Bio-remediation of Most Contaminated Sites by Heavy Metals and Hydrocarbons In Dhiba Port Kingdom of Saudi Arabia Using Chlorella Vulgaris


 Dhiba port has a strategic location near the Neom project. Various anthropogenic activities contributed to the discharge of heavy metals and oil spill in the aquatic system and caused environmental pollution. Microalgae are the best microorganisms in aquatic conditions which known to be capable of eliminating contaminants. We investigate the heavy metals and hydrocarbons contaminations exhibited in five sites of port. Our aim is to determine the most contaminated sites then using Immobilized and fresh Chlorella vulgaris in bioremediation. The results indicated that Immobilized and fresh C. vulgaris have the capabilities of growing in contaminated seawater and were capable of removing heavy metals completely. Immobilized C. vulgaris is the most efficient in the degradation of hydrocarbons at site one. Overall, Immobilized C. vulgaris is the most effective in removing both heavy metals and hydrocarbon. It is an economic tool due to simplifying harvesting and then retaining for further processing.


Introduction
Since coastal and maritime tourism is a new vital economic activity and pioneer in advancing the economic diversity of the Kingdom of Saudi Arabia, beaches and coastal areas quality are based on the nearby areas' environmental quality, including ports. Ports activities of tourism and transportation, including ship discharges of ballast water, loading and unloading of cargo, and accidental discharge of oil and other chemicals in the sea have various environmental impacts that affect the extent of physiochemical and biological constituents run in the port water (Davis & MacKnight, 1990;Jahan & Strezov, 2017). These impacts are signi cant, ranging from heavy metal contamination, oil pollution, fecal pollution to the introduction of exotic species through ballast water uptake and discharge ( Luna et (Crawford & Crawford, 1996). The bioremediation of contaminants in the marine environment is carried out mainly by diverse microorganisms. Algae are low-cost sorbents for elimination of oil and can impact the fate and vehicle of spilled oil (Mishra & Mukherji, 2012). Algae have a pivotal role in self-removal of organic pollution in natural water (Şen et al., 1988). Many studies have depicted that algae eliminate nutrients such as nitrogen and phosphorus (Craggs et al., 1996), heavy metals (Tam et al., 2001), toxic hydrocarbon, inorganic toxins, and pesticides (Shashirekha et al., 1997) from enclosing water by adsorption and absorption (Kızılkaya et al., 2012;El-Naggar et al., 2018) of bioaccumulation abilities of the cells (Oswald, 1988). Several species of microalgae have shown an in uential role in the remediation of both heavy metals and hydrocarbons. Nweze and Aniebonam (2009) reported the probability of using naturally present algae isolated from a puddle near Nsukka Fire Service Station to remove hydrocarbon from water polluted with petroleum products. Microalga Chlorella kessleri could grow at different crude oil concentrations (0.5, 1, and 1.5%), mixotrophically solely and in combination with Anabaena oryzae . Wang et al. (2018) argued that the acclimation process is a potential method of wastewater treatment using Chlorella vulgaris. C. vulgaris showed high e ciency of biodegradation under a low concentration of 0.5% and 1% of crude oil. The growth reached a high level even with the 2% of crude oil in an experiment of 15 days (El-sheekh & Hamouda, 2013). C. vulgaris can be used for the biodegradation of crude and re ned oil in contaminated aquatic environments (Samuel et al., 2020). Ankistrodesmus braunii and Scenedesmus quadricauda were able to eliminate more than 70% of phenol from olive-oil mill wastewaters within ve days (Al-Dahhan et al., 2018). Hamouda et al.
(2016) reported that Scenedesmus obliquus was able to remove heavy metals Pb, Cd, Cu, and Mn, from wastewater under different conditions. Sharma and Khan (2013) noticed that Chlorella minutissima was a better e cient alga in removing heavy metals from polluted habitats than Scendesmus spp and Nostoc muscorum. Matsunaga et al. (1999) observed that Chlorella vulgaris was able to remove 65% cadmium between 7 to 28 days. Chlorella sp. removed effectively by 76% to 96% of cadmium and 78% to 94% of nickel under laboratory condition (Rehman & Shakoori, 2004). Other results showed that C.
vulgaris was able to remove up to 70% and tolerate 200 mg/L of As 5+ present in the growth medium (Jiang, 2011). (Lee et al., 2020) investigated the optimal alginate bead size for the nutrient removal using C. vulgaris and suggested the cell immobilization technology as an e cient technique for the wastewater treatment systems. The main objectives of the current study are to investigate the contamination pattern in Dhiba port marine environment, analyze water in the ve sites inside the port related to contaminations of heavy metals and hydrocarbons, and study the potential of fresh alga C. vulgaris and immobilized C. vulgaris to bioremediate pollutants that exist in the most contaminated two sites in port. It also aims to compare between fresh alga C. vulgaris and immobilized alga for e cient remediation of heavy metals and hydrocarbon in the most contaminated two sites and the effects of these pollutants on chlorophyll and carotene content.

