCO3 as carbon source. Cell densities of 25, 50, and 100 mM NaHCO3 cultures increased rapidly during the first 3 days of cultivation, and were higher than those of 200, 300, and 500 mM cultures. After that, cell density decreased in cultures with NaHCO3 concentrations lower than 100 mM. The lowest cell density was observed in 25 mM NaHCO3 and the highest cell density was recorded in 200 mM NaHCO3 culture, which was 1.65 × 106 cells/ml-1 on Day 7. As far as the DW is concerned, the highest DW was obtained on Day 7 with 200 mM NaHCO3 concentration (0.66 ± 0.02 g/l), whereas the lowest DW of 0.35 ± 0.01 g/l was obtained in culture with 50 mM NaHCO3 (Fig. 2). It was interesting that D. salina was able to grow in culture with 500 mM NaHCO3, although 200 mM NaHCO3 enabled the fastest growth.

Figure 1. Effect of different concentrations of NaHCO3 on (A) D. salina cell density; (B) pH of the culturing broth. Values represented as mean ± SD (n = 3).
Figure 2. Effect of NaHCO3 concentrations on DW, β-carotene content, and β-carotene yield of D. salina. Values represented as mean ± SD (n = 3).

Correspondingly, pH variations of these cultures were displayed in Fig. 1B. The pH values of the six cultures spiked on Day 1 then increased slowly afterwards. At Day 7, the highest and lowest pH values were obtained in 25 mM culture (pH = 10.3) and 500 mM culture (pH = 9.8), respectively. For cultures with ≥100 mM NaHCO3, pHs in these cultures were below 10.0 during the whole cultivation time. When correlating pH with microalgal growth, cultures with higher pH resulted in lower growth rates, indicating that elevated pH inhibited growth of D. salina, although it was able to grow at pH value higher than 10.0. Moreover, these results indicated that NaHCO3 had a strong buffering effect on pH.

When correlating NaHCO3 concentration with DW, the cellular β-carotene content levels of D. salina grown under different NaHCO3 concentrations were displayed in Fig. 2. The highest β-carotene content of 4.20 ± 0.12% DW was obtained in culture with 200 mM NaHCO3, and the corresponding β-carotene yield was 27.72 ± 1.65 mg/l. In contrast, the lowest β-carotene content of 2.1 ± 0.11% was obtained in 25 mM culture, reaching β-carotene yield of 9.77±0.45mg/l. In culture with 500mM NaHCO3 concentration, although high β-carotene content of 3.7 ± 0.11% was obtained, the overall available β-carotene yield was only 12.95 ± 0.46 mg/l owing to limited microalgal growth. From these results, it was implied that NaHCO3 concentration could greatly influence cell growth and β-carotene accumulation of D. salina, and 200 mM was optimal for both biomass and β-carotene production in this study.

Effect of Ca2+ and Mg2+ Concentrations on Cell Growth and β-Carotene Accumulation

The correlation between Ca2+, Mg2+ and growth of D. salina was shown in Fig. 3A. Cell numbers ranged from 1.40 to 1.65 × 106 cells/ml under different Ca2+ and Mg2+ concentrations, and significant differences were observed among all cultures on Day 7 (p = 0.03 < 0.1). The highest cell number was obtained in culture with 3.0 mM Ca2+ and 5.0 mM Mg2+, while the lowest cell number was obtained in culture with 0.3 mM Ca2+ and 0.5 mM Mg2+. pH values of all cultures on Day 7 displayed insignificant differences (p = 0.12 > 0.1), with values around 9.8 (Fig. 3B).

Figure 3. Effect of Ca2+ and Mg2+ concentrations on (A) cell number of D. salina; (B) pH of the culturing broth. Values represented as mean ± SD (n = 3).

As shown in Fig. 4, DW was positively correlated with Ca2 + and Mg2 + concentrations, with the highest and lowest DW (0.69 g/l and 0.61 g/l) respectively at the same culturing conditions regarding cell numbers (Fig. 3A). In contrast, β-carotene content was negatively correlated with Ca2 + and Mg2 + concentrations, with the highest and lowest β-carotene content (respectively 4.5% and 4.1%) obtained at culturing conditions opposite to those of cell density and DW. It was noteworthy that β-carotene yield obtained in cultures with different Ca2+ and Mg2 + were around the same levels (26.2 to 27.5 mg/l), displaying no significant differences (p = 0.87 > 0.10). Thus, low concentration Ca2+ and Mg2+ of 0.3 mM and 0.5 mM respectively, are adequate for β-carotene accumulation.

