DOI:https://doi.org/10.65613/738874
Gherbi Bachir 1, Abdellatif Rahmani 2, Miloud Khenifer 3
1University of Hamma Lakhdar, ElOued, Algeria
2 University of Ouargla, Fac. des sciences appliquées, Lab. dynamique interaction et réactivités des
systèmes BP 511, Route de Ghardaïa, Ouargla 30000, Algeria
3 University of Amar Telidji, Laghouat, Algeria
*Email : Gherbi-bachir@univ-eloued.dz, abdellatif.rahmani@univ-ouargla.dz , drmiloudkhenifer@gmail.com
Received : 11/04/2026 ; Accepted : 03/07/2026 ; Published : 08/07/2026
Abstract
This study evaluates the potential of the microalga Chlorella vulgaris, which has been gradually adapted, for the comprehensive bioremediation of wastewater produced from highly polluted oil fields, with subsequent lipid extraction for use in renewable biofuel applications. The alga was cultivated in a 50-liter open tank under the harsh arid climatic conditions of southern Algeria, taking advantage of the vast flatlands and high solar irradiance and ambient temperatures to stimulate biomass growth and metabolic stress responses. Over 18 days of cultivation, Chlorella vulgaris demonstrated exceptional resistance and high pollutant removal efficiency, successfully reducing the chemical oxygen demand (COD), total petroleum hydrocarbons (TPH), ammonium nitrogen (NH₄⁺), total nitrogen (TN), and total phosphorus (TP) by 85.50%, 89.40%, 100%, 97.65%, and 98.91%, respectively. Despite the high salinity and inherent toxicity of the raw industrial wastewater, biomass production stabilized at 1.6 ± 0.1 g/L by the end of the experiment. Subsequent lipid extraction using the Soxhlet extraction process successfully recovered a remarkable 38% lipid content, confirming the high potential of the harvested feedstock for conversion into biodiesel. These results underscore the viability of a sustainable circular economy framework that effectively integrates advanced oilfield wastewater treatment with clean bioenergy generation in arid environments.
Keywords: Bioremediation, Oilfield Wastewater, Chlorella vulgaris, Arid Regions, Lipid Extraction, Biofuel Production.
Introduction
Global warming and the depletion of conventional fossil fuels represent the most critical challenges facing humankind in the twenty-first century (Wu et al., 2022). To address this twin crisis of energy security and environmental degradation, microalgae-based technologies have emerged as a highly integrated and promising approach, offering a dual solution for advanced wastewater treatment and sustainable biodiesel production (Sivaramakrishnan et al., 2022). Due to the rapid expansion of industrial and petroleum activities globally, there is an unprecedented generation of hazardous industrial effluents, particularly petroleum produced water (PW), which poses a severe threat to arid ecosystems and scarce freshwater resources (Fakhru’l-Razi et al., 2009). Microalgae cultivation has emerged as an efficient bioremediation strategy capable of mitigating this industrial pollution and combatting climate change through high rates of photosynthetic carbon dioxide sequestration (Sayre et al., 2010).
This technology gains vital importance in arid regions, such as southern Algeria, where severe freshwater scarcity necessitates the utilization of non-conventional water sources. In this context, petroleum produced water presents a strategic alternative. Despite its significant environmental hazards resulting from hyper-salinity and toxic heavy metal and hydrocarbon contents (Jiménez et al., 2020), cultivating resilient and tolerant microalgal strains like Chlorella vulgaris in this matrix not only mitigates oilfield pollution through biological mechanisms but also exploits these harsh conditions as metabolic stressors. This stress actively triggers microalgal cells to enhance lipid accumulation, thereby optimizing biomass quality for high-efficiency biofuel conversion (Al-Gheethi et al., 2021).
Numerous studies have demonstrated that robust microalgae strains are highly efficient at removing heavy metals, total petroleum hydrocarbons (TPH), and excessive nutrients from diverse industrial wastewaters, meeting strict international discharge standards (Abdel-Raouf et al., 2012). For large-scale applications, cultivation in open raceway ponds is widely preferred because it requires low capital investment and effectively utilizes the abundant solar irradiance characteristic of arid regions (Borowitzka, 2005). Among the various green microalgae, the genus Chlorella, and specifically Chlorella vulgaris, has been extensively utilized due to its exceptional tolerance to environmental stress (Wang et al., 2010).
