Effects of sodium acetate and ammonium acetate on the growth and production of cellular components by Chlorella vulgaris 31

doi:10.21203/rs.3.rs-3033260/v1

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Effects of sodium acetate and ammonium acetate on the growth and production of cellular components by Chlorella vulgaris 31

https://doi.org/10.21203/rs.3.rs-3033260/v1

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Mixotrophic cultivation using organic carbon sources has become increasingly important for large-scale production and application of microalgae, as it can overcome the current commercial bottleneck of low yield and high cost associated with photoautotrophic and heterotrophic cultivation. In this study, we investigated the effects of adding two acetates, sodium acetate (NaAc) and ammonium acetate (NH₄Ac), at different concentrations (2, 4, 6, 8, 10 and 12 g L^-1) on the biomass, biochemical components content (pigments, proteins, soluble sugars and lipids) and fatty acid composition of Chlorella vulgaris 31 (Cv-31) under mixotrophic cultures. Our results showed that the addition of NaAc significantly increased the biomass and lipid content of microalgae compared with NH₄Ac, with 10 g L^-1 NaAc having the best effect on the growth and lipid synthesis. Furthermore, only 2 and 4 g L^-1 NaAc promoted the synthesis of pigments in algal cells, while all different concentrations of NH₄Ac were inhibitory. In contrast to pigments, the effect of two acetates on protein synthesis was opposite, which was promoted by low concentrations of NH₄Ac. Both acetates had a "low concentration promotion, high concentration inhibition" effect on the synthesis of soluble sugars. Moreover, the percentage of saturated fatty acids in the fatty acid profile increased with the amount of NaAc supplementation, while stearic acid and oleic acid appeared. Our findings suggest that regulating the type and concentration of acetate can improve the biomass and lipid yield of Cv-31 to promote the microalgal biomass production.

Chlorella vulgaris 31

sodium acetate

ammonium acetate

biomass production

cellular components

Microalgae are a diverse group of unicellular or structurally simple multicellular autotrophic microorganisms that can be found in a wide range of aquatic environments, such as freshwater, marine, and terrestrial habitats. They possess several advantages over other microorganisms, such as fast growth rate, short reproduction cycle, and adaptability to various environmental conditions. Furthermore, microalgae are a valuable source of various bioactive compounds, including polyunsaturated fatty acids, astaxanthin, phycocyanin, vitamins (Morais Junior et al. 2020; Russell et al. 2022), making them highly valuable for commercial applications in fields such as bioenergy, pharmaceuticals, aquaculture, animal feed, and food industries (Kumar et al. 2022; Zhuang et al. 2022). Commercially, Spirulina and Chlorella are the most commonly utilized microalgae species due to their ease of cultivation and high yield of bioactive compounds.

Chlorella vulgaris is a unicellular eukaryotic microorganism that belongs to the Chlorellaceae family, with a cell diameter ranging from 2–10 µm. When faced with unfavorable growth conditions such as nitrogen and phosphorus limitation, as well as salt stress, the lipid and carbohydrate content of Chlorella increases while biomass yield remained constant or decreased (Cheng and He 2014; Chen et al. 2017). On the other hand, suitable culture conditions promote protein accumulation. Under optimal culture conditions, the protein and lipid content of Chlorella can reach 42–58% and 5–40% of the dry weight (Safi et al. 2014), respectively, making it a valuable resource for food supplements and as a feedstock for biodiesel production. Additionally, C. vulgaris contains important polysaccharides such as starch and β-1, 3-glucan, which possess high nutritional value.

Indeed, high cell density culture is essential for large-scale industrialization of microalgae. There are three main types of microalgal culture: autotrophy, heterotrophy, and mixotrophy. Autotrophy relies on the photosynthesis of microalgae, which has a ten-fold higher efficiency compared to terrestrial plants (Sathasivam et al. 2019), with a supply of only 1–2% CO₂ being sufficient for growth (Loke Show 2022). However, this model has a low growth rate and is vulnerable to bacteria contamination. Heterotrophy accelerates cell growth rate by eliminating light limitation, but the addition of glucose-based organic carbon sources increases costs. Furthermore, dark conditions of culture lead to a limited number of adapted microalgae species and a reduced level of light-induced metabolites or even none at all (Castillo et al. 2021). Therefore, more attention has been given to the mixotrophic model, which have been found to obtain greater specific growth rates and biomass yields (Wang et al. 2014; Yu et al. 2022) while reducing costs. However, the the microalgal mixotrophic pattern still needs to be further elucidated. Some studies suggest that mixotrophy is a simple combination of autotrophy and heterotrophy, while others propose that it is the result of synergistic effects of both (Heredia-Arroyo et al. 2011).

The current challenges of high cost and low yield present significant obstacles to large-scale microalgae cultivation. To address these issues, the construction and optimization of the mixotrophic culture systems have emerged as a key approach to improving yields, directly influencing the growth characteristics and cellular composition of microalgae. Commonly employed organic carbon sources, such as glucose, acetate, glycerol, fructose, sucrose, xylose (Song and Pei 2018), and even organic compounds found in wastewater (Khalid et al. 2019), exhibit varying effects on growth and metabolism. Nitrate, ammonium, urea, yeast extracts and peptone are nitrogen sources commonly used in cultures, and they are closely associated with lipid synthesis (Adesanya et al. 2014) and influence fatty acid composition (Rtsa and Djg 2018). Moreover, culture conditions including temperature, light, and pH play pivotal roles. In conclusion, strict control of the culture system is essential for achieving high cell density and maximizing the yield of bioactive substances.

