Effective Removal of Alkanes and Polycyclic Aromatic Hydrocarbons by Bacteria from Soil Chronically Exposed to Crude Petroleum Oil Eman Afkar ( [email protected] ) Dept. of Basic Sciences, Preparatory Year Deanship (PYD), King Faisal University, Al-Ahsa, 31982, Saudi Arabia. https://orcid.org/0000-0002-7442-4880 Aly M. Hafez Dept. of Chemistry, College of Science, King Faisal University, Al-Ahsa, 31982, Saudi Arabia. Rashid I.H. Ibrahim Dept. of Biological Sciences, College of Science, King Faisal University, Al-Ahsa, 31982, Saudi Arabia. Dept. of Botany, Faculty of Science, University of Khartoum, PO Box 321, PC 11115, Sudan Munirah Aldayel Dept. of Biological Sciences, College of Science, King Faisal University, Al-Ahsa, 31982, Saudi Arabia. Research Keywords: n-alkanes, GC-MS, crude oil degradation, bacteria, contaminants, hydrocarbons Posted Date: February 16th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-207407/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/18 Abstract In this study, two bacterial strains isolated from an oil-contaminated soil, designated as AramcoS2 and AramcoS4 were able to degrade crude oil, long-chain n-alkanes of C10 to C20; (n-decane, n-undecane, n- dodecane, n-tridecane, n-tetradecane, n-pentadecane, n-hexadecane, n-heptadecane, n-octadecane n- nonadecane, and n-eicosane) and polycyclic aromatic hydrocarbons (PAHs) including biphenyl, naphthalene, and anthracene. Gas chromatography-mass spectrometry (GC-MS) technique was conducted to analyze and identify the crude oil residues after biodegradation. AramcoS2 and AramcoS4 were able to reduce the concentration of long-chain n-alkanes of C10-C20 eciently on average by 77% of the original concentration. Both isolates could also degrade PAHs on average by 67% of the original concentration within 7 and 14 days of incubation at 30ºC, pH=6.8±0.2. The 16S rRNA gene sequences of AramcoS2 and S4 classied these isolates as Actinobacteria; well-known alkanes and PAHs degraders. The nucleotide sequences of AramcoS2 and AramcoS4 were submitted to the GenBank database under the accession numbers MN142506 and MN142551, respectively. Both isolates can be used to restore the environments contaminated with crude oil components. They should be of great practical signicance both in bioremediation of soil contaminated with crude oil and bio-treatment of oil spills on surface water. Introduction Biodegradation is the breakdown of organic substances by bacteria or microbes to its nal products as water and carbon dioxide. Crude oil is an excellent energy source and provides starting materials for petrochemical industries. It is composed of a mixture of different hydrocarbons and non-hydrocarbon; the most commonly crude oil hydrocarbon molecules are n-alkanes (paran) represent 22%, cycloalkanes (naphthenes) represent 50%, PAHs represent 17% (Acosta-González et al., 2015; Moubasher et al., 2015). Contamination with crude oil severely affects both human and environmental health (Bao et al., 2012; Ganesh Kumar et al., 2014; Bacosa and Inoue, 2015). Generally, the process of oil contamination is dependent on many parameters such as exploitation, transportation, and rening. When these parameters are combined with accidental spills, substantial environmental harm will happen. The high potential of environmental risks from crude oil spills encouraged us to search for local bacterial isolates inhabiting the oil-contaminated soils and able to utilize the contaminant as an energy source. Crude oil hydrocarbons (n-alkanes toluene and PAHs) are prevalent organic contaminants (Lee et al., 2018; Huang et al., 2019). They are usually produced as results of the pyrolysis of organic compounds or incomplete combustion. PAHs have mutagenic, carcinogenic, and cause endocrine system problems (Dutta et al. 2017). The carcinogenic effect of those organic pollutants on body organs appears after prolonged periods of exposure (Ljunggren et al., 2014; Hernández et al., 2017; Koual et al., 2019). Intrinsic biodegradation of crude oil was investigated in batch cultures. Short and long-chain alkanes and PAHs were the most common components of crude oil. Many organisms utilize these components as carbon and energy sources (Zawierucha et al., 2014; Adetutu et al., 2015; Ahmad et al., 2015). Despite their hydrophobicity, long-chain alkanes and PAHs can be degraded by microorganisms but at a slower rate (Chen et al. 2017b). The discovery of new genera and species of microorganisms that are capable of Page 2/18 degrading crude oil is the core of petroleum microbiology. However, the Gulf area, particularly the Kingdom of Saudi Arabia is considered one of the largest countries for crude oil production; Saudi Arabia is the second-largest oil reservoir globally. Shipping and oil processing also contribute to crude oil contamination in the eastern province of Saudi Arabia. Biodegradation and Bioremediation are useful methods to remove hydrocarbon pollutants from the environment. Saudi Arabia is an