Time-Course Bioremediation of Lead, Nickel, and Chromium from Water by the Green Alga Chlorococcum humicola

2.1 Cultivation and growth medium of algae

Chlorococcum humicola was grown in BG11 culture medium (Figure 1) in optimized laboratory conditions at a light intensity of 3000 lux, a photosystem of 8:16 of lighting: dark, and a temperature of 25±2℃, with daily shaking. The PH of both the culture media and stored solutions was measured daily using a Cond 3320 device to ensure stable chemical conditions. The shaking was performed manually, and the PH was regularly monitored and controlled during the experiment. Growth was monitored by calculating the number of cells and reading absorbance daily, in duplicate to ensure the accuracy [21, 22].

Figure 1. Characteristics of Chlorococcum humicola under the microscope (40x)

2.2 Preparation of solutions of lead, chromium, and nickel elements

The stock solutions for heavy elements were prepared by dissolving 1 gram each of lead acetate Pb(CH₃COO)₂, potassium dichromate K₂Cr₂O₇, and nickel sulfate NiSO₄ in 100 mL of distilled water separately, while continuously stirring with a glass rod for 30 minutes to obtain solutions with a concentration of 10.000 mg/L for lead, chromium, and nickel, respectively [23, 24].

2.3 Biomass measurement and adsorption capacity calculation

The initial biomass for each algal species was monitored over a 45-day period using cell density measurements to establish a baseline before the biosorption experiments. During the treatment, metal concentration and cell densities were recorded every three days. Adsorption capacity was calculated at each time point using the equation:

$q=\frac{\left(C_0-C_t\right) \cdot V}{m}$

where, C₀ and C_t represent the initial and residual metal concentrations (mg/L). V is the volume of the solution (L), and m corresponds to the biomass in terms of cell density. This procedure allows the performance of each species in metal uptake to be directly related to the available biomass over time.

2.4 Experiment design for elements determination

The experiment was designed to evaluate the efficiency of C. humicola in removing heavy metals: lead (Pb), chromium (Cr), and nickel (Ni), each prepared at the same initial concentration. Metal stock solutions were added to achieve a final concentration of 125 ppm foreach metal in the culture flasks. Algal cultures were grown in 500 mL flasks containing 100 mL of GB-11 medium, supplemented with 10 mL of actively growing algae inoculum, giving a total working volume of 110 mL. The flasks were incubated under the same conditions described above, and the remaining metal concentrations in the medium were measured using atomic absorption spectrophotometry every three days over a 12-day period to determine the removal efficiency.

2.5 Blank control

Each heavy metal had a blank control prepared by using 100 mL of the metal solution (125 ppm) added to a sterile 500 mL Erlenmeyer flask containing no algal biomass. All blanks followed identical incubation conditions as the treatment groups (3000 lux, 8:16 light/dark photoperiod, 25 ± 2℃), which included daily manual movement. The samples for every blank were taken on days 1, 3, 6, 9, and 12. Following the collection of the samples, the samples were chemically treated with nitric acid, and they underwent analysis for metal concentration using AAS, just as was done for the flasks containing algal biomass. The blank controls confirmed that any detected changes in metal concentration were not due to environmental factors or contaminants.

2.6 Estimation of the residual concentration of heavy metals in polluted aqueous media

The concentrations of the elements under study were estimated as stated in study [25], where the method included the following:

A volume of 50 mL was taken from the samples, placed inside a clean 100 mL glass flask, and 5 mL of concentrated nitric acid was added to digest the samples. The flask was heated on a hot plate, and heating continued on the hot plate until just before drying. An additional 5 mL of concentrated nitric acid was added while continuing to heat, in order to obtain a precipitate. The solution was left to cool, and then the volume was completed to 25 mL with distilled water. The solution was filtered using a filtration membrane (0.45 µm), making the samples ready for estimating heavy metal concentrations. Filtration through this membrane completely removed the algal cells, ensuring that only the unadsorbed dissolved metals remaining in the supernatant were determined by atomic absorption. The absorbance of these digested samples was measured using an atomic absorption device (SHEMADZU AA 7000).

Figure 2. The standard calibration curve for lead (Pb) using Atomic Absorption Spectroscopy (AAS)

Figure 3. The standard calibration curve for nickel (Ni) using Atomic Absorption Spectroscopy (AAS)

Figure 4. The standard calibration curve for chromium (Cr) using Atomic Absorption Spectroscopy (AAS)

The standard calibration curves for lead, nickel, and chromium are shown in Figures 2, 3, and 4, respectively.