Figure 4. Effect of Ca2+, Mg2+ concentrations on DW and β-carotene accumulation of D. salina. Values represent as mean ± SD (n = 3).

Effect of Micronutrients on Cell Growth and β-Carotene Accumulation

Optimization of significant factors. For central composite design, the central point values and level ranges of three significant factors were selected according to the PB design results (Table S1). As shown in Table 3, the central point in the central composite design was repeated five times, and standard deviation of these five replicates used to determine the experimental errors were 0.008 g/l for DW, and 0.07% for β-carotene content. The experimental data of DW and β-carotene content in Table 3 were correlated as functions of the three variables by a second-order polynomial equation using the Design-Expert software. The coefficient values in Eq. (1) and their p-values and F-values were listed in Table 4. The optimal value of the three variables were derived by Design-Expert software, and the maximum dry cell weight of 0.71 g/l was obtained in culture with 1.85 μM FeCl3·6H2O, 1.6 μM CoCl2·6H2O, and 1.48 μM NaVO3 concentrations. The maximum β-carotene content of 4.76% was obtained in culture with 5.92 μM FeCl3·6H2O, 2.23 μM CoCl2·6H2O, and 2.05 μM NaVO3. From p-levels in Table 4 and the response surfaces in Fig. 5, it is evident that microelements, especially Fe3+ and Co2+ and their concentrations, can have significant influences on D. salina growth and β-carotene accumulation.

Table 4 . The values of coefficients in the second-order polynomial and the associated statistical test for DW and β-carotene..

VariableDWβ-carotene


F-valuep-valueEstimateF-valuep-valueEstimate
Model12.060.00680.712.110.00673.96
A- FeCl3•6H2O36.440.0018-0.0293.050.04120.1
B- CoCl2•6H2O3.750.1104-9.19E-035.520.06570.14
C- NaVO37.60.040.0134.70.0824-0.13
AB2.150.20279.83E-037.660.0395-0.23
AC6.010.0579-0.0163.09E-030.9578-4.61E-03
BC3.140.1366-0.0121.348E-070.9997-0.00003048
A^227.20.0034-0.01870.580.00040.36
B^215.770.0106-0.01415.270.01130.17
C^25.230.0709-7.82E-030.0040340.95180.002685

Figure 5. Three-dimensional response surface plot of (A) DW and (B) β-carotene content as a function of FeCl3·6H2O and CoCl2·6H2O.

Three-dimension surface responses were plotted to illustrate the relationship between the variables and their responses. Because the statistical analysis indicated that FeCl3·6H2O and CoCl2·6H2O concentration had more significant effects on the responses than NaVO3 concentration (p-level, Table 4), the responses (DW and β-carotene content) were plotted as the functions of FeCl3·6H2O and CoCl2·6H2O. As shown in Fig. 5A, low concentrations of FeCl3·6H2O and CoCl2·6H2O led to an increase in DW, but β-carotene content increased with augmentation of CoCl2·6H2O and FeCl3·6H2O concentrations (Fig. 5B).

Verification of Optimized Culture Conditions

The optimal conditions determined from the central composite design were verified by comparing the experimental data obtained at these conditions with that predicted from central composite design (Eq. (1) and Table 4). Three verification experiments were conducted, which were respectively optimal for DW, β-carotene content, and both DW and β-carotene content (Table 5). For DW experiment under optimized condition, the experimental data was 0.77 ± 0.01 g/l, while the predicted value was 0.86 g/l, indicating 9–10% deviation. As for the condition optimal for β-carotene content, experimental data was 4.78% ± 0.04, while the predicted value was 4.73%, indicating a deviation of less than 5% (Table 5). With the second-order polynomial equation obtained in this study, the calculated DW (before optimizing the microelements) was 0.67 g/l, and the β-carotene content was 4.2%. After the microelement optimization, DW was calculated as 0.78 g/l, and β-carotene content was 4.78%(Table 5). These experiments verified the effectiveness of the model developed in this study.

Table 5 . Comparison of experimental and predicted DW and β-carotene content at optimal culture conditions..