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  (Table 1).
Water samples were collected from the water surface on 25 th January 2020 from ve 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.

Preparation of immobilized microalga in alginate beads
For each ask, 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 speci ed 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 asks (250 ml) contained 150 ml of sterilized seawater were enriched with nitrogen and phosphate source (0.225 g of NaNO 3 and 0.006 g of K 2 HPO 4 ). Under a laminar ow cabinet, three asks were cultivated with the algal beads, and the other three were cultivated with the residue of 30 ml of centrifuged algal cells of each ask. 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. 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. Signi cance was determined using Duncan's multiple range tests (p≤0.05). Analysis was carried out using MS Excel (2016) and SPSS (Version 16).

Results And Discussion
Results in Table 1 show the number of vessel arrivals from various ports worldwide that reached Dhiba port. 12029 vessels, through 14 years ago, denoting anthropogenic activities during these years and hence accumulation of waste products. Results in Table 2 show nineteen heavy metals that were investigated in ve sites of Dhiba port. The results demonstrated that among nineteen heavy metals were investigated, different concentrations of As, Be, and Se. The Be and Se were found at all ve locations.
Site no. 1 was contaminated by Sb 3+ , As 3+ , Be 2+ , and Se 2+ with concentrations 0.03168, 0.04126, 0.08985, and 0.199 ppm respectively where the site no. 3 was contaminated by the previous heavy metals Sb3 + , As 3+ , Be 2+ , Se 2+ , in addition to Zn 2+ with concentrations 0.07546, 0.05709, 0.09326, 0.4618, and 0.00979 ppm respectively. The concentrations of metals in surface seawaters varied from one site to another. Zinc metal has been depicted only in the third site, which has a high total concentration of heavy metals (HMs) compared with other sites, so it was chosen for the bioremediation experiment. The hydrocarbons concentrations were estimated before experiment ( Table 3). The level of total hydrocarbons ranged from 0.21 ppm to 0.55 ppm. The rst site was the most highly polluted with hydrocarbons. It showed particular compounds that were not found in the other sites (1,1,3-Trimethylcyclopentane and Diethyl Phthalate), so it was chosen for the biodegradation experiment.

Estimation of C. vulgaris growth
Green microalgae C. vulgaris is halotolerant, proliferating, and growing in marine environments and favorably using it for bioremediation and biodegradation experiments. Luangpipat  Immobilization technique can offer higher micro-algal cell density, which is useful for diminishing lag period (Ide et al., 2016), due to it is less sensitive to stress conditions (Lee et al., 2020). Results in Table 4 showed that the effect of seawater was taken for both sites on chlorophyll-a, content of both immobilized and suspension C. vulgaris cells. Chlorophyll-a contents are more promotive in suspension C. vulgaris that was grown in seawater taken from site 3 within ten days. Contaminations in site 3 were more abundant with heavy metals and less content of hydrocarbons, so the alga was grown under photoautotrophic conditions. Chlorophyll-a in autotrophic was promoted by alga growth, which revealed the production of necessary pigments by C. vulgaris for photosynthesis, the only pathway for the metabolism of phototrophic microalgae (Mohammad Mirzaie et al., 2016). The same trends were observed for Chlorophyll-b contents but within seven days with suspended alga (Table 5). After 10 days of cultivation, Chlorophyll-a and b were decreased. This may be due to decrease of nutrients content in media such as nitrogen and phosphorus. Chlorophyll contents decrease could be due to decreasing of nitrogen in media (Li et al., 2008). The highest level of carotenoid contents of C. vulgaris was 666.14 μg ml -1 recorded at day 14 with suspension cells that was grown in seawater sample taken from site one followed by carotenoid contents of C. vulgaris that was grown at the same days but in site three. Both sites at the day 14 of growth had the stress conditions such as site three that had most contaminations by metals, site one was mostly contaminated by hydrocarbons, and when incubations period to day 14, the nutrients of media decreased and accumulation of toxic compounds (Table 6) Table 7 demonstrated the variable among different sites, alga treatments and the incubation periods in relation to Chl a, Chl b and carotenoid. The results indicated that there were signi cant interaction among sites, alga treatments (immobilized and suspended), and incubation times in relation to pigments contents in C . vulgaris. In site 1 there were signi cant interactions among the types in treatments (suspension, alga, and immobilized) and incubations periods and also in case of site three (Table 8).

