Effect of Carbon Dioxide on the Growth and Nutrient Uptake of the Microalgae Chlorella sorokiniana from Digestate

Abstract

Microalgae are photosynthetic microorganisms capable of capturing CO₂ from both the atmosphere and industrial emissions while producing valuable biomass. Among the various factors influencing microalgal growth, CO₂ availability plays a critical role. This study examined how different CO₂ flow rates affect the growth and nutrient assimilation of Chlorella sorokiniana cultivated in diluted digestate from a biogas plant with nitrogen concentrations up to 5 g/L. Results showed that biomass productivity increased with CO₂ supply up to a threshold, beyond which it declined. The highest mean productivity was observed at a CO₂ flow rate of 0.025 LPM, which did not differ significantly from the 0.050 LPM treatment, indicating comparable performance. In contrast, the highest flow rate (0.100 LPM) led to reduced productivity, although still higher than the control (no CO₂). A similar trend was observed in ammonium removal, whereas phosphorus uptake remained relatively unaffected by CO₂ supply. Overall, elevated CO₂ levels appeared to shift microalgal metabolism towards biomass with lower nitrogen content and increased lipid and carbohydrate accumulation.

1. Introduction

Algae are classified into macroalgae and microalgae [1]. Microalgae constitute an untapped resource, with over 25,000 species identified, yet just 15 are presently exploited commercially [2]. These microscopic organisms are found in both seawater and freshwater and are classified as eukaryotic microorganisms [3]. Rightly referred to as nature’s ‘green gold’ [4], algae is a promising raw material with the potential to mitigate global warming and water pollution, and at the same time providing premium products with significant added value [5,6]. They accumulate high amounts of nutrients by assimilating them from the growing medium [7] and can be cultivated without the use of herbicides or fertilizers [8].

Microalgae are mainly composed of lipids, carbohydrates, and proteins [9]. Depending on their growth conditions, species, strain, and growth phase, microalgae are also capable of producing various non-nutritive metabolites [10] of high added value, such as phenols, alkaloids, and flavonoids [6], which may exhibit antioxidant, anti-inflammatory, antimicrobial, antiviral, and anticancer activities [11]. They also contribute to enhancing sustainable agriculture by providing an alternative bio-fertilizer [12]. Finally, microalgae are used in wastewater treatment, with simultaneous recovery of water, nitrogen, and phosphorus [13].

The approximate molecular formula CO_0.48H_1.83N_0.11P_0.01 reflects the minimal nutritional requirements for microalgal growth [14,15]. Growth is influenced not only by nutrient concentrations but also by various environmental and physiological parameters, including the type of microalgae, light intensity, pH, salinity, nutrient availability, nitrogen-to-phosphorus (N:P) ratio, temperature, dissolved oxygen levels, and the type of culture system used [9,16,17]. Among essential nutrients, carbon, nitrogen, and phosphorus are particularly important for autotrophic growth [9]. Carbon must be supplied in relatively high amounts, as it constitutes a fundamental building block of all cellular organic compounds, including carbohydrates, proteins, nucleic acids, vitamins, and lipids [9]. These nutrients, along with water, can be sourced from waste materials such as wastewater and exhaust gases, which are low-cost or even freely available. This makes microalgae a compelling option for bioenergy production, supporting environmental sustainability and contributing to carbon neutrality, while also conserving finite natural resources [9,18,19].

Carbon dioxide (CO₂) is a crucial factor in microalgal growth, accounting for approximately 50% of biomass by weight based on the biomass molecular formula [20]. Through photosynthesis, microalgae capture CO₂ and convert it into biomass, which can then be used to produce lipids for bioenergy, as well as other valuable products such as food and pharmaceuticals, which strengthens the market potential and financial sustainability of such projects [21,22,23]. Microalgae are significantly more efficient than terrestrial plants in capturing CO₂; while terrestrial plants are estimated to capture only 3–6% of annual anthropogenic CO₂ emissions due to their slow growth, microalgae can capture CO₂ 10 to 50 times more effectively [21,24], having the capacity to incorporate up to 100 gigatons (GT) of CO₂ into biomass per annum [25].

Given the high demand for CO₂ in microalgae cultivation—approximately 1.8 to 2.0 kg of CO₂ is required per kg of biomass produced [9,26]—atmospheric CO₂ (0.03%) alone is insufficient. Instead, utilizing CO₂ from industrial exhaust emissions presents a practical and sustainable alternative, offering a low-cost carbon source while simultaneously reducing atmospheric CO₂ levels [9,17]. Moreover, wastewater is recognized as a readily available source of both water and nutrients, addressing two major limiting factors in algae cultivation [27,28]. Using wastewater not only reduces input costs, with estimated savings of over 50% [29], but also enhances sustainability by treating and purifying waste streams [9,30]. This integrated approach supports the water-energy-environment nexus [26,31]. Microalgae cultivation is a renewable and environmentally friendly process that makes use of non-arable land year-round [32,33], and unlike conventional secondary treatment systems, it actively assimilates CO₂, including that released during sludge dewatering processes [27,34].