2.7 Statistical analysis

The statistical analysis was conducted using GraphPad Prism by means of t-test, ANOVA, and the results were presented as mean ± standard error of the mean at a significance level of p ≤ 0.05.

The results of the statistical analysis showed no significant differences between the studied minerals on the same day, while significant differences were recorded between the days for the same mineral at a significance level of P ≤ 0.05. The alga C. humicola showed significant differences between the first day and each of the third, sixth, ninth, and twelfth days. Significant differences were also found between the third and ninth days, as well as between the sixth and ninth days, and between the ninth and twelfth days, at a significance level of P ≤ 0.05 (Table 3).

It can be observed that the C. humicola algae have a low effectiveness in treating the three metals, with a slight difference in chromium removal compared to the other metals, where the removal percentages (Pb, Ni, Cr) were 16.2, 13, and 23.4 ppm, respectively, on the third day of the experiment. This decrease is attributed to the Chlorophyta demonstrating high physiological flexibility that enables them to survive and adapt under environmental stress conditions, including exposure to high concentrations of pollutants (Table 1) [7, 26].

During the early stages of exposure to pollutants, algae may show a decrease in metabolic activity or growth, which is interpreted as a necessary dormant phase for adaptation. Over time, algae activate detoxification systems, modify membrane permeability, and reorganize their metabolic pathways to cope with chemical stresses. This flexibility is a key factor in their use in bioremediation and environmental pollution monitoring (Tables 1 and 2) [26].

The experiment included a blank control, and the blank control maintained a value of zero or close to zero (in almost all cases), therefore proving that the decrease in metal concentration was primarily a result of algal activity. This was confirmed in the abstract because the blank control showed that there was no considerable abiotic loss throughout the entire experiment.

The limitation is that the initial algal biomass was not measured; therefore, metal removal could only be expressed as concentration changes (ppm) and not as adsorption capacity (mg/g biomass). This limits the ability to compare the results with other studies. This limitation restricts the ability to make quantitative comparisons with previously published work. Future studies should include precise biomass measurements (e.g., dry weight or ash-free dry weight) to allow accurate estimation of adsorption capacity and to strengthen cross-study comparability.

The high concentration of metals is considered a toxic factor that can inhibit enzyme activity and cause damage to cellular membranes, negatively affecting the absorption capacity of algae initially [27, 28]. In addition, the active binding sites on the cell wall, such as carboxyl and phosphate groups, require time to become fully active under new environmental conditions [29].

On the sixth day, a slight improvement in metal removal efficiency was observed compared to the early days; however, the extraction levels remained relatively low, indicating that the C. humicola algae are still at the end of the cellular adaptation phase [29]. At this stage, some functional groups on the cell surface, such as carboxyl and phosphate groups, begin to gradually release and open, enabling the cells to gradually restore their metabolic activity, but this activity is not yet at a full level. The slow metabolic response is due to the continued toxic effect of the heavy metals and the slow activation of the enzymes and transporters responsible for the uptake of these metals. Also, some binding sites may lack sufficient charge due to changes in pH value or reduced efficiency of photosynthesis. This period represents a transitional phase in which cells are biologically prepared and reconfigure their internal structure to maximize uptake efficiency in subsequent days, which is consistent with what has been suggested in previous studies [30].

On the sixth day, a clear clustering of algal cells was observed, while retaining the green color indicative of chlorophyll. This behavior indicates a collective physiological response of the cells to heavy metal stress, as their aggregation helps to reduce the area directly exposed to the metal's toxicity. The cells at this stage also showed remarkable resistance, with no visible signs of degradation or pigment loss, reflecting their continued biological activity and photosynthetic capacity (Tables 1 and 2) [31]. This is attributed to the secretion of extracellular polymeric substances (EPS) that trap metals outside the cells, as well as the role of the cell wall in the initial biosorption, followed by slower accumulation inside the cell. These findings are consistent with recent studies showing that algae can activate antioxidant enzymes and produce proteins such as phytochelatins and metallothioneins that bind to toxic metals and reduce their impact [31, 32]. When analyzing the metal removal efficiency on the sixth day, it was observed that the algae removed nickel more efficiently compared to lead, followed by chromium. This difference is attributed to the chemical properties and toxicity of each metal. Nickel is characterized by low toxicity and a small ionic size that facilitates its binding to adsorption sites, while lead shows a gradual and moderate effect on cells. Chromium, especially in its Cr⁺⁶ isoform, is highly toxic and rapidly affects cellular processes, reducing the ability of algae to absorb it. Algae also show a differential response depending on the type of metal [33], supported by biological mechanisms such as the secretion of EPS and the activation of internal defenses, making the absorption of nickel and lead more effective compared to chromium (Tables 1 and 2).

