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Article

Enhanced Production of Microalgal Metabolites Through Aeration Coupled with Stirring

by
Yangyingdong Wu
1,
Yuqing He
1,
Tuo Zhao
1,
Yang Zhao
1,
Ze Yu
1 and
Haiyan Pei
1,2,*
1
Department of Environmental Science and Engineering, Fudan University, Shanghai 200433, China
2
Shandong Provincial Engineering Center on Environmental Science and Technology, Jinan 250061, China
*
Author to whom correspondence should be addressed.
Sustainability 2024, 16(20), 9001; https://doi.org/10.3390/su16209001
Submission received: 5 August 2024 / Revised: 15 October 2024 / Accepted: 15 October 2024 / Published: 17 October 2024
(This article belongs to the Section Environmental Sustainability and Applications)
Browse Figures
Versions Notes

Abstract

:
Adequate mixing is a key factor for microalgal cultivation to achieve high biomass production, so it is essential to clarify the comparative effects of different mixing methods on microalgal productivity, which has rarely been studied previously. This work therefore aimed to investigate the effects of different mixing methods (stirring, aeration, and aeration coupled with stirring) on the growth and metabolite composition of Chlorella sorokiniana SDEC-18, a strain with potential for large-scale application. The results showed that mixing was beneficial for carbohydrate accumulation, while dual mixing (aeration coupled with stirring) promoted growth and achieved the highest dry mass and metabolite productivities (including carbohydrates, proteins, and lipids) through enhancement of light energy capture in the entire system. The stirring speed in the dual mixing approach of aeration coupled with stirring was also considered: the optimal condition was found to be 800 rpm. The maximum biomass was 3.56 g L−1, and the carbohydrate productivity was as high as 119.45 mg L−1 d−1, which was the highest metabolite productivity (higher than proteins or lipids), obtained from aeration coupled with stirring at 800 rpm. Our study suggests that aeration coupled with stirring provides a feasible strategy for microalgal production, due to the optimal availability of CO2 and light achieved through effective mixing.

