10 © 2025 The Author(s). Published by College of Education for Pure Science (Ibn Al-Haitham), University of Baghdad. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License Biological Treatment of Crude Oil by Synechococcus sp. Marwa Saad Al-Khazraji1* , Altaf Abed Alwahed Al-Rawi2 and Abdul Hameed M. Jawad Al-Obaidy3 1,2Department of Biology, College of Science for Women, University of Baghdad, Baghdad, Iraq. 3Department of Civil Engineering, University of Technology. Baghdad, Iraq. *Corresponding Author. Received: 31 May 2023 Accepted: 7 August 2023 Published: 20 April 2025 doi.org/10.30526/38.2.3489 Abstract Crude oil is known globally as one of the major causes of environmental pollution. This chemical compound exerts exhausting impacts on cultivable lands and water surfaces, leading to profound damage to agriculture and aquatic life. Over the years, several bioremediation approaches have evolved to manage this pollution problem properly. A phylum of microorganisms known as cyanobacteria offers an efficient alternative to established bioremediation methods. We conducted the present study to investigate the effectiveness of cyanobacteria in eliminating residual total petroleum hydrocarbon (TPH). Synechococcus algae treated three concentrations of crude oil (0.6, 1.8, and 3 ppm) over four time periods (on days 3, 6, 9, and 12). The gas chromatography test showed that on days 3, 6, 9, and 12 of treatment, the percentages of petroleum hydrocarbon removal were 34.66, 65.47, 83.98, and 93.12%, respectively, at a concentration of 0.6 ppm of crude oil. We recorded removal rates of 29.44, 57.42, 80.60, and 90.41% for 1.8 ppm of crude oil, and 30.67, 51.52, 76.38, and 90.74% for 3 ppm on days 3, 6, 9, and 12, respectively. Ultimately, we discovered that cyanobacteria (Synechococcus sp.) are effective biological pollutant removers, effectively eliminating hydrocarbon compounds from the water. Keywords: Crude oil, Cyanobacteria, Synechococcus, Bioremediation. 1. Introduction Crude oil has remained the world's primary energy source over the past half century (1). Crude oil serves as the primary component of petroleum, alongside natural gas. Hydrocarbons, which are chemical compounds composed of hydrogen and carbon in a 2:1 ratio with varying molecular mass values, form the overall backbone of crude oil, along with various amounts of nitrogen, oxygen, sulfur, phosphorus, and heavy metals (2, 3). Accidental or anthropogenic exposure to hydrocarbons in aquatic or soil environments is one of their most serious pollution sources (4). The cross-oceanic annual transport of approximately 35 million barrels of oil is one https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ https://orcid.org/0000-0001-8463-1763 mailto:marwa.saad1202a@csw.uobaghdad.edu.iq https://orcid.org/0000-0003-3293-5508 mailto:altafaw-bio@csw.uobaghdad.edu.iq https://orcid.org/0000-0002-8024-3672 mailto:abdulhameed.m.alobaidy@uotechnology.edu.iq IHJPAS. 2025, 38(2) 11 example of the vulnerability of aquatic environments to pollution due to oil spills, leading to serious threats to forms of life in these and other types of water bodies (5). Since food webs and chains bio magnify toxic compounds and elements, large-scale oil spills cause severe damage to biological systems (6, 7). Researchers have developed mechanical and physicochemical solutions to oil contamination issues, which involve burying, evaporating, dispersing, or washing out contaminants. Nevertheless, such solutions have the disadvantages of high costs and insufficient elimination of contaminants. Bioremediation is the technology of the biological breakdown of pollutants using microorganisms. Bioremediation of ecosystems that \are finely engineered and done in situ is thought to be the best way to treat hydrocarbons because it releases non-toxic materials. It is also less costly and more environmentally friendly than classical detoxification systems (8). Over time, researchers have studied and utilized various fungal and bacterial species in this context. Still, microalgae and cyanobacteria are the best because they are very flexible and can grow in a number of different ways, including autotrophic, heterotrophic, and mixotrophic (9, 10). Cyanobacteria are Gram-negative bacteria capable of performing oxygenic photosynthesis (11). Following the Gulf War in Kuwait, researchers noticed a close association between a massive crude oil spill and a cyanobacteria bloom. The intensive growth of this bloom was considered an early marker of a self-cleaning mechanism along the vast area of oil pollution. Such an observation provided supportive evidence for the ability of cyanobacteria to bioremediate chemical compounds in large crude oil spills (12). By using different types of cyanobacteria (13-15), more research showed how crude oil and other complex organic compounds (like surfactants) can be broken down. Among these, Lyngbya, Oscillatoriasalina, Plectonematerebrans, Aphanocapsa sp., and Synechococcus sp. have demonstrated their ability to develop mats in aquatic environments, which has led to their successful utilization in the degradation of oil spills in various regions globally (16, 17). Naturally occurring associations of cyanobacteria with other bacteria can achieve remediation of oil-polluted water bodies and soils (18). Therefore, we conducted this study to explore the potential of the cyanobacterium Synechococcus sp. variables in the biodegradation of crude oil at various concentrations of petroleum hydrocarbons, and to assess the impact of oil on their growth. 2. Materials and Methods 2.1. Algae sampling and growth rate evaluation Cultures of Synechococcus sp were identified and collected from the Advanced Environmental Laboratory at the College of Science for Women, University of Baghdad, Baghdad, Iraq. BG-11 culture medium, the constituents of which were described elsewhere (19) , was the specific growth culture utilized. The growth of the isolate was achieved at 25 ±2˚C and in the cooled incubator at a constant illumination intensity of 250-268 𝜇E m– 2 with a 16/ 8 light-dark cycle (20). The impact of pH on growth was examined at a pH of 6,5-8 (21). The cyanobacterial suspension was prepared, and spectrophotometric measurement of the optical density of the cyanobacterium suspension was achieved at an absorbance of 750 nm, where the BG11 culture medium served as a blank. 2.2. Cyanobacteria cultivation with crude oil Iraqi medium crude oil was collected from the Al-Dora Refinery in Baghdad for the experiment. Table 1 shows crude oil's physical-chemical properties. To determine the ability of IHJPAS. 2025, 38(2) 12 Synechococcus sp to remove hydrocarbons from the aquatic environment, three different concentrations of crude oil were chosen (0.6, 1.8, and 3.0 ppm) (22). Suitable amounts of crude oil were mixed in 250 mL Erlenmeyer flasks with 100 mL BG11 medium. The cyanobacterial culture was inoculated into three different flasks with the respective concentrations of crude oil, followed by incubation (25 ±2˚C) with shaking at 150 rpm. After adding Synechococcus sp. separately, the analysis was conducted gradually (at 3, 6, 9, and 12 days) to determine the concentrations of the TPH compound. Table 1. Physical-chemical characteristics of crude oil used in the experiment (23). Properties and component Value The density of crude oil 0.84 Specific Gravity at 60/60 F 0.886 API 31.4 Water content, Vol% 0.025 Water &Sediment, vol % Trace Salt content, Ib/1000brl 