Estimation of Bacterial Biodegradability of PAH in Khor Al-Zubair Channel, Southern Iraq

Estimation of Bacterial Biodegradability of PAH in Khor Al-Zubair Channel, Southern Iraq Fadhil N.A. Alkanany, Satar A. Gmais, Anwar A. Maki, Asaad M.R. Altaee Marine Science Center, University of Basra, Iraq Corresponding author email: amraltaee@yahoo.com International Journal of Marine Science, 2017, Vol 7, No 42 doi: 10.5376/ijms.2017.07.0042 Received: 01 Sep., 2017 Accepted: 27 Sep., 2017 Published: 20 Oct., 2017 Copyright © 2017 Alkanany et al., This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Alkanany F.N.A., Gmais S.A., Maki A.A., and Altaee A.M.R., 2017, Estimation of bacterial biodegradability of PAH in Khor Al-Zubair channel, Southern Iraq, International Journal of Marine Science, 7(42): 399-410 (doi: 10.5376/ijms.2017.07.0042)


Introduction
Polycyclic aromatic hydrocarbons (PAHs) are compounds consisting of two or more aromatic rings, the USEPA has reported sixteen unsubstituted PAHs as main concern pollutants, eight of them may be human carcinogens (Yan et al., 2004). The concern is that some PAHs are toxic, carcinogenic or teratogenic. PAHs are a resource of carbon and energy and being exploited by some bacteria as growth substrates. The presence of PAH in the marine environment is largely attributed to oil spills, discharge and natural river infiltration, import or transfer of airflow. Therefore, global increased human activity has increased risks to the marine environment (Latimer and Zheng, 2003).

Collection of samples
Samples were collected from Khor Al-Zubair channel, southern Iraq ( Figure 1) from five stations. Water samples (500 ml) were collected at two depths in sterile glass bottle, all samples transferred to an ice box and transported to the laboratory.

Isolation and identification of bacteria
One milliliter of each sample was cultured in a conical flask containing 100 ml mineral salts medium (MSM), the composition of the MSM was 0.3 gm of KCl, 1.0 gm of K 2 HPO 4 , 0.5 gm of KH 2 PO 4 , 0.01 gm of FeSO 4 .7H 2 O, 30.0 gm of NaCl, 0.5 gm of MnSO 4 .7H 2 O, 0.2 gm of CaCl 2 and 1000 ml DW (Fujisawa and Murakami, 1980). Four concentrations of crude oil as 0.25 ml, 0.5 ml, 1 ml and 2.0 ml (Provided by Al-Shua'aba Refinery) were added separately to the medium. Decimal dilutions of 7 days grown culture was cultivated at 30°C. Nutrient agar (Hi media-India) with 30 ppt sodium chloride and marine agar (Difco, USA) were used to isolate Vibrio vulnificus, Brevundimonas diminuta/ vesicularis, Sphingomonas paucimobilis and Ochrobactrum anthropic and identified by the Vitek II system (VK2C8300, USA).

Degradation of PAH
One millilitre of broth culture of each bacterial isolate was incubated separately in 250 ml Erlenmeyer flask containing 50 ml of MSM at 20ᵒC for 7 days at 120 rpm using a cooling incubator shaker (Al-Sulami et al., 2014). All the experiments were carried out in two duplicates, and the residual crude oil was performed after 7 days.

Gas chromatographic analysis of residual crude oil
The residual crude oil was extracted by liquid-liquid extraction, as described by Adebusoye et al. (2007). The aqueous phase was removed by separating funnel and the residual oil dried in the oven at 40ᵒC to remove CCl 4 .
The aromatic fraction was separated using separation column (25 cm length, 3 cm diameter) containing 8 gm of silica gel over a little amount of glass cotton (Farid, 2006). The residual oil dissolved in 25 ml of benzene and poured into the separation column and drawn off the aromatic fraction in 50 ml beaker. Control flasks were also extracted similarly, aromatic fraction hydrocarbons estimated by FID gas chromatography technique (Agilent Chem Station).