Acetate holds great promise as a carbon feedstock for the mixotrophic model. Firstly, acetate offers advantages over glucose as it is cost-effective and easily obtainable, being derived from C₁ gases such as CO, CO₂ and CH₄ (Kim et al. 2021). Additionally, acetate possesses antibacterial properties (Dragone 2022), thereby reducing the risk of contamination. Acetate stimulates biomass and lipid synthesis by enhancing carbon metabolic fluxes in the tricarboxylic acid cycle following acetyl coenzyme A production within algal cells (Cheng et al. 2021).

This study aimed to investigate the effects of different concentrations of two acetates, sodium acetate (NaAc) and ammonium acetate (NH₄Ac), on the biomass and biochemical composition of Chlorella vulgaris 31 (Cv-31) in the mixotrophic cultivation. Specifically, the study examined the impact of these acetates on the content of pigments, proteins, soluble polysaccharides, lipids, and the fatty acid composition of Cv-31. Furthermore, the research explored the dynamic trends of growth and metabolic parameters under the mixotrophic culture mode, focusing on identifying the optimal concentration and type of carbon source. The ultimate goal was to determine the carbon source that would enable achieving the highest cell density and lipid content, providing insights into the metabolic mechanism and facilitating the industrial-scale cultivation of this strain.

Microalga strain, chemicals, and culture medium

The Cv-31 strain used in this study was purchased from the Freshwater Algal Culture Collection, Institute of Hydrobiology, Chinese Academy of Sciences. Analytically pure reagents, including vanillin, phenol, anhydrous ethanol, citric acid, ferric ammonium citrate, EDTA disodium, NaNO₃, K₂HPO₄, and other inorganic compounds were purchased from specific chemical reagent companies in China.

The microalgae were cultivated in BG11 medium, which was formulated based on the composition provided at http://algae.ihb.ac.cn/. The BG11 medium contained the following components (mg L^− 1): NaNO₃ 1500, K₂HPO₄ 40, MgSO₄·7H₂O 75, CaCl₂·2H₂O 36, citric acid (C₆H₈O₇) 6, ferric ammonium citrate (C₆H₈FeNO₇) 6, EDTA disodium (C₁₀H₁₄N₂Na₂O₈) 1, NaCO₃ 20, H₃BO₃ 2.86, MnCl₂·4H₂O 1.86, ZnSO₄·7H₂O 0.22, Na₂MoO₄·2H₂O 0.39, CuSO₄·5H₂O 0.08, Co(NO₃)₂·6H₂O 0.05. The finished medium was adjusted to an initial pH of 7.0 using 1 mol L^− 1 HCl or NaOH.

Culture conditions

The Cv-31 strain, stored in a 4°C light incubator, was diluted and then spread on solid medium. It was then incubated in a constant light incubator at 25°C for one week. Afterward, single colonies of microalgae were selected and streaked for 7 days to obtain pure cultures. Subsequently, the liquid medium was inoculated with the obtained culture as the inoculum.

For the experiments, 100 mL of BG11 liquid medium was prepared into 250 mL flasks, containing different concentrations of NaAc or NH₄Ac: 2, 4, 6, 8, 10 and 12 g L^− 1. The medium was autoclaved, and then 10% (v/v) of Cv-31 at logarithmic phase were inoculated into each flask. The cultures were maintained at 25 ± 1°C, 150 r/min, with a light intensity of 95 µmol photons m^− 2 s^− 1, and a 12: 12 h light/dark cycle. A control group without the addition of acetate was also included. Each experimental group had three replicates, and samples were collected at 24-hour intervals for the analysis of biomass and other physiological and biochemical parameters.

Determination of growth curve and kinetic parameters

Biomass content

The biomass of Cv-31 was determined using the turbidimetric method (Kong et al. 2013, 2020). Samples were collected every 24 hours and diluted appropriately. The absorbance values at 680 nm were measured using a spectrophotometer. The algal solution was then subjected to centrifugation at 6000 r/min for 10 minutes, followed by freeze-drying until a constant weight was achieved. The algal cell concentration was calculated according to the linear regression equation of OD₆₈₀-biomass concentration

$$Y=0.5473X-0.0763 ({R}^{2}=0.9945)$$

where X is OD₆₈₀, Y is the cell dry weight concentration (g L^− 1), and R² is the correlation coefficient.

Specific growth rate and Biomass productivity

Microalgal growth kinetics were assessed using calculated specific growth rate (µ, d^− 1) and biomass productivity (P, g L^− 1 d^− 1) according to Eq. (2) and Eq. (3)

$${\mu }=(\text{l}\text{n}{X}_{t}-ln{X}_{0})/({t}_{x}-{t}_{0})$$

$$P=({X}_{t}-{X}_{0})/({t}_{x}-{t}_{0})$$

where X_t and X₀ are the absorbance values at 680 nm at t_x and t₀ of culture, respectively.

