Comparative Phycoremediation Performance of Two Green Microalgal Strains Under Four Biomass Conditions for Industrial Wastewater Treatment

Abstract

This study uses industrial wastewater from an aluminum factory to evaluate the phycoremediation efficiency of two green microalgal strains, Dictyosphaerium sp. and Tetradesmus obliquus. The industrial wastewater contained high levels of pollutants, including COD, ammonium, nitrate, phosphate, and heavy metal ions (Al³⁺, Cu²⁺, Cr³⁺, Zn²⁺, Mn²⁺, Cd²⁺). Four biomass conditions were tested: free-living cells (active living cells), immobilized cells (entrapped within alginate), dried biomass (non-living dried cells), and acid-treated dried biomass (chemically modified for enhanced adsorption). Both strains demonstrated significant pollutant removal, with living biomass (free and immobilized) achieving the highest nutrient and organic pollutant removal, and non-living biomass (dried and acid-treated) being more efficient for rapid heavy metal removal. Tetradesmus obliquus showed superior performance across most parameters, while Dictyosphaerium sp. exhibited the highest aluminum removal (99.4%, reducing Al from 481.2 mg/L to 10.2 mg/L). These findings highlight the potential of microalgae-based approaches and support species-specific strategies for cost-effective and sustainable phycoremediation of industrial wastewater.

1. Introduction

Water is an important and indispensable natural resource that supports life on Earth. It is found in almost 70% of the Earth’s surface, and only 1% is usable or safe for human consumption [1]. With the expansion of the world economy and accelerating urbanization, water demand has soared, and the gap between water supply and demand has further widened. If current trends persist, we could face a 40% global water supply deficit by 2030 [2]. Growing amounts of untreated wastewater released from municipal, industrial, and agricultural sources worsen this crisis. Due to its complex and highly variable composition, which is frequently rich in hazardous heavy metal contents such as copper, zinc, chromium, cadmium, and aluminum, industrial wastewater is one of the most dangerous of these [3]. These pollutants contaminate surface and groundwater and pose serious risks to ecosystems and public health. They are frequently released by industries such as metalworking, electronics, and electroplating [4,5].

For industrial wastewater management, conventional treatment technologies like ion exchange, chemical precipitation, membrane filtration, electrochemical treatment, and reverse osmosis are frequently used [6]. However, these techniques are frequently expensive and energy-intensive and produce secondary waste, such as toxic sludge, limiting their long-term sustainability and efficiency [7]. Furthermore, because the efficacy of these methods depends on concentration, they are ineffective when used for low concentrations of heavy metal ions (between 10 and 100 mg/L) [8]. In light of these limitations, there is a clear and urgent need for eco-friendly and cost-effective alternatives [9]. Among the emerging eco-friendly approaches, phycoremediation has gained increasing attention. This process involves using microalgae to remove or transform various pollutants, including nutrients, heavy metal ions, and organic contaminants from wastewater [10]. Integrating this method with the biorefinery concept enhances its sustainability by enabling the valorization of harvested microalgal biomass. This biomass, obtained after heavy metal remediation, can produce bioenergy and a range of high-value bioproducts [11,12]. Such an approach not only supports the simultaneous removal of contaminants from wastewater but also facilitates the generation of green energy. Aligned with circular economic principles, it promotes a zero-waste framework, wherein waste streams are transformed into valuable resources, supporting environmental protection and resource efficiency.

Microalgae facilitate heavy metal removal via two primary mechanisms: biosorption, a passive process involving metal ions binding to functional groups on the algal cell surface, and bioaccumulation, an active, energy-dependent process where metal ions are taken up into the cells [13]. Biosorption can occur with both living and non-living biomass, largely due to negatively charged groups such as carboxyl, hydroxyl, and phosphate on the algal cell wall [14]. In contrast, bioaccumulation is restricted to living cells, as it relies on intact metabolic pathways for metal uptake [15]. Each type of biomass has its particular advantages and limitations in terms of efficiency, cost, and resilience under different environmental conditions [16,17]. Living algal biomass requires essential nutrients for sustained growth, many of which are in wastewater. Under such conditions, pollutant removal efficiency is closely linked to the algal growth rate and resulting biomass production uptake [18]. Alternatively, using non-living dried algal biomass for metal adsorption presents a viable option for certain wastewater treatment applications. Since dried biomass does not require nutrients or oxygen, issues related to pollutant toxicity are largely mitigated by uptake [19].

One of the primary challenges of using living microalgal cells in wastewater remediation is the recovery and harvesting of biomass from the treated effluent. An effective separation system is essential not only for efficient wastewater recycling but also for enabling subsequent biomass utilization. Immobilization methods in which live microalgae are entrapped within polymeric matrices have become increasingly popular to improve treatment effectiveness and biomass recovery. These systems preserve cell viability, bolster tolerance to pollutants, assist in reactor operation, and enable biomass retrieval [20]. Once pollutants are absorbed, the treated water diffuses from the polymer matrix and can be collected and reused. This process can be repeated across multiple treatment cycles, making it a sustainable and efficient approach for wastewater remediation. On the other hand, chemical pretreatment of non-living biomass, such as acid modification, greatly increases surface functionality and the capacity to bind metal ions [21].

The main objectives of this study are to (i) evaluate and compare the phycoremediation efficiency of two green microalgal strains, Dictyosphaerium sp. and Tetradesmus obliquus, for the treatment of industrial wastewater from an aluminum factory; (ii) investigate the pollutant removal performance of four biomass conditions (free-living cells, immobilized cells, raw dried biomass, and acid-treated dried biomass); (iii) determine the capacity of each biomass type to remove or reduce key pollutants, including nutrients (ammonium, nitrate, phosphate), organic load (COD), and heavy metals (Al³⁺, Pb²⁺, Cu²⁺, Cr³⁺, Ni²⁺, Zn²⁺, Mn²⁺, Cd²⁺); and (iv) identify the most effective and sustainable biomass strategy for potential large-scale phycoremediation applications.

