Effects of Trophic Modes, Carbon Sources, and Salinity on the Cell Growth and Lipid Accumulation of Tropic Ocean Oilgae Strain Desmodesmus sp. WC08

Abstract

The effects of trophic modes, carbon sources, and salinity on the growth and lipid accumulation of a marine oilgae Desmodesmus sp. WC08 in different trophic cultures were assayed by single factor experiment based on the blue-green algae medium (BG-11). The results implied that biomass and lipid accumulation culture process were optimized depending on the tophic modes, sorts, and concentration of carbon sources and salinity in the cultivation. There was no significant difference in growth or lipid accumulation with Na₂CO₃ amendment or NaHCO₃ amendment. However, Na₂CO₃ amendment did enhance the biomass and lipid accumulation to some extent. The highest Desmodesmus sp. WC08 biomass and lipid accumulation was achieved in the growth medium with photoautotrophic cultivation, 0.08 g L⁻¹ Na₂CO₃ amendment and 15 g L⁻¹ sea salt, respectively.

Introduction

Microalgae are a kind of unicellular photosynthetic microorganisms which can absorb solar energy to digest water and CO₂, and then release O₂ into atmosphere. Its cell is tiny, diverse in morphology, strong in adaptability, and one of the most ancient primary producers in the world [1]. The lipid accumulation of microalgae varies from species to cultivation conditions, ranging from 50 to 70 % of their dry weight [2]. Meanwhile, there are many nutritious elements of high levels, which proved to be significantly practical in development.

At present, the energy workers have paid more attention to the oilgae, because of the shortage of fossil fuels and the pollution. Biodiesel, also known as fatty acid alkyl esters which are made form renewable biological sources such as vegetable oils or animal fats, is a sort of environment friendly, renewable energy [3]. Although microalgae has many advantages such as high growth rate, high oil production rate, and no occupation of farmland over traditional oil crops, the cost of highly densed cultivation is still the bottleneck issue if microalgae become commercial biodiesel raw material [4]. Therefore, it is significant to research the different environments and nutritional factors on the microalgae cell growth and the influence of oil accumulation.

Lipid accumulation is typically induced by fluctuation of growth conditions or various types of stress and it can be expressed as variation in fatty acids compositions or total lipids [5]. Zhang et al. and Luo et al. indicated that fast Desmodesmus sp. WC08 growth under optimal conditions followed by some specific stress factors, such as nitrogen starvation, phosphors starvation, iron starvation, calcium starvation, and magnesium starvation, to obtain maximum lipid content [6, 7].

Carbon is the most important element in microalgae cells and can make up to 50 % of the dry biomass [8]. Some studies have examined the use of different organic carbon sources for algae cultivation. Malorie et al. used residual corn crop hydrolysate and silage juice as alternative carbon sources in microalgae production, which showed that the use of residual corn hydrolysate represented an interesting and efficient alternative as an organic carbon source while silage juice needed additional treatments to be implemented as a culture medium [9]. In addition, about inorganic carbon sources, Kesaano et al. showed that there was no significant difference in growth rates and lipid accumulation were observed in the algal biofilms with or without bicarbonate amendment. However, the influence of bicarbonate on photosynthetic and respiration rates was especially noticeable in biofilms. Rajdeep et al. indicated that Na₂CO₃ increased the bio-oil yield for high carbohydrate-containing algae at higher temperatures [10].

Furthermore, various studies have focused on the influence caused by salinity. In these studies, salinity stress has been reported to enhance lipid accumulation in various microalgae [11]. Microalgal growth could be affected by salinity through osmotic pressure and ionic pressure. Pancha et al. showed that Scenedesmus sp. CCNM 1077 grown with 400 mM salinity stress under single stage cultivation accumulated higher lipid contents but resulted in lower biomass, during two-stage cultivation, 3-day stress of 400 mM NaCl enhanced the lipid contents with negligible biomass reduction [12].

