Carbon Dioxide Bio-Sequestration and Biomass Production for Energy Purposes During C. vulgaris Cultivation Powered by Real Exhaust Gases from a Municipal Thermal Power Plant

Abstract

This study assessed the potential of Chlorella vulgaris to biosequester carbon dioxide (CO₂) and produce microalgal biomass using real exhaust gases from a municipal heating plant. Experiments were conducted in vertical tubular photobioreactors (V-PBRs) in three series: a control with atmospheric air as the CO₂ source (S1), exhaust gases containing SO_x (S2), and fully desulphurised exhaust gases (S3). The highest productivity of C. vulgaris was recorded in S3, where 2120 ± 123 mg VS/L was achieved with an exponential growth rate of 281.0 ± 16.2 mg VS/L·d. The presence of SO_x in the gases caused the culture to die off as early as day 8 of the cultivation cycle, resulting in a decrease in biomass concentration and acidification of the culture medium. In S2, compared to the other experimental series, significantly lower organic carbon, lipid, and sugar contents were also observed in the microalgal biomass. However, protein content remained stable regardless of the CO₂ source tested. Carbon Dioxide Utilisation Efficiency (CO₂UE) was 53.8% (S1), 24.1% (S2), and 41.4% (S3), respectively. The results indicate that the presence of SO_x in exhaust gases negatively affects the growth and survival of C. vulgaris, while its removal improves both biomass productivity and CO₂ sequestration efficiency. The research demonstrates the potential for integrating microalgae cultivation with industrial CO₂ emission management.

1. Introduction

The ongoing rise in atmospheric carbon dioxide (CO₂) concentration, resulting primarily from the combustion of fossil fuels, presents one of the greatest challenges for contemporary energy and climate policy. Strategic documents such as the European Green Deal and the Paris Agreement clearly indicate the need to achieve climate neutrality within the coming decades [1]. Achieving this objective requires the implementation of effective carbon capture, storage, and utilisation (CCUS) technologies [2]. Conventional methods such as amine absorption, membrane separation, and mineral carbonation, although technically advanced, are often associated with high operating costs and significant energy consumption [3].

In this context, CO₂ biosequestration using microalgae is a particularly attractive alternative, as it combines emission reduction with the simultaneous production of biomass, offering valuable functional and economic benefits [4]. Unlike classical CCUS technologies, in which CO₂ is only captured and deposited in geological formations, microalgae bind this gas in stable organic compounds (proteins, lipids, and carbohydrates), which can be stored long-term in biomass [5]. Depending on further development, this biomass can be used as a substrate for the production of liquid biofuels, biogas, or biohydrogen [6]; as a raw material in biotechnological processes [7]; for the production of high-value-added bioproducts [8]; or for the creation of modern construction and building materials [9]. In scenarios where microalgae products are used in non-energy sectors, including composite materials, organic fertilisers, or feed additives, CO₂ remains removed from the carbon cycle for longer [10]. The facts presented above make biosequestration a viable element of a sustainable CO₂ storage strategy within CCUS.

Numerous studies have shown that microalgae, in addition to CO₂ assimilation, are capable of binding other gaseous pollutants, such as nitrogen oxides (NO_x) and sulphur oxides (SO_x), commonly present in waste gases from power plants [11,12]. This mechanism would further enhance the attractiveness of microalgae technologies for reducing multicomponent atmospheric emissions. However, research results are inconclusive. Some reports indicate a beneficial effect of low SO₂ and NO_x concentrations on biomass growth [11,13,14], while others demonstrate a strong inhibition of photosynthetic processes and a reduction in culture efficiency [15,16]. A significant limitation of current knowledge is that most studies to date have been conducted on a laboratory or pilot scale, most often using synthetic gas mixtures [17,18]. Full-scale experiments integrating microalgae cultures with real energy installations are still rare. The lack of such studies significantly limits the ability to reliably assess the technology’s effectiveness, cost-effectiveness, and potential for implementation in industrial settings.

In studies on CO₂ biosequestration performed under real operating conditions, the choice of microalgal strains with high tolerance to environmental variability and flue gas impurities is crucial [19]. These organisms must demonstrate both efficient CO₂ assimilation and adaptability to elevated levels of nitrogen oxides (NO_x), sulphur oxides (SO_x), variable pH, and temperature. From a technological perspective, species used in industrial biosequestration systems should also maintain stable growth over time, show predictable productivity, and have well-defined physiological characteristics [20]. Chlorella vulgaris fulfils these requirements. It is a eurybiontic and well-studied species, widely recognised for its robustness and versatility in environmental biotechnology [21]. This microalga tolerates CO₂ concentrations up to 15–20%, exhibits rapid growth, and efficiently assimilates carbon even in the presence of gaseous pollutants. Its ability to adapt to changing light, temperature, and medium conditions makes C. vulgaris a model organism for evaluating real flue gas utilisation in biosequestration systems [22]. The species also offers a favourable biochemical profile, with biomass rich in proteins, lipids, and carbohydrates, suitable for renewable energy production such as anaerobic digestion and liquid biofuel synthesis [21,23]. Owing to its stable growth, extensive literature background, and reproducible results across pilot- and semi-technical-scale studies, C. vulgaris is considered a reference species for integrating CO₂ biosequestration with energy biomass production using real exhaust gases from municipal heating plants [24].

Therefore, the aim of this study was to determine the potential of Chlorella vulgaris microalgae to biosequesteration CO₂ under real-world conditions, using exhaust gases from an existing municipal heating plant. Two experimental variants were analysed: (i) a culture fed with gases containing SO₂ at concentrations compliant with applicable emission standards, and (ii) a culture fed with fully desulphurised exhaust gases. The studies verified the effect of low SO₂ concentrations on the growth dynamics and stability of C. vulgaris cultures, microalgae biomass production efficiency, and CO₂ sequestration efficiency. The experimental results allow for a better assessment of the realistic possibilities of integrating microalgae technologies with commercial energy production and indicate their role as an innovative path within CCUS systems, combining emission reduction with carbon storage in biomass and the potential for further energy and industrial use.