2. Materials and Methods

2.1. Water Sampling, Isolation, Purification, and Identification of Microalgae Species

Water samples (pH = 7.5 ± 0.5) were collected in well-stoppered, sterilized bottles during the fall season of 2022 from the Nile River in Giza, Egypt, and streaked with an inoculation needle across the surface of sterilized solid BG-11 agar plates and incubated for 12 days at 25 ± 2 °C under white, fluorescent lamp (3000 Lx, Philips TL-D 36W, Shanghai, China). Following growth, approximately 12–15 visible algal colonies were obtained, from which the two dominant morphotypes were selected and subsequently purified through three successive subcultures to ensure axenic cultures before being transferred to sterilized liquid media. The morphological identification of the two selected isolated microalgae species was performed under a light microscope (Olympus CX21, Olympus Corporation, Tokyo, Japan) using the following references: [22,23,24], and the identification was confirmed using the AlgaeBase (www.algaebase.org), which exhibits updated taxonomical information about the classification of algal species [25]. To verify the identity of isolated microalgae, molecular identification was performed using the universal 18S rRNA gene and specific ITS1 and ITS4 primers, following conventional protocols. PCR amplifications were carried out in a thermal cycler (Techne TC-512, Techne, Staffordshire, UK) under the following conditions: initial denaturation for 5 min at 95 °C, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min, and a final extension at 72 °C for 7 min. PCR products were purified using the AxyPrep DNA Gel Recovery Kit (Axygen Biosciences, Union City, CA, USA) and sequenced using the Sanger sequencing method on an ABI 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA). The obtained sequences were compared with available sequences in GenBank using the BLAST (Basic Local Alignment Search Tool) tool version 2.17.0 (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 15 January 2025) in the National Center for Biotechnology Information (NCBI). Sequence alignment of the phylogenetic tree was conducted via MEGA11 software. Phylogenetic trees were built using multiple approaches by means of the neighbor-joining (NJ) algorithm of parameter distance (PD) [26]. The robustness of the phylogenetic analysis was assessed through 1000 bootstrap replicates, with significant bootstrap values (≥70%) displayed in the final tree [27]. The nucleotide sequences generated in the present study have been deposited in the National Center for Biotechnology Information (NCBI) database for public access.

2.2. Industrial Wastewater Collection

Industrial wastewater was collected from the effluent discharge point of an aluminum factory located in El-Gharbia Governorate, Egypt (30.7144° N, 31.2400° E). Approximately 20 L of wastewater were collected in sterilized polyethylene containers during a single sampling campaign. Immediately after collection, the samples were transported to the laboratory for initial physicochemical characterization and phycoremediation. Prior to use, the wastewater was filtered through Whatman No. 1 filter paper to remove large debris and suspended solids.

2.3. Physicochemical Analysis

Physicochemical characterization of the initial industrial wastewater was performed according to Standard Methods for the Examination of Water and Wastewater, 22nd ed. [28] to assess baseline contamination levels prior to treatment. Hydrogen ion concentration (pH) was measured directly using a calibrated pH meter (JENCO 6173, Jenco Instruments, Inc., San Diego, CA, USA). Chemical Oxygen Demand (COD) was determined using the closed reflux method with a COD reactor (Hach DRB200, Hach Company, Loveland, CO, USA), following APHA Standard Method 5220 C. Ammonia concentrations were determined using the manual phenate method in accordance with APHA Standard Methods 4500-NH₃ F. Nitrate was determined using the cadmium reduction method, following APHA Standard Method 4500-NO₃⁻ C. Phosphate concentration was determined using the ascorbic acid colorimetric method [29]. The heavy metal ions—including aluminum (Al³⁺), copper (Cu²⁺), chromium (Cr³⁺), cadmium (Cd²⁺), nickel (Ni²⁺), lead (Pb²⁺), manganese (Mn²⁺), and zinc (Zn²⁺)—were quantified using an atomic absorption spectrophotometer (PerkinElmer AAnalyst 200, PerkinElmer, Inc., Waltham, MA, USA), in accordance with APHA Standard Methods (22nd edition, Methods 3111 & 3120) [28].

2.4. Biomass Preparation Techniques

Four types of biomass were prepared for use in the phycoremediation experiments: free-living, immobilized-living, raw dried, and acid-treated dried.

Free-Living Biomass: Freshly cultivated cells were grown in BG-11 medium under white, fluorescent light (3000 Lx, Philips TL-D 36W, Eindhoven, The Netherlands) with a 12:12 h light/dark cycle, maintained at 25 ± 2 °C with continuous aeration (0.5 vvm). Cells were harvested in the exponential phase, which was previously determined to occur between days 4 and 12 of cultivation based on preliminary optical density (OD680) and dry weight measurements obtained in a separate study under consideration for publication. These cells were subsequently used as an inoculum for the phycoremediation experiment.

Immobilized-Living Biomass: Fresh algal cells were harvested by centrifugation at 4000 rpm for 10 min (Hettich Rotina 380, Tuttlingen, Germany), washed twice with sterile distilled water, and then suspended in 2% sodium alginate (Sigma-Aldrich, St. Louis, MO, USA). The suspension was dripped through an 18 G sterile needle into a gently stirred 3% calcium chloride solution (Merck, Darmstadt, Germany), forming beads that were cured for 1 h at room temperature. Beads were then washed twice with sterile distilled water and used immediately [30].