Many studies on the trophic modes and factors affecting photosynthesis of various microalgae have been made. Marudhupandi et al. revealed that the heterotrophically cultivated Nannochloropsis salina with various carbon yielded higher biomass and lipid content than that of the photoautotrophical cultivation [13]. Mixotrophic culture is a new method for culturing microalgae in light with organic compound as supplementary carbon source, which can enhance the lipid yield and biomass of the microalgae [8].

Desmodesmus sp. WC08 was isolated from Hainan Island in China with high biomass and high lipid content. At present, the cultivation research of Desmodesmus about natural conditions (temperature, illumination intensity, photoperiod, and pH) and nutritional elements (iron, calcium, magnesium) have been done. At the same times, it can be cultivated by sewage. The study presented here evaluated the effects of adding inorganic carbon and sea salt to medium to grow algae and observe lipid accumulation in order to:

• (1) Obtain the optimal inorganic carbon source and the optimal concentration,

• (2) Determine an optimal salinity, and

• (3) Screen an optimal trophic mode.

Materials and Methods

Microalgae Strain and Preculture Conditions

The microalgae Desmodesmus sp. WC08 used in this study was obtained from Wenchang (Hainan province, China) as described preciously [14]. The microalgae was cultrued at 25 ± 1 °C in 1-L flasks using BG-11 medium with an initial pH of 7.0 ± 0.1, salinity 15 g/L, and 12:12 h light/dark period under 8000∼10000 lx light intensity. The flasks were manually shaken thrice a day.

Photobioreactor and Cultivation Conditions

After preculture in the the flasks, the microalgae was cultivated at 25 ± 2 °C at a light intensity of 10000 lx in a 5-L circular cylindrical laboratory scale luminescent bubble column photobioreactors (PBRs) operated at 150 rpm (there is a mini stirrer in the reactor) with a working volume of 3 L using BG-11 medium with an initial pH of 7.0 ± 0.2 and salinity 15 g L⁻¹. The medium was inoculated (10 % v/v) with an active culture of the microalgae consortium. Aeration with filtered sterile air was provided through an air purifier. Filtered air was supplied to the reactor through a 0.25-μm polytetrafluoroethylene membrane with 0.50 air volume/culture volume/minute. All the autotrophic and mixotrophic experiments were performed in batch culture with 15 days and a 12:12 light/dark cycle. The heterotrophic experiments were performed without light.

The basal BG-11 medium consisted of 1.5 g L⁻¹ NaNO₃, 0.04 g L⁻¹ K₂HPO₄, 0.075 g L⁻¹ MgSO₄ · 7H₂O, 0.036 g L⁻¹ CaCl₂ · H₂O, 0.006 g L⁻¹ citric acid, 0.006 g L⁻¹ ammonium ferric citrate, 0.02 g L⁻¹ Na₂CO₃, 0.001 g L⁻¹ EDTA · Na₂, 2.860 g L⁻¹ H₃BO₃, 0.220 g L⁻¹ ZnSO₄ · 7H₂O, 1.810 g L⁻¹ MnCl₂ · 4H₂O, 0.079 g L⁻¹ CuSO₄ · 5H₂O, 0.049 g L⁻¹ Co(NO₃)₂ · 6H₂O, and 0.390 g L⁻¹ Na₂MoO₄ · 2H₂O.

Experimental Design Method

The Influences of Trophic Modes on Biomass and Lipid Accumulation

Screening of the best organic carbon sources for biomass and lipid production was done in one variable at a time manner. Briefly, 12:12 h light/dark cycle was provided by the fluorescent lamps for the photoautotrophic and mixotrophic cultures while heterotrophic cultures were kept in the dark. Carbon (67 mmol L⁻¹) in the organic carbon sources (glucose, sodium acetate, and glycerol) was controlled to be added into the BG-11 medium (only for mixotrophy and heterotrophy). All experiments were performed in triplicate and the salinity was 15 g L⁻¹.

The Influences of Inorganic Carbon Sources on Biomass and Lipid Accumulation

Cultures were carried out in triplicate in the reactor containing BG-11 medium with various carbon sources (Na₂CO₃ and NaHCO₃) and the carbon molarity of Na₂CO₃ and NaHCO₃ were equal to the molarity of carbon source in the BG-11 medium (2.3 mmol L⁻¹). In all the parallel experiments, the salinity was determined as 15 g L⁻¹.