57.3 Asphaltene content, wt % 2.1 Sulpher content, wt % 2.9 H2S Dissolved, ppm 14.1 Wax content, wt% 3.8 Carbon Residue, wt% 6.2 Pour point, c Below -25 The heat of combustion, cal/g 10800 RVP@100F, psi 9.4 Kinematic Viscosity. Cst @100F @140F 8.588 5.548 Flashpoint c Flammable Vanadium ppm 30.75 Nickel ppm 6.5 2.3. Analysis of Petroleum Hydrocarbons The analysis was carried out in the Ministry of Science and Technology laboratories to detect and identify total petroleum hydrocarbon (TPH) compounds using gas chromatography (GC, Shimadzu 2010, Japan). A volume of 1 µL was injected with a known concentration of a mixture of standard compounds to determine each standard compound's retention time and area, as shown in Figure 1. A separating flask (1000 ml) was used to mix 500 ml of the samples with 50 ml of Dichloromethan (DCM), followed by shaking and regular pressure release. After letting the sample stand for several minutes, two layers were recognized. The lower layer, i.e., the extract, was filtered with filter paper and kept in a baker. The filtrate was evaporated at room temperature to achieve a concentration of 1 ml(24), utilizing the gas chromatography protocols. The temperature values of the injector and detector Flame Ionization Detector (FID), respectively, were 280 and 330 °C, while the column (KB-5) oven program temperature was 100–300 °C (10 °C/ min). The carrier gas was N2 at 120 Kpa. IHJPAS. 2025, 38(2) 13 Figure 1. Chromatographic chart of standard TPH by GC 3. Results After determining the retention time and area for each standard compound, we diluted 1L of crude oil to determine the concentration of each petroleum compound, as illustrated in Figure 1. Table 2 and Figures 2, 3 and 4 illustrate the concentrations of hydrocarbon compounds prepared after low dilution, along with their retention time values, in the crude oil prior to the addition of algae and before the biological treatment process. Table 2. Concentrations of petroleum hydrocarbon compounds detected in each of the three crude oil concentrations before adding algae. Crude oil concentrations (ppm) No Name Rt 0.6 1.8 3 1 Hexane 3.1 86.5 182.6 279.8 2 Heptane 3.7 90.8 192.5 416.8 3 Octane 4.5 105.8 215.9 396.5 4 Nonane 5.1 99.8 114.5 208.9 5 Decane 5.4 70.8 159.8 366.9 6 Undecane 6.4 80.9 167.4 365.8 7 Dodecane 6.9 136.5 274.5 625.4 8 Tetradecane 7.4 90.8 190.6 386.5 9 Hexadecane 8.4 88.9 186.5 360.2 10 Octadecane 9.6 105.8 219.8 405.8 11 Eicosane 10.6 114.5 235.9 425.9 12 Tetracosane 11.7 125.9 260.5 601.5 13 Tetratrtracontane 12.2 108.9 215.8 436.5 14 Dotriacontane 12.6 658.5 1356.5 2546.2 15 Hexatriacontane 13.5 854.6 1895.6 3569.5 16 Tetracontane 14.3 896.8 2145.6 3652.0 IHJPAS. 2025, 38(2) 14 Figure 2. Chromatographic chart of control 0.6 ppm of crude oil before adding algae by GC. Figure 3. Chromatographic chart of control 1.8 ppm of crude oil before adding algae by GC. IHJPAS. 2025, 38(2) 15 Figure 4. Chromatographic chart of control 3 ppm of crude oil before adding algae by GC. The results in Table 3 show the lowest, highest, and average concentrations of hydrocarbon compounds found during the analysis. The average concentrations of total hydrocarbons were 1858.910, 4251.395, and 8188.275 in the concentrations of 0.6, 1.8, and 3 ppm of crude oil, respectively. Table 3. Statistical description of TPH concentration in three different concentrations of crude oil (0.6, 1.8 and 3 ppm). Statistical description TPH Con. Ppm 0.6 1.8 3 Mean 1858.910 4251.395 8188.275 Std. Deviation 1294.808 2744.444 4947.670 Minimum 255.400 768.450 1392.450 Maximum 3715.800 8014.000 15044.200 The concentrations of TPH compound residues each day. Before and after treatment, we determined the cell density of the algal culture. The results demonstrated a decrease in the concentrations of TPH residues across the three prepared concentrations, as depicted in Figures 5 and 6, over the course of the experimental days. The tested cyanobacteria used these hydrocarbons as their sole carbon source, leading to this decrease, as evidenced by the algal culture's increased cell density after incubation. IHJPAS. 