Extraction and determination of photosynthetic pigments

A three-wavelength colorimetric method (Kong et al. 2013, 2020) was employed for the determination of chlorophyll and carotenoid concentrations. Five milliliters of the algal culture were centrifuged at 5000 r/min for 10 min. The resulting precipitate was washed with deionized water, followed by centrifugation. Subsequently, 10 mL of 95% ethanol was added to the precipitate and thoroughly mixed. After extracting overnight (about 12 h) at 4°C avoiding light, the extract was collected by centrifugation at 5000 r/min for 10 min. Then, 5 mL of 95% ethanol was added for a second extraction for 6 h until the algal cells turned white. The two extracts were centrifuged and diluted appropriately to measure the absorbance at 470 nm, 649 nm, and 665 nm. The chlorophyll a (Ca), chlorophyll b (Cb), and carotenoid (Cc) concentration (mg L^− 1) were calculated according to the following Eqs. (4) to (6):

$${C}_{a}=13.95{OD}_{665}-6.88{OD}_{649}$$

$${C}_{b}=24.96{OD}_{649}-7.32{OD}_{665}$$

$${C}_{c}=(1000{OD}_{470}-2.05{C}_{a}-114.8{C}_{b})/245$$

Extraction and determination of protein

The protein content of the algal samples was determined using the Kjeldahl method, as described by Patel et al. (2022). Initially, 100 mg of dried algal powder was precisely weighed into a dry digestion tube. The digestion process involved adding 0.4 g of mixed catalyst and subsequently 10–20 mL of digestion solution to the tube. The sample was then placed on a graphite digestion apparatus until it reached the same state as the clarified light green blank group. The digestion parameters were set as follows: the first stage involved heating at 380°C for 30 minutes, followed by the second stage at 420°C for 60 min. After allowing the sample to cool, distillation and titration were were performed.

The protein content in dry biomass was calculated according to the Eqs. (7):

Protein content$（\%）=[C\times ({V}_{1}-{V}_{2})\times 0.014]\times 100\times 6.25/W$ (7)

where C is the concentration of standard HCl solution (mol L^− 1); V₁ is the volume consumed when titrating the standard solution of hydrochloric acid (mL); V₂ is the volume consumed when titrating the blank (mL); W is the sample mass (g).

Extraction and determination of total soluble sugar content

The total soluble sugar content in the algal cells was determined using the phenol-sulfuric acid method, as described by Dubois et al. (1956) and Shi et al. (2007). The disrupted algal powder was resuspended in distilled water in a water bath at 80°C for 1 hour to obtain the extract. After two extractions, the supernatant obtained by centrifugation (4°C, 5000 r/min, 20 min) was used as the sample for the total soluble sugar assay. The sample solution was appropriately diluted and sequentially mixed with phenol and concentrated sulfuric acid for color reaction. The absorbance value of the resulting solution was measured at 490 nm. The total soluble sugar mass and content were calculated using a mass-OD₄₉₀ standard curve, with glucose as the standard, and a linear regression equation described by Eqs. (8) and (9):

$$Y=0.0078X+0.0044 ({R}^{2}=0.9984)$$

where X is the sugar mass (µg), Y is OD₄₉₀, and R² is the correlation coefficient.

Total soluble sugar content$（\%）=\frac{C\times {V}_{T}\times N}{W\times {V}_{S}\times {10}^{6}}\times 100$ (9)

where C is sugar mass (µg) calculated from the standard curve; V_T is total volume of the sample (mL); N is dilution multiple of the sample; V_S is the volume of sample (mL) taken for the determination; W is sample mass (g).

Extraction and determination of lipid

The lipid content of the algal cells was determined using the sulfo-phospho-vanillin (SPV) colorimetric method, as described by Byreddy et al. (2016). 25 mg of freeze-dried algal powder was suspended in 5 mL of distilled water. From the well-mixed algal solution, 100 µL was taken as the sample. To the sample, 2 mL of 98% concentrated sulfuric acid was added, and the mixture was boiled in a water bath for 10 minutes. Subsequently, it was cooled in an ice bath for 5 minutes. Afterward, 5 mL of prepared chromogenic agent was added and left at room temperature for 20 minutes. The absorbance value of the resulting solution was measured at 530 nm. The lipid concentration was calculated using a standard curve of OD₅₃₀-lipid concentration, with refined olive oil used as the standard. The relationship between OD₅₃₀ and lipid concentration was determined using Eqs. (10):

$$Y=2.1585X-0.0159 ({R}^{2}=0.9971)$$

where X is OD₅₃₀, Y is lipid concentration (g L^− 1), and R² is the correlation coefficient.

Analysis of fatty acid composition

For lipid extraction, the chloroform-methanol method described by Wahidin et al. (2013) was employed. 100 mg of algal powder was accurately weighed and placed in a 15 mL stoppered centrifuge tube. To the tube, 6 mL of chloroform-methanol mixture (2:1, v/v) was added, and the mixture was subjected to extraction for 1 h. The organic phase at the bottom of the tube was collected by centrifugation at 5000 r/min for 10 minutes. The extraction process was repeated once. The collected extracts were subjected to rotary evaporated to ensure complete evaporation of chloroform. Subsequently, the resulting samples were methyl esterified using the BF₃-CH₃OH method. The methyl esterified samples were analyzed using gas chromatography-mass spectrometry (Rtsa and Djg 2018).

Statistics analysis

All experimental data were analyzed and presented as mean ± standard deviation (SD). The experiments were conducted with three independent biological replicates. Statistical analysis was performed using IBM SPSS Statistics 22 software, and significance was determined with a p value less than 0.05. Graphs and plots were generated using Origin 2018 software.