2. Materials and Methods

2.1. Organisation of Experimental Work

Experimental work was carried out at the Municipal District Heating Company (MDHC, Olsztyn, Poland), while analytical work took place at the laboratories of the University of Warmia and Mazury in Olsztyn. The study was divided into three experimental series (S), based on the composition of gases supplied to the operating vertical tube photobioreactor (V-PBR), which provided a source of carbon dioxide (CO₂) for the microalgae Chlorella vulgaris. In series 1 (S1), the control, the culture medium was supplied only with atmospheric air. In series 2 (S2), exhaust gases treated using a system operated at MDHC were added to the V-PBR. These gases met the emission standards for SO_x, NO_x, and dust applicable to this system. In series 3 (S3), the CO₂ source for the growing C. vulgaris biomass was exhaust gases completely free of SO_x, which were removed using a scrubber with calcium carbonate sorption. The study focused on assessing the efficiency and dynamics of microalgae biomass production and determining the CO₂ utilisation efficiency (CO₂UE).

2.2. Research Location

Experimental work related to the operation of the tested V-PBRs was conducted at the Municipal District Heating Company (MDHC) LLC (Limited Liability Company) in Olsztyn, Poland (53°74′78.20″ N, 20°44′79.70″ E). MDHC operates six water boilers (WR-25), including three modernised with sheet pile technology, adapted for co-firing coal and biomass. The total nominal capacity of the installation is 174.45 MW. MDHC has a complete exhaust gas treatment installation and a 70 m high emitter. All analytical work was carried out at the Centre of Aquaculture and Ecological Engineering of the University of Warmia and Mazury in Olsztyn, Poland (UWM in Olsztyn) (53°45′14.03″ N, 20°27′43.33″ E).

2.3. Materials

2.3.1. Microalgae Biomass

The C. vulgaris strain BA-166 was acquired from the Collection of Baltic Algae Cultures (CCBA) at the University of Gdańsk. Prior to the experimental runs, the culture was expanded in 1 L flasks containing F/2 medium (8 ppt). The preculture was maintained at 25 °C, under continuous cool-white illumination (~12,000 lx, approximately to 210–300 µmol/m²∙s), with orbital mixing at 100 rpm. Inoculum material was withdrawn once the cells reached the exponential growth phase, ensuring a metabolically active biomass supply. Subsequently, the inoculum was transferred to the V-PBR operated under the defined process conditions. The initial biomass concentration in the reactor was standardised to 50 mg VS/L.

2.3.2. Cultivation Medium

The base medium (BM) was prepared by combining tap water with a nitrogen–phosphorus fertiliser (Azofoska, Grupa INCO SA, Warsaw, Poland) and a microelement mixture (MicroPlus, Intermag, Olkusz, Poland). According to the manufacturer, Azofoska contains 13.3% total nitrogen, distributed as 5.5% nitrate-N and 7.8% ammonium-N, as well as 6.1% P₂O₅ bound as ammonium citrate, 4.0% water-soluble P₂O₅, 17.1% K₂O, 4.5% MgO, and 21.0% SO₃. The MicroPlus supplement provides trace elements in chelated form, including 2.3 g/L boron, 1.2 g/L copper, 22.3 g/L iron, 9.5 g/L manganese, 0.6 g/L molybdenum, and 3.5 g/L zinc. For medium preparation, the fertiliser and the microelement solution were added at 1.35 g/L and 0.1 mL/L, respectively. Tap water used for medium preparation was not sterilised. This approach was intentionally adopted to better reflect real cultivation conditions of microalgae. The absence of sterilisation allowed for the simulation of the natural microbial background and potential interactions typical of semi-open or open systems used in practical CO₂ biosequestration and large-scale biomass production.

2.3.3. CO₂ Source

In S1 (control series), the CO₂ source was atmospheric air. Air was continuously introduced into the V-PBR using an ACO-208 diaphragm pump (Hailea, Chaozhou, China) with a capacity of approximately 5000 ± 50 L/h. This maintained a CO₂ concentration in the culture medium of 0.6 ± 0.2 mg/L, corresponding to the maximum equilibrium concentration of CO₂ in water. In S2 and S3, the CO₂ source was exhaust gases from the MDHC power plant. In S2, the exhaust gases, after pretreatment in the industrial plant, were introduced using an ACO-208 diaphragm pump (Hailea, China) with a capacity of 100 ± 15 L/h. The exhaust gas supply was combined with gas recirculation from the V-PBR gas phase using an ACO-208 diaphragm pump (Hailea, China) with a capacity of approximately 4000 ± 45 L/h. The flow of new exhaust gases into the system was controlled by a pH metre with a voltage controller (1000 L VWR, International, Radnor, PA, USA). The operating pH of the culture medium was 7.0 with a hysteresis of 0.2. When the pH in the culture medium dropped below 6.6, the system shut off the exhaust gas flow. The recirculation pump only drew gas from the gas phase of the V-PBR. If the pH exceeded 7.0, the exhaust gas flow to the recirculation pump intake was activated. Fresh and recirculated exhaust gases were pumped from the bottom of the V-PBR. In S2, the gases came directly from the pipe leading to the emitter. These exhaust gases were reduced in NO_x concentration using Selective Non-Catalytic Reduction (SNCR) technology, and the SO_x and dust content were reduced using a semi-dry method with Ca(OH)₂ lime milk and bag filters.

