Monitoring of oil pollution at Gemsa Bay and bioremediation capacity of bacterial isolates with biosurfactants and nanoparticles

Marine Pollution Bulletin xxx (2014) xxx–xxx

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Marine Pollution Bulletin

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Monitoring of oil pollution at Gemsa Bay and bioremediation capacityof bacterial isolates with biosurfactants and nanoparticles

http://dx.doi.org/10.1016/j.marpolbul.2014.07.0590025-326X/� 2014 Published by Elsevier Ltd.

⇑ Corresponding author. Address: Egyptian Petroleum Research Institute (EPRI), 1Ahmed El-Zomor Street, El-Zohour Region, Nasr city, 11727 Cairo, Egypt. Tel.: +20 222745902; fax: +20 2 227727433.

E-mail address: [email protected] (H.S. El-Sheshtawy).

Please cite this article in press as: El-Sheshtawy, H.S., et al. Monitoring of oil pollution at Gemsa Bay and bioremediation capacity of bacterial isolatbiosurfactants and nanoparticles. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.07.059

H.S. El-Sheshtawy ⇑, N.M. Khalil, W. Ahmed, R.I. AbdallahEgyptian Petroleum Research Institute, Nasr City, 11727 Cairo, Egypt

a r t i c l e i n f o a b s t r a c t

Article history:Available online xxxx

Keywords:Oil pollutionBiodegradationBiosurfactantsNanoparticles

Fifteen crude oil-degrading bacterial isolates were isolated from an oil-polluted area in Gemsa Bay, Red Sea,Egypt. Two bacterial species showed the highest growth rate on crude oil hydrocarbons. From an analysis of16S rRNA sequences, these isolates were identified as Pseudomonas xanthomarina KMM 1447 and Pseudo-monas stutzeri ATCC 17588. Gas Chromatographic (GC) analysis of the crude oil remaining in the culturemedium after one week at 30 �C showed that the optimum biodegradation of crude petroleum oil was dem-onstrated at 50% in medium containing biosurfactant with two types of nanoparticles separately and twobacterial species. The complete degradation of some different members of polyaromatics and the percent-age biodegradation of other polyaromatics increased in microcosm containing two different types of nano-particles with biosurfactant after 7 days. In conclusion, these bacterial strains may be useful for thebioremediation process in the Gemsa Bay, Red Sea decreasing oil pollution in this marine ecosystem.

� 2014 Published by Elsevier Ltd.

1. Introduction Petroleum hydrocarbons can be degraded by microorganisms

Petroleum hydrocarbons are the most common environmentalpollutants, and oil spills pose a great hazard to terrestrial and mar-ine ecosystems. Oil pollution may arise either accidentally or oper-ationally whenever oil is produced, transported stored, processedor used at sea or on land. Oil spills are a major menace to the envi-ronment because they severely damage the surrounding ecosys-tem (Shahian et al., 2012). The majority of PAHs have strongtoxicity, carcinogenicity, teratogenicity and mutagenicity (Ruizet al., 2011; Liu et al., 2012a). The alkanes and PAHs pollutants inthe water environment cause serious pollution to the water eco-systems and are harmful to the health of the living creatures andhuman bodies (Zhang and Kang, 2009). Polycyclic aromatic hydro-carbons (PAHs) are widespread and mainly originate from fossilfuel combustion and the release of petroleum and petroleum prod-ucts (Andres et al., 2010; Chao et al., 2010). Some PAHs with four ormore benzene rings, such as benzo[a]anthracene, chrysene andbenzo[a]pyrene produce mutagenic and carcinogenic effects.Moreover, PAHs are persistent in the environment and can causelong-term adverse effects. Thus, 16 PAHs have been listed as prior-ity pollutants by the U.S. Environmental Protection Agency (USEPA).

such as bacteria, fungi, yeast, and microalgae (Atlas, 1981; Leahyand Colwell, 1990). The individual microorganisms metabolize ofthe crude oil only to a limited range of hydrocarbon substrates(Britton, 1984). Biodegradation of crude oil requires mixture of dif-ferent bacterial groups or consortia functioning to degrade a widerrange of hydrocarbons (Bordenave et al., 2007). Contaminatedmarine environments are inhabited by a range of selected microor-ganisms able to tolerate and remediate pollutants that impactedthe environment, leading to the dominance of pollutant-tolerantbacteria. Hence, bacterial communities in contaminated sites aretypically less diverse than those in nonstressed systems(Harayama et al., 2004).