During the ninth day, a clear increase was observed in the algae's ability to remove heavy metals from the medium, with a removal rate of 53.6 ppm for lead, 56.1 ppm for nickel, and 63.5 ppm for chromium. The ninth day started with a significant decrease in metal concentration, which completely disappeared by the twelfth day, indicating a peak in the algae's activity.

This is partly attributed to a deficiency in essential nutrients such as nitrogen (N) and phosphorus (P), which has led to the stimulation of an environmental stress response in algal cells. When algae are exposed to nutrient deficiency, they increase the exposure or activation of functional groups (carboxyl and phosphate) on the cell walls and enhance the production of extracellular polymers (EPS) that have a high capacity to bind metal ions. Therefore, an increase in bioremediation efficiency has been observed as a defensive mechanism to obtain alternative sources for the deficient element or to eliminate toxins [34].

The mechanisms behind this efficiency include: cellular adaptation and chemical stress response, which confirm that microalgae increase the production of metal-associated compounds and interact to enhance bioavailability [35]. Additionally, the secretion of extracellular polymeric substances (EPS) increases with continuous exposure to metals, which enhances surface adsorption (biosorption) [36]. On the ninth day, it was observed that the highest removal rate was for chromium and the lowest for nickel. This shift is attributed to the completion of environmental adaptation of the algae, which led to an expansion in its vital activity and an increase in the efficiency of heavy metal binding, especially chromium, known for its strong interaction with surface groups, after the cell stabilized physiologically. In contrast, the efficiency of nickel began to decline due to the saturation of its binding sites, or the cells transitioning to internal pathways for isolating or precipitating the element (Tables 1 and 2) [37, 38].

On the twelfth day, the removal of metals was sequentially Pb, Ni, Cr (35, 24.6, 20.6), where the algae C. humicola effectively removed the metals as its concentration decreased from 125 ppm to a value below the laboratory detection limit. Recent studies have shown that the use of algae in the bioremediation of water contaminated with heavy metals leads to a reduction in the concentrations of these metals to levels below the detection limit due to the large amounts of metals that algae can absorb and remove efficiently and effectively. Additionally, algae produce low waste and possess good biodegradation capabilities (Tables 1 and 2) [33].

It should be emphasized that the values presented in Tables 1 and 2 refer to the amount of metal removed rather than the residual concentrations in the medium. The decline in removal efficiency observed on the twelfth day does not indicate an increase in residual metals but reflects the onset of physiological stress and possible site saturation after prolonged exposure. Such late-stage decreases in biosorption performance have been previously reported in green microalgae under extended heavy metal stress, where loss of cellular integrity and metabolic inhibition limit further uptake ]39-41[. The algal cells showed significant morphological changes under the light microscope (Figure 8), with a clear aggregation observed in cells exposed to lead, nickel, and chromium elements, indicating a potential physiological response to metal stress. This aggregation may be related to the initiation of the secretion of extracellular polysaccharides (EPS) to reduce metal absorption or to the activation of defensive mechanisms such as the production of antioxidant enzymes (like glutathione) and metal-binding proteins (metallothioneins), which are known for resisting oxidative stress and safely storing toxic metals inside the cell [38-41].

In addition, the continued presence of the green pigment within the cells indicates that the damage has not reached the point of complete breakdown of the three metals, suggesting the possibility of partial adaptation or the activation of cellular repair pathways. These responses may be delayed as a result of reaching an internal metal balance after a period of exposure, which is consistent with what recent studies have shown on this type of algae when exposed to lead, nickel, and chromium, where it was observed that the cells begin to adapt after several days of exposure, especially through the activation of cellular defense systems [38, 39, 41, 42].

Although this study did not include direct biochemical or molecular analyses to confirm the proposed mechanisms (EPS secretion, antioxidant enzymes, functional groups, metabolic pathway reorganization), these interpretations are based on previously published findings on similar algal species and are consistent with the observed removal patterns and morphological responses in the current work.

The results indicate that the alga Chlorococcum humicola exhibited a gradual increase in growth during the initial days of exposure to pollutants, as shown in the optical density (OD) curves. This increased growth was correlated with higher pollutant removal efficiency, as removal percentages rose alongside the algal metabolic activity. A slight decline in growth and removal efficiency was observed after the ninth day, which can be attributed to the saturation of adsorption sites on the cell surface or the physiological adaptation of the algae under metal stress. The algae demonstrated a clear ability to continue uptake even after prolonged exposure, confirming their effectiveness in the bioremediation of aquatic pollutants [8].