The Influences of Concentration of Carbon Source on Biomass and Lipid Accumulation

Cultures were carried out in triplicate in the reactor containing BG-11 medium with Na₂CO₃ concentrations of 0, 0.02, 0.04, 0.06, and 0.08 g L⁻¹. In all the parallel experiments, the salinity was 15 g L⁻¹.

The Influences of Salinity on Biomass and Lipid Accumulation

Cultures were carried out in triplicate in the reactor containing BG-11 medium with sea salt concentrations of 10, 15, 20, and 30 g L⁻¹.

Analytical Methods

Determination of Growth

Microalgal growth was monitored by direct sampling directly using a sterile disposable syringe and measuring optical density (OD) at 680 nm every 24 h intervals at a fixed time over a period of 18 days. Each parameter was tested in triplicates.

Determination of Biomass

After 15-day cultivation, microalgae was harvested by centrifugation at 8000 rpm for 6 min and cleaned by deionized water for three times. Then, the microalgae paste was dried by a vacuum freeze drier. Total biomass was expressed as gram dry weight per liter. Based on this information theoretical biomass by each litre of microalgae can be obtained from equation (1) as below:

$$ P\mathrm{biomass}\left(\mathrm{g}/\mathrm{L}\right)=\frac{N0\left(\mathrm{g}\right)}{V\left(\mathrm{L}\right)} $$

(1)

where P is biomass, N ₀ is the mass of dried microalgae powder, and V is the volume of culture medium.

Determination of Total Lipid Extraction

Total lipid yield was also expressed as gram dry weight per liter while the lipid content was expressed as percentage. Lipid content was determined using a modified method of Axelsson and Gentili (2014) which was chloroform-methanol solvent base [5, 15]. In brief, 2 mL of chloroform:methanol (2:1 v/v) was added to the 0.1-g lyophilized microalgae powder (the liquid-solid ratio was 20:1) in 15 mL solvent resistant falcon tubes and placed in an ultrasound bath extraction in the ambient temperature for 30 min then homogenized using a magnetic stirring apparatus for 3∼6 h (keep sealing). The mixture was centrifuged at 4000 rpm for 5 min. After centrifugation, the supernatant was transferred to a clear tube. For rest of the microalgal dreg, the experiments above were carried out in duplicate for 5 times and pooled all the supernatant together then 0.9 % NaCl (1:0.2 v _supernatant/v _NaCl) was added to produce a layered solvent system. The layered solvent system was vortexed for 5 min. After a few minutes of standing, the lower layer was transferred to a clear tube and dried to constant weight using pressure blowing concentrator.

The total lipid content and total lipid yield was calculated according to Eqs. (2) and (3):

$$ \mathrm{Total}\ \mathrm{lipid}\ \mathrm{content}\ \left(\%\right)=\frac{\mathrm{Algal}\ \mathrm{lipid}\ \left(\mathrm{g}\right)}{\mathrm{Mass}\ \mathrm{of}\ \mathrm{Algal}\ \left(\mathrm{g}\right)}\times 100 $$

(2)

$$ \mathrm{Total}\ \mathrm{lipid}\ \mathrm{yield}\ \left(\mathrm{g}/\mathrm{L}\right)=\mathrm{Algal}\ \mathrm{biomass}\ \left(\mathrm{g}/\mathrm{L}\right)\times \mathrm{Total}\ \mathrm{lipid}\ \mathrm{content}\ \left(\%\right) $$

(3)

Statistical Analysis

All results were expressed as mean values ± standard deviation. The statistical differences between experimental groups were analyzed by analysis of variance (ANOVA). The analysis of least significant digit (LSD) and the graphic plotting were used by statistical product and service solutions (SPSS) and Oringin 8.0, respectively.