Identification of bacteria
Sixty five isolates of gram negative bacilli was isolated from Khor Al-Zubair channel southern Iraq, Fifty-six of them were identified correctly to the species level as V. vulnificus, Sphingomonas aucimobilis, Brevundimonas diminuta/ vesicularis and Ochrobactrum anthropi, while 9 strains were not identified. Direct identification, reporting time of the VITEK II was 4.5 to 10 hours after incubation (Table 1). Four species were isolated by the VITEK II system and their ability to degrade four concentrations of crude oil was examined.

Degradation study of PAH
In the present study, four species were examined their capability to degrade 4 concentrations of crude oil. Table 2 shows the percentage of degradation of PAH for each genus. Sphingomonas paucimobilis was the most effective bacteria to degrade PAH (97.39%) especially in concentration 2.0 mg/l, while Ochrobactrum anthropi was the lowest (13.15%) in the same concentration. Figure Figure 12; Figure  13). The genus of Vibrio was first described as a degrading phenanthene organism from West and colleagues, who isolated large numbers of strains by spreading Chesapeake Bay samples onto complex media each with a superficial layer of phenanthrene West et al., 1984).
Sphingomona paucimobilis showed high degradation percentage (97.39%) in concentration 2.0 mg/l and low degradation (27.26%) in concentration 0. 5 mg/l (Table 2; Figure 14; Figure 15; Figure 16; Figure 17). This is consistent with a report by Tao et al. (2007) who found that this bacterium has unique genes to degrade a wide range of PAH and related compounds. The gene is related from other Pseudomonas genera and reports so far in sequence homology and gene organization. Also Pinyakong et al. (2003a;2003b) observed that, many sphingomonads degrade naphthalene, anthracene, and phenanthalene by common pathways w e r e found in other gram-negative bacteria. In addition, many researchers (Kim et al., 1996;Feng et al., 1997;Romine et al., 1999;Ogram et al., 2000;Basta et al., 2004) found large plasmids in strains of Sphingomonas xenobiotic contributing to the degradation of PAH.   (Mrozik et al., 2003).
While Ochrobactrum anthropic showed low degradation percentage of all concentrations (Table 2; Figure 18; Figure 19; Figure 20; Figure 21). Sulaiman et al. (2016) found that, out of 35 isolates obtained from petroleumcontaminated soil only five isolates have the ability to degrade phenanthrene and anthracene. Whereas Katsivela et al. (2002) found that, 97% of 2; 2; 4; 4; 6; 8; 8-heptamethylnonane, 55% of toluene, 71% of acenaphthylene and 72% of acenaphthene were depleted after 9 days of growth. The ability of these bacteria to remove different PAHs looks promising for use in bioremediation technologies.  Biodegradation of PAHs as described by Johnsen et al. (2005) can serve three different functions: 1-Assimilative biodegradation that yields carbon and energy for the microorganism and goes along with the mineralization of the compound.
2-Intracellular mechanism of PAHs is soluble in water and therefore causes excretion.
3-Cometabolism which is the degradation of PAHs without production of energy and carbon for the organism metabolism. Figure 19 Crude oil 0.5% with Ochrobac. anthropi Figure 20 Crude oil 1.0% with Ochrobac. anthropi These differences between the bacterial degradation of only the PAH environment probably reflect the diversity in the catabolic systems PAH and general physiology and possibly mediated ecological differences that are not understood (Brian et al., 2001).
The degradation of aliphatic hydrocarbons and PAH was mainly of the mono-and dioxygenase produced by bacteria. Therefore, the presence of six key enzymes coding genes, including both monooxygenase and dioxygenase in the ASU-06 strain of S. koreensis the existence of monooxygenase genes (alkB and alkB1) dioxygenase (naháč) and Catechol dioxygenase (C12O and C23O) genes was confirmed. Therefore, one possible reason may be that these genes are strong evidence between different conserved Gram-negative bacteria (Hesham et al., 2014).

Conclusion
In this study, 4 oil degrading bacterial strains were isolated from brackish water. The bacteria were identified as Brevundimonas diminuta/ vesicularis, Vibrio vulnificus, Sphingomonas paucimobilis and Ochrobactrum anthropic based on morphological properties and by Vitek II system. We found a variation in their capabilities to degrade PAH and most effective in higher concentrations. Perhaps, this is related to the types of bacteria and the mechanisms which to degrade crude oil.