While CO₂ is essential for microalgal growth, excessive concentrations can be detrimental. When CO₂ supply exceeds the metabolic capacity of the microalgae, it can lead to acidification of the culture medium, negatively affecting growth [20]. Concentrations above 5% are considered toxic for some species [9]. Tolerance to elevated CO₂ levels varies among species and should be a key criterion when selecting microalgae for CO₂ sequestration. For instance, Scenedesmus sp. can grow at CO₂ levels up to 20%, though its optimal concentration is around 2%, whereas Chlorella sp. can tolerate up to 40% CO₂ [26]. In addition to nitrogen and phosphorus limitation, high CO₂ concentrations can also enhance lipid accumulation in microalgal biomass [20].

It should also be noted that tolerance to environmental stressors, such as high concentrations of ammonium nitrogen (N-NH₄⁺), varies significantly between species. Ammonium toxicity—typically observed at concentrations above 100 mg/L—can impair growth by inducing oxidative stress and disrupting cellular metabolism, although some strains have shown greater resilience depending on cultivation conditions [15,35].

Interventions in variables such as growth temperature [9], light intensity [9,18,20,36], pH [9], alkalinity [11,16,32], and nutrient supply or limitation [18,21,22,24] have been widely studied for their effects on both biomass growth rate and composition of C. sorokiniana. However, relatively few studies have specifically examined the influence of the carbon source—particularly carbon dioxide—on biomass production [37]. The majority of relevant studies, whether on C. sorokiniana [20,38,39] or other microalgae [26], report CO₂ supply as a percentage of the total gas mixture, without specifying the actual flow rate in absolute volumetric units, thereby limiting the evaluation of CO₂ quantity as an independent variable. Since CO₂ is essential for photosynthesis but can inhibit microalgal growth at high levels, defining its supply is crucial. To our knowledge, Montoya-Vallejo et al. [20] have employed similarly high CO₂ flow rates, reaching up to 0.2 LPM. In contrast, Do Thi et al. [38] used a 20% CO₂ concentration with a flow rate below 0.02 LPM, whereas Gabrielyan et al. [39] reported a maximum concentration equivalent to 0.008 LPM.

Considering that the efficiency of CO₂ uptake by microalgae is directly affected by the cell density of the suspension [21], the aim of this study was to investigate the growth behavior and macronutrient composition of C. sorokiniana under varying CO₂ concentrations. Additionally, the study assessed the effect of CO₂ levels on nutrient removal efficiency from polluted wastewater streams. A secondary objective was to evaluate how different CO₂ concentrations affect the carbon and nitrogen content of C. sorokiniana biomass. To the best of our knowledge, the carbon and nitrogen composition of C. sorokiniana biomass under these specific conditions has not yet been reported in international scientific texts.

2. Materials and Methods

2.1. Initital Chlorella sorokiniana Cultivation

The green microalga C. sorokiniana employed in this study was obtained from an existing culture previously maintained at the International Hellenic University in Sindos, Greece, derived from anaerobic digestion effluent [22]. Cultivation occurred in 2 L Erlenmeyer flasks under a steady incubation temperature of 25 ± 1 °C. The growth medium used was BG-11, supplemented as described by Psachoulia et al. [22]. All cultivation materials and media were autoclaved at 121 °C for 20 min to ensure sterility. However, the ammonium ferric citrate and trace element solutions were sterilized using 0.2 μm Whatman PTFE syringe filters (Whatman Inc., Piscataway, NJ, USA). Compressed air, filtered through the same type of syringe filters, was introduced into the cultures at a flow rate of 1 L per minute (LPM) for aeration purposes. Illumination was provided from both sides using LED lights (6000 K, 7.2 W/m), delivering cool white light at an intensity of 20 µmol/m²/s under a 16:8 h light-to-dark photoperiod [22,36,40], a lighting setup shown to enhance biomass yield [40]. Light levels were monitored with a QSO-E PAR Photon Flux Sensor (Apogee Instruments Inc., Logan, UT, USA) linked to a Fluke Model 177 True-RMS Digital Multimeter (Everett, WA, USA). Every 15 days, mother cultures were transferred to fresh BG-11 medium at a 1:10 inoculation ratio to maintain culture vitality. This practice helped ensure that all main cultures were initiated from physiologically consistent populations.

