Bioremediation of Tartrazine Dye Using the Green Microalga Chlorella vulgaris in Aqueous Systems

Water is an essential element for the existence of human civilization. Water and life are closely intertwined, as humans have historically been drawn to water bodies and have depended on them for various aspects of life [1]. However, rapid population growth and technological advancements have exacerbated global water pollution. One of the major contributing factors is the widespread use of synthetic dyes in various industries, including textiles, food, leather tanning, paints, printing, paper manufacturing, cosmetics, and pharmaceuticals [2]. These industries consume large volumes of water and often discharge untreated or partially treated wastewater into rivers, thereby contributing significantly to water pollution. These pollutants alter the physicochemical characteristics of water, posing serious threats to aquatic ecosystems and their inhabitants, which ultimately impacts human health [3].

Synthetic dyes are complex chemical compounds with carcinogenic, toxic, and mutagenic properties, which accumulate within the food chain [4]. Textile dyes fall under the category of azo dyes, which break down into aromatic amines that can cause cancer and skin sensitization in people [5]. Tartrazine is a synthetic dye with the chemical formula C₁₆H₉N₄Na₃O₉S₂, a molecular weight of 534.36 g/mol, and consists of two benzene rings. It is classified as a yellow azo dye and is used as a food additive to enhance flavor and color in foods. Tartrazine is a biologically active substance that is soluble in water [6]. As soon as an artificial food coloring agent is consumed, the body begins to experience a number of detrimental health effects. In addition to heart disease and cancer, studies show that these additives cause respiratory illnesses like asthma, neurological disorders such as attention deficit hyperactivity disorder (ADHD), and metabolic issues like obesity [7]. Cancer research has shown that prolonged exposure to tartrazine induces toxicity in eukaryotic cells, leading to mutations and cancer [8]. Beyond its application in food, tartrazine is also widely used in non-food industries, including pharmaceuticals, cosmetics, soaps, shampoos, stamp dyes, and coloring pens [9]. 

Algae are a large group of living organisms, most of which are eukaryotic and a few of which are prokaryotic. They play an important role in maintaining the balance of the ecosystem [10, 11]. There are many different types of algae. Microalgae are unicellular microscopic organisms that can perform photosynthesis. They are visible under a microscope and typically range in size from 1 to 400 µm [12]. Protein, carbohydrates, fats, and vitamins are all abundant in algae, and the majority of species exhibit high growth efficiency in contaminated waters [13]. Waste from human activities has a significant impact on how different aquatic organisms, such as algae, behave and are used as indicators of water quality [14]. Most local studies focus on the classification of algae in diverse environments [15-18]. Chlorella vulgaris is considered a green unicellular alga that ranges in diameter from 2 to 10 µm. It is characterized by a regular spherical shape that primarily spreads in freshwater environments, such as rivers and lakes, in addition to moist soil. It has a thick cellular wall and contains chlorophyll a and b pigments, which give it a distinctive green color. Chlorella vulgaris is identified based on morphological characteristics using a light microscope [19].

Chlorella vulgaris is an efficient, sustainable, and low-cost remediation agent, outperforming chemical and biological methods. It grows rapidly, tolerates diverse conditions, and removes pollutants via functional groups and enzymatic degradation [19, 20]. Additionally, it yields bioresource-rich biomass [21]. Recent studies emphasize C. vulgaris as an effective agent in dye removal via biosorption, bioaccumulation, and enzymatic degradation. Their cell walls, rich in polysaccharides, proteins, and functional groups, aid dye binding and uptake [20, 22]. Chlorella vulgaris withstands high pollutant levels through antioxidant defenses that counter oxidative stress from reactive oxygen species [23]. It has shown strong potential in removing azo dyes, including malachite green, tartrazine, and carmoisine [24]. Additionally, assimilated compounds are converted into protein-, lipid-, and carotenoid-rich biomass [25]. Despite these advances, there remains limited work on the efficiency of locally isolated C. vulgaris strains in the bioremediation of synthetic dyes such as tartrazine, which this study addresses.

