Sampling location and collection
The study location is Dhiba port (27° 34' N to 34° 33' E), located at the north-western corner of the Kingdom of Saudi Arabia. It is the nearest Saudi port to the Suez Canal and the Mediterranean basin countries ports, including Turkey 593 miles, Greece 491 miles, and 988 miles to the nearest French ports (www.mawani.gov.sa). Thus, it acquires unique importance in its strategic location near the NEOM project, which is the Saudi Crown Prince Mohammed Bin Salman's vision and a centerpiece of Saudi Arabia 2030 Vision (www.neom.com). The registration of vessel arrivals from various ports worldwide showed that a number of 12029 vessels had navigated the port during the period 2005-2019 (Table 1).
Water samples were collected from the water surface on 25th January 2020 from five different Dhiba port locations (Figure 1). For heavy metals and hydrocarbons determination, samples were stored in the dark at a low temperature of 4°C until examination.
Isolation and identification of Chlorella vulgaris
The green microalga Chlorella vulgaris was isolated from water samples collected from Thuwal beach, Red Sea, Saudi Arabia (22°16'35.0"N 39°05'22.3"E). The isolation was done through a serial dilution technique followed by plating on modified BG-11 medium (Rippka, 1998; Stanier et al., 1971). The alga was based on AlgaeBase (Guiry & Guiry, 2021), and (Bellinger & Sigee, 2010; Stevenson & Lowe, 1986).
Preparation of immobilized microalga in alginate beads
For each flask, 30 ml of algal suspension in its exponential growth phase were harvested by centrifugation at 3000 rpm for 10 min. The supernatant was then decanted, and the volume of sediment was adjusted to 2 ml with sterilized deionized water. After that, the concentrated algal suspension was mixed with 2% (w/v) sodium alginate solution and dropped into a 2% calcium chloride solution using a sterilized burette. Beads were left to harden overnight then rinsed with distilled water.
Estimation of Growth
Optical density
For microalga growth and pigments measurement, alginate beads should be dissolved in 100 ml of 0.1 M sodium citrate solution with pH 5 that was prepared by adding 10 ml of sodium citrate to a specified number of beads at 45 ˚C with stirring, and the beads would dissolve within one hour. Then, the solution was centrifuged at 5000 rpm for 5 min. After that, the supernatant was decanted, and the volume was adjusted to 3 ml with sterilized water. Alga's biomass was determined every three days by measuring the algal suspension's optical density at 600 nm using a SHIMADZU UV-2600 spectrophotometer.
Pigments determination
A known volume of culture was centrifuged at a speed of 3000 rpm for 10 min. After that, the algal pellets were treated with a known volume of methanol, kept in the water bath for 30 min at 55 ˚C, and then centrifuged again. The absorbance of the pooled extracts was registered by SHIMADZU UV-2600 spectrophotometer at 666, 653, and 470 nm. Calculations were made according to the formulae devised by (Costache et al., 2012) for chlorophyll a, chlorophyll b, and carotenoids.
Experimental Design
Two treatments were conducted triple to study the potential of C. vulgaris in the bioremediation of heavy metals and the biodegradation of hydrocarbons. For each treatment, two Erlenmeyer flasks (250 ml) contained 150 ml of sterilized seawater were enriched with nitrogen and phosphate source (0.225 g of NaNO3 and 0.006 g of K2HPO4). Under a laminar flow cabinet, three flasks were cultivated with the algal beads, and the other three were cultivated with the residue of 30 ml of centrifuged algal cells of each flask. The cultures were incubated under the conditions of 12:12h light: dark and at 25 ˚C temperature and slight aeration for two weeks (Figure 2).
Chemical parameters analysis
Heavy metals
Laboratory analysis was carried out for heavy metals determination before and after the experiment. The Elements: Aluminum, Antimony, Arsenic, Barium, Beryllium, Cadmium, Chromium, Cobalt, Copper, Iron, Lead, Manganese, Molybdenum, Nickel, Selenium, Silver, Titanium, Vanadium, and Zinc were measured using ICP- OES (Inductively Coupled Plasma-Optical Emission Spectrometer) Agilent Technologies 720 ICP-OES Axial.
Determination of petroleum hydrocarbons
Petroleum hydrocarbons were extracted from 100 ml of seawater of each sample. The pH was adjusted with 1 M HCl to get pH < 3. Organic compounds were extracted via liquid-liquid phase extraction thrice, using 10 ml and 5 ml of dichloromethane (CH₂Cl₂). The organic lower phase was collected, and the moisture was removed by adding about 2g anhydrous sodium sulfate (Na2SO4). The clear extract was transferred to a test tube and evaporated with a gentle nitrogen gas stream at room temperature. The sample concentrated to about 10 μL (Suhrhoff & Scholz-Böttcher, 2016). The analysis was performed using a gas chromatograph (GCMS-QP2010 Plus, Shimadzu, Japan) equipped with a mass spectrometer with a fuse-silica capillary column (30 m × 0.25 mm ID × 0.25 µm ̶ Rtx®-1, Restek, USA) was used. Helium was used as a carrier gas, and the temperature programming was 60-300 ˚C, 1/5 min. GC-MS internal library search was used to identify the hydrocarbons. The analysis was conducted before and after the experiment.
Statistical Analysis
Experiments were conducted in triplicate and expressed as ± standard error of the mean. The data were compared by analysis of variance one-way and three-way ANOVA. Significance was determined using Duncan’s multiple range tests (p≤0.05). Analysis was carried out using MS Excel (2016) and SPSS (Version 16).