2025, 38(2) 16 Figure 5. Separation patterns of hydrocarbons using gas chromatography throughout the experimental days. Figure 6. The effects of Synechococcus sp. alga on the removal of TPH. 3.1. The removal efficiency of TPH in the concentration of 0.6 ppm of crude oil Table 4 shows the initial and resulting concentrations of hydrocarbon compounds in the concentration of 0.6 ppm of crude oil and the percentage of their removal by the algae Synechococcus sp. over different incubation times. It was found that some compounds, such as Dotriacontane and Hexatriacontane, had lower removal rates (87.79% and 89.44%, respectively). Other compounds such as Decane, Tetracontane, Undecane, Dodecane, Nonane, Hexane, Hexadecane, Heptanen, Tetradecane, Octane, Octadecane, Tetratrtracontane, Eicosane had higher removal rates (91.17%, 93.22%, 93.69%, 94.57%, 94.73%, 99.42%, 99.43%, 99.44%, 99.44%, 99.52%, 99.52%, 99.54%, and 99.56%, respectively). In comparison, the highest IHJPAS. 2025, 38(2) 17 removal rate was exerted against Tetracosane (99.60%). The results show that the total removal rate of the compounds on day 3 was 34.66 %, on day 6 was 65.37 %, and on day 9 was 83.88 %, while the highest removal rate was 93% on day 12 of treatment. Table 4. Removal rates of petroleum hydrocarbon compounds of the crude oil concentration of 0.6 ppm using Synechococcus sp. as detected through GC analysis. No Name Initial con 3day 6day 9day 12day *Total removal 1 Hexane 86.5 32.5 (62.42%) UDL (99.42%) UDL UDL (99.42%) 2 Heptane 90.8 50.6 (44.27%) UDL (99.44%) UDL UDL (99.44%) 3 Octane 105.8 62.8 (40.64%) 18.2 (82.79%) UDL (99.52%) UDL (99.52%) 4 Nonane 99.8 55.8 (44.08%) 20.5 (79.45%) 12.5 (87.47%) 5.25 (94.73%) (94.73%) 5 Decane 70.8 32.5 (54.09%) 16.5 (76.69%) 10.2 (85.59%) 6.25 (91.17%) (91.17%) 6 Undecane 80.9 39.8 (50.80%) 20.5 (74.66%) 12.6 (84.42%) 5.1 (93.69%) (93.69%) 7 Dodecane 136.5 90.8 (33.47%) 33.6 (75.38%) 13.6 (90.03%) 7.4 (94.57%) (94.57%) 8 Tetradecane 90.8 52.9 (41.74%) 22.5 (75.22%) 10.8 (88.10%) UDL (99.44%) (99.44%) 9 Hexadecane 88.9 33.9 (61.86%) UDL (99.43%) UDL UDL (99.43%) 10 Octadecane 105.8 62.5 (40.92%) UDL (99.52%) UDL UDL (99.52%) 11 Eicosane 114.5 71.5 (37.55%) UDL (99.56%) UDL UDL 99.56% 12 Tetracosane 125.9 86.5 (31.29%) UDL (99.60%) UDL UDL (99.60%) 13 Tetratrtracontan e 108.9 44.5 (59.13%) UDL (99.54%) UDL UDL (99.54%) 14 Dotriacontane 658.5 452.6 (31.26%) 214.5 (67.42%) 150.2 (77.19%) 80.4 (87.79%) (87.79%) 15 Hexatriacontane 854.6 635.9 (25.59%) 458.9 (46.30%) 174.5 (79.58%) 90.2 (89.44%) (89.44%) 16 Tetracontane 896.8 622.5 (30.58%) 477.8 (46.72%) 210.5 (76.52%) 60.8 (93.22%) (93.22%) **Total Removal % 34.66 % 65.37 % 83.88 % 93 % *. Total Removal %: Removal percentage of each hydrocarbon compound during experimental days. **. Total Removal %: Removal percentage of all hydrocarbon compounds during experimental days. 3.2. The removal efficiency of TPH in the concentration of 1.8 ppm of crude oil Table 5 displays the initial and resulting concentrations of hydrocarbon compounds in the crude oil at a concentration of 1.8 ppm, along with the percentage of these compounds removed by the algae Synechococcus sp. The results of the removal rate of each hydrocarbon compound on day 12 of treatment showed that some compounds, such as Tetracontane, Dotriacontane, and Hexatriacontane had the lowest values (88.11 %, 88.28 %, and 88.68 %, respectively). Other compounds such as Tetratrtracontane, Decane, Nonane, Eicosane, Tetracosane, Octadecane, IHJPAS. 