2.2. Anaerobic Digestion Effluent Collection and Processing

The anaerobic digestion effluent (ADE) employed in this work originated from a biogas facility (1 MWel capacity) located in Thessaloniki, Greece, which utilizes waste materials and by-products from nearby livestock farms. To determine its viability for supporting microalgal growth, a chemical characterization was performed to analyze its nutrient composition (as shown in

Table 1. Chemical composition of raw ADE.

Parameters	Before Dilution	After Dilution
COD (mg L−1)	6523 ± 34.00	481 ± 3.00
TN (mg L−1)	2097.6 ± 12.50	155.7 ± 2.10
N-NH4 (mg L−1)	1719 ± 12.24	127.3 ± 1.80
N-NO3 (mg L−1)	63.2 ± 3.31	5.0 ± 0.30
N-NO2 (mg L−1)	3.18 ± 0.12	<0.60
P-PO4 (mg L−1)	19.8 ± 0.37	1.53 ± 0.20
pH	8.31 ± 0.20	8.11 ± 0.12

). Initially, the ADE underwent centrifugation at 5000 rpm for 15 min to eliminate suspended solids and decrease turbidity, after which the supernatant was filtered through Whatman filters (Clifton, NJ, USA; 150 mm, Grade 1, pore size 11 μm). For further purification, the sample was subjected to ultrafiltration using a hydrophilic flat sheet membrane (type H-203, pore size 0.4 μm, Kubota, Osaka, Japan), effectively removing viruses and potentially harmful bacteria that might impede microalgal development. The treated ADE was then stored at −18 °C to preserve its chemical integrity.

2.3. Cultivations with Digestate and Different CO₂ Concnetrations

C. sorokiniana was cultivated in an ADE that had been diluted (dilution factor ≈ 13.5) with sterile water to lower the elevated ammonia nitrogen content to non-toxic for microalgal growth [15,41]. To establish a nitrogen-to-phosphorus ratio (NH₄:PO₄) of roughly 10:1.5, potassium dihydrogen phosphate (KH₂PO₄) was added. The experimental setup involved 2 L Erlenmeyer flasks, each containing approximately 920 mL of culture medium and inoculated with 80 mL of pre-culture, resulting in a final volume of 1.000 mL with an initial optical density (OD₆₈₀) of around 0.35, with an average pH level of 7.95 ± 0.10. Cultures were incubated at 25 ± 1 °C in uniformly lit conditions using cool white LED strips (6000 K, 7.2 W/m) delivering 108 µmol/m²/s of light, under a 16:8 h light/dark photoperiod. Aeration was maintained at 1 LPM using atmospheric air filtered through 0.2 µm Whatman PTFE syringe filters (EK-2LR, Kytola Instruments, Lahti, Finland). Controlled CO₂ supplementation was provided at three flow rates—0.025, 0.050, and 0.100 LPM—regulated by precision flowmeters (FL-3845G-HVR, Omega Engineering, Norwalk, CT, USA), with all CO₂ being sterilized by passage through 0.2 µm syringe filters. Each CO₂ level was tested in two independent biological replicates, with analytical samples collected in triplicate per treatment. The cultivation period lasted for 19 days.

2.4. Microalgae Pre-Treatment for Composition Analysis

Biomass collected from the cultivations was centrifuged at 5000 rpm for 10 min using a Hermle Z326K centrifuge (Hermle Labor-technik, Wehingen, Germany) to separate the cells from the culture medium. The supernatant was discarded, and the concentrated biomass paste was immediately frozen prior to undergoing freeze-drying in a GAMMA 1-20 LMC lyophilizer (Martin Christ GmbH, Osterode am Harz, Germany). Lyophilization was carried out at a pressure of 0.010 bar and a temperature of −78 °C for 48 h. The resulting dried biomass was stored at −18 °C for preservation.