In S3, exhaust gases from the industrial MDHC pretreatment system were additionally directed to a separate pretreatment system for complete SO_x removal. The system used the limestone method for flue gas desulphurisation, which involves absorbing SO₂ in a limestone suspension (ground limestone, CaCO₃) in water and reacting in the liquid phase:

CaCO₃(aq) + H₂O → Ca²⁺ + HCO³⁻+ OH⁻

SO₂(aq) + H₂O → HSO³⁻ + H⁺

HSO³⁻ → H⁺ + SO₂³⁻

HSO³⁻ + ½ O₂ → SO₂⁴⁻

and leading to precipitation:

Ca²⁺ + SO₂³⁻ + ½ H₂O → CaSO₃·½ H₂O(s)

Ca²⁺ + SO₂⁴⁻ + 2 H₂O → CaSO₄·2 H₂O(s) (gypsum)

After calcium sulphate and sulphite precipitate in water, subsequent portions of calcium carbonate dissolve according to its solubility product, allowing further SO₂ absorption. In flue gas desulphurisation (FGD) systems, the absorption process was carried out after dust removal and cooling of the flue gas. The system comprised a 30-litre dust removal tank filled with 1 mm thick glass fibre fabric, with fibre diameters ranging from 0.2 to 2 µm and a porosity of approximately 70 to 90%. The dedusted flue gas then flowed into a 30-litre scrubber filled with calcium carbonate. From the scrubber, the gases passed into a recirculation tank, which served as the intake for the pump supplying the flue gas to the V-PBR feed system. Exhaust gases entered and exited the desulphurisation system at a rate of 100 ± 15 L/h, with recirculation occurring at a rate of 4000 ± 45 L/h. The method of dosing the pretreated exhaust gases to the V-PBR in S3 was analogous to that in S2, using pH measurement (6.6 ± 0.2) to activate the exhaust gas dosing pump. The characteristics of the exhaust gases used in the subsequent test series are presented in

Table 1. Characteristics of gases constituting the source of CO₂ in successive experimental series.

Indicator	Unit	Series
		S1	S2	S3
CO2	% obj.	0.042 ± 0.001	5.92 ± 0.31	5.61 ± 0.22
O2	% obj.	21.0 ± 0.2	10.2 ± 2.1	9.8 ± 1.6
CO	mg/m3	0.0 ± 0.0	241 ± 92	233 ± 74
NOx	mg/m3	8.2 ± 0.9	284 ± 21	277 ± 33
SOx	mg/m3	11 ± 1.1	801 ± 91	0.0 ± 0.0
dust	mg/m3	not marked	4.3 ± 0.6	0.0 ± 0.0

. A diagram of the method for feeding the V-PBR with CO₂ in the subsequent test series is shown in Figure 1.

2.4. Experimental Stations and Procedures

C. vulgaris microalgae were cultured in three vertical, cylindrical photobioreactors (V-PBRs) operated on a fractional-technical scale. The total volume of the V-PBRs was 150 L, with a working volume of 120 L filled with culture medium (internal diameter 400 mm, working height 800 mm). The V-PBRs were made of transparent Plexiglas. They were illuminated with fluorescent lamps with a colour temperature of 900 K. The applied luminous flux was 720 lm, measured at photosynthetically active radiation (PAR) of 270–300 μmol/m²·s, at a wavelength of 400–700 nm, using cool white light with a 12:12 light–dark cycle (Osram, Munich, Germany). The culture temperature was maintained at 23.0 ± 2.0 °C using a TERMIO-1 temperature recorder (TER-MOPRODUKT, Bielawa, Poland) with a PT1000 temperature sensor. The adopted environmental conditions reflect the typical operational parameters considered suitable for maintaining stable growth, photosynthetic activity, and cellular integrity of C. vulgaris in controlled photobioreactor systems, ensuring reproducible biomass production throughout the cultivation period.

2.5. Analytical, Computational and Statistical Methods

Samples for volatile solids (VS) and chlorophyll a were collected every 24 h. Volatile solids were determined gravimetrically by combusting the biomass at 550 °C (LAC L muffle furnace, Dąbrowica, Poland) and subsequently weighing the ash (DanLab AX423, Białystok, Poland). The results were expressed as mgVS/L. The concentration of chlorophyll a was determined using a fluorescence method with an Algae Online Analyzer (bbe Moldaenke, Schwentinental, Germany). This device allows continuous and non-invasive measurement of chlorophyll a concentration directly in microalgal suspensions. The analysis is based on the excitation of chlorophyll molecules with modulated blue and red light, followed by detection of emitted fluorescence signals. The intensity of fluorescence is automatically converted to chlorophyll a concentration according to pre-established calibration curves obtained using standard chlorophyll a solutions (Sigma-Aldrich, Burlington, MA, USA, analytical grade). The results were expressed as mg/L. Taxonomic analysis of C. vulgaris biomass was performed using an MF 346 biological microscope with an Optech 3MP camera (Eduko, Warsaw, Poland). Microscopic observations were conducted under bright-field illumination at magnifications of 400× and 1000×. Morphological features of the cells, including shape, size, and colony organisation, were evaluated and subsequently compared with descriptions provided in classical taxonomic keys and relevant databases [25,26]. pH measurements were taken with a 1000 L pH metre (VWR International, Radnor, PA, USA). The pH solenoid valves were controlled using an Aqua Medic computerised pH controller (Aqua Medic, Janikowo, Poland). Illuminance was measured with a HI 97500 luxmeter (Hanna Instruments, Woonsocket, RI, USA). PAR measurements were performed using an HD 2102.2 device with an LP471PAR 400 nm–700 nm probe (Senseca, Remscheid, Germany) with a measurement range of 0.1–10,103 μmol/m²·s. The exhaust and effluent gases from the V-PBR were analysed using a Handheld Multigas Analyzer Optima MRU (MRU, GmbH, Neckarsulm, Germany). The analyser measured the gas content of SO₂, CO₂, CO, and NO_x using photoelectric measuring cells. Carbon Dioxide Utilisation Efficiency (CO₂UE) was calculated according to Equation (1):