However, when grown under the highly saline and toxic conditions of oilfield wastewater, microalgae exhibit significantly varying lipid contents, making downstream processing a critical area of development (Woertz et al., 2009). Recovering these intracellular lipids is highly challenging, as the microalgal cell wall is a complex structure composed of cellulose, proteins, and robust polysaccharides that require optimized extraction and disruption methods (Domozych et al., 2012). Microalgal lipid extraction is crucial for the techno-economic viability of biorefineries. Among available methodologies, Soxhlet extraction remains the gold standard for quantifying total cellular lipids accumulated under environmental stress; its continuous contact between hot organic solvents and dry biomass ensures near-complete quantitative recovery (Halim et al., 2012). This mechanism relies on repeated solvent evaporation and condensation cycles to penetrate the rigid cellulose and glycoprotein cell walls of Chlorella, serving as the fundamental benchmark for evaluating extraction performance in renewable energy research (Mercer & Armenta, 2011).
Therefore, this study aims to evaluate the feasibility of utilizing Chlorella vulgaris for the comprehensive bioremediation of raw petroleum produced water under the arid climate conditions of southern Algeria. Concurrently, this work investigates the efficiency of the conventional Soxhlet extraction process in recovering the accumulated lipid fractions from the hyper-saline stressed biomass, establishing a sustainable, circular-economy framework that links wastewater polishing with optimized bioenergy feedstock preparation
Materials and Methods
2.1 Experimental site and wastewater
The experiment steps were performed outdoors during the fall season under sunlight. The temperatures ranged between 20 and 35 °C during the day and 9-17 °C at night. The isolation period was approximately 12 hour a day (from 7:00 a.m. to 7:30 p.m.). The highest irradiance was about 1200 W/m2.
The produced water (PW) evaluated in this study was sourced from an active oil facility in Bir Sebaa, Touggourt Province (southern Algeria). Effluent samples were collected following three-phase separation (water, gas, and oil) to prepare the wastewater for microalgae cultivation. During the experiments, a submersible circulation pump maintained homogeneity within the open-channel basin, preventing cell sedimentation while ensuring uniform nutrient and pollutant distribution
2.2 Biomass growth for inoculation
Microalgae of the species Chlorella vulgaris, adapted to petroleum-produced water and the region’s climatic conditions, were used. In this research, the algae were cultured in the laboratory.The inoculations were performed in sterile environments. 6 mL
(7.6 × 106 cells/mL) of Chlorella vulgaris. was inoculated into 500 mL of the BG11 medium once an appropriate density.
And the growth was monitored by optical density at 680 nm.
The BG11 medium consists of (g/L) NaNO3 (1.5), K2HPO4 (0.04), MgSO4.7H2O
(0.075), CaCl2. H2O (0.036), Na2CO3 (0.02), citric acid (0.006), EDTA (0.001), and 1 mL prepared from the following composition (g/L): H3BO3 (2.86); MnCl2.4H2O
(1.81); ZnSO4.7H2O (0.22); NaMoO4.2H2O (0.39); CuSO4. 5H2O (0.079);
Co (NO3)2.6H2O (0.05). Mixing was realized by continuous bubbling with air.
The pH was equal to 7–7.5 under aseptic conditions. All cultures were preserved at 22 ± 1 °C in 5L Erlenmeyer flasks. The light was provided by red and blue fluorescent tubes (incident intensity of 150–200 μmol photons m–2 s−1) under a 24-h regime/24 h. (Rahmani et al. 2022).
2.3 Analytical methods
Sunlight irradiance (W/m2) was measured by solarimeter (SL200, instrument, France). Dissolved Oxygen (DO, mg/l), pH, and conductivity (mS/cm) was measured using a multipara meter analyser (Consort C3020, Belgium). Microalgal growth was assessed by daily measurements of the optical density at 680 nm (OD680nm) using Uv/vis spectrophotometry (UV 2300 Spectrophotometer). However, the OD680nm related to suspended solids (SS) in the waste water was subtracted from the total OD 680 nm (with biomass). The final concentration of the algae biomass was determined by subtracting the biomass in the control experiments from the total biomass. Algae biomass was considered as the total volatile suspended solids (Ma et al.2014).
