Optimizing Chlorella vulgaris Bioremediation of Wastewater via Advanced Aeration Systems: A Pilot-Scale Implementation

Abstract

Towards the bioremediation of toxic compounds from aquatic environments using living microalgae, Chlorella vulgaris has emerged as a promising candidate for the removal of heavy metals. The present study advances the scale-up of the microalga’s culture and investigates its efficiency in multi-metal removal (Cu, Cd, Ni, Pb, and Zn at 1 ppm each). Two aeration conditions were investigated: standard/conventional aeration (SA), and an innovative, custom-built micro-bubble aeration (MBA), which optimizes CO₂ residence time to enhance photosynthesis. Conducted in a pilot-scale 30 L photobioreactor (PBR) over a cultivation period of 7 days, control and multi-metal treated cultures were monitored for pH, cell population growth, and pigment content. Heavy metal removal efficiency was evaluated by means of atomic absorption spectroscopy (AAS) on Days 3 and 7 of cultivation. The comparative results reveal that MBA significantly enhances both the population and the photosynthetic pigment content of the cultures. Furthermore, the heavy metal removal efficiency under MBA reached up to 95% even by Day 3 of cultivation, remarkably higher than the 67% of the SA treated culture. These findings not only demonstrate Chlorella vulgaris’s effectiveness in multi-metal treated systems but also highlight the potential of advanced aeration systems to enhance bioremediation efficiency in larger-scale aquatic environments.

1. Introduction

Water pollution is a critical issue in environmental research and related scientific studies. Pollutants such as chemicals, plastics and microplastics, heavy metals, nutrients, microbial contaminants, and even thermal pollution affect aquatic ecosystems globally [1]. Water contamination with heavy metals has emerged as a significant environmental and public health issue, due to their toxicity and bioaccumulation [2,3,4]. Despite the natural occurrence of heavy metals in the Earth’s crust, and consequently in aquatic systems, their concentration can be significantly amplified by several human activities, such as industrial waste, agricultural runoff, and mining procedures.

The remediation of heavy metals in wastewater is performed with several methods, including adsorption, electrochemical treatment, membrane filtration, and bioremediation, the latter employing microorganisms to absorb metals [5]. To this direction, the use of microalgae for wastewater bioremediation is considered as a promising and green solution, due to their low cost, the CO₂ consumption achieved through photosynthesis, and the possibility of reusing both the microalgae biomass and the recovered heavy metals after treatment [6,7,8,9,10].

Until now, heavy metal bioremediation using microalgae cultures has been proven promising for various strains, including Chlorella vulgaris (C. vulgaris) and Arthrospira (Spirulina) platensis, mainly under single metal treatment (mono metal-treated, mono-MT) systems [11,12,13,14]. However, there is still a significant gap in the literature surrounding the use of microalgae in multi metal-treated (multi-MT) systems [8,15,16,17,18].

Despite the microalgae potential in heavy metal bioremediation, such studies are mostly focused on laboratory-scale experiments, with typical cultivation volumes ranging from 120 mL to 1 L [15,19,20,21,22,23]. The problems and challenges associated with scaling up experiments should also be taken into consideration. As their study—and consequently their implementation—is demanding [5,10], pilot-scale and industrial-scale experiments and applications are scarce [24], with the main drawbacks being the high cost and difficulties in monitoring [25]. Nevertheless, scaling up is crucial to be examined, as the aforementioned downsides can be handled with the fast growth rate of microalgae [26] and the reuse of the treated biomass and/or recovered metals, together with recent and still evolving progress in monitoring, such as machine learning and big data analysis [27].

Scaling up in photobioreactors (PBRs) encounters challenges that influence microalgae growth and health, due to phenomena such as sedimentation, cell membrane disruption, and nutrient distribution, while slow movement leads to light depletion [28]. Apart from the conventional aeration of cultures using bubbles—with plain air, or enriched in CO₂ air [29]—advanced aeration systems have also been proposed, such as conventional aeration coupled with stirring [30], and the use of micro- and nano-bubbles [29]. The latter, apart from enhanced aeration due to higher CO₂ dissolving rate, longer retention time, and lower buoyancy [29], also enhances flow mixing, thus improving light and nutrient uptake from the microalgae [28,31].

Based on the above, this preliminary study aims to assess whether micro-bubble aeration improves scaled-up cultivation of C. vulgaris, to evaluate the multi-metal removal efficiency at pilot scale, and to examine the potential of cultivation and removal efficiency due to advanced aeration. A previous laboratory scale study under standard aeration, using both mono- and multi-MT, showed that the presence of multiple heavy metals can lead to synergistic or antagonistic effects on the microalga’s growth and heavy metal removal efficiency [18]. The present study focuses specifically on multi-MT systems, as a realistic model to evaluate the impact of aeration on biomass growth and bioremediation.

2. Materials and Methods

2.1. Microalga Cultivation Conditions

C. vulgaris (strain NIVA-CHL 108), purchased from NORCCA (Oslo, Norway), was cultivated in a 30 L PBR in Bold’s Basal Medium (BBM) (pH = 6.8) [18,32], under a 12 h light:12 h dark illumination cycle, using a full spectrum LED array light source (LUMATEK, London, UK, ATTIS 200 W, ~160 μmol photons m⁻²·s⁻¹) at 28 ± 0.5 °C.

Two aeration conditions were applied. The first one, referred to as standard aeration (SA), involved a continuous aeration using a gas mixture consisting of 30% CO₂ in air, at a flow rate of 1.67 L/min, using two pumps of 50 L/h each. The second, micro-bubble aeration (MBA), operated at the same flow rate using the same gas mixture. In this case, aeration was applied for 10 min every hour. The setup and the specifications of the MBA system are described in Appendix A.

Multi-MT cultures were subjected to both aeration conditions, while untreated cultures (Control) were also cultivated under the same conditions for comparison. For the multi-MT cultures, Cu, Cd, Ni, Pb, and Zn were added at 1 ppm each (a concentration typically found in moderately contaminated industrial wastewater [33]), in the form of nitrate salt solutions, and the cultivation period was 7 days.

2.2. Analytical and Imaging Methods for Biomass Growth, Pigment Content and Heavy Metal Removal

Cell population and morphological characteristics were determined daily by optical microscopy (CX43RF microscope equipped with an EP50 camera, Olympus Corporation, Tokyo, Japan) using an improved Neubauer hemocytometer. The pigment content of the cultures was estimated on Days 0, 3, and 7 of cultivation by means of UV-Vis spectrophotometry (Lambda 25, Perkin Elmer, Waltham, MA, USA), using methanol for the extraction [34]:

$$\mathrm{C}\mathrm{h}\mathrm{l}.a=16.72A_{665.2}-9.16A_{652.4}$$

(1)

$$\mathrm{C}\mathrm{h}\mathrm{l}.b=34.09A_{652.4}-15.28A_{665.2}$$

(2)

$$\mathrm{C}\mathrm{h}\mathrm{l}.tot=1.44A_{665.2}+24.93A_{652.4}$$

(3)

$$\mathrm{C}\mathrm{h}\mathrm{l}.x={\displaystyle \frac{1000A_{470}-1.63\mathrm{C}\mathrm{h}\mathrm{l}.a-104.96\mathrm{C}\mathrm{h}\mathrm{l}.b}{221}},$$

(4)

where Chl.a, Chl.b, Chl.tot, and Chl.x are the concentrations of chlorophyll a, chlorophyll b, total chlorophylls, and total carotenoids, respectively, while A is the absorbance measured at different wavelengths, after the subtraction of A₇₅₀, for turbidity correction [35]. The heavy metal residual concentration was determined by atomic absorption spectroscopy (AAS) (AAnalyst 800, Perkin–Elmer, Waltham, MA, USA) and the total % removal was calculated on Days 3 and 7 of cultivation compared to the initial concentration at Day 0 (also determined with AAS and found to range between 0.98 to 1.03 ppm). All measurements were performed in duplicate; the results are expressed as the mean of the two values with their standard deviation (SD).