2025, 38(2) 18 Undecane, Tetradecane, Dodecane, Hexane, Hexadecane, and Heptane showed rates of 90.50 %, 90.73 %,91.04 %, 91.30 %, 91.78 %, 93.08 %, 93.18 %, 93.44 %, 95.04 %, 99.72 %, 99.73 %, and 99.74 %, respectively. The highest removal rate was exerted by Octane (99.76 %). The results also show that the total removal rate of compounds on day 3 was 29.44 %, on day 6 was 57.42 %, and on day 9 was 80.58 %, whereas the highest removal rate was 90.38 % on day 12 of treatment. Table 5. Removal rates of petroleum hydrocarbon compounds of crude oil with a concentration of 1.8 ppm using Synechococcus sp., as detected through GC analysis. No Name Initial con 3day 6day 9day 12day *Total Removal 1 Hexane 182.6 85.6 (53.12%) 20.5 (88.77%) UDL (99.72%) UDL 99.72 % 2 Heptane 192.5 104.9 (45.50%) 55.8 (71.01%) UDL (99.74%) UDL 99.74 % 3 Octane 215.9 120.6 (44.14%) 60.8 (71.83%) UDL (99.76%) UDL 99.76 % 4 Nonane 114.5 92.5 (19.21%) 44.5 (61.13%) 22.5 (80.34%) 10.25 (91.04%) 91.04 % 5 Decane 159.8 96.5 (39.61%) 39.8 (75.09%) 21.5 (86.54%) 14.8 (90.73%) 90.73 % 6 Undecane 167.4 87.4 (47.78%) 50.4 (69.89%) 29.8 (82.19%) 11.4 (93.18%) 93.18 % 7 Dodecane 274.5 120.6 (56.06%) 74.5 (72.85%) 30.5 (88.88%) 13.6 (95.04%) 95.04 % 8 Tetradecane 190.6 110.5 (42.02%) 60.9 (68.04%) 22.8 (88.03%) 12.5 (93.44%) 93.44 % 9 Hexadecane 186.5 101.5 (45.57%) 55.8 (70.08%) UDL (99.73%) UDL 99.73 % 10 Octadecane 219.8 123.6 (43.76%) 96.5 (56.09%) 30.5 (86.12%) 15.2 (93.08%) 93.08 % 11 Eicosane 235.9 152.9 (35.18%) 97.4 (58.71%) 50.6 (78.55%) 20.5 (91.30%) 91.30 % 12 Tetracosane 260.5 196.5 (24.56%) 135.6 (47.94%) 50.9 (80.46%) 21.4 (91.78%) 91.78 % 13 Tetratrtracontane 215.8 185.4 (14.08%) 112.5 (47.86%) 60.8 (71.82%) 20.5 (90.50%) 90.50 % 14 Dotriacontane 1356.5 956.2 (29.50%) 680.5 (49.83%) 360.5 (73.42%) 158.9 (88.28%) 88.28 % 15 Hexatriacontane 1895.6 1262.5 (33.39%) 956.8 (49.52%) 368.5 (49.52%) 214.5 (88.68%) 88.68 % 16 Tetracontane 2145.6 1856.9 (13.45%) 869.8 (59.46%) 505.1 (76.45%) 254.9 (88.11%) 88.11 % 17 **Total Removal% 29.44 % 57.42 % 80.58 % 90.38 % *. Total Removal %: Removal percentage of each hydrocarbon compound during experimental days. **. Total Removal %: Removal percentage of all hydrocarbon compounds during experimental days. 3.3. The removal efficiency of TPH in the concentration of 3 ppm of crude oil Table 6 shows the initial and resulting concentrations of hydrocarbon compounds in the concentration of 3 ppm of crude oil and the percentage of their removal by the algae Synechococcus sp. After 12 days of the experiment, it was found that Tetratrtracontane had the lowest value (79.35%). Other compounds such as Tetracosane, Hexatriacontane, Dotriacontane, IHJPAS. 2025, 38(2) 19 Tetradecane, Octadecane, Nonane had values of 84.95 %, 85.39 %, 85.82 %, 88.58 %, 89.13 %, 89.22 %, respectively, while Hexadecane, Eicosane, Tetratrtracontane, Dodecane, Decane, Tetracontane, Undecane, Hexane, and Octane had values of 91.53 %, 91.57 %, 93.36 %, 94.38 %, 94.58, 98.06 %, %,99.82 %, 99.87 %, respectively. The highest removal rate was recorded at Heptane (99.88 %). The table shows that the total removal rate of compounds on day 3 was 30.67 %, on day 6 was 51.52 %, and on day 9 was 76.37 %, whereas the highest removal rate was 90.73 % on day 12 of incubation. Table 6. Total removal rates of petroleum hydrocarbon compounds of crude oil of concentration of 3 ppm using Synechococcus sp., as detected through GC analysis. NO Name Initial con 3 days 6 days 9 days 12 days *Total Removal 1 Hexane 279.8 155.9 (44.28%) 96.8 (65.40%) UDL (99.82%) UDL 99.82 % 2 Heptane 416.8 259.8 (37.66%) 174.5 (58.13%) UDL (99.88%) UDL 99.88 % 3 Octane 396.5 284.5 (28.24%) 168.9 (57.40%) UDL (99.87%) UDL 99.87 % 4 Nonane 208.9 164.8 (21.11%) 96.8 (53.66%) 48.9 (76.59%) 22.5 (89.22%) 89.22 % 5 Decane 366.9 145.8 (60.26%) 99.8 (72.79%) 44.5 (87.87%) 20.6 (94.38%) 94.38 % 6 Undecane 365.8 235.9 (35.51%) 130.8 (64.24%) 40.5 (88.92%) 19.8 (94.58%) 94.58 % 7 Dodecane 625.4 425.8 (31.91%) 208.9 (66.59%) 90.8 (85.48%) 41.5 (93.36%) 93.36 % 8 Tetradecane 386.5 152.6 (60.51%) 105.9 (72.60%) 81.4 (78.93%) 44.1 (88.58%) 88.58 % 9 Hexadecane 360.2 195.8 (45.64%) 126.5 (64.88%) 50.2 (86.06%) 30.5 (91.53%) 91.53 % 10 Octadecane 405.8 245.9 (39.40%) 186.8 (53.96%) 69.8 (82.79%) 44.1 (89.13%) 89.13 % 11 Eicosane 425.9 269.8 (36.65%) 166.2 (60.97%) 77.8 (81.73%) 35.9 (91.57%) 91.57 % 12 Tetracosane 601.5 358.9 (40.33%) 324.8 (46.001% 153.6 (74.46%) 90.5 (84.95%) 84.95 % 13 Tetratrtracontane 436.5 320.5 (26.57%) 198.7 (54.47%) 144.5 (66.89%) 90.1 (79.35%) 79.35 % 14 Dotriacontane 2546.2 1985.6 (22.01%) 1365.8 (46.35%) 895.5 (64.82%) 360.8 (85.82%) 85.82 % 15 Hexatriacontane 3569.5 2658.9 (25.51%) 1856.5 (47.98%) 952.2 (73.32%) 521.4 (85.39%) 85.39 % 16 Tetracontane 3652.0 2568.6 (29.66%) 1985.2 (45.64%) 902.8 (75.27%) 70.65 (98.06%) 98.06 % 17 **Total Removal% 30.67 % 51.52 % 76.37 % 90.73 % *. Total Removal %: Removal percentage of each hydrocarbon compound during experimental days. **. Total Removal %: Removal percentage of all hydrocarbon compounds during experimental days. 4. Discussion Bioremediation using cyanobacteria is characterized by sustainability and being friendly to the environment, which is widely favorable in the middle of the global trend toward cleaner energy sources. The utilization of fungal and bacterial species in the biological treatment of IHJPAS. 2025, 38(2) 20 hydrocarbons has been an attractive research topic for several decades. But better benefits, like being able to treat a wider range of hydrocarbons and releasing fewer greenhouse gases, have made cyanobacteria, in which blue-green algae power the remediation system, more popular. Cyanobacteria are better at collecting oil and making it available to the environment more quickly than other bacteria, and they don't need as much food to grow (25, 26). In the present study, we utilized a local isolate of Synechococcus sp., which showed a good ability for petroleum hydrocarbon degradation, which could be due to the activities of certain enzymes produced by these microorganisms (27, 28). The results revealed that Synechococcus sp. provided 99% of total petroleum hydrocarbon degradation within twelve days, indicating its potential advantages as a bioremediating agent. Although not fully explored and exploited, this approach of bioremediation has the potential to go in harmony with existing green energy practices, particularly in the context of growing global concerns related to climate change and the continuous search for green energy options (29). 5. Conclusion This investigation revealed that the utilized cyanobacterial species could be an excellent bioremediation agent that can be utilized to clean environments contaminated with crude oil. High rates of degradation of various hydrocarbons were recorded. The availability, sustainability, and environmentally-friendly features of this treatment source, combined with the efficient enzymatic mechanism of hydrocarbon degradation, make it an excellent tool for achieving global goals of sustainable development. Acknowledgment Many thanks to the Department of Biology at the College of Science for Women, University of Baghdad, for their invaluable assistance in facilitating the practical sections of this article. Conflict of Interest The authors declare that they have no conflicts of interest. Funding No funding. References 1. Zhang HY, Ji Q, Fan Y. What drives the formation of global oil trade patterns. Energy Econ. 2015;49:639–648. https://doi.org/10.1016/j.eneco.2015.03.004. 