Graphical Abstract

1. Introduction

Microalgae have recently received widespread attention due to their numerous advantages, such as their fast growth rate and abundance of value-added products—including carbohydrates, lipids, pigments, and other bioactive compounds [1]. At the same time, the applications of microalgae are not limited to their use as a feedstock for third-generation biofuels; they can also be used in a wide range of other industries, such as pharmaceuticals, food, feed, cosmetics, and the environment [2,3,4]. Microalgae are also known as one of the future food sources that will be able to relieve the food crisis arising from increases in the global population [5]. Generally, the level of carbohydrates is high in microalgae cultivated in suitable conditions, and those carbohydrates contain high amounts of energy [6]. The carbohydrates extracted from microalgae also play important roles in antioxidative, anti-aging, and anticancer products, inter alia [7,8]. While producing these carbohydrates, microalgae absorb a large amount of carbon dioxide from the atmosphere through photosynthesis, slowing down the earth’s greenhouse effect. However, few previous studies have focused on production of microalgal carbohydrates compared to the number published on proteins and lipids—mainly accumulated under unfavorable growing conditions. To wit, there have been approximately 5349 papers published on the production of microalgal lipids, 3286 papers on proteins, and only 1910 papers on carbohydrates since 2019. Thus, it is necessary to enhance the productivity of microalgae that have high carbohydrate contents to provide sufficient functional substances for alleviation of the food crisis, to reduce the greenhouse effect, and to promote the sustainable development of the earth’s environment and human society.
Many environmental factors—including pH, light, and mixing—have an impact on the cultivation of microalgae, and furthermore on the production of carbohydrates [6,9]. In particular, the mixing method greatly affects the photosynthesis, growth, and biochemical composition of microalgae [10,11]. Adequate mixing is crucial in microalgal cultivation to help maintain the uniformity of culture, to ensure that CO2, nutrients, and light are obtained by each cell, and to avoid the settling of microalgae; it also helps remove O2 [12,13,14]. Aeration, stirring, and shaking are the main mixing modes at present, and single modes of mixing (aeration alone, stirring alone, or shaking alone) are largely employed during the cultivation of microalgae and bacteria [14,15,16,17]. Aeration can provide enough CO2 for microalgae and enhance the fixation of CO2, thereby contributing to carbon neutrality. When the scale of cultivation becomes large, the microalgae will sediment if only aeration is used [18]. Even if the air flow rate is raised to mix the culture adequately, the shear stress faced by microalgae will increase and the microalgae will collect at the top of the photobioreactor due to a flotation effect, adversely affecting growth [11,19]. Stirring is usually used in heterotrophic cultivation to avoid sedimentation and to ensure an even distribution of nutrients, but the level of dissolved CO2 would be deficient when stirring for microalgal growth in autotrophic cultivation at a large scale [20]. Finding a suitable mixing strategy would therefore be helpful for large-scale cultivation of microalgae.
The dual mixing method offers a novel approach for microalgal cultivation; however, most previous studies have focused on Dunaliella salina, Porphyridiaceae, and Phaeodactylum tricornutum [10,21,22,23]. In a dual mixing approach, such as aeration coupled with stirring, the bubbles provided by aeration can be uniformly distributed with the help of stirring, and high amounts of CO2 can be captured per algal cell. Meanwhile, the problems of sedimentation and the damage to microalgae caused by shear stress are alleviated. There are few studies of dual modes of agitation that pay attention to biomass production or the amount of CO2 fixed by microalgae (Table 1). In addition, in our previous studies, C. sorokiniana SDEC-18 was confirmed to be an ideal unicellular alga with large-scale cultivation potential for biomass production, exhibiting remarkable productivity regardless of whether it was cultivated in BG11 medium, monosodium glutamate production wastewater, seawater, or other media [24,25,26]. In the present research, different mixing modes are therefore chosen for the cultivation of Chlorella sorokiniana SDEC-18 to evaluate suitable mixing methods.
The main objectives of this study are as follows: (i) to clarify the contribution of different mixing methods (aeration, stirring, and aeration coupled with stirring) to microalgal growth, as well as pigment levels and biochemical composition, to obtain the optimal mixing method; and (ii) to find the optimal stirring speed for aeration coupled with stirring in order to obtain the highest biomass production—especially carbohydrate accumulation.
Table 1. Biomass and biochemical composition of microalgae cultivated with different mixing methods.
Table 1. Biomass and biochemical composition of microalgae cultivated with different mixing methods.
MicroalgaMixing MethodCulture Volume (L)Culture Time (d)Dry Mass (g/L)Carbohydrates (%)Lipids (%)Proteins (%)Reference
Mychonastes homosphaeraAeration3.8215.3 ± 0.425.8 ± 0.742.5 ± 1.2 [27]
Nannochloropsis sp.Aeration + stirring3.2753.2 [28]
Arthrospira platensis3.9
Tetradesmus obliquus CPCC05Aeration1.542.8 [29]
Chlorella sorokinianaAeration0.02107.5 [30]
Chlorella vulgaris25.9
Chlorella vulgarisAeration8.667.21 [31]
Chlorella sp.Aeration200061.83 [32]
Scenedesmus dimorphus2.23
Dunaliella salinaAeration + stirring392.423321625[10]
Nannochloropsis oculata3.06111634
Ecdysichlamys minutaStirring1.535312116
Neochloris conjuncta0.644221334
Chlorella sorokiniana SDEC-18Aeration2.5202.08 25.1 [33]
Chlorella sorokiniana SDEC-18Aeration + stirring3143.6241.421.728.4This study

2. Materials and Methods

2.1. Microalgal Strain

The Chlorella sorokiniana SDEC-18 strain used in this study was isolated from Quancheng Lake in Jinan, China [34]. The microalga was cultivated in BG11 medium, containing 1500 mg L−1 NaNO3, 40 mg L−1 K2HPO4, 75 mg L−1 MgSO4·7H2O, 36 mg L−1 CaCl2·2H2O, 6 mg L−1 citric acid (C6H8O7), 6 mg L−1 ferric ammonium citrate (C6H8FeNO7), 1 mg L−1 EDTA-Na2, 20 mg L−1 Na2CO3, 2.86 mg L−1 H3BO3, 1.86 mg L−1 MnCl2·4H2O, 0.22 mg L−1 ZnSO4·7H2O, 0.39 mg L−1 Na2MoO4·2H2O, 0.08 mg L−1 CuSO4·5H2O, and 0.05 mg L−1 Co(NO3)2·6H2O. The C. sorokiniana SDEC-18 was cultured under a constant white light intensity of 60 μmol photons m−2 s−1 for 24 h at 25 ± 1 °C until it reached the exponential stage of growth.