3. Results

The effect of the aeration conditions on the growth, pigment content and heavy metal removal efficiency of C. vulgaris is described below.

Figure 1 shows the normalized cell population of all cultures, as well as under both aeration conditions. In the Control cultures (Figure 1a), under SA, cell population increases up to Day 3, after which the decline phase follows until Day 7. On the other hand, when MBA is applied, the population increases rapidly up to Day 2, presents a stationary phase until Day 4, and then increases again until Day 7. With the addition of 1 ppm of heavy metals, the pattern of cell growth shifts (Figure 1b): under SA, population starts to increase slightly on Day 1 and then firmly declines until the end of the experiment. Under MBA, a stationary phase is observed during the first day, probably due to cell adjustment to metal exposure and at the same time to enhanced aeration. After that, there is a gradual but steady increase until Day 6, and on the final day, there is a sharp increase, indicating that cell division prevails. In both cases, the beneficial effects of MBA can probably be attributed to the enhanced CO₂ availability and nutrient transport that may enhance the efficiency of photosynthesis and detoxification processes. Furthermore, the findings suggest that while heavy metals exert a toxic effect on the growth of C. vulgaris [36], MBA can enhance cellular tolerance and adaptation to the toxic environment [37].

As presented in Figure 2, microscopic observation of C. vulgaris cells on Day 7 of cultivation reveals distinct morphological differences depending on the aeration condition and heavy metal exposure. Under SA, control cells exhibit healthy morphology, whereas cells exposed to 1 ppm of heavy metals show reduced density and signs of structural deformation [18]. Under MBA, the control cells appear denser and uniform, while metal-treated cells preserve their structural integrity. These findings suggest that MBA enhances cellular resistance to the stress induced by the heavy metal effect. The aeration mode significantly influences C. vulgaris morphological features, heavy metal tolerance, and removal capacity.

During the cultivation period, as presented in Figure 3, pH increases both for Control and 1 ppm multi-MT cultures, with variations between the two aeration conditions. In the case of the Control cultures, SA triggers a continuous rise in pH from 8.64 on Day 1, to 10.91 on Day 7, indicating significant alkalinization, apparently as a result of high-level photosynthetic CO₂ uptake and minimal replenishment. The Control culture under MBA exhibits a less pronounced rise, from 8.95 to 9.85, pointing to increased CO₂ availability and buffering capacity, due to enhanced gas–liquid exchange. In multi-MT cultures, pH also increases with time but follows a different pattern. Under SA, pH grows from 8.9 to 10.18, and under MBA from 8.84 to 9.50. The presence of heavy metals most probably suppresses photosynthesis, or changes nutrient assimilation, thereby moderating the pH change [38]. The overall trend suggests the coupling of photosynthetic CO₂ fixation with nitrate assimilation, both of which are responsible for medium alkalinization. MBA seems to have a stabilizing effect to the carbonate system balance, potentially mitigating metal-induced stress by improving gas exchange and buffering capacity [39].

The pigment content of the microalga was estimated as a health indicator of the cultures [40]. Figure 4 shows the Chl.tot and Chl.x content for both aeration conditions. In general, the pigment content follows the population results. Regarding the Control cultures (Figure 4a,b), considerable differentiations between the two conditions are observed; Chl.tot and Chl.x are both significantly higher under MBA than SA. This enhancement indicates increased photosynthetic activity and, consequently, higher biomass accumulation under MBA [41]. The improvement can be explained by the increased CO₂ solubility and enhanced nutrient transport provided by microbubbles, which allows better photosynthesis capacity in C. vulgaris cells. In the case of the multi-MT cultures (Figure 4c,d), Chl.tot is decreased for both aeration conditions, in comparison to the Control cultures because of the metal exposure [42]. Nevertheless, the elevated carotenoid content under MBA is noted. Increased carotenoid levels are a stress indicator, due to a number of factors, such as high light intensity [43]. Other factors such as nutrient depletion, oxidative stress due to heavy metal exposure, salinity stress, and temperature fluctuations cannot be taken into consideration, as the same conditions were applied to the SA one.