Growth characteristics of Cv-31

The growth curves of Cv-31 under two acetates treatments with varying concentrations are depicted in Fig. 1. The addition of NaAc at different concentrations exhibited similar growth trends, significantly promoting the growth of Cv-31 (p < 0.05) (Fig. 1a). Except for the concentration of 12 g L^− 1, Cv-31 treated with different concentrations rapidly entered the logarithmic phase after a one-day or two-day lag phase. Initially, the growth of Cv-31 was slower when cultivated with 12 g L^− 1 NaAc, as the high substrate concentration required a long adaptation period. However, by the fourth day of cultivation, the biomass began to accumulate rapidly, slightly surpassing that of the 10 g L^− 1 concentration on the seventh day. The growth of Cv-31 exhibited a concentration-dependent response to NaAc within a certain concentration range (0–10 g L^− 1), with the most pronounced growth promotion observed at 10 g L^− 1 NaAc in mixotrophic culture, followed by 12 g L^− 1 and 8 g L^− 1. Conversely, the growth promotion effect of NH₄Ac on Cv-31 was significantly weaker compared to NaAc (Fig. 1b). On the second day of culturing, almost all concentrations of NH₄Ac showed a promoting effect on the growth of Cv-31 (p < 0.05); whereas the growth rate of algal cells at 12 g L^− 1 concentration was lower than that of the control group. Except for 12 g L^− 1, NH₄Ac inhibited the growth of cell and entered the decline phase after four days of culture. The photosynthetic autotrophic group exhibited a slow gradual growth increase throughout the culture period. The most significant growth-promoting effect was observed with the addition of 2 g L^− 1 of NH₄Ac, while the effect of higher concentration treatment was relatively diminished. According to the pH change trend of the corresponding culture medium shown in Fig. 2, it can be observed that the different concentrations of NaAc-treated groups generally exhibit an increasing trend with increasing culture time. With the exception of the blank control group and the 2 g L^− 1 treated group, the pH tends to stabilize at the end of the culture. In the NH₄Ac-treated group, the trend is mostly similar to that of NaAc. However, in the 10 g L^− 1 and 12 g L^− 1 treated groups, the pH of the culture medium remains weakly alkaline and unchanged during the first six days of cultivation, and then shows an upward trend.

Table 1 presents the biomass concentration, specific growth rate, and productivity of Cv-31 subjected to different concentrations of two acetates in mixotrophic culture for 7 days. The biomass concentration, specific growth rate, and productivity of Cv-31 cultivated with the NaAc addition were significantly higher (p < 0.05) compared to the photosynthetic autotrophic group, consistent with the growth curve trend. The highest biomass concentration and productivity were obtained at 12 g L^− 1 NaAc, which were 6.45 and 6 times higher than those of the control group, respectively. However, these three growth kinetic parameters showed a roughly decreasing trend with increasing NH₄Ac concentration. The values at 6, 10, and 12 g L^− 1 NH₄Ac were drastically lower than those of the control group (p < 0.05). These findings indicate that during the 7-day culture period, different concentrations of NaAc had a significant promoting effect on the growth and biomass accumulation of Cv-31; the concentration of NH₄Ac promoted the growth within the threshold and inhibited it when the concentration exceeded the threshold.

Table 1

Effects of two acetates on the biomass concentration, specific growth rate and productivity of *C. vulgaris* 31.
Concentration (g L^− 1)	Biomass concentration (g L^− 1)		Specific growth rate (d^− 1)		Productivity (g L^− 1 day^− 1)
Concentration (g L^− 1)	NaAc	NH₄Ac	NaAc	NH₄Ac	NaAc	NH₄Ac
0	0.84 ± 0.02^g	0.84 ± 0.02^c	0.49 ± 0.01^f	0.49 ± 0.01^c	0.13 ± 0.003^g	0.13 ± 0.003^c
2	2.47 ± 0.04^f	1.45 ± 0.04^a	0.64 ± 0.01^e	0.57 ± 0.01^a	0.36 ± 0.005^f	0.21 ± 0.005^a
4	3.51 ± 0.01^e	0.97 ± 0.01^b	0.69 ± 0.01^d	0.51 ± 0.01^b	0.51 ± 0.002^e	0.14 ± 0.002^b
6	4.36 ± 0.02^d	0.70 ± 0.03^d	0.72 ± 0.01^c	0.47 ± 0.01^d	0.63 ± 0.003^d	0.11 ± 0.004^d
8	4.83 ± 0.06^c	0.87 ± 0.03^c	0.73 ± 0.004^b	0.50 ± 0.01^c	0.70 ± 0.008^c	0.13 ± 0.005^c
10	5.29 ± 0.01^b	0.47 ± 0.02^e	0.75 ± 0.01^a	0.42 ± 0.01^e	0.76 ± 0.002^b	0.07 ± 0.003^e
12	5.42 ± 0.11^a	0.47 ± 0.03^e	0.75 ± 0.003^a	0.42 ± 0.004^e	0.78 ± 0.016^a	0.07 ± 0.004^e
Values are mean ± SD, n = 3. Different minuscule denoted the significant differences between treatments (p < 0.05).