2.2. Experimental Design

In order to investigate the effects of different mixing modes on the microalgal growth, batch cultivation was carried out. Three different mixing methods were employed in this study, namely aeration, stirring, and dual mixing (aeration coupled with stirring); the control system had no mixing (Figure 1). The aeration rate was controlled at 0.1 VVM (air volume/culture volume/min), and the default stirring speed was 800 rpm. We also evaluated the influence of stirring speeds on the growth of C. sorokiniana SDEC-18 for aeration coupled with stirring; three different stirring speeds were used: 500 rpm, 800 rpm, and 1000 rpm. A model SH-3 magnetic stirrer was used (Huzhou Larksci Lab Intrument Co., Huzhou, China); the diameter of the magnetic stir bar was 8 mm and the length was 30 mm. All treatments were performed in 5 L photobioreactors, each with a working volume of 3 L, using BG11 medium, and the initial concentration of algae was 0.2 g L−1. The cultivation lasted 14 days at 25 ± 1 °C, illuminated with white light (60 μmol photons m−2 s−1). Samples were taken every day for the analysis of dry mass, every 48 h for the analysis of cell density, and on days 2, 6, 10, and 14 for the analysis of pigments. The microalga was harvested for biochemical composition analysis on the last day.

2.3. Microalga Biomass and Biochemical Composition Determination

2.3.1. Dry Mass and Cell Density

The biomass was determined from OD680 measured on a spectrophotometer (Evolution 220, Thermo Scientific, Waltham, MA, USA), and the dry mass was calculated according to Equation (1):
Dry   mass   ( g   L − 1 ) = 0.657   ×   O D 680 −   0.0841 ,   R 2 = 0.9926
The cell density of the microalga was measured with a hemocytometer under an optical microscope (BX53, Olympus, Tokyo, Japan).

2.3.2. Cellular Pigments

The cellular pigments (chlorophyll a, chlorophyll b, and carotenoids) were quantified according to a spectrophotometric approach [35]. Briefly, 1.5 mL of culture was centrifuged at 10,000× g for 5 min, and then 1.5 mL of methanol was added after discarding the supernatant. The absorption values at 665.2 nm (A665.2), 652.4 nm (A652.4), and 470 nm (A470) were measured by spectrophotometer after incubation in a water bath at 45 °C for 24 h. The chlorophyll and carotenoids were calculated according to Equations (2) and (3):
Chl   ( mg   g − 1 ) = 1.44   ×   A 665.2 −   24.93   ×   A 652.4 Dry   mass
Caro   ( mg   g − 1 ) = 1000   ×   A 470 −   3561.11   ×   A 652.4 + 1575.62   ×   A 665.2 221   ×   Dry   mass
where Chl and Caro represent the contents of chlorophyll and carotenoids, respectively.

2.3.3. Lipid and Fatty Acids

The content of total lipids was determined with an organic solvent extraction method [6]. The calculation of lipid productivity was performed according to Equation (4):
Lipid   productivity   ( mg   L − 1 d − 1 ) = Dry   mass   ( mg   L − 1 ) ×   Lipid   content   ( % ) Culture   time   ( d )
The fatty acid (FA) contents were determined by methyl esterification [6]. Fatty acid methyl esters (FAMEs) were analyzed by gas chromatography–mass spectrometry (7890B-5977A, Agilent Technologies Inc., Santa Clara, CA, USA).

2.3.4. Carbohydrates

The measurement of carbohydrates was carried out using a phenol–sulfuric acid method [6]. The calculation of carbohydrate productivity was conducted with an equation similar to Equation (4).

2.3.5. Protein

The protein content of the microalga was determined by elemental analysis [36]. Approximately 3 mg of freeze-dried algal powder was put into a tin capsule, which was then inserted into an elemental analyzer (UNICUBE, Elementar, Langenselbold, Germany). The protein content was calculated according to Equation (5):
Protein   content   ( % ) = C N   ×   4.44
where CN (%) represents the content of nitrogen in algal powder.
The calculation of protein productivity was conducted with an equation similar to Equation (4).

2.4. Data Analysis

All results are presented as the respective mean ± standard deviation (SD) from three replicates. The data were analyzed by one-way ANOVA using the SPSS software application (version 19.0), and statistical significance was considered to have been demonstrated when p < 0.05.