COD was measured using cuvette test kits LCK 514 (100–2000 mg COD/L), following DIN 38,049–4.Ammonium–N (NH4 + –N) kits LCK303 (2.0–47.0 mg/L N-NH4 +) were used following standards DIN38406-E5–1 and nitrate–N (N-NO3–). LCK 339 (0.23–13.50 mg/L N-NO3–) was used following standards DIN 38,402–1 A51 where total phosphorus (TP) was achieved according to ISO 6878–1-1986 standards, DIN 38,405 D11-4. test kit LCK 349 ranges of P-PO4– 3(2–20 mg TP/L) (Rahmani et al. 2022). The spectrophotometer DR 2800 (Hach Lange, Germany) was used for absorbance measurement. However, all, the samples were heated for 2 h at 148 °C for COD.
Examination about 60 min at 100ºC for TP measurement, before being placed in the spectrophoto meter. The heavy metal contents were measured using an atomic absorption
Spectrophotometer (AAS Varian, Walnut Creek, CA).
Removal (%) = ( ) x100 (1)
Ci Initial Concentration: The initial concentration of the pollutant in the sample before the treatment process begins (or at time t = 0).
Cf Final Concentration: The final or residual concentration of the pollutant in the sample after the treatment process is complete.
(2)
mh: represents the mass of the extracted oil.
ma: represents the mass of the dry microalgal biomass.
Data analysis
All the tests were realized in triplicate, graphs were generated with GraphPad Prism version 6.
- Results & Discussion
Outdoor experiments were conducted in a galvanized open raceway pond (100 cm length, 50 cm width, and 20 cm depth).
Open raceways were inoculated with 10% algal suspensions (v/v) of the working volume (50 L). The initial inoculum cell concentrations. Were 7.5 x 106 cells/ml.
Experiments were run for 18 days (External conditions). The mixing and recirculation of the culture medium. was achieved by a stirring system.
- Characterization of Wastewater before and after algae treatment
The characteristics of the wastewater were analyzed in order to identify the concentrations of nutrient.
The physico-chemical parameters of the wastewater are given in Table 1.
Table 1. The physico-chemical parameters of the wastewater.
| Environmental Indicator / Parameter | Symbol / Unit | Extracted Value / Removal Efficiency | Experimental Observations & Statistics |
| PH | PH | 4.6-7.8 | |
| Chemical Oxygen Demand | COD | 85.50% | Significant reduction in the organic load of the water. |
| Total Petroleum Hydrocarbons | TPH | 89.40% | High efficiency in degrading petroleum pollutants. |
| Ammonium-Nitrogen | N-NH4+ | 100.00% | Complete removal of ammonium compounds. |
| Total Nitrogen | TN | 97.65% | Excellent consumption of nitrogenous nutrients. |
| Total Phosphorus | TP | 98.91% | Near-complete removal efficiency of phosphates. |
| Iron | Fe | 76.74% | Decrease from.. 4.0 to 0.93 mg L-1. 0.1mg L-1. |
| Lead | Pb | 72.80% | Decrease from 1.1 mg L-1. to 0.3mg L-1. |
| Copper | Cu | 73.39% | Decrease from 1.12 to 0.295 mg L-1. 0.01mg L-1. |
| Cadmium | Cd | 44.44% | Decrease from 0.08 mg L-1. to 0.045mg L-1. |
| Maximum Biomass Productivity | g L-1 | 1.6 ± 0.1 | Excellent growth despite the presence of heavy metals and toxicity. |
| Cultivation & Evaluation Period | Days | 18 Days | In an open raceway pond under climatic conditions. |
3.3 Microalgae Cultivation and Growth Kinetics in Oilfield Produced Water
The temporal profiles of microalgal biomass production within the oilfield produced water (OPW) matrix are depicted in Fig. 1. Microalgae growth dynamics are intricately governed by a synergistic interplay of nutrient availability, light intensity, temperature, and initial inoculation density (Chokshi et al., 2020). Upon shifting the microalgal culture from the optimized, nutrient-rich BG11 medium to the raw OPW, the cells were subjected to a distinct shift in the nutrient profile, alongside chemical stressors such as high salinity and residual petroleum hydrocarbons.
Figure 1 shows the growth kinetics of microalgae in water produced from oil fields.