Effect of acetates on photosynthetic pigments of Cv-31

Figure 3 illustrates the impact of acetate on the photosynthetic pigments of Cv-31. After eight days of incubation in the photosynthetic autotrophic group, the contents of chlorophyll a, chlorophyll b, and carotenoids were recorded as 44.06, 21.47, and 7.86 mg g^− 1, respectively. As the NaAc addition increased, the total pigments content in Cv-31 gradually decreased. Notably, at high concentrations of 10 g L^− 1 and 12 g L^− 1, the total pigment content was significantly lower than that of the control group (p < 0.05) (Fig. 3a). At the concentration of 2 g L^− 1, the total pigment content of algal cells peaked at 20.79% higher than that of the control group. The most abundant photosynthetic pigment was chlorophyll a, with significant synthesis observed in the medium at concentrations of 2, 4, 6 and 8 g L^− 1. The chlorophyll b peaked at 30.72 mg g^− 1 at the concentration of 2 g L^− 1, while different concentrations of NaAc were not sensitive to carotenoid synthesis. In terms of productivity, the total pigment productivity showed an increasing tend followed by a decrease as the concentration of NaAc increased. The maximum productivity of 4.06 mg L^− 1 d^− 1 was obtained at an addition dosage of 8 g L^− 1.

The impact of different concentrations of NH₄Ac on photosynthetic pigments of Cv-31 is presented in Fig. 3b. After eight days of incubation with 2 g L^− 1 NH₄Ac, the total pigment content was comparable to that of the photoautotrophic group, with chlorophyll a, chlorophyll b, and carotenoid content of 43.51, 16.37 and 13.66 mg g^− 1, respectively. However, the addition of the remaining concentrations of NH₄Ac significantly inhibited the synthesis of photosynthetic pigments in Cv-31 (p < 0.05). As the NH₄Ac concentration increased, both the pigment content and productivity of algal cells gradually decreased.

Effect of acetates on protein and carbohydrate of Cv-31

The impact of the addition of two acetates on the protein and soluble sugar content of Cv-31 after 8 days of incubation is depicted in Fig. 4. Different concentrations of acetate exhibited varying effects on protein synthesis in Cv-31. The protein content displayed an increasing and then decreasing trend at different concentrations of NaAc, which were lower than that of Cv-31 under photosynthetic autotrophic culture (20.58%) (Fig. 4a). Conversely, the addition of NH₄Ac significantly improved the protein synthesis of Cv-31 (Fig. 4b). As the NH₄Ac concentration in the medium increased, the protein content of algal cells exhibited an overall decreasing trend. At the concentrations of 2 and 4 g L^− 1, the protein content was 18.08% and 21.09% higher than that of the control group, respectively. In terms of productivity, the protein productivity of the different NaAc treated groups was 12.56, 26.52, 28.64, 29.72, 29.87 and 28.59 mg L^− 1 d^− 1, respectively, while those of the different NH₄Ac treated groups were 16.04, 11.38, 2.82, 2.55 and 1.43 mg L^− 1 d^− 1, respectively. Overall, the microalgae in the NaAc treated group exhibited significantly higher protein accumulation efficiency compared to the NH₄Ac treated group and the control group.

The soluble sugar content of Cv-31 after 8 days of photosynthetic autotrophic culture was measured at 19.43%. The addition of NaAc to the cultured algal cells resulted in a dynamic change in soluble sugar content, characterized by an initial increase followed by a decrease. Only at the concentration of 4 g L^− 1 of NaAc, the soluble sugar content (22.50%) was significantly higher than of the photosynthetic autotrophic group. Conversely, the addition of different concentrations of NH₄Ac led to a continuous decrease in soluble sugar content. Apart from the highest sugar content (20.02%) at 2 g L^− 1 NH₄Ac, the soluble sugar content of the remaining treatment groups was significantly lower than that of the control group. Although the stimulation of soluble sugar content in algal cells by high concentration of acetate treatment in the medium was not significant, it had a positive effect with the accumulation of algal cell biomass, ultimately contributing to productivity. The overall analysis of productivity revealed that different concentrations of NaAc significantly facilitated the bioaccumulation of soluble sugars in Cv-31, reaching maximum productivity (33.25 mg L^− 1 d^− 1) at 10 g L^− 1 NaAc. In contrast, the productivity of NH₄Ac treatment gradually decreased and peaked at 13.22 mg L^− 1 d^− 1 at the concentration of 2 g L^− 1. Therefore, NaAc, as an organic carbon source, promoted the accumulation of soluble sugar in Cv-31 to a greater extent than NH₄Ac.

Effect of acetates on lipid of Cv-31

The effect of adding two acetates in mixotrophic culture for 8 days on lipid accumulation in Cv-31 was investigated and the results are presented in Table 2. The addition of NaAc to the medium resulted in higher lipid content, production, and productivity of algal cells (p < 0.05). At a NaAc concentration of 12 g L^− 1, Cv-31 exhibited a lipid content of 18.46%, lipid production of 0.35 g L^− 1, and lipid productivity of 43.28 mg L^− 1 d^− 1, indicating that the carbon source concentration had not reached the threshold of algal cell consumption. The lipid producing capacity of Cv-31 was enhanced within a certain concentration range of NH₄Ac, beyond which lipid synthesis was inhibited. The mixotrophic culture with 2 g L^− 1 NH₄Ac demonstrated the highest lipid content (14.69%), second only to the medium supplemented with 10 g L^− 1 NaAc, but with lower production and productivity. In general, NaAc can be used as an organic carbon source for achieving high biomass concentration and high lipid content of Cv-31 in mixotrophic cultivation.