3. Results and Discussion

3.1. Effects of Mixing Method on Microalgal Growth and Biochemical Composition

3.1.1. Microalgal Biomass and Photosynthetic Pigments

The dry masses and cell densities of C. sorokiniana SDEC-18 are presented in Figure 2 for different mixing methods. The results illustrate that aeration coupled with stirring promoted the growth of microalgae with the highest dry mass of 3.56 g L−1 and the highest cell density of 6.44 × 107 cells mL−1 after 14 days of cultivation, which were, respectively, 183.7% and 70.2% higher than those of the control group. The dry masses of microalgae cultivated in the stirring-only batches and aeration-only batches were significantly lower than what was obtained when aeration was coupled with stirring, although they caught up to a similar level on the last day, and stirring alone was slightly beneficial to growth (at least in the first 12 days of cultivation) and algal cell division (with a high cell density compared to the aeration-only batches). Thus, aeration helped cell division and biomass accumulation, while stirring only encouraged the biomass production (compared with the control group). It was also found that the dry masses with stirring alone and with aeration alone were 0.90 g L−1 and 0.92 g L−1 higher than in the control group, while it was 2.3 g L−1 when aeration was coupled with stirring.
The pigments—principally including chlorophyll and carotenoids—capture light, reflecting the strength of photosynthesis, and also have an important correlation with microalgal growth [37]. As shown in Figure 3a,b, the chlorophyll and carotenoid contents increased in the early stage of cultivation and then dropped. For the mixed batches, the pigment contents peaked on day 2, whereas in the control group the maximum contents were obtained on day 6. The highest chlorophyll and carotenoid contents, amounting to 25.83 mg g−1 and 6.96 mg g−1, respectively, were observed for aeration alone. The lowest chlorophyll level was 12.02 mg g−1 for stirring alone, similar to the value for dual mixing and the lowest carotenoid was 2.34 mg g−1 for dual mixing on the last day. The results imply that dual mixing had discernible adverse effects on the accumulation of pigments. The ratios Chl a/Chl b and Caro/Chl reflect the activity of PS II [35,38]. The Chl a/Chl b ratio for all mixed systems first increased slightly and then decreased, while the Caro/Chl ratio reduced during cultivation. The lowest Chl a/Chl b and Caro/Chl ratios were observed under conditions of aeration combined with stirring, followed by aeration alone, stirring alone, and then the control. This suggests that the dual mixing promoted a significant increase in the proportion of Chl in both light-harvesting complex II and reaction center complex II, thereby enhancing the PS II activity [39,40].
It is important to mix adequately in the cultivation of microalgae. Generally, a single mixing mode, such as aeration or stirring alone, is used to cultivate microalgae. When using a large-scale columnar photobioreactor, a dead zone can appear during aeration, possibly leading to insufficient mixing [18]. The dual mixing approach (aeration with stirring) not only helped to mix the culture adequately, but also provided sufficient CO2 for microalgal growth [14] in the present study, so lower pH values in the culture and high carbon contents in C. sorokiniana SDEC-18 were obtained for dual mixing compared with stirring alone (Figures S1 and S2). The dry mass obtained from aeration coupled with stirring was about five times higher than the highest biomass of C. sorokiniana SDEC-18 reported previously [24]. With regard to the pigments, C. sorokiniana SDEC-18 accumulated more chlorophyll and carotenoids in the early stage, with the help of an adequate concentration of nitrogen to promote the rapid growth of microalgae. As the microalgal biomass increased, the pigment content gradually decreased, which could enhance the light permeability of the cultivation system, facilitating the utilization of light energy by some microalgal cells and subsequently increasing biomass [40].
According to Figure 3c,d, the photophosphorylation of chloroplast in C. sorokiniana SDEC-18 was promoted [41]. A further consequence found was that individual mixing methods (aeration or stirring) on their own had a similar influence on pigment accumulation [14]. It could therefore be supposed that the dual mixing used herein would not cause damage to C. sorokiniana SDEC-18, the reaction center pigment was sufficient to convert light energy into chemical energy, and microalgal cells preferred to decrease the pigment content to make more cells absorb light for growth. By regulating the pigment composition, the proportion of light-harvesting pigment was facilitated and more light energy and CO2 were captured, leading to an increased production of microalgal biomass in the system, indicating that dual mixing provided an ideal strategy for the cultivation of C. sorokiniana SDEC-18.