This study is innovative in utilizing a locally isolated strain of C. vulgaris from the Tigris River, Iraq, for the bioremediation of the carcinogenic dye tartrazine. This study investigates the adaptive capacity of native microalgae under controlled laboratory conditions, in contrast to previous research utilizing commercial strains. The study methodically assesses the efficacy of tartrazine removal across different concentrations (25–100 ppm) and exposure durations (1–12 days), achieving total elimination at 25 ppm within nine days. A major step forward is the use of high-performance liquid chromatography (HPLC) to accurately measure how much dye has broken down. The study also shows that algae can adapt physiologically, such as by grouping cells together, storing lipids, and becoming more tolerant to oxidative stress. This is a long-term, eco-friendly way to control dye pollution and learn more about how algae-based bioremediation works.

The present study aimed to quantify the removal efficiency of tartrazine by C. vulgaris under different initial concentrations (25, 50, and 100 ppm) and contact times (1-12 days) and to elucidate the potential mechanisms involved.

As shown in Table 1, the experimental results demonstrated a clear increase in the absorbance of Chlorella vulgaris during the treatment period, indicating enhanced cell density, chlorophyll content, and photosynthetic activity, which directly influenced the efficiency of tartrazine removal. At a low concentration of 25 ppm, the residual dye concentration decreased from 23.55 ppm on day zero to 7.00 ppm on the sixth day, and reached undetectable levels by the ninth day, corresponding to a removal efficiency of 100% on day nine and complete removal thereafter [30, 31]. This rapid removal is attributed to the high metabolic activity during the exponential growth phase, when cells exhibit optimal enzymatic function and nutrient assimilation, thereby facilitating effective dye uptake and transformation. The correlation between increased absorbance and dye removal suggests that active growth and photosynthetic efficiency are key determinants in the early stage of bioremediation.

Table 1. Residual concentration and removal efficiency of tartrazine by Chlorella vulgaris

At an intermediate concentration of 50 ppm, the residual tartrazine concentration decreased more gradually from 48.77 ppm at day zero to 35.08 ppm on day three, and 21.00 ppm on day six, ultimately reaching 5.01 ppm by day nine, with a corresponding removal efficiency of 89.98%. This slower initial removal reflects the time required for cells to adapt to higher pollutant concentrations, triggering biochemical defense mechanisms such as the production of unsaturated fatty acids and antioxidant enzymes to mitigate oxidative stress [32, 33]. The gradual increase in removal over the treatment period highlights the importance of cellular adaptation in overcoming moderate stress conditions, although the decline in absorbance toward the later stage indicates that nutrient depletion and waste accumulation begin to limit growth and photosynthetic activity.

At the highest tested concentration of 100 ppm, the residual dye concentration decreased from 97.01 ppm on day zero to 71.45 ppm on day three, 55.70 ppm on day six, and 17.09 ppm by day nine, corresponding to removal efficiencies of 26.33%, 42.58%, and 82.91%, respectively. Maximum removal efficiency of 93.02% was observed on day twelve (Figure 2). This pattern is attributed to the aggregation of algal cells and their encapsulation in lipid layers, which enhances the adsorption and retention of tartrazine molecules [34]. However, prolonged exposure to high dye concentrations induced cellular stress and cytotoxicity, leading to cell death and a subsequent decline in absorbance, which resulted in a temporary plateau in removal rates [35, 36].

Overall, these data confirm a strong positive correlation between algal growth, photosynthetic activity, and tartrazine removal efficiency, particularly during the early and exponential growth phases. Figure 3 illustrates this relationship by plotting residual dye concentration and removal percentage alongside the absorbance of C. vulgaris over the treatment period. As the experiment progressed, limiting factors such as nutrient depletion, waste accumulation, and cellular stress gradually reduced algal growth and photosynthetic efficiency, which in turn decreased the removal rate. These findings underscore the importance of optimizing initial dye concentrations, nutrient availability, and cultivation conditions to maximize bioremediation efficiency.