$${CO}_{2}UE={\displaystyle \frac{1.88·(M_{PBR}-M_0)}{M_{{CO}_2}}}$$

(1)

where the coefficient 1.88 is derived from the adopted empirical formula for microalgae biomass (CO0.48H1.83N0.11P0.01), where (M_PBR − M₀) represents the increase in dry biomass mass in the V-PBR, and M_CO2 is the mass of supplied CO₂. The value of M_CO2 was calculated as the total gas volume multiplied by the density ρCO₂ = 1.8 g/L (28–30 °C, 105 Pa). The total volume of CO₂ (V_CO2) was determined based on the efficiency of the pumps supplying gases containing CO₂ (flow rate R_GAM) and the gas analyser (molar fraction n_CO2) as shown in Equation (2):

$$V_{{CO}_2}=n_{{CO}_2}· R_{GAM}·(t_2-t_1)$$

(2)

Experiments were performed in quadruplicate. Statistical analysis was conducted using Statistics 13.3 software (Statsoft, Inc., Tulsa, OK, USA). The significance level for differences between variables was set at 0.05. Normality of distribution was assessed using the Shapiro–Wilk test, differences between means were determined using one-way analysis of variance (ANOVA), and homogeneity of variance within groups was tested using Levene’s test. The significance of differences between analysed variables was assessed using the honest significant difference (HSD) test.

3. Results and Discussion

3.1. Process and Efficiency of C. vulgaris Cultivation

The biomass production efficiency of C. vulgaris varied significantly between the experimental series. In the control (S1), the microalgae concentration increased steadily from 50 ± 7 mg VS/L on day 0 to a maximum of 1980 ± 101 mg VS/L on day 15 of culture (Figure 2a,b). In S1, the lag phase lasted 3 days, and the exponential phase lasted 9 days (Figure 2c), allowing biomass growth at a rate of 186.6 ± 7.4 mg VS/L·d (Figure 2d,

Table 2. C. vulgaris biomass growth rate, characterised by vs. and Chl-a concentration during the linear growth phase, according to the experimental series.

Series	Unit	Value
S1	mg VS/L·d	186.6 ± 7.4
S2		147.2 ± 12.1
S3		281 ± 16.2
S1	mg Chl-a/L·d	7.72 ± 0.34
S2		6.37 ± 0.32
S3		12.92 ± 0.41

).

In S2, where the CO₂ source was non-desulfurized exhaust gases, the initial growth of the C. vulgaris population was faster than in the control sample. Already on day 2, the biomass reached 144 ± 13 mg VS/L, and on the next day 297 ± 17 mg VS/L (Figure 2a). The obtained values were significantly (p < 0.05) higher than those observed in S1. The lag phase was shorter and amounted to 2 days (Figure 2c), and the exponential growth rate was 147.2 ± 12.1 mg VS/L·d (Figure 2d, Table 2), which is significantly (p < 0.05) lower than those observed in both S1 and S3. The highest C. vulgaris biomass concentration in S2, at 910 ± 71 mg VS/L, was achieved on day 9 (Figure 2b). After 2 days of stable growth, a die-off phase occurred and the biomass density significantly decreased to 470 ± 42 mg VS/L on day 16 (Figure 2a). The observed growth curve indicates that although additional CO₂ stimulated C. vulgaris population growth at the beginning of the culture cycle, the presence of contaminants, primarily SO₂, caused acidification of the medium, oxidative stress, and toxic sulphate/sulphite accumulation, which led to population destabilisation and decreased productivity later.

The highest technological performance was observed in S3, where the CO₂ source consisted of desulfurized flue gases. In this series, the lag phase was short, lasting only 2 days, followed by an exponential growth phase of 6 days, during which C. vulgaris biomass increased at the highest rate of 281.0 ± 16.2 mg VS/L·d (Figure 2c,d). By day 13, the biomass reached 2120 ± 123 mg VS/L, which was approximately 11% higher than in the control S1 (1910 ± 97 mg VS/L) and more than twice that observed in S2 (Figure 2b). These differences were statistically significant (p < 0.05). The stationary phase lasted for 5 days, indicating a stable culture. The slight decrease in biomass to 2020 ± 120 mg VS/L on day 16 (Figure 2a) was minor and within the expected range of natural culture stabilisation at high cell density.

The results obtained confirm literature reports indicating that SO₂ is the main barrier to exhaust gas utilisation in microalgae cultivation and that the desulphurisation process is a key step enabling effective CO₂ biosequestration [27,28,29,30]. The observed phenomenon can be interpreted in light of several complementary biological and physicochemical mechanisms. SO_x present in the aquatic environment dissolves and dissociates, forming sulphuric acid and lowering the pH of the culture medium [12]. The toxic influence of exhaust gas components on C. vulgaris KR-1 was described by Lee et al. [31], who demonstrated that increasing SO₂ concentration to 150 ppm (with 15% CO₂) rapidly reduced carbon fixation and biomass production within 12 h. Similarly, Fu et al. [15] found that the presence of SO₄²⁻ ions up to 800 mg/L decreased biomass by 58%, while concentrations above 400 ppm completely inhibited algal growth due to pH decline and chloroplast degradation. Studies using exhaust gases containing SO_x confirmed that exposure to 150 ppm H₂SO₄ reduced algal dry matter from >0.2 g VS/L to approximately 0.05 g VS/L, with the critical threshold at 90 ppm [16]. Literature data consistently indicate that excessive acidification induces physiological stress, inhibits photosynthesis and macromolecule biosynthesis, and reduces the accumulation efficiency of organic substances, directly determining the vs. level [32]. Under low pH conditions, microalgae adjust carbon assimilation mechanisms, switching from active bicarbonate uptake to passive CO₂ diffusion [33]. Abinandan et al. [33] also demonstrated metabolic shifts in Desmodesmus sp. MAS1 and Heterochlorella sp. MAS3, with downregulation of carbohydrate pathways and upregulation of protein and lipid pathways [34]. Additionally, the presence of sulphates disrupts ionic balance and nutrient/microelement transport [35], leading to slower growth rates and a lower proportion of organic matter in total cell mass.