Raw Dried Biomass: Fresh algal cultures were harvested by centrifugation at 4000 rpm for 10 min, washed twice with distilled water to remove residual medium and impurities, and then oven-dried at 60 °C for 48 h (Memmert UN55, Schwabach, Germany). The dried biomass was ground into a fine powder and stored in airtight containers until use.

Acid-Treated Dried Biomass: Dried algal biomass was subjected to acid treatment following Almomani and Bhosale [21]. Briefly, 1 g of dried algal biomass was suspended in 20 mL of 0.5 M H₂SO₄ (Sigma-Aldrich, St. Louis, MO, USA) and mixed at 500 rpm using a magnetic stirrer (Torrey Pines Scientific, Inc., Carlsbad, CA, USA) at 35 ± 1 °C for 60 min. The mixture was centrifuged at 5000 rpm for 15 min, and the recovered biomass was washed repeatedly with deionized water until a neutral pH (7.0 ± 0.5) was reached. Finally, the biomass was oven-dried at 60 °C for 24 h and stored until use.

2.5. Phycoremediation Experimental Designs

In this study, four different industrial wastewater treatment techniques were used to compare the capability of the two selected microalgae strains (Dictyosphaerium sp. and Tetradesmus obliquus (homotypic synonym: Scenedesmus obliquus) to treat the collected industrial wastewater sample using both living algal cultures (free and immobilized) and dead dry algal biomass (raw and treated). Many previous studies have reported that the optimal pH for the growth of the selected microalgae strains and maximal heavy-metal removal efficiency ranges from 6 to 8 [31,32]. For all treatments, the industrial wastewater pH was adjusted to 7.5 ± 0.2 by using 0.1 M NaOH and 0.1 M HCl solutions prior to inoculation, and experiments were conducted under controlled laboratory conditions at room temperature (25 ± 2 °C). The initial concentrations of heavy metals in the wastewater were measured before treatment and are presented in

Table 1. Physicochemical compositions of the collected industrial wastewater sample.

Parameter	Value
Temp (°C)	24 ± 0.5
pH	12.07 ± 0.4
COD (mg/L)	160 ± 3.1
Ammonia (mg/L)	31.2 ± 1.4
Nitrate (mg/L)	430.38 ± 5.3
Phosphate (mg/L)	26.01 ± 0.82
Cd2+ (µg/L)	165 ± 2.2
Cr3+ (µg/L)	260 ± 3.1
Cu2+ (µg/L)	448 ± 6.2
Ni2+ (mg/L)	ND
Pb2+ (mg/L)	ND
Al3+ (mg/L)	646.1 ± 6.5
Mn2+ (µg/L)	75 ± 1.5
Zn2+ (µg/L)	106 ± 2.4

ND: Not Detected.

. All experiments were performed in triplicate.

2.5.1. Phycoremediation by Free-Living Algal Culture

In 2000 mL sterile Erlenmeyer conical flasks containing 1000 mL of the collected industrial wastewater sample, 150 mL of an actively growing algal culture was inoculated separately into each flask. The experiment was conducted in triplicate under control conditions using a shaking incubator at 25 ± 2 °C, and white, fluorescent illumination (3000 Lx) with a 12:12 h light/dark cycle over 12 days. At regular intervals every two days, the samples were subjected to analysis to determine the physicochemical parameters, which were previously described.

2.5.2. Phycoremediation by Alginate-Immobilized Microalgae (Algal Beads)

Immobilized algal strains (beads) were inoculated into the pre-filtered industrial wastewater (1000 mL medium containing 100 g fresh weight of beads). The batch cultures were maintained in Erlenmeyer flasks and incubated under controlled conditions at 25 ± 2 °C, with illumination (3000 Lx, 12:12 h light/dark cycle) and agitation at 150 rpm in a shaking incubator. The experiment was conducted over a 12-day period, during which physicochemical parameters of the wastewater were monitored at two-day intervals to evaluate treatment performance and nutrient removal efficiency.

2.5.3. Phycoremediation by Raw Algal Biomass

Equilibrium experiments were conducted by adding 1 g of oven-dried algal biomass to 1000 mL of industrial wastewater in a 2500 mL conical Erlenmeyer flask. The mixture was placed in a shaking incubator at 150 rpm at 25 ± 2 °C for 150 min, as this time was proven enough to attain equilibrium [33], with samples collected at 0, 30, 60, 120, and 150 min.

2.5.4. Phycoremediation by Acid-Treated Algal Biomass

The adsorption experiments were carried out in batch mode in a 2500 mL conical flask containing 1000 mL of industrial wastewater, and 1 g of the adsorbent was added. The mixture was agitated on a mechanical shaker (150 rpm) at 25 ± 2 °C for 150 min to ensure thorough homogenization and adequate contact between the biomass and pollutants, during which samples were collected every 30 min to capture the rapid adsorption process.

All treatments were performed with equivalent biomass loadings. For immobilized and fresh cultures, biomass was standardized on a fresh weight basis, while for raw and acid-treated biomass, the dosage was based on dry weight to ensure consistency across treatments.

The percentage removal and metal uptake efficiencies (removal efficiency %) of all adsorbents were determined using Equation (1) [34]:

$$\mathrm{R}\mathrm{e}\mathrm{m}\mathrm{o}\mathrm{v}\mathrm{a}\mathrm{l} \mathrm{e}\mathrm{f}\mathrm{f}\mathrm{e}\mathrm{c}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{y} \left(\mathrm{\%}\right)=\left[\right(\mathrm{C}\mathrm{i}-\mathrm{C}\mathrm{f})/\mathrm{C}\mathrm{i}]\times 100$$

(1)

where Ci is the initial metal concentration in solution (mg/L) and Cf is the final metal concentration in the supernatant after adsorption (mg/L).