3.1.2. Carbohydrate Production

When C. sorokiniana SDEC-18 was cultivated in BG11 medium, the main metabolite was carbohydrates, which accounted for over 39% of the dry mass and was affected by application of different mixing techniques (Figure 4a). The results revealed that, compared with the control conditions, an increase in carbohydrate content was measured, whether with a single mixing method (stirring or aeration alone) or with dual mixing (aeration coupled with stirring). Aeration alone yielded the highest carbohydrate content (49.68%). In terms of the carbohydrate productivity, as shown in Figure 4b, due to the highest dry mass being obtained from dual mixing at the end of experiment, the corresponding carbohydrate productivity was as high as 119.45 mg L−1 d−1, statistically significantly higher than for the other batches (p < 0.05). As a result, the best approach with respect to carbohydrate production was provided by aeration coupled with stirring.
There was more CO2 present in the culture with the dual mixing method, and C. sorokiniana SDEC-18 cultivated in this favorable condition preferentially synthesized carbohydrates, resulting in a higher abundance of carbohydrates compared with the static condition. The microalga’s productivities were mainly dependent on the biomass accumulation, and the carbohydrate yield in this study was higher than those of C. sorokiniana SDEC-18 cultivated in anaerobically digested effluent from kitchen waste and C. sorokiniana UTEX1230 subject to sulfur limitation, and the same as that from C. sorokiniana CMBB276 harvested in aquaculture wastewater [6,42,43]. The results obtained herein indicate that C. sorokiniana SDEC-18 is a strain that has potential to be applied to produce value-added products and slow down the Earth’s greenhouse effect, especially when cultivated with dual mixing, due to the high dry mass and carbohydrate productivity.

3.1.3. Protein, Lipid, and Fatty Acid Production

Similar contents of proteins and lipids were obtained in C. sorokiniana SDEC-18 with the different types of mixing. The protein contents exhibited little reduction when stirring was introduced during cultivation (Figure 4a). The protein content was 28.75% for aeration alone, which was significantly higher than with other mixing methods; the highest protein productivity (79.74 mg L−1 d−1) was obtained from dual mixing, with a statistically significant difference from the other batches (p < 0.05) (Figure 4b). It was also noted that the highest lipid productivity (58.46 mg L−1 d−1) was observed with dual mixing, because it yielded the maximum biomass. Aeration is beneficial for the production of microalgal cells high in proteins, while the protein content changes slightly with changes in the aeration rate at a low concentration of CO2 [44,45]. Although there was little difference in lipid contents with different types of mixing, the fatty acid compositions exhibited a difference, just as in previous studies [13,44,46].
As shown in Figure 5a, more than 92% of fatty acids were C16–C18, of which palmitic acid (C16:0), linoleic acid (C18:2), and linolenic acid (C18:3) were the main fatty acids. Similar profiles and contents of fatty acid, and high contents of saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs), were measured for stirring alone and aeration alone after 14 days of cultivation. The comparatively high content of polyunsaturated fatty acids (PUFAs) obtained with dual mixing implied that aeration coupled with stirring had a positive effect on the accumulation of PUFAs (mainly C18:2), and single modes of mixing had a small influence on the fatty acid content (Figure 5b). A similar result of an increase in PUFAs was recorded when Tetradesmus obliquus was cultivated with 350 rpm agitation coupled with aeration of 0.1 VVM compared with the level for agitation alone [13]. As the main component of the chloroplast membrane, PUFA plays a crucial role in maintaining photosynthetic function, and its accumulation could improve the fluidity of the thylakoid membrane, thereby promoting the electron flow rate involved in photosynthesis [47,48]. With dual mixing, the microalgae may have had an increase in photosynthesis, which was in accordance with the highest PS II activity and PUFA levels being observed, resulting in abundant biomass. In conclusion, aeration coupled with stirring promoted the accumulation of biochemical components—including lipids, proteins, carbohydrates and PUFA—by contributing to the biomass production.