Culture conditionsDW (g•L-1)β-carotene content (%)
Optimal for DWPredicted0.864.11%
200 mM NaHCO3, 0.45 mM MgCl2•6H2O,0.05 mM MgSO4•7H2O, 0.3 mM CaCl2•2H2O, 1.85 µM FeCl3, 1.5 µM NaVO3, 1.6 µM CoCl2 •6H2OExperimental data0.77±0.014.23%±0.01
All other conditions were as described in section Materials and MethodsDeviation (%)-10.00+2.90
Optimal for β-carotene contentPredicted0.764.86%
5.92 µM FeCl3, 2.0 µM NaVO3, 2.2 µM CoCl2 •6H2O, all other conditions were as described for “optimal DW”Experimental data0.69±0.024.78%±0.04
Deviation (%)-9.20-1.60
Optimal for DW & β-carotenePredicted0.784.73%
5.92 µM FeCl3, 2.0 µM NaVO3, 2.2 µM CoCl2 •6H2O, all other conditions were as described for “optimal DW”Experimental data0.71±0.054.52%±0.04
Deviation (%)-9.00-4.60


Comparisons between Cultivations with NaHCO3 and CO2

After 5 days of cultivation with supply of two different carbon sources, the growth showed significant differences in both OD680 (p < 0.01) and DW (p < 0.01). The initial ODs were similar (0.78 for NaHCO3-based and 0.79 for CO2-based) and then increased to 5.65 ± 0.17 and 6.58 ± 0.23, respectively (Table 6), and the corresponding final DWs were 0.89 ± 0.10 and 1.09 ± 0.08 g/l, respectively (Table 6). The productivity in cultures with NaHCO3 and CO2 were 0.18 and 0.21 g/l-1/d-1, respectively. Clearly, the CO2-based mode provided a better growth environment for D. salina, resulting in a 14.2% and 22.4% higher OD and DW than the NaHCO3-based mode. The pH varied from 6.7 ± 0.1 to 8.3 ± 0.1 in the CO2-based mode, while the pH varied from 8.0 ± 0.11 to 9.5± 0.1 in the NaHCO3-based mode (Fig. 6B).

Table 6 . Comparison of biomass, β-carotene yield, biomass productivities in cultivation with different carbon sources..

TreatmentDayOD680Dry weight (g/l)β-carotene yield (mg/l)Biomass productivity (g/l/d-1)
2% - CO200.78 (0.01)0.16 (0.02)1.04 (0.03)
2% - CO255.65**(0.17)1.09* (0.08)23.8** (0.5)0.21
200 mM NaHCO300.79 (0.02)0.16 (0.02)1.04 (0.03)
200 mM NaHCO356.58**(0.23)0.89* (0.10)41.5** (0.2)0.18

Values are mean (±SD) of n = 3 cultivations per treatment, *represent the significant effect (p < 0.05) and ** represent the very significant effect (p < 0.01).


Figure 6. NaHCO3-based cultivation vs. CO2-based cultivation by online monitoring (A) Growth curve of D. salina; (B) pH curve; (C) Fv/Fm curve; (D) β-carotene content. The shaded areas indicate the standard error of the line values.

Moreover, the 200 mM bicarbonate had positive effects on the productivity of target value chemicals, β-carotene, showing the highest carotenoid concentration in the NaHCO3-based condition, 4.7% (41.5 ± 0.2 mg/l)(Fig. 6D, Table 6), this value was significantly higher than that with CO2-based condition, 2.2% (23.8 ± 0.3 mg/l). Also, the difference between the DW and β-carotene content in Figs. 6 and 2 should be due to microelement optimization, as evidenced in Fig. 5 and Table 4.

The changing patterns in Fv/Fm are depicted in Fig. 6C, and the Fv/Fm of D. salina changed periodically following changes in light under two carbon supply conditions, but exhibited similar patterns. Notably, the Fv/Fm followed ‘sine’ trends during the whole culture light/dark period. As for 2% CO2-based condition, during the light period, the Fv/Fm decreased quickly from 0.73 to the lowest value 0.68 within the first 3 h and then increased gradually to the highest value (0.77) until the darkness period. During the 10 h darkness period, the Fv/Fm decreased gradually but was significantly higher than that in the 14 h illumination period. For the NaHCO3-based culture condition, it was found that lowest Fv/Fm values were significantly lower than CO2-based mode (p = 0.0003), and the lowest value of Fv/Fm in the CO2-based mode was 0.05 higher than in the NaHCO3-based culture.