2.5. Analytical Measurements

2.5.1. Growth Determination and Nutrient Analysis

To assess both culture growth and nutrient uptake under each experimental condition, samples were taken at intervals of 24 to 48 h. Growth was tracked by measuring the optical density at 680 nm [42] by a UV–Vis spectrophotometer (DR 3900, HACH, Loveland, CO, USA). In parallel, biomass concentration was quantified as dry cell weight (DCW). For this, 15 mL of culture were filtered through pre-weighed glass microfiber filters (Whatman 934-AH, Piscataway, NJ, USA; pore size 1.2 μm), which were then dried at 60 °C for 24 h and reweighed using a high-precision analytical balance (XP 105, Mettler Toledo, Greifensee, Switzerland) [22,43]. Biomass dry weight was calculated using Equation (1) [44].

$$\mathrm{D}\mathrm{C}\mathrm{W}\mathrm{ }\left({\displaystyle \frac{\mathrm{g}}{\mathrm{L}}}\right)={\displaystyle \frac{\left({\mathrm{W}}_1-{\mathrm{W}}_0\right)}{\mathrm{V}}}\times 100$$

(1)

DCW refers to the concentration of cells, with W₁ indicating the combined mass of the dry cells and their supporting hollow fiber membrane (g), W₀ representing the mass of the hollow fiber membrane alone (g), and V specifying the total volume occupied by the hollow fiber membrane.

2.5.2. Nutrient Analysis

Ammonium nitrogen (N-NH₄) and soluble Phosphorus (P-PO₄) concentrations in the filtrate’s liquid phase were quantified using standardized HACH cuvette tests, performed with a UV–Visible spectrophotometer (DR 3900, HACH, Loveland, CO, USA).

2.5.3. Lipids Determination

Lipid extraction was performed on approximately 0.2 g of freeze-dried C. sorokiniana biomass following the Bligh and Dyer method [45]. Briefly, methanol (2 mL), chloroform (1 mL), and distilled water (0.8 mL) were added in a 2:1:0.8 (v/v) ratio, and the mixture was sonicated at 20 kHz and 400 W/L for 30 min (Sonopuls UW 3400, Bandelin, Berlin, Germany). After centrifugation at 5000 rpm for 10 min, the organic phase was collected. This extraction was repeated twice to maximize lipid recovery [20,22]. Then, chloroform (3 mL) and distilled water (3 mL) were added (2.2:1.8 v/v), followed by centrifugation under the same conditions for phase separation. The lower chloroform layer containing lipids was collected, dried overnight at 60 °C, and weighed with an analytical balance (XP 105, Mettler Toledo, Greifensee, Switzerland)

2.5.4. Proteins Determination

Approximately 0.2 g of freeze-dried microalgal biomass was rehydrated in 100 mL of distilled water for protein extraction. The cells were disrupted via sonication at 20 kHz and 400 W/L for 30 min, after which the mixture was centrifuged at 10,000 rpm for 10 min at ambient temperature. The resulting supernatant was then subjected to protein quantification using the Lowry method [46]. In brief, 1 mL of the sample was combined with 5 mL of modified Lowry reagent and incubated for 10 min. Subsequently, 0.5 mL of 1 N Folin–Ciocalteu reagent was added, and the mixture was stirred and incubated for an additional 30 min. Absorbance was read at 750 nm using a UV–Vis spectrophotometer (DR 3900, HACH, Loveland, CO, USA), and protein concentration was determined based on a BSA standard curve ranging from 0 to 1500 μg/mL [44].

2.5.5. Carbohydrates Determination

The supernatants obtained from the biomass–water suspensions prepared for protein assays were also employed to assess carbohydrate content using the phenol–sulfuric acid method [47]. In brief, 1 mL of 1% (w/v) phenol and 5 mL of 96% (w/w) sulfuric acid were added to the sample. Absorbance was recorded at 483 nm using a UV–Vis spectrophotometer (DR 3900, HACH, Loveland, CO, USA). Neutral sugars were quantified as glucose equivalents based on a calibration curve prepared with D-glucose.

2.5.6. Elemental Analysis

Total nitrogen, carbon and phosphorus were assessed using a CHNS elemental analyzer (FlashSMART 1112, Thermo Fisher Scientific, Waltham, MA, USA). Approximately 2–3 mg of microalgal biomass were loaded into tin capsules and subjected to combustion at 925 °C, with pure oxygen serving as the oxidizing gas and pure helium used as the carrier gas.

2.5.7. Statistical Analysis

Each treatment was measured in triplicate, and the results are expressed as mean values ± standard deviation (SD). Statistical analyses were performed using Minitab software (version 21.4.2, Minitab LLC, State College, PA, USA). Differences among treatments were assessed via analysis of variance (ANOVA) followed by Tukey’s post hoc HSD test, considering p ≤ 0.05 as the threshold for statistical significance.