Results and Discussions

The Effects of Trophic Modes on Growth, Biomass, and Lipid Accumulation of Desmodesmus sp. WC08

Many researches have elaborated the effects of trophic modes on biomass and lipid accumulation of microalgae. But most of the researches indicated that the mixotrophic cultivation with glucose was the best trophic mode. In this research, at the first 6 days, growth period of autorophic cultivation of Desmodesmus sp. WC08 was short while compared to mixotrophic (glucose, sodium acetate, glycerinum) and heterotrophic (glucose, sodium acetate) clutivation. After 10 days, the growth of autotrophic cultivation exceeded all the other trophic cultivations. The result showed that glycerinum in mixotrophic cultivation and heterotrophic showed inhibitory effect on the growth of Desmodesmus sp. WC08. Microalgal growth was enhanced at the exponential growth phase with mixotrophic (glucose) cultivation compared to autotrophic cultivation at the control condition. Although the optical density of mixotrophic (glucose) cultivation was higher than any other trophic modes at the first 10 days, after experienced an exponential enhancement in the previous days, the subsequent growth entered into a steady phase and inclined to decline (Fig. 1).

Fig. 1

Growth curves of Desmodesmus sp. WC08 in different trophic modes

From the Table 1, Desmodesmus sp. WC08 with autotrophic cultivation showed the maximum P _biomass of 1.83 ± 0.18 g L⁻¹ and there was significant difference between autotrophic cultivation and other trophic cultivation (p < 0.05). The biomass was lowest when glycerinum was added in the mixotrophic and heterotrophic cultivation, respectively. The result attested to the inhibiting effect of glycerinum on Desmodesmus sp. WC08’s biomass.

Table 1 Biomass of microalgae Desmodesmus sp. WC08 in different trophic modes

The highest lipid content (42.11 ± 0.61 %) and lipid yield (3.01 ± 0.34 g L⁻¹) were observed in the autotrophic cultivation and there was highly significant difference between autotrophic cultivation and other trophic cultivation (p < 0.01). The lipid yield of mixotrophic cultivation with glucose and sodium acetate was 1.02 ± 0.52 and 1.15 ± 0.37 g L⁻¹, respectively and had no significant difference. The lipid content and lipid yield of heterotrophic cultivation with glucose and sodium acetate had no significance either. Glyerinum in mixotrophic and heterotrophic cultivation showed considerable enhancement in lipid content, whereas it induced the lowest lipid yield in micotrophic and heterotrophic cultivation, respectively (Fig. 2).

Fig. 2

The effects of various trophic methods on lipid content and lipid yield of Desmodesmus sp. WC08. Different letters indicate values are significantly different (p < 0.05) for each trophic modes. Where A is autotrophy, M1 is mixotrophy (glucose), M2 is mixotrophy (sodium acetate), M3 is mixotrophy (glycerinum), H1 is heterotrophy (glucose), H2 is heterotrophy (sodium acetate), and H3 is heterotrophy (glycerinum)

Overall, the growth rate of mixotrophic (glucose) cultivation showed an upward trend, which was far higher than the rates of other groups at the first few days. Nevertheless, the growth rate would decline slightly subsequently. Glycerinum could increase lipid content, however, inhibiting the growth and biomass of Desmodesmus sp. WC08. Results of the seven trophic mode tests indicated that autotrophic cultivation were optimistic trophic mode for growth enhancement and biomass production of Desmodesmus sp. WC08, resulting in a significant increase (p < 0.05) in lipid accumulation after 18 days of culture compared to control at all tested trophic modes.

The Effects of Two Inorganic Carbon Sources on the Growth, Biomass, and Lipid Accumulation of Desmodesmus sp. WC08