Consequently, a distinct lag phase spanning the first 3 days was observed, during which the microalgae underwent physiological acclimation and metabolic reprogramming to tolerate the complex toxicological matrix of the industrial effluent. Following this adaptation period, an accelerated growth phase occurred between days 4 and 8. The culture subsequently entered a robust exponential growth phase from day 8 to day 14, where biomass density culminated at its peak. , driven by the rapid bio-fixation of dissolved inorganic carbon.
Between days 14 and 17, the culture gradually transitioned into the stationary phase, reflecting an equilibrium where the rate of cellular replication matched that of mortality, typically induced by nutrient depletion and self-shading effects. From the 18th day onward, a discernible decline in biomass was recorded, signaling the onset of the death phase.
Extensive literature has investigated the feasibility of utilizing Chlorella and Scenedesmus species for the bioremediation and valorization of oilfield produced water (Al-Ketife et al., 2019; Tan et al., 2022). Our findings corroborate the paradigm that utilizing (OPW) which inherently contains functional thresholds of nitrogen and phosphorus compounds presents a highly viable techno-economic strategy. This configuration significantly minimizes the overall cost of microalgae-based biorefineries by eliminating the reliance on expensive external synthetic nutrients, while simultaneously offering an advanced circular treatment pathway for complex oilfield effluents.
3.4 Removal of Organic Load and Petroleum Hydrocarbons
The experimental configuration demonstrated an outstanding capability for remediating highly recalcitrant petrochemical effluents under realistic environmental conditions. Within the 18-day cultivation period, the system achieved a Chemical Oxygen Demand (COD) removal efficiency of 85.50%, accompanied by an 89.40% reduction in Total Petroleum Hydrocarbons (TPH). This simultaneous degradation highlights the robust metabolic plasticity and enzymatic machinery of the biological system in breaking down complex, long-chain carbonaceous components and polycyclic aromatic hydrocarbons (PAHs) typically inherent in raw oilfield produced water.
These findings align closely with contemporary studies by Usha et al. (2022) and Praveen et al. (2024), who reported that specialized microalgae strains exhibit remarkable physiological tolerance to hyper-saline and hydrocarbon-rich matrices, utilizing emulsified organic fractions as supplementary carbon sources via mixotrophic metabolic pathways. Furthermore, the substantial TPH reduction achieved in this study corroborates a multi-step remediation mechanism: initial rapid bio-sorption onto the functional groups of the microalgal extracellular polymeric substances (EPS), followed by progressive intracellular accumulation and subsequent enzymatic biodegradation.
3.5. Nutrient Assimilation Pathways (Nitrogen and Phosphorus)
A striking feature of the current configuration is the near-complete exhaustion of macronutrients within the hyper-saline industrial matrix, with removal efficiencies reaching 100.00% for Ammonium-Nitrogen N-NH+4, 97.65% for Total Nitrogen (TN), and 98.91% for Total Phosphorus (TP). The preferential and absolute assimilation of N-NH4+ is a well-documented bio-kinetic phenomenon in microalgal systems; ammonium serves as the most energetically favorable nitrogen species, as it is directly incorporated into cellular transamination pathways without requiring the energy-intensive enzymatic reduction steps associated with nitrate or nitrite assimilation. According to Wang et al. (2021) and Kshirsagar et al. (2023), such superior nutrient recovery rates from hyper-saline oilfield produced water are driven by highly efficient assimilatory metabolic pathways, where dissolved nitrogen and phosphorus are actively transported across the cell membrane and incorporated into structural proteins, nucleic acids, and cellular energy shuttles (ATP). Furthermore, this rapid consumption under arid, open-pond conditions underscores a critical operational synergy: while high evaporation rates are typical in such climates, they did not hinder biological uptake. Instead, elevated solar irradiance and optimal ambient temperatures actively accelerated photosynthetic phosphorylation, thereby driving hyper-accumulation and rapid nutrient fixation processes despite the complex chemical background of the petroleum effluent
3.6. Heavy Metals Bioremediation and Toxicity Resilience
Industrial produced water is notoriously characterized by inhibitory concentrations of heavy metals. In this study, despite the initial toxic matrix, significant removal efficiencies were recorded for Lead (Pb: 72.80%), Copper (Cu: 73.39%), Iron (Fe: 76.74%), and to a lesser extent, Cadmium (Cd: 44.44%). The reduction profiles (e.g., Pb dropping to 0.01 ppm) demonstrate effective polishing of the effluent.
Figure 2: This represents the efficiency of microalgae in the adsorption of heavy metals.