Biosurfactants (BS) can emulsify hydrocarbons via enhancingtheir water solubility, decreasing surface tension and increasingthe displacement of oily substances from soil particles (Banat,1995; Banat et al., 2000). Surfactants can also increase the rate ofthe biodegradation of slightly soluble contaminants by increasingtheir bioavailability (Guha and Jaffe, 1993).

In the last two decades, nanotechnology has attracted a greatinterest due to its expected impact on areas of catalysis and/orwater treatment (Azim et al., 2009). Nanoparticles have beenwidely used to improve various reactions as reductants and/or cat-alysts in chemistry field due to their high specific surface areas andcharacters (Hildebrand et al., 2008; Nezahat et al., 2009). On theother hand, nanoparticles effect on microbes has also caught agreat attention. Nanoparticles are capable of assisting microbeactivities however, so far, very limited studies have been reported

es with

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Fig. 1. Location of the sample collection sites in the Gemsa Bay, in the Red Sea,Egypt.

2 H.S. El-Sheshtawy et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx

on nanoparticle effect on the microbiological reaction rates (Shinand Cha, 2008). The higher activity of nanoparticles is usuallyreferred to their unique properties and high available active spe-cific surface areas. Generally, nanoparticle catalysts increase themicrobiological reaction rates by locating on the cells to stimulatethe activity of microbes (Shan et al., 2005).

The Egyptian Red Sea region which is about 1080 km from Suezto Mersa Halayab has unique characteristics of hot and dry climatethus produces a very high water temperature and salinities. It hasvery little rain fall with its coastal areas being exclusively of majorstrategic economic and scientific importance. The Red Sea regionshave the highest density and frequency of oil tanker traffic of anyregional seas or other oceanic areas. Due to these characteristicsand the extensive off-shore oil exploration and production activi-ties, probabilities of major oil spills are high. There are regularlosses of oil to the sea during routine transfer operations involvingloading, deballasting of oil tankers in preparation of loading duringoff-shore drilling and from coastal refineries and other sources(Gladstone et al., 2013). In the present paper, Gamsa Bay at55 km from north of Hurghada, between Hurghada and Ras Gharibin the Gulf of Suez at the coasts of Red Sea in Egypt is studied(http://www.almasryalyoum.com/node/502797). Petroleum spills,occurred in five locations on-shore and off-shore, in the Gulf ofGemsa leak petroleum in six ancient wells. This area was exposedto leaks previously since 2009 till now. The layers in the leakagearea were proved to be of kind salt limestone. The oil spills result-ing from the fountains explode from time to time are due to non-perceptible earthquakes in the land of the seabed in the region thatwas characterized by geological installed the vulnerability of theearth’s crust and there were several trap leaking oil (http://www.greencleannow.org/index.php/news/arab_news/4023.htlm).That area of ‘‘Gemsa’’ had 24 oil wells since 1919 which had beenclosed with the exception of three wells closed only since twoyears as a result of non-economic output (http://www.greenclean-now.org/index.php/feed/news/arab_news/4023.txt.).

The present paper is directed toward the attainment of concen-trations of pollutants in sea water at Gemsa Bay. The degradationcapacity of crude oil samples by pure and mixed bacterial culturesisolated from contaminated water sample is studied. Also, theenhancement of crude oil biodegradation (bioremediation) byusing two different types of synthetic nanoparticles and biosurfac-tants is evaluated.

2. Materials and methods

2.1. Samples collection

The crude oil and water samples were collected from GemsaBay, in the Red Sea, Egypt (Fig. 1). The water sample was collectedfor microbiological analysis and petroleum assessment. The crudeoil sample was collected from limestone caves from Gemsa Bay.