3. Results

3.1. Effect of CO₂ Flow Rate on Chlorella sorokiniana Growth

A series of different CO₂ flow rates (0.025 LPM, 0.050 LPM and 0.100 LPM) in mixture with air were applied to examine the effect of surplus CO₂ on the growth rate of the C. sorokiniana culture. The referenced culture was supplied only with air. At the lowest CO₂ flow rate (0.025 LPM), the concentration was approximately 2.5% of the supplied gases. Higher flow rates of 0.050 LPM and 0.100 LPM corresponded to concentrations of approximately 5.0% and 10%, respectively. Throughout the cultivations, the pH of the culture medium fluctuated within 7.3 ± 0.3 in all CO₂-supplemented variants. In the cultivation supplied with air only, without additional CO₂, the pH stabilized at 8.1 ± 0.2.

Biomass growth of C. sorokiniana is presented in Figure 1a,b, based on optical density at 680 nm (OD₆₈₀) and DCW, respectively. In all cultures supplemented with CO₂, the biomass growth rate was significantly higher than in the reference culture, which received CO₂ only from the supplied air (~0.03–0.04%). Specifically, cultures with CO₂ flow rates of 0.025 and 0.050 LPM showed OD₆₈₀ values that were 41.95% and 38.37% higher, respectively, compared to the reference. A similar trend was observed in the DCW measurements. The 0.025, 0.050, and 0.100 LPM CO₂ cultures produced 37.24%, 36.55%, and 21.38% more biomass, respectively, than the reference culture. Furthermore, the growth curves for both OD₆₈₀ and DCW followed a similar pattern in the 0.025 and 0.050 LPM cultures, indicating comparable and consistently enhanced biomass production at these CO₂ levels.

Changes in CO₂ concentration were found to be critical for the productivity of C. sorokiniana in the study of Do Thi et al. [38] and Gabrielyan et al. [39]. Similarly, Montoya-Vallejo et al. [20] achieved high growth rates of the microalga C. sorokiniana with a CO₂ supply of 10% (0.100 LPM), compared to low CO₂ provided exclusively from the supplied air. In the same study, a CO₂ concentration of 20% (0.200 LPM) resulted in a significantly higher biomass productivity than in the absence of exogenous CO₂ supply but not compared to the 10% CO₂ supply which resulted in reduced productivity.

Similar results are supported by studies on other microalgae as well. In particular, Khan et al. [48] in a study using two strains of the species Dictyosphaerium sp. concluded that the growth rate for both microalgal strains increased with CO₂ supplementation. A CO₂ concentration of 4% led to increased biomass production compared to 2% or to CO₂ provided only through the supplied air (0.03–0.04%). Chlorella vulgaris, even at extreme CO₂ concentrations, not only showed increased biomass yield, but also exhibited a protective effect on the photosynthetic mechanism from photoinhibition and photodamage. In the same study, the inhibitory effects of CO₂ occurred at concentrations of 60% and above, corresponding to 0.84 LPM CO₂, with the optimal growth rate of cultivation occurring at CO₂ concentrations between 30 and 40% or between 0.42 and 0.56 LPM CO₂ [49]. Similarly, Li et al. [50] reached similar conclusions, albeit with much lower CO₂ concentrations. They reported that the optimal growth rate was observed at a CO₂ concentration of 5% or 0.01 LPM, compared to concentrations of 0.04% (0.0004 LPM), 2% (0.004 LPM), 10% (0.02 LPM), and 15% (0.03 LPM).

The optimal CO₂ concentration range for microalgae cultivation is likely species-dependent, and excessive levels may result in reduced productivity or even inhibitory effects. In this study, C. sorokiniana cultures grown under CO₂ flow rates of 0.025 and 0.050 LPM achieved the highest biomass productivity, with mean DCW values of 0.1047 and 0.1042 g/L/day, respectively. These values did not differ significantly from each other, indicating comparable performance. In contrast, the 0.100 LPM CO₂ supply led to a lower biomass yield (0.0992 g/L/day), although still higher than that of the control culture supplied only with ambient air (0.0763 g/L/day).

3.2. Effect of CO₂ Flow Rate in the Macronutrient and Vital Elements Concentration of Biomass

The chemical composition of microalgae can vary considerably when they are cultivated under different environmental conditions [51,52,53]. In many microalgae, lipids are the most responsive macronutrient to environmental stress, particularly nutrient limitation. However, aside from lipids, microalgae also contain carbohydrates and proteins, and any shift in one of these components typically correlates with adjustments in the others [54]. Also, as the cultivation process advances, the levels of specific macronutrients within the biomass undergo alterations [55]. To monitor these variations, macronutrient levels of C. sorokiniana cells were measured on the first, tenth, and the last day of the cultivation series.