The catalysts of carbon source supplementation on the growth of various microalgae were well documented. Supplementation of inorganic carbon, namely Na₂CO₃ [10] and NaHCO₃ [16], in photoautotrophic cultures were shown to be beneficial in enhancing biomass and lipid productivity. Results of the various inorganic carbon experiments indicated that Na₂CO₃ and NaHCO₃ were potential inorganic carbon sources for growth enhancement and lipid accumulation of Desmodesmus sp. WC08. As shown in Fig. 3 and Table 2, Desmodesmus sp. WC08 grew the fastest with NaHCO₃ as the organic source at the first 5 days and then was exceeded by Na₂CO₃ group to get a higher growth rate and reach highest biomass of 2.94 ± 0.25 g L⁻¹, while the lowest biomass of 2.66 ± 0.37 g L⁻¹ was obtained in the cultivation using NaHCO₃ as the carbon source. The measured lipid content and lipid yield were 33.66 ± 0.45 % and 0.90 ± 0.13 g L⁻¹, respectively, for Desmodesmus sp. WC08 with NaHCO₃ amendment. Similarly, final total lipid content and lipid yield measured from Na₂CO₃ were 32.35 ± 0.24 % and 0.95 ± 0.01 g L⁻¹, respectively. No significant difference in lipid accumulation was observed in the cultivation of Desmodesmus sp. WC08 with Na₂CO₃ or NaHCO₃ amendment (Fig. 4). The Desmodesmus sp. WC08 possibly did not experience carbon-limited conditions because of the large reservoir of dissolved inorganic carbon in the medium. However, a higher increase in growth rate, biomass, and lipid yield were observed in experiment amended with Na₂CO₃. The reason why CO₃ ²⁻ and HCO₃ ⁻ could enhance the biomass and lipid accumulation had been well demonstrated [17,18]. Briefly, the active transport of two ions (CO₃ ²⁻ and HCO₃ ⁻) and the mutual transformation between two ions and CO₂ controlled by carbonic anhydrase could increase the concentration of CO₂ which surrounded ribulose 1,5-bishosphate carboxylase/oxidase. One the one hand, these combined actions could benefit the competition of ribulose 1,5-bishosphate carboxylase/oxidase’s binding site to CO₂ and increase carboxylation rate; on the other hand, these combined actions could inhibit photorespiration and enhance net photosynthetic rate, however limited research on the comparison of the mechanisms between CO₃ ²⁻ and HCO₃ ⁻. On the basis of Zhang et al. [19], the increasing rate of pH caused by HCO₃ ⁻ was faster than that of CO₃ ²⁻ supplement, and the increasing of pH could inhibit the biomass and the lipid accumulation of microalgae.

Fig. 3

Growth curves of Desmodesmus sp. WC08 with two different inorganic carbon sources

Table 2 Biomass of microalgae Desmodesmus sp. WC08 with two different inorganic carbon sources

Fig. 4

The effects of two inorganic carbon sources on lipid content and lipid yield of Desmodesmus sp. WC08. Different letters indicate values are significantly different (p < 0.05) for each carbon sources

The Effects of Different Concentrations of Na₂CO₃ on the Growth, Biomass, and Lipid Accumulation of Desmodesmus sp. WC08

Desmodesmus sp. WC08 cells had a full growth cycle, as shown by the sigmoid growth models. These growth cycles comprised a more dynamic pattern in different concentrations of Na₂CO₃. Growth rate in Desmodesmus sp. WC08 was enhanced at the exponential growth phase with Na₂CO₃ amendment compared to the cultivation without Na₂CO₃ amendment at the control condition. Although the overall growth rates obtained were relatively similar, the highest growth, with 0.08 g L⁻¹ Na₂CO₃, was achieved 17 days before. Figure 5 showed that the growth rate increased with the increasing of the concentration of Na₂CO₃.

Fig. 5

Growth curves of Desmodesmus sp. WC08 with different concentrations of Na₂CO₃

In terms of biomass (Table 3), Na₂CO₃ (0.08 g L⁻¹) resulted in approximately 0.5-fold increase compared to control group (0.00 g L⁻¹). Supplementation of culture medium with 0.08 g L⁻¹ Na₂CO₃ resulted in a final dry biomass weight of 0.99 ± 0.25 g L⁻¹, which was higher than for Desmodesmus sp. WC08 supplemented with 0.02, 0.04, and 0.06 g L⁻¹ Na₂CO₃ groups, which produced approximately 0.89 ± 0.23, 0.83 ± 0.18, and 0.89 ± 0.14 g L⁻¹, respectively, and there was no significant difference between them (p > 0.05).