2.6. Principal Component Analysis (PCA)

To assess the patterns of pollutant removal by Dictyosphaerium sp. and Tetradesmus obliquus under different biomass conditions (free-living, immobilized, raw dried, and acid-treated dried forms), Principal Component Analysis (PCA) was performed. The dataset included removal efficiency values for chemical oxygen demand (COD), ammonium, nitrate), phosphate, and heavy metals (Cd, Cr, Cu, Al, Mn, Zn).

Prior to analysis, data were normalized (mean-centered and scaled to unit variance) to minimize bias due to differences in magnitude among parameters. PCA was conducted using OriginPro 2024, OriginLab Corporation, Northampton, MA, USA to reduce the dimensionality of the dataset and to identify associations between biomass types and pollutant removal efficiencies.

The first two principal components (PC1 and PC2) were extracted and plotted as a biplot to visualize treatment clustering and the direction of pollutant vectors. Eigenvalues were used to determine the percentage of variance explained by each component. The loading scores of pollutants (vectors) and the positioning of treatments (scores) were interpreted to identify the main contributors to variation in removal efficiency.

2.7. Statistical Analysis

All analyses were conducted in triplicate, and results were expressed as means ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism v10.4.2 (GraphPad Software, San Diego, CA, USA), applying two-way ANOVA at p ≤ 0.05.

3. Results

3.1. Identification of Microalgae Species

Two microalgae strains were isolated from the Nile River in Egypt. Morphological and molecular examination showed these microalgal species belong to the green algae division (Chlorophyta): Dictyosphaerium sp. and Tetradesmus obliquus. Dictyosphaerium sp. is colonial, consisting of small spherical to ovoid cells connected by branching threads. T. obliquus features spindle-shaped cells that form colonies of two or four cells. Some solitary cells were also observed in the culture, as shown in Figure 1.

The Neighbor-Joining (NJ) phylogenetic tree (Figure 2) identifies the isolated Dictyosphaerium and Tetradesmus obliquus species based on 18S rRNA nucleotide sequences. The tree reveals two major clades: one corresponding to Dictyosphaerium and the other to Tetradesmus. Within the Dictyosphaerium clade, the newly identified Dictyosphaerium sp. (PP949296) groups closely with Dictyosphaerium ehrenbergianum strains (CCAP 222/57 and YN28-2) with strong bootstrap support (100), suggesting a close evolutionary relationship. This cluster is distinct from other Dictyosphaerium strains, including Dictyosphaerium sp. 8-1, Dictyosphaerium sp. Iso 6, D. strain A20, Dictyosphaerium sp. 8-6, and Dictyosphaerium sp. SMUS-2018.

In the Tetradesmus clade, Tetradesmus obliquus (PP949294), this cluster is clearly separated from T. dimorphus strains (S6, S2, and S10) with high bootstrap support (90) and from other Coelastrella species (C. oocystiformis and C. terrestris). Similar phylogenetic approaches using 18S rRNA have been applied to identify new Chlorophyta strains, supporting the reliability of this method for species delineation. The high bootstrap values across the tree (mostly 100) indicate the reliability of these phylogenetic relationships.

The distinct clustering of the newly identified species confirms their classification within their respective genera and highlights the genetic divergence between Dictyosphaerium and Tetradesmus. Dictyosphaerium sp. and Tetradesmus obliquus were selected for detailed experiments since they represented the most dominant isolates obtained from the Nile River sample. These strains are also known to be widely distributed in freshwater environments and have been previously reported as efficient candidates for nutrient and heavy metal removal, which justifies their selection for this study.

3.2. Industrial Wastewater Analysis

The physicochemical analysis results of the industrial wastewater sample collected from the effluent of an aluminum factory located in El-Gharbia governorate, Egypt, are presented in Table 1. Since the optimum pH value ranges between 6 and 8 for the growing microalgae, prior to microalgal cultivation, the pH of the collected industrial wastewater sample was adjusted to 7.5.

3.3. Evaluation of Industrial Wastewater Treatment (Phycoremediation)

3.3.1. Phycoremediation Using Dictyosphaerium sp.

The impact of Dictyosphaerium sp. in various forms, either living (free culture and immobilized) or non-living (raw dried biomass and acid-treated biomass), on industrial wastewater physicochemical properties and nutrients and heavy metal removal percentages was estimated and recorded in Figure 3 and Figure 4. Dictyosphaerium sp. free culture demonstrated the highest COD removal rate at 66.5%, as it decreased from 160 mg/L before treatment to 53.6 mg/L within 12 days, with immobilized microalgae closely behind at 63.6%, while acid-treated and dry raw biomass achieved removal rates of up to 43.7% and 37.9%, respectively. Similarly to the COD removal rate, Dictyosphaerium sp. free culture had the highest ammonia and nitrate removal rates (up to 99.4% and 58.8%, respectively), followed by immobilized microalgae (79.5% and 53.6%, respectively), acid-treated biomass (42.3% and16.5%, respectively), and raw dried biomass (33.3% and 18.6%, respectively). On the other hand, the immobilized Dictyosphaerium sp. was able to remove the highest percentage of phosphate (71.5%), exceeding the removal rates of free culture, acid-treated, and raw dried biomass, which removed 65.8%, 50%, and 47.7%, respectively. On the other hand, complete removal (100%) of Cd²⁺ and Cr³⁺ was achieved in the free culture after 10 days of treatment. Interestingly, this complete adsorption was achieved by raw dried biomass within 120 min and by acid-treated Dictyosphaerium sp. within 120 min for Cd²⁺ and 150 min for Cr³⁺. Immobilized Dictyosphaerium sp. showed slightly lower efficiency after 12 days, with removal rates of 89.8% for Cd²⁺ and 95.4% for Cr³⁺. Cu²⁺ removal rates were close among free culture, acid-treated biomass, and immobilized Dictyosphaerium sp. at 78.3%, 77.7%, and 76.6%, respectively. In comparison, raw dried biomass achieved the lowest Cu²⁺ removal rate at 64.8%, decreasing its concentration from 484 µg/L to 157.9 µg/L on the twelfth day. In contrast, raw dried biomass showed the highest Al³⁺ removal at 98.4%, decreasing its concentration from 646.1 mg/L to 10.2 mg/L, followed closely by immobilized microalgae at 98.2% (11.6 mg/L), while acid-treated biomass and Dictyosphaerium sp. free culture removed up to 97.9% and 94.7%, respectively. Total removal of manganese (Mn²⁺) reached 100% in the immobilized Dictyosphaerium sp. after 10 days of industrial wastewater treatment, surpassing the free culture, acid-treated, and raw dried biomass of Dictyosphaerium sp., which recorded 97.9% after 12 days, 92.9% after 150 min, and 88.7% after 150 min of treatment, respectively. Zinc (Zn²⁺) was completely removed (100%) in the free algal culture after 10 days of industrial wastewater treatment and in the acid-treated Dictyosphaerium sp. after 150 min, outperforming the removal rates of raw dried biomass and immobilized Dictyosphaerium sp., which removed 96.6% and 93.9%, respectively.