Figure 2 shows the macronutrient (carbohydrates, proteins and lipids), nitrogen and carbon content of C. sorokiniana cells during cultivation with different concentrations of CO₂. Carbohydrates showed a slight increase, but no statistical difference was observed in their concentration over time. Their levels were not affected by CO₂ supply across all cultivation series. Similar results were reported by Mortensen and Gilserød, where the starch concentration of C. sorokiniana was unaffected by increased CO₂ levels, while a change from artificial light to daylight led to a significant increase in starch concentration from 1.5% to 6% [56]. Moreover, Sun et al. reported that elevated CO₂ levels during the cultivation of C. sorokiniana, activated carbohydrate pathways, are likely to provide precursors (pyruvate and acetyl-CoA) for triacylglycerol (lipids) synthesis [57]. Higher lipid concentrations were observed in the cultures supplied with 0.025, 0.050 and 0.100 LPM CO₂ and this will be discussed further in this section below.

In other microalgae species, the literature reports an increase in carbohydrate content with CO₂ supply. For instance, increasing the CO₂ concentration in Parachlorella kessleri cultures raised the carbohydrate content of the biomass from 33.7% to 44.2% [58]. Similarly, Patil and Kaliwal [59] observed a significant increase in the carbohydrate content of Scenedesmus bajacalifornicus biomass under high CO₂ supply, reporting a fourfold increase from 6.88% to 26.19%. However, in C. sorokiniana cultivations no statistically significant effect on biomass carbohydrate content was observed from the interaction between cultivation duration and CO₂ supply (Figure 2).

The protein content in the biomass of the microalgae C. sorokiniana was initially 31.1 ± 1.9%. Over time, it exhibited a declining trend across all cultivations. Statistically significant differences were observed only in the cultures supplied with 0.050 and 0.100 LPM of CO₂. In these two cultures, the protein content decreased significantly by approximately 11% after 10 days. However, a total reduction of about 27% from the initial protein content was recorded only in the culture supplied with 0.100LPM of CO₂. Similar results were reported by Varshney et al., showing that the protein content in C. sorokiniana cells decreased over time during cultivation with different CO₂ concentrations (0.04%, 5%, 10%, and 15%) [60]. However, different results presented by Cecchin et al. showed that a 2.5% CO₂ supply significantly increased protein levels during the cultivation of C. sorokiniana when compared to air supply [60]. Cultivation of C. sorokiniana with a higher CO₂ supply (0.050 and 0.100 LPM) led to a reduction in protein levels over time, likely due to nutrient depletion (e.g., nitrogen or phosphorus) that channeled the excess carbon from CO₂ into lipid production [60].

Contrary results were obtained for lipids. Both CO₂ supply and the duration of cultivation favored lipid synthesis in microalgal biomass. The highest lipid production after 19 days, compared to the initial lipid content, was observed in the culture with 0.500 LPM CO₂ (20%), followed by those with 0.025 LPM (17%) and 0.100 LPM (14%). A possible cause for the variation in lipid content under different CO₂ supplies is the induction of metabolic and functional changes in microalgal biomass, converting high CO₂ levels into biomass with higher lipids content [61]. A previous study by Sun et al. [57] demonstrated that increased CO₂ supply enhances lipid composition by enhancing the biosynthesis of Acetyl-CoA. Similar results, such as increased lipid content in the presence of high CO₂ concentrations, were also observed in C. vulgaris, reaching up to a 19% increase. The researchers attributed this increase to the cells’ attempt to adapt to the stress caused by the high CO₂ concentration [61]. Similarly, in another study, CO₂ supply at a concentration of 2.6% significantly increased the lipid content of C. vulgaris microalgal biomass, along with a marked rise in Acetyl-CoA concentration [37].

The total nitrogen content in the C. sorokiniana biomass showed a decreasing trend over time across all cultures (Figure 2). On the first day of the experiment, nitrogen content was measured at 7.13 ± 0.07%. Statistically significant differences were observed only in cultures supplied with CO₂. Specifically, after 10 days, the total nitrogen content had decreased by approximately 7% in the culture with 0.025 LPM CO₂, and by about 12% in the cultures with 0.050 and 0.100 LPM CO₂. By the end of the experiment, the total nitrogen reduction in the 0.100 LPM CO₂ culture had reached around 27% compared to the initial value, indicating a strong relationship between increased CO₂ supply and reduced nitrogen content in C. sorokiniana biomass. In the other CO₂-supplemented cultures, the nitrogen content at the end of the experiment (day 19) had decreased by about 11% and 13% for 0.025 LPM and 0.050 LPM CO₂, respectively. A possible explanation for this trend—namely, the reduced N-NH₄ fixation by microalgae grown under high exogenous CO₂ supply, despite an increased dry cell weight and, consequently, a higher cell population compared to cultures without exogenous CO₂—is that CO₂ enhances the activity of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) [62]. An increase in this enzyme has been shown to reduce nitrogen use efficiency [63]. Therefore, the additional nitrogen taken up, as a fundamental structural component of proteins, was utilized for protein formation [9]. This trend was also reflected in the nitrogen and protein content of the biomass under the various cultivation conditions. The average nitrogen-to-protein (N-protein) conversion factor was calculated as 4.51, which aligns well with values reported in the literature for microalgae grown under a range of environmental conditions (4.78) [64].