Another mechanism described in the literature is the toxic effect of free SO₂ forms, which can penetrate the cell membrane and generate reactive oxygen species (ROS), such as superoxide radicals, hydrogen peroxide, and hydroxyl radicals [36,37,38,39]. Oxidative stress causes damage to lipid structures, proteins, and nucleic acids, limiting the ability of cells to divide and effectively biosynthesise organic compounds [38,40,41,42]. Consequently, a decrease in biomass quality is observed, indicated by a lower vs. content

In the control series (S1), the initial concentration of C. vulgaris biomass was 3.10 ± 0.20 mg Chl-a/L and increased systematically to a maximum of 79.20 ± 3.30 mg Chl-a/L on day 15 of culture (Figure 3a,b). The microalgae growth curve was typical, with a 3-day adaptation phase (lag), a dynamic exponential phase lasting 7 days, and a stable stationary phase (6 days), without a distinct death phase (Figure 3c).

In S2, the chlorophyll a concentration reached a maximum of 35.60 ± 2.40 mg Chl-a/L; however, after day 9, growth was inhibited and a decrease in pigment content was observed, reaching 17.41 ± 1.40 mg Chl-a/L on day 16 (Figure 3a). The exponential phase in S2 lasted only 4 days (Figure 3d). The stationary phase lasted 3 days, followed by a distinct death phase lasting 7 days (Figure 3c). The biomass growth rate of 6.37 ± 0.32 mg VS/L·d was significantly (p < 0.05) lower than in both S1 and S3 (Table 2). These results indicate that the presence of not fully desulphurised exhaust gases negatively affected the metabolism and stability of the culture, leading to premature cell senescence. This was most likely a consequence of oxidative stress induced by the presence of SO₂ and other gaseous pollutants, which impaired the photosynthetic apparatus [43].

The initial biomass concentration in S3 was 2.95 ± 0.40 mg Chl-a/L, but the increase was significantly (p < 0.05) more dynamic than in the other variants (Figure 3d). By day 6, a level of 54.80 ± 3.10 mg Chl-a/L was reached, and the maximum occurred between days 11 and 13, reaching 93.30 ± 3.70 mg Chl-a/L (Figure 3a). The lag phase was short (2 days), and the exponential phase lasted 5 days, characterised by the highest growth rate among all series, amounting to 12.92 ± 0.41 mg VS/L·d (Figure 3d, Table 2). This value was significantly (p < 0.05) higher than in S1 and S2. The stationary phase lasted 4 days, followed by a gradual die-off phase, during which the biomass decreased slightly to 88.90 ± 4.10 mg Chl-a/L on day 16 (Figure 3c).

It should be noted that only chlorophyll a was determined in this study, as it represents the principal photosynthetic pigment and a direct indicator of photosynthetic efficiency in C. vulgaris. Although chlorophyll b is also involved in light harvesting, its quantitative variations are typically proportional to those of chlorophyll a, and therefore the omission of this parameter does not affect the interpretation of biomass growth dynamics or photosynthetic activity. Chlorophyll a content can be considered a direct indicator of the toxic effects of various factors, including SO₂, on C. vulgaris culture [44,45]. Fu et al. [15] observed that as the dissolved SO₂ concentration increases, the concentration of hydrogen ions (H⁺) in the culture medium rises rapidly, resulting in a rapid drop in pH and thus inhibiting the activity of enzymes and pigments necessary for photosynthesis. The negative mechanism of SO₂ involves its dissolution in water and the formation of SO₃²⁻ and H_SO₃⁻ ions, which are toxic to microalgae cells [46]. It has been documented that these ions can damage photosynthetic pigments by oxidising the porphyrin ring of chlorophyll a, leading to pigment degradation [47]. Inhibition of chlorophyll biosynthetic enzymes, such as protochlorophyllide reductase, has also been confirmed, as well as disruption of the integrity of thylakoid membranes, where chlorophylls are associated with photosystem protein complexes [48]. Induction of oxidative stress through the generation of ROS is also widely suggested, which enhances pigment degradation [40]. In the presence of SO₂, damage to the photosystem II reaction centre, reduced RuBisCO enzyme activity, and decreased electron transport efficiency also occur [49,50]. These disruptions result in limited carbon assimilation and, consequently, lower production of organic compounds in C. vulgaris cells. In practice, this means that the presence of SO_x in untreated exhaust gases (S2) not only limits chlorophyll a synthesis but also accelerates its degradation. This explains the shortened exponential phase and the rapid transition of biomass to the die-off phase in this series.

3.2. Characteristics of C. vulgaris Biomass

C. vulgaris cultivation in the presence of SO_x in exhaust gases (S2) was characterised by a significantly (p < 0.05) lower vs. content in the biomass. The vs. value in this series was 86.3 ± 1.5% TS, while in S1 and S3 it reached 88.9 ± 1.4% TS and 88.7 ± 1.6% TS, respectively (

Table 3. Characteristics of C. vulgaris biomass depending on the test series.