Discussion

Although several studies have indicated that D. salina grew well in high concentration of bicarbonate, this study is the first one that reported D. salina can accumulate a good amount of β-carotene under such cultivation conditions. Table 7 compared the β-carotene content and β-carotene yield of Dunaliella strains in available literature. For D. salina CCAP 19/18, the strain used in this study, it accumulated only 2.26 mg/l β-carotene when cultured with 20 mM NaHCO3 [20]. Also, the β-carotene yield obtained in this study is much higher than previous studies with other strains in Dunaliella. For example, it is about 4-fold of the yield of D. salina V-101 with 100 mM NaHCO3 [12], and about 1.6-fold of Dunaliella sp. with 60 mM NaHCO3 [7]. Actually, 200 mM NaHCO3 supported better cell growth than other concentrations, but it is lower than that with 2% CO2 (Table 6). Since 200 mM bicarbonate resulted in a decreased value of Fv/Fm (Fig. 6C), it is considered as a stress, but this is favorable for β-carotene production. However, the commonly observed stagnant growth under stress was not found in this study, since the obtained DW of 0.71 ± 0.05 g/l-1 is quite comparable with those observed without stress [15]. Thus, this study provided a feasible approach for β-carotene production from D. salina with bicarbonate as carbon source.

Table 7 . β-carotene accumulation by strains of Dunaliella under varied cultivation conditions..

MicroalgaeInitial cell densityL/D cycleLight intensity (µmol•m-2•s-1)Culture time (d)β-carotene contentβ-carotene yield (mg/l)Carbon sourceReference
D. salina V-101-16/85070.05%8.25±0.01100 mM NaHCO3Ramachandran Svasanini et al,2018 [12]
D .salina CCAP 19/30-12/122007-1.210 mM NaHCO3Yanan Xu et al,2016
D. salina UTEX 2538-12/1210005-13.210 mM NaHCO3Yanan Xu et al,2018(Xu et al. 2018)
Dunaliella sp.0.1-34017-20.43±2.8460 mM NaHCO3Ga-Yeong Kim et al,2017 [7]
Dunaliella sp.0.116/822280.18%7.10±0.08150 mM NaHCO3Srinivasan et al,2015 [9]
D. salina CCAP 19/180.2×10612/1220074.50%32.0200 mM NaHCO3This study


Precipitation appeared in culture with 200 mM NaHCO3 along with 3.0 mM Ca2+ and 5.0 mM Mg2+, since they react with excessive CO32- at high pH (equilibrium HCO3- + OH- → CO32- + H2O). Reducing the concentration of Ca2+ and Mg2+ to 0.3 mM and 0.5 mM, respectively, was proved to be an effective approach to avoid precipitation, and showed no significant reduction of biomass and β-carotene accumulation. In order to reduce production cost, seawater with NaHCO3 supply may be used for D. salina cultivation, in which Ca2+ and Mg2+ concentration is about 9-12.5 mM, and 80.5 mM, respectively [21, 22]. These are much higher than 0.3 mM and 0.5 mM, thus excessive Ca2+ and Mg2+ need to be removed via pretreatment. Precipitation with carbonate may be used as a simple method, and CO2 bubbling could regenerate bicarbonate afterwards, which may provide inorganic carbon as in this study [23].

This study first reported the effect of microelements on β-carotene accumulation in D. salina when NaHCO3 is used as carbon source. The result indicated FeCl3·6H2O has a negative effect on cell biomass and β-carotene content with bicarbonate as carbon source. These findings were in accordance with previous research with CO2 as carbon source, and the amount of FeCl3·6H2O supplied in this study may induce the generation of active oxygen molecules, and result in a negative effect on biomass and positive effect on β-carotene accumulation [11, 19]. As shown in this study, CoCl2·6H2O has negative effect on cell growth but positive effect on β-carotene accumulation. There was no report on this topic when D. salina is cultivated with CO2, but the study on Platymonas subcordiforus, Chaetoceros curvisetus and Skeletonema costatum did show that Co2+ inhibition to cell growth in that it affects the interactions among the thylakoid membrane protein-pigment complexes, and obstructs the reaction center of PSII [24]. Also, it was reported that Co2+ contributes to the accumulation of carotenoids in Pavlova viridis, since it is an oxidative stress-inducing factor to react with hydrogen peroxide through a Fenton-type reaction to generate hydroxyl radicals [25], and these findings are in accordance with this study. This study also indicated that NaVO3 concentration had positive effect on biomass but had no significant effect on β-carotene accumulation when cultured with NaHCO3, and there was no previous report on this in D. salina cultivated with CO2. It was reported that 2.5 mM NaVO3 promoted astaxanthin production in H. pluvialis, and the possible mechanism is the inhibited expression of PTPases (Protein Tyrosine Phosphatases) by NaVO3 [26-28]. However, the NaVO3 concentration used in this study was only 2.62 μM, which may be too low to induce β-carotene accumulation.