Figure 5 presents the metal-removal efficiency of C. vulgaris under the two aeration conditions, as measured on Days 3 and 7 of cultivation. The results indicate that the removal efficiency was significantly higher in the MBA culture condition compared to SA. By Day 3, the MBA culture achieved nearly 95% removal of Cu and Pb, while Cd, Ni, and Zn showed removal rates of 90% ± 3.0%, 26% ± 1.2%, and 15.4% ± 1.4%, respectively. In contrast, the SA culture exhibited much lower efficiency, with only Cu reaching 87.5% ± 2.1% by Day 7, while the other metals remained below 30%. Over time, metal removal further improved, with the MBA culture maintaining 100% removal for Cu and Pb, while Cd, Zn, and Ni increased to 78.3% ± 3.1%, 52.9% ± 1.8%, and 27% ± 1.2%, respectively. SA culture also showed some improvement, particularly for Cu (87.5% ± 2.1% removal), but still lagged significantly behind the MBA culture [44]. Overall, the presence of MBA culture played a crucial role in enhancing heavy metal removal, especially for Pb and Cd, which showed a significant improvement compared to SA culture.

4. Discussion

This preliminary study evaluates the effect of micro-bubble aeration on scaled-up cultivation of C. vulgaris for heavy metal bioremediation. MBA led to better cell morphological features, higher biomass production and photosynthetic pigment content of the microalga in both Control and multi-MT cultures, in agreement with previous findings [28]. In the 30 L pilot-scale cultivation that was used for the present study, an exponential growth phase was achieved until Day 7 when MBA was applied, while SA resulted in a prolonged decline. In comparison with a previous laboratory scale study (500 mL) using the same cultivation conditions but using SA only [18], scaling up to 30 L in the present study resulted in reduced biomass growth under SA. On the other hand, the introduction of MBA in the present pilot scale Control culture improved growth, thus confirming the positive effect of enhanced aeration in metal-free conditions during scaling up. Nurrusyda et al., who studied the effect of various environmental conditions on C. vulgaris biomass growth under laboratory-scale cultivation, found that aeration, and in particular constant SA, significantly affects the microalga’s growth [45]. In contrast, the present study demonstrates that when scaling up to pilot-scale, SA leads to reduced growth, while MBA significantly enhances biomass growth.

The evaluation of the heavy metal removal efficiency of C. vulgaris during the scale-up of cultivation was performed by comparing the present results with that of a previous study at laboratory (small) scale [18]. Under conventional aeration, the small-scale (500 mL) multi-MT cultures at 1 ppm exhibited a Pb removal efficiency of ~50%, while the other metals (Cu, Cd, Ni, and Zn) showed removal efficiencies above 90%. In the present pilot-scale (30 L) study under SA, Cu exhibited removal efficiency values comparable to those observed at small-scale, whereas the other metals showed values below 45%. This comparison reveals a consistent increase in removal efficiency when C. vulgaris is cultivated under MBA. In both cultivation scales, Cu and Cd exhibited the highest removal efficiencies due to their strong affinity for algal surface functional groups and stable chelation or intracellular sequestration. However, in the present 30 L MBA system, removal rates of Cu and Pb reached nearly 95% by Day 3, and 100% by Day 7, higher than in the previously studied 500 mL cultures, particularly under multi-MT conditions, where competition is expected to reduce efficiency. Cd also showed enhanced removal under MBA (90% on Day 3 and 80% on Day 7), consistent with its proven capacity for stable biosorption and bioaccumulation. Conversely, the more sensitive metals, i.e., Ni and Zn, were removed less efficiently but still significantly more under MBA (27% ± 1.2% and 52.9% ± 1.9%, respectively) than under SA, where their removal remained below 30%. Thus, the large-scale MBA setup not only replicated trends observed in small-scale systems, but also amplified the removal potential across all metals, emphasizing the scalability and robustness of MBA for optimizing heavy metal bioremediation.