Table 2

Effects of two acetates on the lipid synthesis of *C. vulgaris* 31.
Concentration (g L^− 1)	Lipid content (%)		Production (g L^− 1)		Productivity (mg L^− 1 day^− 1)
Concentration (g L^− 1)	NaAc	NH₄Ac	NaAc	NH₄Ac	NaAc	NH₄Ac
0	7.24 ± 0.13^e	7.24 ± 0.13^ef	0.02 ± 0.0003^g	0.02 ± 0.0003^e	2.39 ± 0.04^g	2.39 ± 0.04^e
2	9.19 ± 0.37^d	14.69 ± 0.45^a	0.06 ± 0.002^f	0.08 ± 0.002^a	7.30 ± 0.29^f	9.70 ± 0.30^a
4	9.47 ± 0.25^d	10.65 ± 0.38^b	0.10 ± 0.003^e	0.04 ± 0.001^b	12.93 ± 0.35^e	4.86 ± 0.17^b
6	10.40 ± 0.26^c	9.44 ± 0.27^c	0.13 ± 0.003^d	0.03 ± 0.001^d	15.71 ± 0.39^d	3.53 ± 0.10^d
8	11.01 ± 0.07^c	8.78 ± 0.29^d	0.14 ± 0.001^c	0.03 ± 0.001^c	17.81 ± 0.11^c	3.99 ± 0.13^c
10	15.04 ± 0.58^b	7.75 ± 0.33^e	0.31 ± 0.012^b	0.02 ± 0.001^e	38.88 ± 1.51^b	2.14 ± 0.09^e
12	18.46 ± 0.20^a	6.88 ± 0.22^f	0.35 ± 0.004^a	0.01 ± 0.0004^f	43.28 ± 0.46^a	1.71 ± 0.06^f
Values are mean ± SD, n = 3. Different minuscule denoted the significant differences between treatments (p < 0.05).

The effect of different concentrations of NaAc treatment on the fatty acid composition in algal cells was further investigated, as presented in Fig. 5. The predominant fatty acid fractions in Cv-31 included palmitic acid (C16:0), hexadecatrienoic acid (C16:3), oleic acid (C18:1), and linoleic acid (C18:2). Stearic acid (C18:0) and C18:1 appeared in algal cells after the addition of high concentrations of NaAc (6–12 g L^− 1), while their contents increased with the increase of concentration. Moreover, the addition of NaAc elevated the saturated fatty acid content to some extent. The maximum saturated fatty acid content (40.09%) was reached at an addition level of 12 g L^− 1, significantly higher than that of the control group (33.09%). In conclusion, the carbon chain length of fatty acids ranged from C16-C18 after the addition of NaAc as the organic carbon source, of which the saturation and monounsaturation were basically higher than those of the control group. Furthermore, with the increase of NaAc addition, the composition of fatty acids shifted toward longer carbon chain lengths (from C16 to C18), and the synthesis of saturated and monounsaturated fatty acids was promoted.

Dynamic changes of growth and cellular components of Cv-31 treated with 10 g L^-1 NaAc

The aforementioned findings demonstrate that NaAc exhibited superior promotion of growth and metabolism in Cv-31 compared to NH₄Ac, with an optimal concentration of 10 g L^− 1. Therefore, the dynamics of growth and metabolite accumulation of Cv-31 during 8 days of incubation with the addition of 10 g L^− 1 NaAc were investigated. Table 3 shows the dynamics of growth kinetic parameters of Cv-31. The absorbance values of algal cells at 680 nm exhibited a linear increase with prolonged incubation time. Both dry weight concentration and biomass productivity reached their maximum on day 6. However, the low concentration of NaAc on day 8 of the incubation resulted in a constant dry weight concentration and a significant decrease in biomass productivity.

Table 3

Growth dynamic parameters of *C. vulgaris* 31 treated with 10 g L^− 1 NaAc.
Time (d)	OD₆₈₀	Dry weight concentration (g L^− 1)	Productivity (g L^− 1 day^− 1)
2	1.16 ± 0.03^d	0.31 ± 0.04^c	0.31 ± 0.01^c
4	4.71 ± 0.06^c	1.07 ± 0.09^b	0.64 ± 0.01^b
6	8.94 ± 0.20^b	2.15 ± 0.03^a	0.81 ± 0.02^a
8	9.24 ± 0.05^a	2.15 ± 0.02^a	0.63 ± 0.01^b
Values are mean ± SD, n = 3. Different minuscule denoted the significant differences between treatments (p < 0.05).

The content and productivity of photosynthetic pigments, proteins, and soluble sugars exhibited a characteristic pattern of increase followed by a decrease during the cultivation period, as depicted in Fig. 6. On the sixth day of culture, Cv-31 demonstrated the highest total pigment content of 41.85 mg g^− 1 and a productivity of 4.99 mg L^− 1 d^− 1 (Fig. 6a). Protein and soluble sugar content peaked on day 4, while their yield reached the highest on day 6 (Fig. 6b). It is evident that after more than 6 days, the increase in incubation time is not conducive to the accumulation of pigments, proteins, and soluble sugars in Cv-31.