In conclusion, the present study found that Dictyosphaerium sp. is highly efficient in removing most pollutants detected in industrial wastewater. The alga’s living form outperformed its dried form at removing COD, ammonium, nitrite, and phosphate. Furthermore, Dictyosphaerium sp. demonstrated a significant ability to remove heavy metal ions in various states, successfully removing Cd²⁺ and Cr³⁺ in both free-cultured algae and dried forms (regardless of whether raw or acid-treated). Notably, the removal time using dried biomass was just 120 min (making it preferable when dried biomass is available). Immobilized Dictyosphaerium sp. was more efficient at removing aluminum and manganese.

3.3.2. Phycoremediation Using Tetradesmus obliquus

The effects of T. obliquus in different states on industrial wastewater properties and removing nutrients and heavy metal ions were measured and recorded in Figure 5 and Figure 6. Free-cultured T. obliquus showed the highest COD removal rate of 80.4%, reducing from 160 mg/L prior to treatment to 31.3 mg/L over 12 days, with immobilized microalgae following closely at 74.9%. In contrast, acid-treated and dry raw biomass reached removal rates of 62.44% and 60.38%, respectively. Living forms of T. obliquus had significantly higher removal rates for ammonia and nitrate than dried biomass. Free-cultured T. obliquus also removed ammonia and nitrate efficiently with removal percentages of 95.2% and 58.8%, respectively, while immobilized microalgae achieved removals of 70.8% and 76.3%, respectively. On the other hand, acid-treated dried biomass removed 24.4% and 12.4%, respectively, and untreated biomass removed 8.3% and 14.6%, respectively. The immobilized T. obliquus successfully removed the greatest percentage of phosphate (81.5%), surpassing the removal rates of free-culture and acid-treated biomass (79.6% and 73.6%, respectively). The dry untreated biomass had the lowest phosphate removal rate (64.2%). After a 10-day treatment period, the free-cultured T. obliquus removed 100% of Cd²⁺, Cr³⁺, Cu²⁺, and Zn²⁺, as well as 98.4% of Al³⁺, decreasing its concentration in industrial wastewater from 646.1 mg/L to 10.2 mg/L in 12 days, and 89.3% of Mn²⁺. Cd²⁺ and Zn²⁺ were also completely removed (100%) from industrial wastewater after 10 days of treatment with immobilized T. obliquus and 120 min in both dry biomass (untreated and acid-treated). Cr³⁺ was entirely removed (100%) by using immobilized and acid-treated biomass and 87.1% by untreated dry biomass. The immobilized T. obliquus got rid of all of the Al³⁺ after 12 days of treatment. It also removed 94.9% and 91.5% of the Mn²⁺ and Cu²⁺, respectively. Untreated raw dried biomass exhibited the lowest Cu²⁺ removal rate of 48.8% after 150 min of treatment, while it had a high Al³⁺ and Mn²⁺ removal ability of 99.9% and 90.8%, respectively. On the other hand, acid-treated biomass removed Cu²⁺, Al³⁺, and Mn²⁺ at 95.76%, 99.15%, and 96.53%, respectively.

In summary, the previous observations revealed that T. obliquus effectively removed most of the contaminants found in industrial wastewater. Phycoremediation using live T. obliquus, irrespective of being free or immobilized, was highly effective, with Cd²⁺, Cr³⁺, Cu²⁺, Al³⁺, and Zn²⁺ completely removed. Additionally, acid-treated T. obliquus biomass demonstrated greater contaminant removal efficiency than its raw dried biomass.

3.3.3. Comparison Between the Two Microalgae Phycoremediation Rates

Regarding removal rates between the two tested microalgae, T. obliquus outperformed Dictyosphaerium sp. (Figure 7). With the exception of ammonium, Dictyosphaerium sp. achieved the highest ammonium removal rate, at 99.4%. T. obliquus showed the best performance in removing heavy metal ions, completely removing Cd²⁺ and Zn²⁺ in both living and dry forms, Cu²⁺ in its free-living form, and Al³⁺ in its immobilized form. Utilizing biomass in its raw form resulted in the least effective removal of heavy metal ions, with copper removal notably low at 48.8%. Both free-living and acid-treated biomass of Dictyosphaerium sp. demonstrated the highest efficiency in removing Cd²⁺, Cr²⁺, and Zn²⁺.