Parameter	Unit	S1	S2	S3
Volatile solids (VS)	[%TS]	88.9 ± 1.4 a	86.3 ± 1.5 b	88.7 ± 1.6 a
Total nitrogen (TN)	[mg/gTS]	47.1 ± 1.5 a	46.2 ± 1.3 a	47.4 ± 1.7 a
Total phosphorus (TP)	[mg/gTS]	8.4 ± 0.8 a	8.6 ± 1.1 a	8.5 ± 0.9 a
Total carbon (TC)	[mg/gTS]	540 ± 18 a	573 ± 22 b	549 ± 20 ab
Total organic carbon (TOC)	[mg/gTS]	471 ± 14 a	466 ± 13 b	477 ± 15 a
Proteins	[%TS]	29.2 ± 1.2 a	28.4 ± 1.1 a	29.3 ± 1.4 a
Lipids	[%TS]	7.0 ± 0.7 a	5.9 ± 0.8 b	7.3 ± 0.9 a
Saccharides	[%TS]	36.1 ± 1.5 a	34.3 ± 1.4 b	35.7 ± 1.6 a

Description: Different superscript letters (a, b, ab) within the same row indicate statistically significant differences between the analysed variables. The same letters indicate no significant difference.

). Simultaneously, a decrease in TOC content was observed, from 471 ± 14 mg/g TS (S1) and 477 ± 15 mg/g TS (S3) to 466 ± 13 mg/g TS in S2 (Table 3). A significant reduction in the organic fraction indicates a reduced potential for energy use of the biomass, for example, in biomethane and biohydrogen production processes. The content of bioavailable organic matter determines the technological effects of anaerobic digestion [51,52,53,54].

However, the most pronounced effect of SO_x exposure was observed on storage metabolites. In S2, the carbohydrate content was 34.3 ± 1.4% TS, which was significantly lower (p < 0.05) than in S1 and S3, where it was 36.1 ± 1.5% TS and 35.7 ± 1.6% TS, respectively (Table 3). Lower concentrations were also observed for lipids, with the share in S2 not exceeding 5.9 ± 0.8% TS, while in the remaining samples it reached values above 7% TS (Table 3). These differences, confirmed as statistically significant (p < 0.05), clearly indicate that biomass exposure to SO_x limited the ability of cells to accumulate energy compounds, primarily fats and sugars. Literature data show that changes in the pH of the culture medium lead to significant modifications in metabolic processes in microalgae cells, particularly in the synthesis of key biomolecules such as carbohydrates, proteins, and lipids [55]. Environmental stress induced by low pH disrupts cell homeostasis, leading to an extended lag phase and changes in metabolism [56]. Generally, microalgae respond to environmental stress with increased lipid accumulation [57]. However, under reduced pH conditions, a shift in carbon uptake metabolism occurs in microalgae cells, switching from active bicarbonate transport to passive diffusion, resulting in a significant change in cell metabolic activity, particularly regarding the accumulation of carbohydrates, proteins, and lipids [34]. Buayam et al. [58] observed a significant decrease in carbohydrate and amino acid synthesis in Desmodesmus sp. when the culture medium pH was 4.0. Osundeko et al. [59] reported a significant reduction in carbohydrate content and an increase in lipid concentration in microalgae cells subjected to oxidative stress. According to other studies, the increase in dry weight and carbohydrate content in Chlorella ellipsoidea cells reached their maximum values at alkaline pH, while the protein content was highest at pH 4 [60].

In contrast to the parameters characterising C. vulgaris biomass described above, protein content and total nitrogen and phosphorus levels remained similar regardless of culture conditions (Table 3). No statistically significant differences were found between batches (p < 0.05). TN content ranged from 46.2 ± 1.3 mg/g TS (S2) to 47.4 ± 1.7 mg/g TS (S3), while TP ranged from 8.4 ± 0.8 mg/g TS (S1) to 8.6 ± 1.1 mg/g TS (S2). This indicates that protein metabolism processes were less susceptible to SO_x than the biosynthesis of storage metabolites. Protein content ranged from 28.4 ± 1.1% TS (S2) to 29.3 ± 1.4% TS (S3).

This relationship can be attributed to the physiological effects of SO_x on microalgae. In aqueous media, SO_x hydrolyses to sulphites and sulphates, lowering pH and generating reactive sulphur species that are toxic to photosystem II and inhibit photosynthesis and carbohydrate synthesis [48]. The resulting oxidative stress produces ROS that damage lipids, carbohydrates, DNA, and cell membranes, explaining the reduced lipid and organic carbon fractions [61,62]. In response, cells activate antioxidant defence pathways, shifting metabolism toward amino acid and protective protein synthesis at the expense of fat and sugar accumulation [62]. Increased proline and glutathione production helps maintain the NADP:NADPH redox balance and protects against oxidative damage [63,64,65]. Under stress, H₂S further enhances antioxidant enzyme activity through post-translational modifications such as S-nitrosation and S-glutathionylation [48]. Excess SO_x may also disrupt sulphur metabolism and alter the synthesis of sulphur-containing amino acids (methionine, cysteine), leading to changes in biomass composition. ROS-induced oxidation of amino acid residues, particularly arginine, proline, and cysteine, promotes proteolysis of sulphur-rich proteins [66,67]. The absence of significant variation in protein content between batches suggests that nitrogen–protein metabolism remained stable, while carbohydrate and lipid fractions were most affected.

3.3. Changes in the pH of the Culture Medium and CO₂ Fixation by C. vulgaris

In the S1 series, using air as the CO₂ source, the volume of gas introduced during the cultivation cycle was 1920 m³. The typical CO₂ concentration in air was 0.042 ± 0.001% by volume. Taking into account the efficiency of the feeding system used, a total of 1586.6 g of CO₂ was introduced to the V-PBR over the 16 days of cultivation (

Table 4. Output data and achieved CO₂ utilisation efficiency during the full cultivation cycle by experimental series.