The lipid content, production and productivity of Cv-31 exhibited an upward trend during the initial 6 days of incubation, followed by a decline both production and productivity as the incubation continued (Table 4). This indicates that Cv-31 experienced faster lipid accumulation in the first 6 days of culture, followed by a non-significant increase. The primary fatty acid fractions identified in Cv-31 were C16:0, C18:1 and C18:2 (Fig. 7). Among them, the percentage of unsaturated fatty acids was higher than that of saturated fatty acids. It is evident that the fractions of C16:0, hexadecadienoic acid (C16:2) and C16:3 in the algal cells exhibited an initial increase followed by a subsequent decrease as the culture days progressed. The percentage of saturated fatty acids in algal cells reached a peak of 34.22% on day 4 of the culture. On day 8, all fatty acid fractions tended to stabilize.

Table 4

Dynamic changes of lipid content in *C. vulgaris* 31 treated with 10 g L^− 1 NaAc.
Time (d)	Lipid content (%)	Production (g L^− 1)	Productivity (mg L^− 1 day^− 1)
2	0.43 ± 0.05^d	0.00 ± 0.0002^d	0.65 ± 0.09^d
4	3.01 ± 0.17^c	0.03 ± 0.004^c	8.06 ± 0.95^c
6	8.90 ± 0.19^a	0.19 ± 0.006^a	31.83 ± 0.92^a
8	8.34 ± 0.20^b	0.18 ± 0.004^b	22.41 ± 0.47^b
Values are mean ± SD, n = 3. Different minuscule denoted the significant differences between treatments (p < 0.05).

Microalgae have the ability to utilize both inorganic and organic carbon for growth and lipid synthesis. Organic carbon sources, such as glucose, glycerol and NaAc, have been found to significantly influence the growth and intracellular components of microalgae. Previous studies have demonstrated that the addition of acetate for mixotrophic culture can effectively enhance biomass accumulation and lipid content in microalgae. In this work, we investigated the effect of adding two acetates, NaAc (providing an organic carbon source) and NH₄Ac (providing a carbon and nitrogen source), on the growth and metabolism of Cv-31. Experimental results showed that Cv-31 efficiently utilized NaAc, leading to increased biomass, yield, and intracellular lipid content compared to NH₄Ac.

Although Spalding (2009) found that CO₂ diffusion in an aquatic environment is approximately 10⁴ times slower than in air, which greatly exacerbates the problem of CO₂ supply by Rubisco. NaAc as an alkaline salt can contribute to the CO₂-enriched environment of the medium, thus promoting biomass accumulation by increasing Rubisco activity. Rai et al. (2013) also demonstrated that C. pyrenoidosa obtained maximum biomass and lipid yield at the addition of 10 mg L^− 1 NaAc for mixotrophic culture, which were 6-fold and 32-fold higher than those of photosynthetic autotrophic culture respectively.

In contrast, NH₄Ac, after hydrolysis, becomes weakly acidic, making it less favorable for absorption and utilization by microalgae in the medium. In addition, due to the high nitrogen content in BG11 medium in this study, the growth-promoting effect of additional NH₄Ac on Cv-31was not significant, except at a concentration of 2 g L^− 1, which showed some facilitation. Therefore, further exploration of the effect of NH₄Ac on the physiological metabolism of microalgae in nitrogen-limited medium would be more appropriate for future considerations.

Furthermore, the concentration of carbon source has been found to play a crucial role in regulating the growth and metabolism of C. vulgaris (Heredia-Arroyo et al. 2011). In this study, it was observed that the growth of Cv-31 was dependent on the concentration of NaAc. As the concentration increased, the biomass and yield of the algal cells gradually increased and reached the maximum at 12 g L^− 1. However, since the maximum culture concentration used in this study was set at 12 g L^− 1, the tolerance of Cv-31 to higher concentrations remains unclear and warrants further investigation.

Algal pigments play a crucial role in photosynthesis and provide protection against stress and oxidation, making them highly valuable from a commercial perspective. The composition of photosynthetic pigments in mixotrophic cultures of green algae is influenced by various factors, including algal species, type and concentration of carbon source, light intensity, and incubation time. It has been observed that different carbon sources, such as glucose, xylose, sucrose, maltose, NaAc, and glycerol, can inhibit photosynthesis in algal cells, resulting in lower photosynthetic pigment content than in photosynthetic autotrophic cells (Chu et al. 1995; Kong et al. 2020). Similarly, in our study, low concentrations of NaAc (2 and 4 g L^− 1) promoted pigment accumulation in algal cells, but at concentrations exceeding 10 g L^− 1, the pigment content was significantly lower than that in photosynthetic autotrophic cells. In contrast, the pigment content of algal cells treated with NH₄Ac was basically lower than that of the photosynthetic autotrophic group. This is probably due to the high concentration of nitrogen source in the medium, which led to the decrease of pigment content even with the addition of low concentration of NH₄Ac.