3.2. Effects of Stirring Speeds on Microalgal Growth and Biochemical Composition for Aeration Coupled with Stirring

3.2.1. Microalgal Biomass and Photosynthetic Pigments

Based on the results discussed in Section 3.1, it emerged that aeration coupled with stirring (i.e., dual mixing) could achieve a high production of C. sorokiniana SDEC-18, especially in terms of carbohydrates. In order to further optimize the cultivation conditions of microalgae mixed by aeration coupled with stirring, alternative stirring speeds were explored. Figure 6 shows the associated microalgal growth (dry mass and cell density) and levels of cellular pigments (chlorophyll and carotenoids). There were different responses for dry mass and cell density. When the stirring speed was 1000 rpm, the dry mass was higher than it was for 500 rpm or 800 rpm, whilst the cell densities for 1000 rpm were lower than at the other speeds during the whole cultivation. On the 14th day, the maximum dry mass was obtained at 1000 rpm (3.62 g L−1), followed by 800 rpm (3.56 g L−1) and 500 rpm (2.82 g L−1) (Figure 6a). The cell density slowly increased to 2.38 × 107 cells mL−1 by the end of the experiment at 1000 rpm, and rapidly increased for 500 rpm and 800 rpm (Figure 6b).
These results imply that stirring speeds may have an influence on the dry mass of a single cell of the microalga, and the faster the speed, the heavier the microalgal cell. When the speeds were as high as 800 rpm and 1000 rpm, the changes in levels of the pigments (chlorophyll and carotenoids) were highly similar, and the highest contents of chlorophyll and carotenoids were measured on day 2. In terms of the low speed of 500 rpm, the maximum level of pigments was on day 6. It was also found that the pigment content had a negative correlation with dry mass for different stirring speeds in dual mixing (Figure 6c,d). The curves for Chl a/Chl b and Caro/Chl when stirring at 800 rpm and 1000 rpm were also similar to each other, and were lower than those for stirring at 500 rpm during the cultivation (Figure 6e,f). A slight increase in the dry mass was measured, along with a decrease in the pigment content, when the stirring speed increased from 800 to 1000 rpm.
Silva et al. [21] studied the effects of various fluid stresses caused by stirring and aeration in small-scale stirred vessels on Dunaliella salina. Their research showed that mixing in the reactor would create turbulence at the gas–liquid interface, which would promote the transfer of CO2 from the air to the liquid. At the same time, microalgal cells were subjected to stress during mixing, thereby affecting the growth of microalgae. García Camacho et al. [23] used dual mixing (agitation with aeration) to cultivate Porphyridium purpureum. Their study showed that increasing the mixing intensity appropriately to increase turbulence in the reactor can improve the biomass productivity of microalgae. However, excessive mixing intensity can lead to cell damage or even death, which may be caused by the rupture of bubbles in the liquid.
Sobczuk et al. [22] proposed a similar viewpoint: the team studied the effects of different stirring speeds on the growth of Phaeodactylum tricornutum and Porphyridium purpureum with a given air supply rate. Their results indicated that, for a given aeration rate, the concentration of microalgal biomass first increased and then decreased with the rate of mechanical stirring. The upper limit of the tolerable stirring rate depends upon the type of microalga, and excessive mechanical stirring intensity can cause damage to microalgae. Furthermore, mechanical stirring was not the direct cause of damage to microalgal cells: the main reason was the rupture of bubbles on the surface of the culture, which caused cell damage. Increasing the stirring intensity leads to the production of more bubbles inside the culture. These results were similar to those of the present study, indicating that stirring speed and mechanical force have a significant impact on the growth of microalgae.
Aeration coupled with stirring caused an increase in microalgal biomass, but the growth of algal cells was impacted by shear stress generated by conditions such as mixing, aeration, stirring, and so on, and green algae have greater tolerance toward shear stress than other microalgae [11]. Our study confirmed that C. sorokiniana SDEC-18 had a strong shear resistance and grew rapidly even when experiencing aeration coupled with stirring at 1000 rpm. It is known that photosynthetic pigments such as Chl a are one of the features of microalgae that respond to stress, and the pigment content declines as stress increases [49]. The dry mass and Chl contents were not significantly different for stirring at 800 rpm and 1000 rpm. The reason may be that the optimal stirring speed was between 800 and 1000 rpm, and the dry mass probably first increased and then decreased when the stirring speed increased from 800 to 1000 rpm. The C. sorokiniana SDEC-18 was somewhat stressed at the highest stirring speed in this study, although a similar pigment content was obtained. A single cell obtained from stirring at 1000 rpm was heavier than other samples. The possible reasons relate to two aspects. Firstly, the size of cells was generally big compared with those obtained at 500 rpm and 800 rpm. The size of microalgal cells is related to the cellular stress, and the stress would be apparent from an increase in cell size [50,51]. A more active metabolism and higher chloroplast content were present in larger cells, and more ROS would have been produced to alleviate stress compared to smaller cells [52]. Secondly, the microalgal cells accumulated heavier metabolism. According to the results discussed in Section 3.2.2, the biochemical compositions present in all samples were similar. The microalgal cells became large, and the number of large cells was more than for other stirring rates when we quantified the cell density. Otherwise, the microalgae adhered to the wall of the photobioreactor when the stirring speed was fast for aeration coupled with stirring; thus, 800 rpm should be chosen for culture of C. sorokiniana SDEC-18.