Table 3 Biomass of microalgae Desmodesmus sp. WC08 with different concentrations of Na₂CO₃

A slight increase in total lipid content was observed in cell growth in both 0.04 and 0.06 g L⁻¹ Na₂CO₃ groups compared to control group (0.00 g L⁻¹), and there were no significant differences between them (p > 0.05). Meanwhile, 0.02 and 0.08 g L⁻¹ Na₂CO₃ groups could increase (p < 0.05) lipid content significantly (Fig. 6).

Fig. 6

The effects of different concentrations of Na₂CO₃ on lipid content and lipid yield of Desmodesmus sp. WC08. Different letters indicate values are significantly different (p < 0.05) for each concentrations of Na₂CO₃

There were some appreciable variations in maximum growth rate, biomass, and lipid accumulation observed under photoautotrophic conditions depending upon the different concentrations of Na₂CO₃, with 0.08 g L⁻¹, showing quite high growth rates, biomass, and lipid yield. Regardless of other factors, this measure would be important in choosing the optimal concentration of Na₂CO₃ to develop for a practical production system.

The Effects of Different Salinities on the Growth, Biomass, and Lipid Accumulation of Desmodesmus sp. WC08

Salinity for biomass productivity (P _biomass) with Desmodesmus sp. WC08 was optimized. Salinities of 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, and 40.0 g L⁻¹ were established in cultures under photoautotrophic cultivation, respectively. As shown in Fig. 7, the growth of Desmodesmus sp. WC08 with salinities of 15.0, 20.0, and 25.0 g L⁻¹ reached the apex phase at 14, 14, and 15 days, respectively. The high salinity of 35 and 40 g L⁻¹ resulted in the lowest growth rate. With the increasing of salinity, the growth rate increased firstly and then decreased; the highest growth rate was under the cultivation of 15 g L⁻¹ sea salt. The result revealed that the excessively high salinity could inhibit the growth of Desmodesmus sp. WC08.

Fig. 7

Growth curves of Desmodesmus sp. WC08 with different salinities

Various sea salt stresses in the cultivation were carried out for 18 days to find optimum sea salt stress to accumulate high biomass and lipid yield as shown in Table 4 and Fig. 8. The various sea salt concentrations in the culture for biomass and lipid yield ranging from 10.0 to 40.0 g L⁻¹. The top two biomass, total cellular lipid content, and lipid yield under the salinities of 15.0 and 20.0 g L⁻¹ have no significant difference (p > 0.05), whereas there were significant differences compared with other salinities (p < 0.05). The highest biomass and lipid yield of the Desmodesmus sp. WC08 were 5.35 ± 0.30 and 1.26 ± 0.06 g L⁻¹, respectively, under the cultivation of 15.0 g L⁻¹ sea salt while the lowest biomass and lipid yield were under the cultivation of 40 g L⁻¹ sea salt. These results indicated that the contribution of the culture to biomass and lipid accumulation under low-salt conditions was significant.

Table 4 Biomass of microalgae Desmodesmus sp. WC08 with different salinities

Fig. 8

The effects of different salinities on lipid content and lipid yield of Desmodesmus sp. WC08. Different letters indicate values are significantly different (p < 0.05) for each salinity

Conclusions

For this study, autotrophic cultivation proved to be a useable trophic mode for Desmodesmus sp. WC08. Two inorganic sources (Na₂CO₃ and NaHCO₃) could be utilized by Desmodesmus sp. WC08. Although there was no significant difference in growth rate, biomass, and lipid accumulation between Na₂CO₃ and NaHCO₃ amendment, Na₂CO₃ did enhance the biomass and lipid accumulation to some extent compared to the NaHCO₃ amendment. Meanwhile, the increase of the concentration of Na₂CO₃ could considerably enhance the biomass and lipid accumulation of Desmodesmus sp. WC08. In conditions of high salinity (35 g L⁻¹ and above), the growth of Desmodesmus sp. WC08 was significantly inhibited while low salinity could enhance the biomass and lipid accumulation.