NaAc serves as a suitable carbon source for stimulating lipid accumulation in mixotrophically cultured Cv-31 due to its anaerobic hydrolysis into easily assimilated organic small molecules. Acetate ions cross the microalgal cell membrane via monocarboxyl/proton transport proteins and then are assimilated by enzymes to acetyl coenzyme A in the cytoplasm (Mehta and Chakraborty 2021), thereby enhancing fatty acid synthesis and biomass production. Chandra et al. (2014) also proposed that lipid accumulation is a result of a higher rate of sugar consumption relative to cell division, which promotes the conversion of excess carbon into lipids. This explanation aligns well with the findings of our experiment, where NaAc significantly increased the lipid synthesis of Cv-31; with a more pronounced stimulation effect observed at higher concentrations. Conversely, the protein content of Cv-31 exhibited an inverse trend to lipid accumulation. None of NaAc concentrations showed a promoting effect on the protein content of C. vulgaris, while low concentrations of NH₄Ac resulted in higher protein content. Orús et al. (1991) explained this behavior that C. vulgaris UAM 101 under mixotrophic conditions may compensate for the decrease in protein content through the increase in lipids. Andersen (2013) found that many algae produce pyrenoids attributed to the accumulation of RuBisco, an enzyme that plays a key role in photosynthetic carbon sequestration. Our experimental results indicate that high concentrations of NaAc have an inhibitory effect on photosynthetic pigment synthesis, partially explaining the decrease in protein content with increasing NaAc concentration. In addition, carbohydrate content seemed to be negatively correlated with lipid accumulation. This phenomenon may be owing to microalgae deviating from their original metabolic pathways to produce higher concentrations of lipids. Furthermore, excessive carbon source concentrations probably stopped carbohydrate synthesis as a result of low pH and high solubility characteristics. This is consistent with the inhibition of soluble sugar biosynthesis by high concentrations of acetate in this experiment.

Energy-rich microalgae with rapid growth rate and high oil production are considered ideal feedstock for biodiesel production. Lipids in microalgae can be classified into two main types: structural lipids and storage lipids. Structural lipids, such as phospholipids and sterols, are essential components of the cell. Storage lipids, mainly triglycerides, are synthesized through photosynthesis and serve as the primary lipid reserves. Fatty acids are integral constituents of storage lipids. The composition of saturated, monounsaturated, and polyunsaturated fatty acids, particularly in short-chain alkyl fatty acids, directly impacts the performance of biodiesel. A higher proportion of saturated and monounsaturated fatty acids contributes to improved antioxidant properties, while a lipid profile polyunsaturated fatty acids enhances cold flow properties. Yeh and Chang (2012) found that C. vulgaris ESP-31 obtained higher lipid content and yield under mixotrophic culture, while more than 60–68% of the fatty acid composition was saturated fatty acids (C16:0, C18:0) and monounsaturated fatty acids (C18:1). Consistent with their findings, our study observed that algal cells treated with NaAc exhibited a fatty acid composition primarily composed of C16 and C18 fatty acids, with a noticeable increase in the proportions of saturated and monounsaturated fatty acids.

In summary, the addition of acetates as organic carbon source effectively enhanced the growth and metabolism of Cv-31 compared to photosynthetic autotrophic culture. NaAc exhibited a significantly higher promoting effect on the growth and lipid accumulation of Cv-31 than NH₄Ac, with a dependence on concentration. Conversely, NaAc inhibited intracellular protein synthesis, while NH₄Ac facilitated it within a specific concentration range. The stimulation effect of high concentrations of both acetates on the synthesis of soluble sugars was not significant. In the dynamics of growth and metabolism during the 8-day cultivation of Cv-31 with 10 g L^-1 NaAc, the biomass and biochemical components content in the algal cells demonstrated a pattern of increase followed by a decrease, reaching the optimum on the day 6 of incubation. Our study shows that the use of acetate as an organic carbon source to regulate the growth of microalgae and the synthesis of intracellular active components is economical and effective in microalgal cultivation and valuable biomass production.

Chlorella vulgaris 31 - Cv-31, NaAc - sodium acetate, NH₄Ac - ammonium acetate, C16:0 - palmitic acid, C16:2 - hexadecadienoic acid, C16:3 - hexadecatrienoic acid, C17:0 - heptadecanoic acid, C17:2 - heptadecadienoic acid, C18:0 - stearic acid, C18:1 - oleic acid, C18:2 - linoleic acid, C18:3 - linolenic acid, RuBisco - Rubisco ribulose-1,5-bishosphate carboxylase.

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Authors' contributions

Shuting Peng: Investigation, Data curation, Methodology, Visualization, Writing-original draft, Writing-review & editing. Yueqin Cao: Investigation, Data curation, Methodology, Visualization, Writing-original draft, Writing-review & editing. Zijian Xie: Investigation, Methodology, Formal analysis, Writing-review & editing. Xiaoyun Zhang: Investigation, Methodology, Formal analysis, Writing-review & editing. Saimai Ma: Investigation, Methodology, Formal analysis, Writing-review & editing. Weibao Kong: Conceptualization, Methodology, Resources, Funding acquisition. Resources, Formal analysis, Validation, Project administration, Writing-original draft, Writing-review & editing.

Funding

This work was supported by Higher Education Industry Support Plan Project of Gansu Province, China (2020C-21; 2021CYZC-37), Key Research and Development Program of Gansu Province (22YF7NA118), Incubation Programme for Key Scientific Research Project of Northwest Normal University (NWNU-LKZD2022-02).

Availability of data and material

Data will be made available on request.

Code availability

Not applicable.

Acknowledgements

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Effects of sodium acetate and ammonium acetate on the growth and production of cellular components by Chlorella vulgaris 31

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Abstract

Figures

Introduction

Materials and methods

Microalga strain, chemicals, and culture medium

Culture conditions

Determination of growth curve and kinetic parameters

Extraction and determination of photosynthetic pigments

Extraction and determination of protein

Extraction and determination of total soluble sugar content

Extraction and determination of lipid

Analysis of fatty acid composition

Statistics analysis

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Status:

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