The lower removal efficiency of Cd2+ compared to Pb2+ and Cu2+ can be attributed to competitive binding kinetics on the biomass surface. As discussed by Chojnacka (2010) and Zeraatkar et al. (2016), microalgal cell walls possess functional groups (such as carboxyl, hydroxyl, and amine groups) that exhibit varying binding affinities ordered by the covalent index and ionic radius of the cations (Pb2+>Cu2+>Cd2+). The lower uptake of Cadmium is a classic manifestation of competitive bio-sorption in multi-metal real-world matrices.
3.7. Biomass Yield and System Scalability under Arid Climatic Conditions
Remarkably, despite the presence of heavy metals and high salinity inherent to petroleum produced water, the system yielded a maximum biomass production of 1.6±0.1 g L−1 over an 18-day cultivation and acclimatization period. Producing over 1.6 g L−1 in a 50-liter open pond under real dry climatic conditions represents a highly competitive outcome for large-scale outdoor applications.
This sustained growth profile demonstrates that the microalgal culture successfully established a robust stress-tolerance mechanism. This resilience is likely driven by the upregulation of endogenous antioxidant enzymes and the synthesis of phytochelatins, which effectively mitigate heavy-metal-induced oxidative stress (Kumar, A et al., 2023; Ribeiro, C., et al., 2023). From a techno-economic perspective, achieving such high biomass density within an authentic industrial effluent matrix highlights the dual-benefit paradigm of this configuration: it simultaneously yields a low-cost, sustainable feedstock for biorefineries (e.g., biofuels and bioproducts) while driving advanced, circular-economy-driven wastewater remediation.
3.8. Characteristics of microalgae oils
The oil was characterized using infrared spectroscopy, and the extracted oil was analyzed using the Soxhlet method.
Figure 3: Characterization by IR spectrophotometry (Soxhlet method)
The FTIR spectral profile of the microalgal oil obtained via the conventional Soxhlet extraction method reveals a series of prominent, well-defined vibrational bands, confirming the successful isolation and high structural purity of the lipid fraction (Figure 3). The highly intense stretching vibrations observed in the region correspond directly to the asymmetric and symmetric stretching of aliphatic bonds in and groups, a definitive molecular signature of the long-chain fatty acids constituting the acylglycerol architecture.
Crucially, the sharp and highly intense absorption peak centered at is uniquely assigned to the stretching vibration of ester carbonyl groups. The sharpness and precise positioning of this peak align perfectly with established literature for high-purity microbial lipids. According to Mehariya et al. (2021), the preservation of this specific band confirms that the structural integrity of the fatty acid backbones remains completely intact throughout the thermal cycling of the Soxhlet process. Furthermore, the prominence of this peak indicates the absence of significant hydrolytic degradation into free fatty acids (FFAs), which would otherwise shift or distort the carbonyl footprint.
In the fingerprint region, the stretching vibrations of bonds appearing within the range (specifically around ) further substantiate the ester architecture of the isolated triacylglycerols (TAGs). As demonstrated by Prabakaran and Ravindran (2012), a well-defined ester profile combined with the absolute absence of broad hydroxyl bands ( stretching around ) verifies the high efficiency of the extraction method in recovering intact lipids without co-extracting unwanted polar contaminants, moisture, or causing severe thermal degradation.
4. Conclusion
This study successfully demonstrates the viability of cultivating Chlorella vulgaris in open raceway ponds using raw oilfield produced water under the arid climatic conditions of Southern Algeria. The microalgal system exhibited exceptional bioremediation performance, achieving near-complete macronutrient exhaustion 100% N-NH4+, 97.65% TN, and 98.91% TP) along with substantial reductions in organic and hydrocarbon loads 85.50% COD and 89.40% TPH). Despite the inherent toxicity and hyper-salinity of the petrochemical effluent, the culture established robust stress-tolerance mechanisms, stabilizing a high maximum biomass productivity of (1.6 ± 0.1)g L-1. Furthermore, downstream processing via Soxhlet extraction yielded a remarkable lipid content of 38%, successfully validating the harvested biomass as a high-potential feedstock for biofuel production. Ultimately, these findings confirm that coupling Chlorella vulgaris cultivation with oilfield produced water treatment in desert environments offers a highly effective, circular pathway for advanced industrial wastewater remediation and sustainable bioenergy generation.
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