The initial total carbon content in the biomass of C. sorokiniana was 41.29 ± 0.78%. Over time, the carbon content showed an increasing trend, reaching its peak under a CO₂ supply rate of 0.025 LPM (Figure 2). Statistically significant differences were observed among all cultivation conditions. The culture supplied with air showed the smallest increase, with carbon content rising by 6.6% on day 10 and 5.5% on day 19. The maximum carbon composition was recorded in the culture with 0.050 LPM CO₂, reaching 20% and 24% increases after 10 and 19 days, respectively. In the culture with 0.025 LPM CO₂, carbon content increased by approximately 25% on day 10, but decreased to 20% by the end of cultivation. The cultivation of C. sorokiniana significantly increased the total carbon content, as expected due to the external CO₂ supply. However, the highest carbon accumulation was not observed at the maximum CO₂ supply rate, but rather at 0.050 LPM.

Figure 3 presents the final concentrations of carbohydrates, proteins, and lipids within the microalgal cells after 19 days of cultivation under different CO₂ supply conditions. The carbohydrate content showed no significant differences among the various cultures and appeared unaffected by the additional CO₂ supply. In contrast, the protein and lipid contents exhibited noticeable changes. Specifically, protein concentrations decreased with increasing CO₂ flow rates. The culture supplied with 0.100 LPM CO₂ exhibited the lowest protein content, compared to the culture that received only ambient air. On the other hand, lipid concentrations increased in all cultures that were supplemented with CO₂, indicating a positive correlation between CO₂ enrichment and lipid accumulation in the microalgal biomass. The present findings indicate an inverse relationship between lipid and protein content, implying that lipid accumulation may primarily result from protein degradation or suppression of protein synthesis [9].

3.3. Effect of CO₂ Flow Rate on the Nutrients Removal

Contrary to the old belief that liquid waste is a source of pollution, the goal is now to add value to liquid waste, not only through their de-pollution but also through the recovery of resources from it [31]. Microalgae have demonstrated enormous potential for bioremediation of industrial wastewater [9]. Microalgae-based treatment systems can efficiently remove phosphorus and nitrogen with efficiencies of up to 75% [2,4,54]. Microalgae exhibits a strong preference for assimilating ammonium nitrogen, as its uptake demands less energy [65]. Before being incorporated into biomass, alternative nitrogen forms must first be transformed into ammonium inside the cells, which demands additional energy [51].

Figure 4 illustrates the reduction in N-NH₄ content from ADE through uptake by C. sorokiniana cells, analyzed as a function of cultivation time under four different CO₂ supply conditions. ADE had an initial concentration of N-NH₄ of about 115 mg L⁻¹ and P-PO₄ of about 16 mg L⁻¹. After cultivation of C. sorokiniana in ADE for 19 days, under different amounts of CO₂ supply, showed that the reduction in ammonium from the effluent was highest in the reference cultivation with air supply, removing 90.3% of the initial nitrogen as N-NH₄, compared to 73.3%, 73.0% and 60.7% in conditions with 0.025 LMP, 0.05 LPM and 0.100 LPM CO₂, respectively. Similarly, Hena et al. [66] achieved complete removal of N-NH₄ from dairy wastewater using Chlorella strains. A possible explanation for the increased uptake of N-NH₄ by microalgae growing without exogenous CO₂ supplementation, despite a reduced dry cell weight and a smaller cell population, is that CO₂ enhances the activity of the enzyme RuBisCO, which accelerates the photosynthetic process [62]. Similarly, reducing the RuBisCO enzyme improves nitrogen use efficiency [64]. Therefore, the decreased activity of this specific enzyme in absence of exogenous CO₂ supply probably explains the increased nitrogen usage by the culture, aimed at its growth.

Additionally, other abiotic mechanisms such as nutrient volatilization and precipitation may also contribute to nutrient removal. During photosynthesis, microalgae consume CO₂, which can increase the culture’s pH [67] and promote the conversion of NH₄⁺ to volatile NH₃, thereby reducing nitrogen availability [68]. However, this effect was likely negligible under our conditions. At the recorded pH of 8.13 (in the absence of exogenous CO₂), the equilibrium favors NH₄⁺ over NH₃, resulting in only minimal volatilization [69]. Volatilization and precipitation of nutrients become significant mainly at higher pH values (9–11), supporting the minimal volatilization observed at the pH measured in our study. This conclusion is further reinforced by the comparable consumption rates of NH₄⁺ and PO₄³⁻ in this treatment, suggesting that nitrogen loss through volatilization did not occur to a significant extent.