2.2. Physicochemical properties of water sample

pH value, electrical conductivity, total dissolved solids (TDS),density and specific gravity, salinity value, heavy metals werestudied according to standard tests methods (The American societyor testing and Materials, ASTM).

2.3. Extraction of oil from water samples

The sample was acidified with HCl, so that, the pH is adjusted atpH 2. Then an amount of water sample was treated with carbontetrachloride to extract all oil from water sample and its contentwas calculated from the following equation (Khalil, 2007):

Please cite this article in press as: El-Sheshtawy, H.S., et al. Monitoring of oil pobiosurfactants and nanoparticles. Mar. Pollut. Bull. (2014), http://dx.doi.org/10

Oil content ¼ ððA� BÞ � 1000=ml of water sampleÞmg=ml

where A and B are the weights of the flask empty and containing theextracted oil respectively.

2.4. Synthesis of nanoparticles

Ferric nitrate [Fe(NO3)3�6H2O] and zinc chloride [ZnCl2�9H2O]were used as precursors to synthesize iron oxide (a-Fe2O3) andZn5(OH)8Cl2 respectively. Both precursors were purchased fromSigma–Aldrich. The ammonium hydroxide used for the precipita-tion of Zn complex was supplied by sigma.

The Zn5(OH)8Cl2 nanoparticles were prepared by the precipita-tion technique. In practical, 2.6 g of ZnCl2 was dissolved in 1000 mldistilled water and precipitated using dilute aqueous ammoniumhydroxide solution at ambient temperature until the pH 9. Theslurry was then agitated for 30 min. The precipitate was filteredand washed carefully and eventually the sample was dried at100 �C for 12 h.

The pure a-ferric oxide was also prepared, in order to formulatea comparative study, by the conventional thermal decompositionmethod. In brief, a definite quantity of ferric nitrate was heatedin muffle at 600 �C for 4 h.

2.5. Enumeration of halophilic bacteria in the sea water sample

The enumeration of the total halophilic bacteria was imple-mented by using the plate count technique where the pollutedsea water sample (1 ml) was serially diluted in a sterile saline.Then inoculation in Luria broth (LB) plates medium containing(grams per liter of distilled water) NaCl 10.0; tryptone 10.0; yeastextract 5.0. The medium was adjusted to pH 7.0. The cultures werethen incubated at 30 �C for 48 h. Thereafter, plate count in therange of 30 and 300 colonies was recorded; experiments were con-ducted in three independent replicates. The bacterial count per mlwas calculated from the following equation:

Bacterial count=ml ¼ colony count per plate� dilution factor

The colonies with different morphological features wereselected and purified on LB plates medium (Benson, 1995).

llution at Gemsa Bay and bioremediation capacity of bacterial isolates with.1016/j.marpolbul.2014.07.059

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H.S. El-Sheshtawy et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx 3

2.6. Isolation of crude oil degrading bacterial isolates

A synthetic Bushnell Hass Mineral Salts medium (BHMS) wasused for the isolation of crude oil degrading bacteria. BHMS med-ium contains the following: KH2PO4, 1 g l�1; K2HPO4, 0.2 g l�1;MgSO4�7H2O, 0.2 g l�1; CaCl2, 0.02 g l�1; NH4NO3, 1 g l�1; NaCl,2 g l�1; and 2 droplets of 60% FeCl3. The pH was adjusted 7. TheBHMS medium was supplemented with 1% (v/v) crude oil (GemsaBay light crude oil) as the sole source of carbon and energy. Seawater (1 ml) was added to 250 ml Erlenmeyer flasks containing100 ml of the BHMS medium; the flasks were incubated for 7 daysat 30 �C on a rotary shaker operating at 150 rpm. Then, 5 ml ali-quots were transferred to fresh BHMS medium. After a series offour further subcultures, inoculums from the flask were streakedout, and phenotypically different colonies were purified on BHMSagar medium for 3 days of incubation period. The procedure wasrepeated and only isolates that exhibited pronounced growth oncrude oil were stored for further characterization (Shahian et al.,2012).