Figure 7 illustrates that free-living and immobilized biomass of both algal strains are strongly associated with higher removal efficiencies (80–100%) across multiple pollutants, particularly for COD, NH₄, NO₃, PO₄, and heavy metal ions such as Mn²⁺ and Cr³⁺. Specifically, free-living forms show substantial flows toward high removal efficiency bands (90–100%) for COD and ammonium, consistent with their direct assimilation and active metabolic uptake mechanisms.

Immobilized biomass also demonstrated high removal efficiencies (80–100%), notably for phosphate and manganese, reflecting the effectiveness of immobilization in retaining pollutant removal capacity while facilitating biomass recovery.

In contrast, acid-treated and raw biomass showed broader distributions across lower efficiency ranges (30–70%) for several pollutants yet maintained high efficiencies (90–100%) for selected heavy metal ions, including Cd²⁺, Zn²⁺, and Al³⁺. This indicates that non-living biomass can achieve rapid and complete removal of certain metals, despite lower effectiveness for nutrient removal compared to living forms.

3.3.4. Principal Component Analysis (PCA)

To further evaluate the pollutant removal patterns of Dictyosphaerium sp. and Tetradesmus obliquus under different biomass conditions, PCA was conducted, as shown in Figure 8.

The first two principal components accounted for 64.28% of the total variance (PC1 = 40.19%, PC2 = 23.09%). The biplot revealed clear separation among the treatment groups based on removal efficiency and pollutant types.

Dictyosphaerium sp. in free and immobilized forms clustered on the right side of the plot along the direction of COD, ammonium, nitrate, and phosphate vectors, indicating their strong association with removing these parameters. Notably, Dictyosphaerium sp. immobilized biomass aligned closely with the manganese (Mn²⁺) vector, confirming its superior Mn removal efficiency. In contrast, Dictyosphaerium sp. raw and acid-treated biomass clustered on the left side of the plot, near the Al³⁺ vector, reflecting their association with Al³⁺ removal rather than nutrient parameters. On the other hand, T. obliquus in free and immobilized forms positioned on the upper right quadrant near the COD, phosphate, and Cu²⁺ vectors, suggesting their high efficiency in removing these pollutants. The acid-treated form of T. obliquus aligned near the Zn²⁺ and Cd²⁺ vectors, indicating a strong association with heavy metal removal, particularly zinc and cadmium.

The PCA confirms that living biomass forms (free and immobilized) of Dictyosphaerium sp. and Tetradesmus obliquus are closely associated with nutrient (COD, ammonium, nitrate, phosphate) and manganese removal. Meanwhile, acid-treated biomass forms are more aligned with heavy metal removal, especially cadmium, zinc, and aluminum. Immobilization enhances manganese removal in Dictyosphaerium sp., while Tetradesmus obliquus retains high COD, phosphate, and copper removal capacity in its free and immobilized forms. These findings support the higher pollutant removal efficiencies of living biomass over non-living biomass and clarify the specialization of each treatment type for specific pollutants, strengthening the potential of targeted phycoremediation strategies using these locally isolated strains.

4. Discussion

As stated in the study, the combination of morphological and molecular identification techniques aligns with current strategies and mitigates misidentification problems with microalgae species [35]. The Nile River’s environmental features most likely support the adaptive capabilities of these species. The presence of Dictyosphaerium and Tetradesmus indicates eutrophic waters, highlighting possible nutrient over-enrichment and human impacts in the study area [36,37]. The investigation into industrial wastewater samples taken from the aluminum production facility in El-Gharbia, Egypt, demonstrates an initial pH measurement of 12.07, which exceeds the optimal range of 6–8 for microalgal growth. Alkaline environments interfere with cellular functions while simultaneously blocking nutrient absorption in algae. pH modification is performed to achieve a neutral value of 7.5, which boosts algal biomass output and nutrient removal performance [8]. Although the industrial wastewater collected from the aluminum factory was extremely poor in macronutrients, both Dictyosphaerium sp. and Tetradesmus obliquus survived and maintained growth for 12 days. Two main factors can explain this survival and growth. First, the presence of trace organic and inorganic compounds in the wastewater, which, although not reflected in the bulk nutrient profile, was sufficient to sustain basal metabolic activity. Second, microalgae are well known for their adaptive mechanisms under stress conditions, including high nutrient uptake efficiency, recycling of internal nutrient reserves, and activating tolerance pathways that enable persistence in oligotrophic environments. These findings align with those of Wang et al. [38,39] and Liang et al. [40], where a microalgal strain exposed to similar low nitrogen content of only 240 mg/L still sustained growth and pollutant removal. In their work, even under limited availability of major nutrients, the microalgae leveraged trace elements and efficient internal recycling to maintain growth and functionality. Similar observations have been reported in previous studies, where low-nutrient or stress conditions due to efficient internal nutrient allocation and uptake of trace elements from the medium [18,31].