The mechanism of improved β-carotene content under NaHCO3-based culture was unknown and there is little information available on the responses of photosynthetic electron flow, especially in photosystem II (PSII) to NaHCO3-based in D. salina. In the present study, the variation of Fv/Fm exhibited similar patterns under two carbon supply conditions, but the NaHCO3-based culture resulted in lower Fv/Fm values than that of CO2. Also, the pH value was higher at NaHCO3-based culture than that of CO2. Higher β-carotene accumulation may be attributed to both high concentration NaHCO3 and high pH. It was reported that higher extracellular NaHCO3 concentration leads to a higher intracellular pH, which may damage or inhibit the enzymes involved in photosynthesis and reduce the efficiency of PSII photosystem (Fv/Fm) [29]. Also, it was reported that higher NaHCO3 concentration (above 0.6 mM) in the culture could inhibit the extracellular carbonic anhydrase (CA) activity, which is an important enzyme catalyzing the reversible dehydration of HCO3- to CO2, and decline of CA activities has significant inhibition of effective quantum efficiency of PSII, and thus reduce the value of Fv/Fm [30]. It was reported that decreased PSII activity results in the increase of ROS concentration, and β-carotene is synthesized to scavenge the ROS [31]. This may be the reason why increased β-carotene content was observed in culture with high concentration of NaHCO3. However, the connection between ROS and Fv/Fm under NaHCO3 stress in microalgae is still unknown and further in-depth research is needed to disclose the mechanism.

From the above results, FeCl3·6H2O, NaVO3 and CoCl2·6H2O concentrations significantly influenced D. salina biomass production with NaHCO3 as carbon source, which was not reported in cultivation with CO2. The notable difference in Fv/Fm value between cultivations with bicarbonate and CO2 indicates that NaHCO3 acts as a stress factor for β-carotene production more so than CO2, and may make it useful in an easy and effective β-carotene induction method.

Supplemental Material

Acknowledgments

This research was supported by the Fundamental Research Funds for the Central Universities (No. DUT17RC (3)090).

Conflict of Interest

The authors have no financial conflicts of interest to declare.

Fig 1.

Figure 1.Effect of different concentrations of NaHCO3 on (A) D. salina cell density; (B) pH of the culturing broth. Values represented as mean ± SD (n = 3).
Journal of Microbiology and Biotechnology 2020; 30: 868-877https://doi.org/10.4014/jmb.1910.10035

Fig 2.

Figure 2.Effect of NaHCO3 concentrations on DW, β-carotene content, and β-carotene yield of D. salina. Values represented as mean ± SD (n = 3).
Journal of Microbiology and Biotechnology 2020; 30: 868-877https://doi.org/10.4014/jmb.1910.10035

Fig 3.

Figure 3.Effect of Ca2+ and Mg2+ concentrations on (A) cell number of D. salina; (B) pH of the culturing broth. Values represented as mean ± SD (n = 3).
Journal of Microbiology and Biotechnology 2020; 30: 868-877https://doi.org/10.4014/jmb.1910.10035

Fig 4.

Figure 4.Effect of Ca2+, Mg2+ concentrations on DW and β-carotene accumulation of D. salina. Values represent as mean ± SD (n = 3).
Journal of Microbiology and Biotechnology 2020; 30: 868-877https://doi.org/10.4014/jmb.1910.10035

Fig 5.

Figure 5.Three-dimensional response surface plot of (A) DW and (B) β-carotene content as a function of FeCl3·6H2O and CoCl2·6H2O.
Journal of Microbiology and Biotechnology 2020; 30: 868-877https://doi.org/10.4014/jmb.1910.10035

Fig 6.

Figure 6.NaHCO3-based cultivation vs. CO2-based cultivation by online monitoring (A) Growth curve of D. salina; (B) pH curve; (C) Fv/Fm curve; (D) β-carotene content. The shaded areas indicate the standard error of the line values.
Journal of Microbiology and Biotechnology 2020; 30: 868-877https://doi.org/10.4014/jmb.1910.10035

Table 1 . Different concentrations of Ca2+, Mg2+ investigated in this study..

No.Ca2+ (mM)Mg2+ (mM)
A0.30.5
B0.61.0
C1.22.0
D1.83.0
E2.44.0
F3.05.0

Table 2 . The coded and real values of the independent variables in the central composite design..