3.2.2. Biochemical Composition and Productivity

Figure 7 presents the biochemical compositions and productivities of C. sorokiniana SDEC-18 cultivated with aeration while stirring at different speeds. There were few changes to the levels of lipids and proteins in microalgae obtained with different speeds on day 14 (Figure 7a). An increase in carbohydrate content was demonstrated for a stirring speed of 800 rpm, which provided the maximum productivities of the biochemical components (carbohydrates, proteins, and lipids) (Figure 7b). Lipid contents were similar for the three stirring speeds. However, there was a difference in fatty acid composition (Figure 7c). The stirring speeds had a negative correlation with SFAs, and the MUFA content first increased and then decreased with the increase in stirring speed—opposite to the response of PUFAs (Figure 7d).
In conclusion, stirring speeds may be unable to effectively adjust the biochemical compositions for stirring speeds of 500 to 1000 rpm combined with aeration. On the other hand, a stirring speed of 800 rpm was beneficial for the accumulation of biochemical components when using aeration coupled with stirring. A higher stirring speed could promote the mixing and transport of algal cells, providing them with better gas circulation and a shorter light/dark cycle, with a positive impact on biomass production [53,54]. According to the results of the present study, the biochemical composition of C. sorokiniana SDEC-18 was slightly affected by the stirring speed changing from 500 to 1000 rpm, suggesting that stirring speed is not a feasible strategy to regulate the biochemical composition, which is different from what was concluded in another study [10]. Thus, the optimal configuration to generate microalgal biomass, especially in the form of carbohydrates, was aeration coupled with stirring at 800 rpm.
Many previous research results have shown that microalgae can serve as a sustainable source of nutrients to provide energy for humans and animals. This is mainly due to the low demand for cultivated land during the large-scale production of microalgae, which does not cause significant waste of freshwater resources and absorbs a large amount of carbon dioxide [55,56]. Comparison of power consumption across different cultivation modes revealed that, for the same energy input of one kilowatt-hour, the dual mixing method combining aeration and stirring yielded 0.46 g of biomass, while aeration alone yielded 0.43 g, and stirring alone yielded 0.36 g. This further demonstrates the potential sustainability of this cultivation method. In summary, the combination of aeration and stirring at an appropriate speed—as mentioned in this study—can further enhance the biomass’s dry mass and the yield of carbohydrates from the SDEC-18 microalga, thereby promoting the sustainable application of this microalga.

4. Conclusions

In this study, two single modes of mixing, viz. stirring alone or aeration alone, and a dual mixing method (aeration coupled with stirring) were used for the cultivation of C. sorokiniana SDEC-18. Compared to stirring or aeration alone, mixed agitation led to a significant increase in biomass and the production of various biochemical components, particularly carbohydrates. This was likely due to the accelerated photosynthesis process, which occurs more efficiently in a well-mixed environment that allows for the capture of more CO2 and light energy. The stirring speed needs to be considered. With the increase in stirring speed, the shear stress suffered by the microalgal cells was also elevated, and then the growth was inhibited due to the damage to the cells. The optimal condition for C. sorokiniana SDEC-18 cultivation was aeration coupled with stirring at 800 rpm. Under those conditions, the biomass was 3.56 g L−1 and carbohydrate productivity was 119.45 mg L−1 d−1. In conclusion, the dual mixing method was ideal for cultivating microalgae and providing high fixation of CO2.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/su16209001/s1, Figure S1: The element analysis of C. sorokiniana cultivated in different disturbance modes; Figure S2: The changes of pH in different disturbance modes.

Author Contributions

Y.W.: Conceptualization, methodology, formal analysis, investigation, writing—original draft, writing—review and editing, and visualization. Y.H.: Conceptualization, methodology, formal analysis, investigation, writing—original draft, writing—review and editing, and visualization. T.Z.: Investigation. Y.Z.: Investigation. Z.Y.: Methodology, writing—review and editing, and visualization. H.P.: Conceptualization, methodology, resources, project administration, funding acquisition, writing—review and editing, visualization, validation, and supervision. All authors have read and agreed to the published version of the manuscript.