oil-mining region and it faces this problem. Therefore, the signicance of this work is evident. Indigenous hydrocarbon oxidizing bacteria are known to have advantages in comparison with allochthonous cultures since they are ideally adapted to the local conditions. In the present study, the intrinsic biodegradable power of two bacterial isolates, designated as AramcoS2, and AramcoS4, was applied to degrade crude oil and PAHs. These isolates were obtained from crude oil- contaminated soil nearby Aramco petroleum aquifers located in the eastern province of Saudi Arabia. Scanning electron microscopy (SEM) of pure cultures for both bacterial isolates was captured and the 16S rRNA gene sequences were amplied, sequenced, and submitted to the GenBank database. The biodegradation eciency of both isolates understudy was investigated using GC-MS. Crude oil, long- chain n-alkanes of C10 to C20 (decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane, octadecane, nonadecane, and eicosane), toluene, and PAHs (biphenyl, naphthalene, and anthracene) were the subject materials. Materials And Methods Chemicals Crude oil was obtained from the Saudi Arabian national petroleum and natural gas company (Aramco). Hexadecane and octadecane were purchased from BDH chemicals, England, toluene from Sigma-Aldrich Chemical Co. Germany, biphenyl from Scharlau, Spain, and naphthalene and anthracene from Lobachemie, India. Bacterial medium (mineral salt medium; MSM) components were purchased from Merk KGaA, Darmstadt, Germany. Site description Four soil samples were collected from the Uthmaniyah site at the Aramco gas plant area, around 35 km south-west of Al-Ahsa city, Saudi Arabia. Soil samples were withdrawn at different depths; 5, 15, 25, and 35 cm from the head well of petroleum aquifers. Uthmaniyah is located in the desert, geo-position 25°21'52"N, 49°33'55"E. Climate with 83.3 mm of annual precipitation. The range of average temperature in summer lies between 35°C and 45°C, and the winter temperatures are between 5°C and 25°C. Sampling and Enrichment of crude oil-degrading bacteria For enrichment of bacteria, the soil samples were introduced separately to a standard mineral salt medium (MSM) containing (1gm soil/100 ml of MSM each). The composition of the MSM was as follows: 0.2 MgSO4, 0.02 CaCl2, 1.0 KH2PO4, 1.0 K2HPO4, 1.0 NH4NO3, and 0.05 FeCl3 g/L, where pH was Page 3/18 adjusted to 6.8 ± 0.2. The medium was autoclaved at 120 ºC for 30 min and amended with a 1% (v/v) lter-sterilized (0.22 µm syringe lter) crude oil as the sole carbon and energy source (Parthipan Punniyakotti et al. 2017). This medium was aliquoted into 100 ml in 250 ml Erlenmeyer asks and each was inoculated with 5 gm of soil sample. The enrichment step was carried out for 7 and 14 days with continuous shaking at 30ºC, pH = 6.8 ± 0.2, and 120 rpm. A control sample of 1% crude oil in the MSM was used. Ten ml were withdrawn from the enrichment step and transferred to a fresh MSM medium mixed with 1% (v/v) crude oil. After a series of three further subcultures, inoculums were streaked out on MSM containing agar, and the phenotypically different colonies were characterized. The preliminary identication of the bacterial isolates was carried out based on colony appearance, shape, margin, color, and gram staining (according to Bergey's Manual of Systematic Bacteriology). The genotype identication was done using the 16S rRNA sequences analyses. Scanning electron microscopy (SEM) of AramcoS2 and AramcoS4 isolates AramcoS2 and AramcoS4 isolates were examined using SEM. The samples were prepared by directly applying the bacterial cells onto the copper grid and allowed to dry for 2 hours then examined under a scanning electron microscope (TECHNAI10-Philips). Sequencing of 16S rRNA Genomic DNA from single colonies of both AramcoS2 and AramcoS4 isolates were used separately as a template for PCR amplication of the 16S rRNA gene in the presence of the universal primers 27F and 1492R (Weisburg et al. 1991). The PCR reaction was carried out as follows; one cycle of DNA denaturation at 94°C for 45s, 35 cycles at 94°C for 45s, 55°C for the 60s for annealing of primers, and 72°C for 2min as an extension step. These cycles were followed by a nal elongation step at 72°C for 5min. Sequencing of DNA was performed using the Big Dye terminator sequencing kit in a 3730xl DNA analyzer (Applied Biosystems, USA) using the universal primer pair 785F and 907R. Construction of phylogenetic trees The sequences obtained from the 16S rRNA were edited and assembled using GENETYX software (GENETYX, Tokyo, Japan). The assembled sequences were used in BLASTN for a search of similar sequences on the NCBI (The National Center of Biotechnology Information) database. The most similar sequences were obtained and applied to MEGA7 software to build up phylogenetic relationship trees using the neighbor-joining method and bootstrap support based on 1000 replicates (Felsenstein, 1985; Saitou and Nei, 1987; Kumar et al., 2016).