The phycoremediation potential of the two microalgae species was compared with findings from other studies on microalgae-based industrial wastewater treatment, and the advantages of this sustainable approach were highlighted. The present study found that living forms of both free-living and immobilized microalgae exhibited significantly higher removal efficiencies for COD, ammonium, nitrate, and phosphate than their dried counterparts. This enhanced performance can be attributed to the active metabolic processes of living microalgae, which directly assimilate ammonium and nitrate as nitrogen sources for amino acid and protein synthesis, thereby reducing their concentrations in wastewater [41]. The superior ammonium removal efficiency observed in Dictyosphaerium sp. (99.4%) can be explained by the general preference of microalgae for ammonium as their primary nitrogen source. Ammonium can be directly assimilated into amino acids and proteins via the GS–GOGAT enzymatic pathway, minimizing energy costs and enabling rapid nitrogen assimilation. This metabolic advantage may account for the remarkable ammonium removal capacity exhibited by Dictyosphaerium sp. in the present study [39]. Similarly, living microalgae actively uptake phosphate and store it intracellularly as polyphosphate granules, a mechanism absent in dried biomass that relies solely on surface adsorption for nutrient removal. Free T. obliquus stood out with the highest COD removal rate of 80.4%. Then came free Dictyosphaerium sp. at 66.5%. These results match the study by Andrade et al. [42], who also noted that T. obliquus can get rid of up to 80% of COD, which aligns nicely with some recent studies. For example, Scenedesmus obliquus hit a COD removal efficiency of about 71% under optimal conditions [43]. Even the immobilized forms of both microalgae showed decent performance in reducing COD, with rates varying from 63.6% for Dictyosphaerium sp. to 74.9% for T. obliquus. This goes to show that living microalgae are better at reducing COD; this aligns well with Wang et al. [44], who reported that higher metabolic activity in live cells leads to better breakdown of organic pollutants.

Ammonia, nitrate, and phosphate removal rates varied significantly among the studied microalgae and biomass states. Microalgae assimilate nutrients like ammonium and nitrate for growth. The direct utilization of ammonia for amino acid synthesis, as cited from Ding et al. [45], remains a key mechanism. Ammonia removal rates up to 99.4% for free Dictyosphaerium sp. outperformed T. obliquus (95.2%), which exceeded those reported by Ji et al. [46], who found that the ammonia removal of Tetradesmus obliquus was 78.75% after a 10-day wastewater treatment period; and Pham & Bui [47], where Scenedesmus sp. achieved 93% ammonia removal from fertilizer plant wastewater. Nutrient uptake trends mirror findings by Wang et al. [39], who emphasized the role of algal metabolic activity in ammonia assimilation. Nitrate removal efficiencies showed a different trend; the immobilized form of T. obliquus exhibited the highest efficiency at 76.3%, followed by free-cultured Dictyosphaerium sp. at 58.8%. These findings are consistent with Shaker et al. [48], who found that immobilized microalgae reduced nitrate levels significantly, with immobilized Chlorella vulgaris performing the best, reducing nitrate by 72%. Mollamohammada et al. [49] reported that alginate-immobilized Scenedesmus sp. showed 90% nitrate removal in 9 and 12 days. A study by Alhumairi et al. [50] demonstrated the effectiveness of immobilization techniques for organic pollutant removal. These findings highlight the potential of using immobilized microalgae for industrial wastewater treatment applications. Dry microalgal biomass showed a much lower nutrient removal efficiency than living microalgae, perhaps because dry biomass is inactive; it cannot metabolize or assimilate nutrients; therefore, it only works through passive adsorption, which is less efficient.

Similarly, phosphate removal was most effective with immobilized T. obliquus (81.5%), surpassing Dictyosphaerium sp. (71.5%). However, Severo et al. [51] reported that both immobilized and free cells of T. obliquus exhibited exceptional phosphate removal efficiency. Ansari et al. [52] reported a 94.1 ± 4.7% reduction in phosphate levels when Scenedesmus obliquus was cultured in municipal wastewater.

Heavy metal contamination presents significant environmental risks because of its toxicity and persistence [53]. The initial analysis of the industrial wastewater revealed that most heavy metals (e.g., Cd²⁺, Cr³⁺, Cu²⁺, Zn²⁺) were present at relatively low concentrations (<1 ppm), which are notably below the typical ranges (0.2–10 ppm) reported in previous studies on microalgal bioremediation [13,14]. In contrast, aluminum was detected at an unusually high concentration, which can be directly attributed to the industrial origin of the wastewater (aluminum factory effluent). This observation is consistent with findings from recent reports on metal-contaminated effluents from industrial sectors, where aluminum often dominates the pollutant profile [54,55]. From a regulatory perspective, the detected aluminum concentration far exceeds the guideline values set by international standards, such as the World Health Organization [56], which recommends ≤0.2 mg/L, and the U.S. Environmental Protection Agency [57], which sets a maximum contaminant level of 0.75 mg/L. This highlights the environmental significance of developing effective strategies for aluminum removal and supports the need for microalgal-based bioremediation as a sustainable treatment approach.

The studied microalgae showed exceptional abilities in removing heavy metal ions across different biomass states. This may be due to the removal of pollutants being restricted to passive biosorption onto cell wall functional groups, an effective process for heavy metal removal, but limited in nutrient and COD removal [55]. The complete removal of Cd²⁺, Cr²⁺, and Zn²⁺ by free or immobilized microalgae is noteworthy and aligns with the known biosorption capabilities of microalgal cell walls. These results support earlier studies that emphasize the role of algal cell walls in heavy metal adsorption through ion exchange mechanisms. Recent research reports comparable efficiencies. Xu et al. [58] reported that Scenedesmus obliquus could efficiently remove over 95% of Cd²⁺ from the environment through biosorption and bioaccumulation. The superior performance of living cells correlates with active biosorption and intracellular sequestration mechanisms, as highlighted by Thi Nguyen et al. [59]. However, acid-treated biomass showed high cadmium and chromium adsorption efficiency within 120 min. It consistently outperformed other forms in Mn²⁺ removal across both species, comparable to those reported by Koshariya et al. [60], who demonstrated complete cadmium and chromium removal using acid-treated algal biomass. The rapid removal (≤150 min) by acid-treated biomass aligns with advancements in functionalized algal biomass; for example, Almomani & Bhosale [21] reported that acid-treated Spirulina platensis removed up to 95.0 ± 0.3%, 87.0 ± 0.2%, and 63.0 ± 0.3% of Al, Ni, and Cu, respectively, from industrial wastewater.