Series	Volume of Air Introduced Into the V-PBR [m3]	Volume of Exhaust Gases Introduced Into the V-PBR [m3]	CO2 Content in Gases Introduced Into the V-PBR [% vol.]	Amount of CO2 Introduced into the V-PBR During the Cultivation Cycle [gCO2]	CO2 Content in Gases Discharged from the V-PBR [% vol.]	Amount of CO2 Discharged from the V-PBR During the Cultivation Cycle [gCO2]	CO2 Bound During the Cultivation Process in the V-PBR [gCO2]	Total Amount of C. vulgaris Biomass Obtained During the Cultivation Cycle [g]	CO2 Utilisation Efficiency [%]
S1	1920	-	0.042	1586.6	0.022	793.3	793.3	232.0	53.8
S2	-	19.2	5.92	2231.8	4.63	1745.5	486.3	67.4	24.1
S3	-	38.4	5.61	4229.9	3.87	2917.9	1311.9	294.0	41.4

). The amount of C. vulgaris biomass produced was 232 g VS, resulting in a CO₂UE value of 53.8%. The pH of the culture medium systematically increased from an initial level of 7.03 ± 0.03 to 8.14 ± 0.08 on the 16th day of V-PBR operation. This is a typical effect of photosynthesis by microalgae, which decreases the concentration of H⁺ ions in the culture medium and promotes optimal photosynthetic activity. A stable increase in pH in S1 correlates with steady growth of C. vulgaris biomass and high CO₂UE efficiency. A similar increase in pH, from an initial value of 8.55 to 9.55, was observed by Eloka-Eboka and Inambao [4] during studies of CO₂ sequestration using microalgal biomass. The pH of the culture medium is crucial for regulating the efficiency of carbon sequestration, photosynthetic activity of microalgae, and nutrient uptake [68,69,70,71,72]. An increase in pH can lead to an imbalance of carbon species in the culture medium due to decreased CO₂ solubility and accumulation of bicarbonate and carbonate ions, thus limiting carbon availability to microalgae and hindering the photosynthetic fixation process [73]. Among all forms of inorganic carbon, only dissolved CO₂ and the bicarbonate ion HCO₃⁻ can be used by microalgae as a carbon source for photosynthesis through passive diffusion and active transport [74]. Hence, CO₂ absorption increases at higher pH values due to the reaction between OH⁻ and CO₂, leading to the formation of bicarbonate [75], while low pH favours CO₂ sequestration by microalgae by increasing the concentration of free CO₂ [76]. Due to the low atmospheric CO₂ concentration, in air-fed algae cultures, CO₂ assimilation by microalgae cells can cause an increase in pH even to values above 9.5–10 [77]. However, the recent literature shows that in the pH range from 7.0 to 10.6, the reaction has no significant effect on carbon absorption [72].

In the S2 series, a total of 19.2 m³ of exhaust gases containing SO_x were introduced into the V-PBR during a single cultivation cycle. The initial CO₂ concentration in the gases in this experimental variant was 5.92 ± 0.31% by volume, resulting in a total of 2231.8 g of CO₂ being introduced into the culture medium. Taking into account the composition of the gases leaving the V-PBR, the total CO₂ fixation efficiency was 486.3 g CO₂, and the total amount of C. vulgaris biomass was 67.4 g. The obtained CO₂UE value was the lowest of all analysed series at 24.1% (Table 4). The pH in this series decreased from the initial value of 7.01 ± 0.07 to a minimum of 5.86 ± 0.06 on day 15, indicating clear acidification of the medium after day 8 of cultivation. The pH drop was associated with the accumulation of excess CO₂ and the presence of SO_x, which limited microalgae metabolism and photosynthesis, contributing to the early termination of the growth phase and the transition to the death phase. Such rapid acidification of the medium is a typical factor limiting CO₂UE efficiency, as excess CO₂ was not incorporated into the biomass and some of it was chemically converted to bicarbonates or lost in the exhaust gases.

When analysing the cause of the low CO₂UE value in the S2 series in detail, several physicochemical and biological mechanisms should be considered. The introduction of exhaust gases containing SO_x led to significant acidification of the culture medium after day 8, which correlated with the death of microalgae (Figure 2 and Figure 4). Excess CO₂ that was not incorporated into the biomass could undergo partial chemical conversion in the medium as bicarbonate ions (HCO₃⁻) or carbonate ions (CO₃²⁻), depending on the current pH of the medium [75]. In the S2 series, as the pH dropped below 6, most of the CO₂ remained in the dissolved form of carbonic acid (H₂CO₃), which limited its availability for photosynthesis [73]. Literature data indicate that microalgae prefer HCO₃⁻ ions as a carbon source [68,78]. Additionally, the presence of SO_x in the culture medium could react with water to form sulphuric acids (H₂SO₄ and HSO₄⁻) [77]. These reactions have been shown to further lower pH and cause osmotic stress for microalgae cells, inhibiting the activity of photosynthetic enzymes and consequently reducing effective CO₂ capture [11].

As previously reported [79,80], excess CO₂ in the presence of SO_x can be partially released from the medium back into the gas phase, particularly with dynamic mixing and gas flow in the V-PBR. This process may also have contributed to the reduction in total CO₂UE. In the S2 series, the observed decrease in pH from 7.01 to 5.86 indicates intensive H⁺ accumulation in the medium, resulting in a significant reduction in the rate of photosynthesis and carbon incorporation into biomass. This phenomenon further demonstrates that CO₂UE efficiency depends not only on the amount of CO₂ introduced but also on the chemical form of carbon available to microorganisms. Acidification of the growth medium occurs when CO₂ is converted into carbonic acid (H₂CO₃) and bicarbonate (HCO₃⁻) [81]. Additionally, the SO_x content in the exhaust gases fed to the reactor significantly increases substrate acidification [15]. A literature review shows that increasing CO₂ concentration in the microalgae cultivation system to approximately 15% can stimulate growth, while exceeding this value leads to inhibition [82,83]. According to other data, microalgae effectively capture carbon from the cultivation medium fed with exhaust gases at a CO₂ concentration of 10–20%, but SO₂ present in the exhaust gases is toxic to algal cells and hinders carbon fixation [84].