2.7. Selection of the most predominate bacterial isolates

The bacterial isolates (I1, isolate no. 1 and I2, isolate no. 2) wereselected for further studies due to most predominated growth onBHMS medium.

2.8. Molecular identification of the most promising bacterial strains

An analysis of 16S rRNA was performed to taxonomically char-acterize the isolated strains (Sigma Scientific Services Co., Egypt).The cells of the two bacterial strains were harvested through theenrichment medium up to 2 � 109 bacterial cells. DNA wasextracted using protocol of Gene Jet genomic DNA purificationKit (Thermo) (Sigma Scientific Services Co., Egypt). To amplify the16S rDNA genes, a polymerase chain reaction (PCR) was performedusing (50-AGA GTT TGA TCC TGG CTCA-30) (50-GGT TAC CTT GTTACG ACT-30) as forward and reverse primer respectively. PCR wascleaned up to the PCR product using GeneJET™ PCR PurificationKit. A 45 ll of Binding Buffer was added to the completed PCR mix-ture. The mix was then thoroughly transferred from step 1 to theGeneJET™ purification column. The mixture was then centrifugat-ed for 30–60 s at > 12,000�g, then the flow was discarded. A 100 llwash buffer was added to the GeneJET™ purification column, cen-trifuged for 30–60 s, discarded the flow-through and place thepurification column back into the collection tube. The mixturewas centrifuged at empty purification column for an additional1 min to completely remove any residual wash buffer. The purifica-tion column was transferred to a clean 1.5 ml micro centrifugetube. A 25 ll of elution buffer were then added to the center ofthe column membrane which then centrifuged for 1 min, discardthe column and store the purified DNA at �20 �C. Following puri-fication of the PCR products, the DNA sequence of the positiveclone was subjected to a similarity search BLAST on the NCBI web-site (http://www.ncbi.nlm.nih.gov), and deposited into GenBank.Many relevant 16S rRNA gene sequences with validly publishednames were selected as references from the Gen-Bank.

2.9. Crude oil degradation trial

The ability of the isolated microorganisms to degrade crude oilunder aerobic condition was evaluated. 100 ml of mineral saltmediums Minto 250 ml flasks supplemented with 1% crude oilwas prepared. The medium contained (g l�1): Na2HPO4 2.0, KH2PO4

2.0, MgSO4�7H2O 0.01, NaNO3 2.5, NaCl 0.8, CaCl2, 0.2, KCl, 0.8,FeSO4�7H2O, 0.001, yeast extract, 3%, using crude oil as carbonsource and 1 ml of a trace element solution. Trace element solution


contained ZnSO4�7H2O, 525 mg/l, MnSO4�4H2O, 200 mg/l,CuSO4�5H2O 705 mg/l, NaMnO4�2H2O, 15 mg/l, CoCl2�6H2O,200 mg/l, H3BO3, 15 mg/l, NiSO4�6H2O, 27 mg/l (Haghighat et al.,2008). The pure bacterial isolate (I1 and I2) (2 ml, 2 � 107 CFU/ml) was inoculated into the MSM. Also, mixture of this bacterialisolate (1 ml of each isolate) was inoculated into the same typeof medium. Hence, the flasks were incubated at 30 �C, 150 rpm,pH 7.5 for 7 days. Total viable count (TVC) of cells was determinedby agar plate every 3 days, modified method (Bao et al., 2012). Theremaining crude oil samples were extracted from different micro-cosm and gravimetric analysis was also performed.

2.10. Analysis of produced biosurfactant

Synthesis of biosurfactant by the most promising bacterial iso-lates (I1 and I2) was cultured on MSM (the constituent of mediumas mentioned above). The respective carbohydrate (glucose) wasadded to make a final concentration 2%. Glucose is widely usedfor biosurfactant production by many bacteria (Mulligan andGibbs, 1993; Ismail et al., 2013a,b). Cultivation studies have beendone in 500 ml flasks containing 150 ml medium at 30 �C for 72 h.