Heavy metal removal by microalgae, including Dictyosphaerium sp. and Tetradesmus obliquus, is influenced by multiple factors, with the negative surface charge and the composition of cell wall functional groups playing pivotal roles. The algal cell wall typically contains abundant functional groups such as carboxyl, hydroxyl, sulfate, phosphate, and amino groups, which act as active sites for the adsorption of heavy metal ions. The presence of these groups imparts a net negative charge on the algal surface under neutral to slightly alkaline pH conditions, enhancing electrostatic attraction towards positively charged heavy metal cations such as Cd²⁺, Cr³⁺, Cu²⁺, and Zn²⁺ [61].

Specifically, Dictyosphaerium sp. has a cell wall rich in polysaccharides and glycoproteins, providing a high density of carboxyl and hydroxyl groups that facilitate metal binding through ion exchange and complexation mechanisms [62,63]. T. obliquus possesses a robust multilayered cell wall composed of algaenan, cellulose, and hemicellulose, containing carboxyl, phosphate, and sulfate groups capable of binding heavy metal ions efficiently [64]. This structural complexity contributes to their high heavy metal removal efficiency in living and non-living biomass.

Additionally, the removal capacity is affected by the ionic radius and valence state of the metal ions, with divalent cations such as Cd²⁺ and Cu²⁺ showing strong affinity for the negatively charged sites due to their smaller hydrated radius and higher mobility, facilitating rapid biosorption [55]. Living microalgae provide dynamic and adaptable remediation and superior nutrient removal efficiencies; their performance is influenced by environmental factors such as pH, temperature, and nutrient availability; controlled conditions are required; and there is a risk of overgrowth if not managed properly. In contrast, dried biomass provides a more controlled and stable approach, as well as simpler storage and handling, with the potential for chemical modifications to enhance metal-binding capacities. The choice between using living or dried microalgae is determined by the specific application requirements, including the nature of the wastewater, optional conditions, and desired outcomes. According to this study and Faruque et al. [54], using living or non-living algal biomass is becoming more widely recognized as a cost-effective and user-friendly approach to large-scale bioremediation of industrial wastewater (IW). The current study also highlights species-specific variations and identifies T. obliquus as an effective strain overall, achieving the highest removal rates for COD, nitrate, and heavy metal ions such as Cd²⁺, Cr³⁺, Cu²⁺, Al³⁺, and Zn²⁺, most likely due to its robust enzymatic machinery for organic matter assimilation, as noted in a recent review by Li et al. [65]. Recent works by Xu et al. [58] and Balzano et al. [66] corroborate T. obliquus resilience in high-metal environments, attributing this to upregulated metallothionein production. Conversely, Dictyosphaerium sp. excelled in ammonium removal (99.4%). These findings are consistent with previous research demonstrating strain-specific efficiencies based on nutrient uptake mechanisms and environmental adaptability. This may also stem from species-specific cell wall composition, which influences metal-binding capacity [67,68,69].

Overall, this study confirms that both strains’ living biomass (free and immobilized) is highly effective in removing organic and nutrient pollutants from industrial wastewater, achieving removal rates primarily in the 80–100% range. Non-living biomass forms, while less efficient in nutrient removal, excel in heavy metal removal, particularly for cadmium, zinc, and aluminum, achieving near-complete removal within short contact times. T. obliquus demonstrates slightly broader pollutant removal capacity across forms compared to Dictyosphaerium, aligning with its suitability for diverse phycoremediation applications. This integrated visualization complements the PCA findings, supporting the selection of living biomass for nutrient and organic pollutant removal and non-living biomass for targeted heavy metal removal in cost-effective phycoremediation strategies. However, it should be noted that no control treatment of “industrial wastewater alone” was included, which may have limited the ability to fully separate the effects of indigenous microbiota from those of the tested microalgae. Future studies should incorporate such controls to provide a more precise evaluation.

5. Conclusions

This study provides robust evidence supporting the use of microalgae for industrial wastewater treatment, aligning with broader research trends emphasizing the ecological and economic benefits of microalgae-based systems. The comparative analysis highlights the superior performance of living microalgal cultures in removing organic pollutants (COD), nutrients (ammonium, nitrate, phosphate), and heavy metal ions from industrial wastewater. Among the tested biomass states, living forms consistently demonstrated the highest removal efficiencies for COD and nutrients, with Tetradesmus obliquus generally outperforming Dictyosphaerium sp. in most parameters except for ammonium removal, where Dictyosphaerium sp. achieved the highest efficiency (99.4%).

The study also introduces novel insights, such as the exceptional aluminum removal by Dictyosphaerium sp. at 98.4%, decreasing its concentration from 646.1 mg/L to 10.2 mg/L, and the rapid and efficient metal uptake exhibited by acid-treated biomass, indicating its potential for applications requiring short contact times. Overall, this research highlights the feasibility of integrating both living and non-living algal biomass into advanced, eco-friendly wastewater treatment strategies. In a separate study currently under submission, we also analyzed the biomass composition, including chlorophyll content, for both Dictyosphaerium sp. and Tetradesmus obliquus. Such assessments further support the integration of pollutant removal with biomass valorization, reinforcing the sustainability of phycoremediation within a circular economy framework.

Future efforts should focus on optimizing growth conditions, co-culturing strategies, and exploring hybrid approaches combining multiple algal species. In particular, the simultaneous use of both selected strains (Dictyosphaerium sp. and Tetradesmus obliquus) in co-cultivation systems could be explored in future studies, as this approach may provide complementary pollutant removal capacities (e.g., one strain favoring ammonium assimilation while the other enhances phosphate or specific metal uptake) and increase system resilience. Additionally, integrating these systems into larger biorefinery frameworks could further enhance pollutant specificity, treatment outcomes, and sustainability, supporting their scale-up for large-scale industrial application.