Figure 3. Residual tartrazine concentration, removal percentage, and Chlorella vulgaris absorbance over time at initial concentrations of 25, 50, and 100 ppm

Since there were notable variations between the first day and the third, sixth, and ninth days, the statistical analysis's findings demonstrated a significant difference (p ≤ 0.05) between the treatment days for all three dye concentrations. The third day showed significant differences (p ≤ 0.05) with the sixth day of the experiment at a concentration of 25 ppm. Significant differences (p ≤ 0.05) were found between the first day and the third, sixth, and ninth days, as well as between the third and the twelfth days, when the tartrazine concentration was 50 ppm. Additionally, there were notable variations between the sixth and ninth days of the experiment and the twelfth day. At 100 ppm, significant differences (p ≤ 0.05) were observed between day 1 and days 3, 6, 9, and 12, as well as between day 3 and days 9 and 12. Further significant differences (p ≤ 0.05) were noted between day 6 and days 9 and 12, and between days 9 and 12. The faster decomposition of tartrazine can be attributed to its simple structure and the absence of metal complexes, which makes it more susceptible to enzymatic degradation. However, at higher concentrations, the decomposition rate decreases due to the toxic effects on microorganisms [37]. Table 1 shows that on the first day of the experiment, the processing rate for all concentrations dropped, indicating that the algae had become acclimated to tartrazine. On the third day, it evidently increased, demonstrating the algae's biological activity during the three days. Because the algae use tartrazine as a carbon source, the concentration of tartrazine causes the concentration of nutrients in the medium to rise, which explains the increase in processing rate [20]. The removal rate varies slightly among the three concentrations, but the treatment rate keeps increasing steadily until the sixth day. Due to the lower concentration of the contaminant and the smaller cell size of the algae, which helped remove the contaminant more quickly than the other concentrations, complete treatment of tartrazine was observed on the ninth day at a concentration of 25 ppm. The size of the cells influences how well the algae remove pollutants from the environment, and one of the most significant factors affecting the biological treatment efficiency of microalgae is the ratio of surface area to volume. This is consistent with a study [38], which reported that smaller cells, having a higher surface area-to-volume ratio, exhibit greater pollutant absorption capacity and are less prone to damage at low contaminant concentrations. In contrast, at 50 ppm, complete removal was delayed until day 12.

According to a study by Ghosal et al. [23], true green algae have membrane defense systems against oxidation that help them fend off pollutants and eliminate free radicals that the algae produce. High pollutant concentrations result in the generation of reactive oxygen species (ROS) in significant quantities, including O^-, OH, and H₂O₂, which oxidize and damage membrane lipids. Consequently, it was observed that the treatment rate was postponed until the experiment's twelfth day. Conversely, a higher density of accessory pigments like carotenoids, beta-carotene, and triacylglycerols results from a nitrogen shortage and an increase in carbon sources in the medium. Because carotenoids have antioxidant qualities, their buildup in cells shields against the effects of free radicals [39]. As the concentration of pollutants decreases, microalgae regulate the concentration of carbon, phosphorus, and nitrogen by internally accumulating fats, carbohydrates, and proteins in their biomass [24, 40]. The highest processing rate was recorded on the ninth day at a concentration of 100 ppm, which may have been caused by cell aggregation and the fatty material that surrounded them, as depicted in Figure 3. Environmental pollutants cause noticeable changes in the qualitative characteristics of water, which in turn affect the living things that are present in it [38, 41]. This was supported by certain investigations that discovered the presence of pollutants and their distribution within the cells has an impact on the fat content of algae. Furthermore, since the majority of pollutants can be retained in the fatty structures of the algal cells or their surroundings, the presence of fatty substances inside or around the algae increases their consumption and resistance to pollutants (Figure 4) [25]. However, Table 1 also demonstrates that the processing rate decreased as the dye concentration increased, which could be connected to the algal cells becoming saturated once they achieve equilibrium [22]. As observed in Figure 3, high concentrations of tartrazine prevented eukaryotic cells from dividing by causing toxicity, mutations, and cancer if exposed for extended periods of time [42]. Physiological changes were observed in the algal cells, including increased cell size, reduced aggregation, and evidence of cell death. By day 12 at 100 ppm, the algal population had declined to 10.11, reflecting the toxic impact of elevated tartrazine levels.