VariablesUnit-2-1012
FeCl3•6H2OμM1.855.929.9914.118.1
CoCl2•6H2OμM0.340.971.602.232.86
NaVO3μM0.330.91.482.052.62

Table 3 . The central composite design of the significant (in coded level) with DW and β-carotene yield as responses..

RunFeCl3•6H2OCoCl2•6H2ONaVO3DW (g/l)β-carotene (%)
111-10.6454.35
21-110.6374.27
3-1110.6854.76
4-1-1-10.6644.27
5-2000.714.28
62000.6294.59
70-200.6914.16
80200.6654.55
900-20.6714.21
100020.7083.85
110000.6953.97
120000.6963.94
130000.7073.86
140000.694.02
150000.7093.86

Table 4 . The values of coefficients in the second-order polynomial and the associated statistical test for DW and β-carotene..

VariableDWβ-carotene


F-valuep-valueEstimateF-valuep-valueEstimate
Model12.060.00680.712.110.00673.96
A- FeCl3•6H2O36.440.0018-0.0293.050.04120.1
B- CoCl2•6H2O3.750.1104-9.19E-035.520.06570.14
C- NaVO37.60.040.0134.70.0824-0.13
AB2.150.20279.83E-037.660.0395-0.23
AC6.010.0579-0.0163.09E-030.9578-4.61E-03
BC3.140.1366-0.0121.348E-070.9997-0.00003048
A^227.20.0034-0.01870.580.00040.36
B^215.770.0106-0.01415.270.01130.17
C^25.230.0709-7.82E-030.0040340.95180.002685

Table 5 . Comparison of experimental and predicted DW and β-carotene content at optimal culture conditions..

Culture conditionsDW (g•L-1)β-carotene content (%)
Optimal for DWPredicted0.864.11%
200 mM NaHCO3, 0.45 mM MgCl2•6H2O,0.05 mM MgSO4•7H2O, 0.3 mM CaCl2•2H2O, 1.85 µM FeCl3, 1.5 µM NaVO3, 1.6 µM CoCl2 •6H2OExperimental data0.77±0.014.23%±0.01
All other conditions were as described in section Materials and MethodsDeviation (%)-10.00+2.90
Optimal for β-carotene contentPredicted0.764.86%
5.92 µM FeCl3, 2.0 µM NaVO3, 2.2 µM CoCl2 •6H2O, all other conditions were as described for “optimal DW”Experimental data0.69±0.024.78%±0.04
Deviation (%)-9.20-1.60
Optimal for DW & β-carotenePredicted0.784.73%
5.92 µM FeCl3, 2.0 µM NaVO3, 2.2 µM CoCl2 •6H2O, all other conditions were as described for “optimal DW”Experimental data0.71±0.054.52%±0.04
Deviation (%)-9.00-4.60

Table 6 . Comparison of biomass, β-carotene yield, biomass productivities in cultivation with different carbon sources..

TreatmentDayOD680Dry weight (g/l)β-carotene yield (mg/l)Biomass productivity (g/l/d-1)
2% - CO200.78 (0.01)0.16 (0.02)1.04 (0.03)
2% - CO255.65**(0.17)1.09* (0.08)23.8** (0.5)0.21
200 mM NaHCO300.79 (0.02)0.16 (0.02)1.04 (0.03)
200 mM NaHCO356.58**(0.23)0.89* (0.10)41.5** (0.2)0.18

Values are mean (±SD) of n = 3 cultivations per treatment, *represent the significant effect (p < 0.05) and ** represent the very significant effect (p < 0.01).


Table 7 . β-carotene accumulation by strains of Dunaliella under varied cultivation conditions..

MicroalgaeInitial cell densityL/D cycleLight intensity (µmol•m-2•s-1)Culture time (d)β-carotene contentβ-carotene yield (mg/l)Carbon sourceReference
D. salina V-101-16/85070.05%8.25±0.01100 mM NaHCO3Ramachandran Svasanini et al,2018 [12]
D .salina CCAP 19/30-12/122007-1.210 mM NaHCO3Yanan Xu et al,2016
D. salina UTEX 2538-12/1210005-13.210 mM NaHCO3Yanan Xu et al,2018(Xu et al. 2018)
Dunaliella sp.0.1-34017-20.43±2.8460 mM NaHCO3Ga-Yeong Kim et al,2017 [7]
Dunaliella sp.0.116/822280.18%7.10±0.08150 mM NaHCO3Srinivasan et al,2015 [9]
D. salina CCAP 19/180.2×10612/1220074.50%32.0200 mM NaHCO3This study

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