2. Okafor UC, Orji MU, Agu KC, Awah NS, Okeke BC, Okafor OI, Okoro NCN. Bioremediation of crude oil-polluted soil using broiler-chicken droppings. J Appl Environ Microbiol. 2016;4(4):75-84. https://doi.org/10.18502/jaem.v4i4.597. 3. Anaukwu CG, Ezemba CC, Anakwenze VN, Agu KC, Okeke BC, Awah NS, Ekwealor IA. Effect of biosurfactant produced by Citrobacter murliniae AF025369 and a synthetic surfactant on degradation of crude oil. Edorium J Microbiol. 2016;2:1–6. https://doi.org/10.1007/s10095-016- 0168-9. 4. Holliger C, Gaspard S, Gold G, Heijman C, Schumacher W, Schwarzenbach RP, Vazquez F. Contaminated environments in the subsurface and bioremediation: organic contaminants. FEMS Microbiol Rev. 1997;20(3-4):517–523. https://doi.org/10.1016/S0168-6445(97)00019-6. https://doi.org/10.1016/j.eneco.2015.03.004 https://doi.org/10.18502/jaem.v4i4.597 https://doi.org/10.1007/s10095-016-0168-9 https://doi.org/10.1007/s10095-016-0168-9 https://doi.org/10.1016/S0168-6445(97)00019-6 IHJPAS. 2025, 38(2) 21 5. Macaulay B. Understanding the behavior of oil-degrading microorganisms to enhance the microbial remediation of spilled petroleum. Appl Ecol Environ Res. 2015;13(1):247–262. https://doi.org/10.15666/aeer/1301_247262. 6. Dillard LA, Essaid HI, Herkelrath WN. Multiphase flow modeling of a crude-oil spill site with a bimodal permeability distribution. Water Resour Res. 1997;33(7):1617-1632. https://doi.org/10.1029/97WR00912. 7. Head IM, Jones DM, Röling WF. Marine microorganisms make a meal of oil. Nat Rev Microbiol. 2006;4(3):173–182. https://doi.org/10.1038/nrmicro1348. 8. Mansur AA, Adetutu EM, Makadia T, Morrison PD, Ball AS. Assessment of the hydrocarbon degrading abilities of three Bioaugmentation agents for the bioremediation of crude oil tank bottom sludge contaminated Libyan soil. Int J Environ Bioremediation Biodegradation. 2015;3(1):1-9. https://doi.org/10.12691/ijebb-3-1-1. 9. Neilson A, Lewin R. The uptake and utilization of organic carbon by algae: an essay in comparative biochemistry. Phycologia. 1974;13(3):227–264. https://doi.org/10.2216/i0031-8884-13-3-227.1. 10. Subashchandrabose SR, Ramakrishnan B, Megharaj M, Venkateswarlu K, Naidu R. Consortia of cyanobacteria/microalgae and bacteria: biotechnological potential. Biotechnol Adv. 2011;29(6):896– 907. https://doi.org/10.1016/j.biotechadv.2011.07.007. 11. Waterbury JB, Stanier RY. Patterns of growth and development in pleurocapsalean cyanobacteria. Microbiol Rev. 1978;42(1):2. https://doi.org/10.1128/MMBR.42.1.2-24.1978. 12. Sorkhoh N, Al-Hasan R, Radwan S. Self-cleaning of the Gulf. Nature. 1992;359:109-115. https://doi.org/10.1038/359109a0. 13. Hamouda RAEF, Sorour NM, Yeheia DS. Biodegradation of crude oil by Anabaena oryzae, Chlorella kessleri and its consortium under mixotrophic conditions. Int Biodeterior Biodegrad. 2016;112:128–134. https://doi.org/10.1016/j.ibiod.2016.05.004. 14. Raghukumar C, Vipparty V, David J, Chandramohan D. Degradation of crude oil by marine cyanobacteria. Appl Microbiol Biotechnol. 2001;57(3):433–436. https://doi.org/10.1007/s002530100738. 15. Mansy AER, El-Bestawy E. Toxicity and biodegradation of fluometuron by selected cyanobacterial species. World J Microbiol Biotechnol. 2002;18(2):125–131. https://doi.org/10.1023/A:1013798424097. 16. Raghukumar C, Vipparty V, David J, Chandramohan D. Degradation of crude oil by marine cyanobacteria. Appl Microbiol Biotechnol. 2001;57(3):433-6. https://doi.org/10.1007/s002530100800. 17. Cohen Y. Bioremediation of oil by marine microbial mats. Int Microbiol. 2002;5(4):189-93. https://doi.org/10.1007/s10123-002-0089-5. 