It should be noted that the enhanced heavy metal removal efficiency that was observed under MBA may be related to physiological and biochemical defense mechanisms that are activated by the microalga in response to the induced stress conditions. One of the main pathways evolves enhancing its antioxidant defenses, which help protect the cells from damage caused by excess reactive oxygen species (ROS), which are produced under stress. Significant enzymes like superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) are activated to neutralize harmful ROS and protect cellular components from oxidative damage [8,46]. Furthermore, C. vulgaris accumulates proline, an amino acid that functions both as an osmoprotectant and as an antioxidant, which helps to neutralize harmful ROS and maintains water balance, and protects cellular proteins and membranes [47,48,49]. Furthermore, C. vulgaris produces metal-binding peptides such as phytochelatins and metallothioneins, which capture and isolate toxic metal ions, reducing their concentration in the cytoplasm and minimizing key metabolic processes from disruption [50]. In response to metal stress, cells adjust their membrane permeability and activate transporters that remove toxic ions from the cytoplasm, maintaining intracellular metal homeostasis and protecting vital metabolic processes [46]. Ultrastructural analyses have shown that exposure to heavy metals can lead to significant alterations in the cellular architecture of microalgae, notably affecting chloroplast integrity and cytoplasmic organization. Despite these structural disruptions, C. vulgaris demonstrates resilience by maintaining essential metabolic functions through adaptive mechanisms. These adaptations include the activation of antioxidant defenses and the modulation of metal ion transport systems, which collectively mitigate the detrimental effects of metal-induced stress [46,51]. Another important adaptive response is the alteration of lipid metabolism. Under stress conditions, C. vulgaris increases the accumulation of neutral lipids, as part of its survival strategy. This shift not only serves as a reservoir of carbon and energy, but also stabilizes cellular membranes against oxidative and metal-induced damage. Changes in membrane lipid composition help maintain membrane integrity and functionality during stress exposure [52,53].

Finally, it should be noted that C. vulgaris not only has the ability to be cultivated under extreme CO₂ concentrations [54], but also presents enhanced bioremediation performance, as supported by the present study. MBA aeration led to improved cell health, population, and removal efficiency in comparison to SA.

The scale-up of the experiment, from laboratory (500 mL) to pilot-scale, presented results that are promising even for large-scale applications. Apart from cultivation experiments at large-scale for feasibility evaluation, future work should exploit various microalgae strains, for application under various water and environmental factors (i.e., salinity and temperature fluctuations). Moreover, microalgae removal efficiency of various combinations of heavy metals should be also studied, together with the potential for biomass reuse after metal treatment. Preliminary studies have shown that lipid, protein, and saccharide content is retained after bioremediation, supporting potential applications in energy production [18,55,56,57]. Regarding the feasibility of this approach, studies on the use of real wastewater at pilot and large scales, and the potential of recycling the used biomass and recovery of biosorbed metals, are in progress. Future work should also include a detailed energy and cost analysis comparisons between SA and MBA, to further evaluate the economic viability of scale-up.

5. Conclusions

This preliminary study introduces the use of advanced aeration in pilot-scale C. vulgaris cultivation using a 30 L PBR. In comparison to SA conditions, MBA significantly improved the microalga cell morphology, health, and biomass growth, as indicated by pigment content and cell population measurements. MBA also led to the complete removal of Cu and Pb by Day 7 of cultivation, and improved Cd, Zn, and Ni removal in multi-MT systems. In comparison to previous results contacted at the laboratory-scale (500 mL), MBA showed the same or improved removal efficiencies despite the scaling up challenges. These results demonstrate the successful role of advanced aeration in scaling up C. vulgaris cultivation for bioremediation purposes. Towards the feasibility of wastewater bioremediation using C. vulgaris cultures, ongoing work includes the application of MBA to real wastewater and further upscaling the PBR volume.