In the S3 series, the volume of fully desulphurised exhaust gases introduced into the V-PBR was 38.4 m³. The exhaust gases contained 5.61 ± 0.22% by volume of CO₂, corresponding to 4229.9 g of CO₂ supplied to the culture medium during the full culture cycle. The technological conditions used enabled the production of 294 g of C. vulgaris biomass and a CO₂UE value of 41.4% (Table 4). The pH of the culture medium gradually increased from 7.12 ± 0.08 to 8.27 ± 0.06 by day 16, promoting stable photosynthetic activity and CO₂ incorporation into the biomass. The higher CO₂UE efficiency compared to S2 resulted from both the absence of SO_x in the exhaust gases and more favourable pH dynamics, which did not inhibit microalgae metabolism. The absence of SO_x eliminated an additional acidifying factor, allowing most of the CO₂ could be converted into HCO₃⁻ and effectively used in photosynthesis, which directly resulted in a higher CO₂UE value compared to S2.

The nature of the V-PBR feed gas significantly altered the relationship between pH and CO₂ concentration in the exhaust gases, with this dynamic also determined by the physiological state of the biomass. In the case of S1 (air), a very weak positive correlation was observed (R² = 0.2097), indicating that changes in pH did not significantly affect CO₂ content in the exhaust gases. This is due to limited substrate availability—the low CO₂ concentration in atmospheric air (~0.04%) restricts the intensity of photosynthetic assimilation, making the pH effect secondary to substrate limitation. In the S3 series (desulphurised exhaust gases), a strong negative correlation was observed (R² = 0.8773), indicating that increasing pH systematically decreased CO₂ concentration in the exhaust gases. The absence of acidic sulphur impurities meant that the observed relationship directly reflected carbonate equilibrium. At higher pH, HCO₃⁻ ions predominate, which are readily absorbed by microalgae, leading to intensified photosynthetic processes and a reduction in CO₂ content.

The most complex situation was observed in S2 (exhaust gases containing SO_x, ~800 mg/m³), where the negative correlation was moderate (R² = 0.6231). In the initial phase of cultivation (days 0–7), the pH–CO₂ relationship partially reflected the mechanism observed in S3; however, from day 8, the biomass death phase began, accompanied by a rapid drop in pH to 5.92 ± 0.06. Previous research has demonstrated that the presence of SO₂ may further intensify the acidification of the culture medium through the formation of sulphuric acid and sulphites. A literature review shows that immediately after sulphur dioxide (SO₂) present in exhaust gases dissolves in water, hydration reactions produce sulphurous acid (H₂SO₃), and subsequent dissociation and oxidation reactions lead to the accumulation of hydrogen ions (H⁺), bisulphite (HSO₃⁻), sulphite (SO₃²⁻), and sulphate (SO₄²⁻) [85]. Sulphate ions can be assimilated by algal cells as a source of sulphur for the synthesis of amino acids and sulphur-containing thylakoid lipids, while bisulphite and hydrogen ions are responsible for inhibiting cell growth [86]. After the collapse of microalgal population growth, photosynthetic activity is often replaced by degradative processes that lead to the release of CO₂ and organic acids [87]. As a consequence, pH values drop even further and changes in CO₂ concentration become less predictable, which lowers the coefficient of determination.

4. Conclusions

Experimental studies have demonstrated that the presence of SO_x in exhaust gases significantly negatively affects the production efficiency, growth rate, and stability of C. vulgaris cultures. Supplying the culture medium with sulphur-laden exhaust gases resulted in inhibited growth after only eight days of the production cycle, with the microalgae culture entering a dying phase. In this case, the lowest CO₂ utilisation rate was also observed, at 24.1%.

It was found that exposure of C. vulgaris to SO_x in exhaust gases significantly reduced the energy content of microalgae biomass, specifically lipids and carbohydrates. Protein, nitrogen, and total phosphorus content remained stable regardless of the gaseous CO₂ source tested.

The highest C. vulgaris biomass production efficiency, 2120 ± 123 mg VS/L, was achieved in the series where the exhaust gases were completely desulphurised. The exponential phase was the shortest, and the biomass growth rate was the highest, reaching 281 ± 16.2 mg VS/L·d, indicating the crucial importance of flue gas desulphurisation for effective microalgae cultivation. CO₂ utilisation efficiency in this technological variant averaged 41.4%.

Based on these results, the optimal cultivation period for C. vulgaris under the tested conditions is 12–14 days when using fully desulfurized flue gases. This period encompasses the exponential growth phase and allows the culture to reach maximum biomass before the onset of natural stabilisation or stress-related decline, highlighting the importance of flue gas desulfurization and controlled dosing for maintaining culture stability and maximising CO₂ utilisation.

The results confirm that C. vulgaris microalgae can effectively capture CO₂ from real industrial exhaust gases, provided that pre-desulphurisation is implemented. Integrating microalgae cultivation with flue gas desulphurisation installations can be an effective CO₂ biosequestration strategy, simultaneously generating valuable biomass for further use in the production of biofuels, biogas, or high-value-added materials. The results indicate the need to optimise the desulphurisation processes and controlled flue gas dosing to increase the stability of the cultivation and the efficiency of CO₂ utilisation.