18. Sorkhoh N, Al‐Hasan R, Khanafer M, Radwan S. Establishment of oil‐degrading bacteria associated with cyanobacteria in oil‐polluted soil. J Appl Microbiol. 1995;78(2):194-9. https://doi.org/10.1111/j.1365-2672.1995.tb02840.x. 19. Stanier RY, Kunisawa R, Mandel M, Cohen-Bazire G. Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol Rev. 1971;35:171-205. https://doi.org/10.1128/br.35.2.171-205.1971. 20. Thompson P, Guo G, Harrison P. Effects of variation in temperature on the biochemical composition of eight species of phytoplankton. J Phycol. 1992;28:481-8. https://doi.org/10.1111/j.0022- 3646.1992.00481.x. 21. Jusu A, Kong X, Qing B, Tan J, Han B. Time course biochemical responses of green algae Scenedesmus obliqus to Aluminium and low pH. Bull Environ Contam Toxicol. 2004; 73: 1001– 1008. https://doi.org/10.1007/s00128-004-0525-7. 22. Nweze NO, Aniebonam C. Bioremediation of petroleum products impacted freshwater using locally available algae. Bioresearch. 2009;7(1):484-90. http://dx.doi.org/10.4314/br.v7i1.45477. 23. Al-Dora Refinery in Baghdad. Specification of crude oil. Baghdad; 2022. https://doi.org/10.15666/aeer/1301_247262 https://doi.org/10.1029/97WR00912 https://doi.org/10.1038/nrmicro1348 https://doi.org/10.12691/ijebb-3-1-1 https://doi.org/10.2216/i0031-8884-13-3-227.1 https://doi.org/10.1016/j.biotechadv.2011.07.007 https://doi.org/10.1128/MMBR.42.1.2-24.1978 https://doi.org/10.1038/359109a0 https://doi.org/10.1016/j.ibiod.2016.05.004 https://doi.org/10.1007/s002530100738 https://doi.org/10.1023/A:1013798424097 https://doi.org/10.1007/s002530100800 https://doi.org/10.1007/s10123-002-0089-5 https://doi.org/10.1111/j.1365-2672.1995.tb02840.x https://doi.org/10.1128/br.35.2.171-205.1971 https://doi.org/10.1111/j.0022-3646.1992.00481.x https://doi.org/10.1111/j.0022-3646.1992.00481.x https://doi.org/10.1007/s00128-004-0525-7 http://dx.doi.org/10.4314/br.v7i1.45477 IHJPAS. 2025, 38(2) 22 24. Alinnor IJ, Nwachukwu MA. Determination of total petroleum hydrocarbon in soil and groundwater samples in some communities in Rivers State, Nigeria. Acad J. 2013;5(11):292-7. http://dx.doi.org/10.5897/JECE2013.0298. 25. Singh JS, Kumar A, Singh M. Cyanobacteria: a sustainable and commercial bio-resource in production of bio-fertilizer and bio-fuel from waste waters. Environ Sustain Indic. 2019;3:100008. https://doi.org/10.1016/j.indic.2019.100008. 26. Nawaz T, Saud S, Gu L, Khan I, Fahad S, Zhou R. Cyanobacteria: Harnessing the power of microorganisms for plant growth promotion, stress alleviation, and phytoremediation in the era of sustainable agriculture. Plant Stress. 2024;11:100399. https://doi.org/10.1016/j.stress.2024.100399 27. Das N, Das A, Das S, Bhatawadekar V, Pandey P, Choure K. Petroleum hydrocarbon catabolic pathways as targets for metabolic engineering strategies for enhanced bioremediation of crude-oil- contaminated environments. Fermentation. 2023;9(2):196. https://doi.org/10.3390/fermentation9020196. 28. Hamouda RA, Alhumairi AM, Saddiq AA. Simultaneous bioremediation of petroleum hydrocarbons and production of biofuels by the micro-green alga, cyanobacteria, and its consortium. Heliyon. 2023;9(6). https://doi.org/10.1016/j.heliyon.2023.e19143. 29. Hou D, O’Connor D, Igalavithana AD, Alessi DS, Luo J, Tsang DC. Metal contamination and bioremediation of agricultural soils for food safety and sustainability. Nat Rev Earth Environ. 2020;1(7):366-81. https://doi.org/10.1038/s43017-020-0070-x. http://dx.doi.org/10.5897/JECE2013.0298 https://doi.org/10.1016/j.indic.2019.100008 https://doi.org/10.1016/j.stress.2024.100399 https://doi.org/10.3390/fermentation9020196 https://doi.org/10.1016/j.heliyon.2023.e19143 https://doi.org/10.1038/s43017-020-0070-x