Figure 5 illustrates the time-dependent reduction in P-PO₄ from ADE. Regarding the removal of phosphorus from the wastewater, it did not appear to be affected by the additional CO₂ supply, removing in all conditions between 95.8% and 96.6% of P-PO₄. Depending on the selected microalgae species, the duration, and the cultivation environment, up to 70% of the organic load and 90% of the inorganic load can be removed from the initial levels, respectively [70,71]. Specifically, it was reported that Navicula sp. removed 80% of N-NH₄ and 70% of P-PO₄ from contaminated streams [2].

Figure 6 illustrates the rates of N-NH₄ and P-PO₄ assimilation normalized to biomass, expressed in mg of nutrients per gram of cells over time. As demonstrated in Figure 1, the intensity of CO₂ supply affects biomass accumulation. Therefore, a more accurate interpretation of nutrient assimilation rates requires accounting for the corresponding biomass concentration [55]. The highest rates of N-NH₄ and P-PO₄ uptake were recorded during the early days of cultivation, likely due to the abundance of nutrients and the low biomass present. As the culture developed and cell density increased, the assimilation rate per unit biomass progressively decreased. This observation indicates that the impact of CO₂ on nutrient uptake is largely mediated by its effect on biomass productivity rather than direct alterations in cellular metabolism. Similar findings were reported by Palikrousis et al. [44] and Delgadillo-Mirquez et al. [72], who noted that microalgal nutrient assimilation is more closely linked to biomass production than to light availability. Nonetheless, Figure 6 shows that the pattern of nutrient assimilation per unit dry biomass was consistent across all CO₂ treatments, suggesting that CO₂ concentration did not substantially modify the mechanism by which nutrients were incorporated into the cells.

4. Conclusions

This study primarily aimed to examine how CO₂ supply influences the cultivation of C. sorokiniana in biogas plant effluent, with the objective of determining the conditions that maximize biomass production, enhance nutrient removal from the liquid phase, and generate cells with a targeted composition. The key conclusions drawn from the results are as follows: (1) The optimal conditions for high growth and biomass production of C. sorokiniana were achieved under moderate CO₂ supply levels, with the two most effective flow rates showing no significant difference in performance. A higher CO₂ flow rate resulted in slightly reduced productivity, while cultures without additional CO₂ showed clearly limited growth, confirming that insufficient carbon availability is a major constraint for microalgal cultivation. The greatest average productivity occurred at a CO₂ supply of 0.025 LPM, which was not statistically different from the 0.050 LPM condition, suggesting similar efficiency. By contrast, supplying CO₂ at 0.100 LPM resulted in lower productivity, though it remained above the level recorded in the control without CO₂ supplementation. (2) The chemical profile of C. sorokiniana biomass was influenced by CO₂ supply, indicating that adjusting the CO₂ flow rate can steer the biomass toward a preferred composition. Over the course of cultivation, lipid and carbohydrate levels increased, while protein content declined correspondingly. A similar effect was observed regarding CO₂ supply, although it did not result in statistically significant changes in carbohydrate levels. An interaction between culture duration and CO₂ supply was observed only in the lipid content. (3) CO₂ influenced the rate of nutrient assimilation in ADE and had a negative impact on the N-NH₄ removal rate, depending on the CO₂ levels. More efficient N-NH₄ removal was observed in the absence of CO₂ supply, with a direct negative relationship between the removal rate and CO₂ levels. Regarding phosphorus removal, CO₂ supply levels appeared to have no effect on the rate of phosphorus assimilation. (4) The influence of CO₂ concentration on ADE nutrient assimilation was primarily mediated by its impact on biomass productivity. These findings support the potential for improved biomass yields in large-scale microalgal cultivation when ADE with suitable chemical characteristics is used as the nutrient source.

Future research could consider developing indigenous algae–bacteria consortia adapted to the challenging conditions of full-strength ADE. Such synergistic systems have demonstrated enhanced nutrient removal, biomass productivity, and tolerance to pollutants compared to monocultures, through mutual metabolic interactions and coordinated O₂/CO₂ exchange [73]. Selecting native strains pre-adapted to ADE conditions may optimize consortia performance and enable more efficient treatment of nutrient-rich and inhibitory waste streams.