Funding

This work was partially supported by the Department of Science and Technology of Shandong Province (Key R&D Program (International Cooperation)) 2019GHZ030 and Agricultural Science and Technology Innovation Project of Shanghai, grant number 2023-02-08-00-12-F04613.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Figure 1. The photobioreactors operating with the different mixing methods used in this study.
Figure 1. The photobioreactors operating with the different mixing methods used in this study.
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Figure 2. (a) Dry mass and (b) cell density of C. sorokiniana SDEC-18 cultivated with different mixing methods.
Figure 2. (a) Dry mass and (b) cell density of C. sorokiniana SDEC-18 cultivated with different mixing methods.
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Figure 3. (a) Chl, (b) Caro, (c) Chl a/Chl b, and (d) Caro/Chl for C. sorokiniana SDEC-18 cultivated with different mixing methods.
Figure 3. (a) Chl, (b) Caro, (c) Chl a/Chl b, and (d) Caro/Chl for C. sorokiniana SDEC-18 cultivated with different mixing methods.
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Figure 4. (a) Biochemical component and (b) productivity of C. sorokiniana SDEC-18 cultivated with different mixing methods. Columns with no letters in common have statistically significant differences (p < 0.05).
Figure 4. (a) Biochemical component and (b) productivity of C. sorokiniana SDEC-18 cultivated with different mixing methods. Columns with no letters in common have statistically significant differences (p < 0.05).
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Figure 5. (a) Fatty acid content (as percentages of total fatty acids) and (b) the PUFA, MUFA, and SFA contents (as percentages of total fatty acid) for C. sorokiniana SDEC-18 cultivated with different mixing methods; C, S, A, and AS represent control, stirring, aeration, and aeration + stirring, respectively.
Figure 5. (a) Fatty acid content (as percentages of total fatty acids) and (b) the PUFA, MUFA, and SFA contents (as percentages of total fatty acid) for C. sorokiniana SDEC-18 cultivated with different mixing methods; C, S, A, and AS represent control, stirring, aeration, and aeration + stirring, respectively.
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Figure 6. (a) Dry mass, (b) cell density, (c) Chl, (d) Caro, (e) Chl a/Chl b, and (f) Caro/Chl for C. sorokiniana SDEC-18 cultivated with aeration while stirring at different speeds.
Figure 6. (a) Dry mass, (b) cell density, (c) Chl, (d) Caro, (e) Chl a/Chl b, and (f) Caro/Chl for C. sorokiniana SDEC-18 cultivated with aeration while stirring at different speeds.
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Figure 7. (a) Biochemical component, (b) metabolite productivity, (c) fatty acid composition (as percentages of total fatty acid), and (d) PUFA, MUFA, and SFA contents (as percentages of total fatty acid) for C. sorokiniana SDEC-18 cultivated with aeration while stirring at different speeds. Columns with no letters in common have statistically significant differences (p < 0.05).
Figure 7. (a) Biochemical component, (b) metabolite productivity, (c) fatty acid composition (as percentages of total fatty acid), and (d) PUFA, MUFA, and SFA contents (as percentages of total fatty acid) for C. sorokiniana SDEC-18 cultivated with aeration while stirring at different speeds. Columns with no letters in common have statistically significant differences (p < 0.05).
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Wu, Y.; He, Y.; Zhao, T.; Zhao, Y.; Yu, Z.; Pei, H. Enhanced Production of Microalgal Metabolites Through Aeration Coupled with Stirring. Sustainability 2024, 16, 9001. https://doi.org/10.3390/su16209001

AMA Style

Wu Y, He Y, Zhao T, Zhao Y, Yu Z, Pei H. Enhanced Production of Microalgal Metabolites Through Aeration Coupled with Stirring. Sustainability. 2024; 16(20):9001. https://doi.org/10.3390/su16209001

Chicago/Turabian Style

Wu, Yangyingdong, Yuqing He, Tuo Zhao, Yang Zhao, Ze Yu, and Haiyan Pei. 2024. "Enhanced Production of Microalgal Metabolites Through Aeration Coupled with Stirring" Sustainability 16, no. 20: 9001. https://doi.org/10.3390/su16209001

APA Style

Wu, Y., He, Y., Zhao, T., Zhao, Y., Yu, Z., & Pei, H. (2024). Enhanced Production of Microalgal Metabolites Through Aeration Coupled with Stirring. Sustainability, 16(20), 9001. https://doi.org/10.3390/su16209001

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