The bioremediation of heavy metals
The results in Table 9 demonstrated that when applied suspension C. vulgaris and immobilized C. vulgaris, heavy metals were completely disappeared. The removal e ciencies of these metals were affected by their initial concentrations. C. vulgaris presented a high e cacy in removing 100% of Sb, As, Se, and Zn. This nding is consistent with the work of (Zou et al., 2020) where their results showed that C. vulgaris was highly e cient in removing Se and Cr collectively and separately. The bioremediation process was effective using both suspended and immobilized C. vulgaris cells. Thus, our results may also be explained by enhancing growth rate of C. vulgaris during exponential phase. This result is in agreement with (Li et al., 2019) who studied the biotreatment of mixed wastewaters with MnO 2 industry by C. vulgaris. However, the removal of heavy metals (Cu, Cr, Pb, and Cd) from dyes by C. vulgaris was signi cantly enhanced when endophytic bacterial strain MN17 inoculum was applied (Mubashar et al., 2020). Marine green alga Chlorella sp. NKG16014 exhibited the highest elimination of Cd due to cell adsorption and intracellular accumulation (Matsunaga et al., 1999).
Sorption capacities of heavy metals such as Cu, Zn, Cd and Ni by C. vulgaris were attained at the lowest biomass concentration (Fraile et al., 2005). The heavy metals in the current study's contamination levels can be correlated to contamination caused by the port activities.
The biodegradation of petroleum hydrocarbons Results in Table 3 investigated the hydrocarbons that were existent in ve sites in Dhiba port. The results demonstrated that the site no .1 was much contaminated by hydrocarbons, so it was shown for applied C. vulgaris and immobilized C. vulgaris for possible bioremediation and cleaning. Results in Figs 4a,b and Table 10 revealed the effect of C. vulgaris and immobilized C. vulgaris on removing hydrocarbon that exhibited in site one. Both treatments were effective of biodegradation of hydrocarbons but the highest biodegradation rate of hydrocarbons was observed with immobilized C. vulgaris. Muñoz et al., (2003) suggested that the microalgae release biosurfactants that could improve phenanthrene degradation. Madadi et al., (2016) recommended using C. vulgaris and surfactants to treat wastewaters from petroleum industries. C. vulgaris had a high ability in remediation of crude oil hydrocarbons within 14 days (Kalhor et al., 2017). The results showed a complete absence of the previous hydrocarbons and a presence of new compounds. This new compounds may be due to the conversion of hydrocarbons into intermediate compounds (Okoh, 2006) This result is in agreement with El-sheekh and Hamouda, Immobilized cells have ampli ed reaction rates due to superior cell density (Mallick, 2002).

Conclusions
Dhiba's port is a strategic location and one of the most vital ports in Saudi Arabia where humans' activities are expected to be increased when NEOM project will release. There were some contaminations indicated by heavy metals and hydrocarbons appeared in ve sites of Dhiba's port. Suspension and immobilized microgreen alga C. vulgaris were proved e ciency for bioremediate both heavy metals and hydrocarbons. Immobilized C. vulgaris was the most effective in removing heavy metals and also hydrocarbons that existed in two sites than suspension alga, and hence harvesting beads from media are very simple, and could be applied in biofuel production after bioremediation processes.  Figure 1 Geographical map focusing on sampling sites in Dhiba port Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.