2.10.1. Emulsification activity (E24)E24 of the produced biosurfactant in the supernatant was mea-

sured by adding kerosene (6 ml) (Dearomatized kerosene was sup-plied by Alexandria Petroleum Refining Company, Alexandria,Egypt) to the aqueous phase (supernatant culture) and vortexingfor 2 min. After 24 h, the emulsion index (E24) was calculatedaccording to the following equation (Cooper and Goldenberg,1987).

ðE24Þ ¼ 100 ðheight of the emulsion layer=the total heightÞ

2.10.2. Surface tensionSurface tension was measured by a Du Nouy platinum ring

method with Krüss K6 tensiometer. The bacterial supernatant solu-tion (50 ml) was tested at 25 �C to evaluate the surface tension ofbio and chemical surfactant (Nitschke and Pastore, 2006). Thevalue of surface tension was expressed as mN/m.

2.11. Biodegradation of crude oil in presences of two differentnanoparticles and biosurfactants

This experiment was conducted to assess the impact of com-mercially available Fe2O3 and Zn5(OH)8Cl2 nanoparticles on growthand crude oil biodegradation. The bacterial strain (7.2 log count) analiquot of 2 ml of inoculum was inoculated into MSM (100 ml) in a250 ml Erlenmeyer flask. The cultures were incubated on a temper-ature controlled shaker incubator at 150 rpm at 30 �C for 7 days,using (1 g) crude petroleum oil as a sole carbon source and/oradded (0.1 g) of different nanoparticles separately. The biosurfac-tant production using the medium MSM with (1 g) glucose as asole carbon source for 72 h followed by addition of (1 g) crudepetroleum oil and/or added (0.1 g) of different nanoparticles sepa-rately until the end of incubation period. A sample without inocu-lum was taken as a control. Hence, the flasks were incubated at30 �C, 150 rpm, pH 7.5 at 7 days (Haddad et al., 2008). The bacterialcount, surface tension and emulsification power were determinedevery 3 days and the crude oil samples were extracted from differ-ent microcosms and were gravimetrically analyzed.

2.12. Extraction of crude oil after treatment by bacterial strain andgravimetric estimation

At the end of incubation period, 7 days, the polluted bacterialbroth (100 ml) was thoroughly shaken with carbon tetrachloride


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(50 ml 3 times) in a separating funnel and the three fractions werecollected in case of crude oil sample. The collected organic layerwas dried over anhydrous sodium sulphate. The solvent wasremoved on a rotary evaporator until a constant weight (Khalil,2007). The oil sample was accurately weighed, percentage of thebiodegraded oil was calculated and alterations in its chemical com-position were studied by chromatographic analysis (GC and HPLC)(Zakaria, 1998; El-Sheshtawy, 2011).

2.13. Gas chromatographic analysis (GC)

The oil extracted from the studied water sample and the biode-graded crude oil were monitored using Agilent 6890 plus, Gas chro-matograph attached to computerized system with chemstationsoftware condition of operation according to the standard testmethod IP 318/75 (Institute of Petroleum, 1995). The componentseparation was completed on HP-1 capillary column (100% methylsilicone siloxane, 30 m length, 0.35 mm internal diameter and0.25 mm thickness film). For a typical chromatogram, a 0.5 llcrude oil sample was introduced into a splitter injector whichwas previously heated at 350 �C. The oven temperature was pro-gramed 100–320 �C at a fixed rate of 3 �C/min. The nitrogen (oxy-gen-free) was used as a carrier gas with a flow rate of 2 ml/min. Amixture of pure n-paraffins was used as standard. The peak area ofeach resolved component (consisting of either n- and iso-paraffins)was determined individually. While, the unresolved complex mix-tures (humps) composed of non n-paraffins presumably mainlycycloparaffins and aromatics with long side chains, were deter-mined only as a total.

2.14. High performance liquid chromatographic analysis (HPLC)

The oil extracted from the studied water sample and the crudeoil remained after the biodegradation and the corresponding con-trol sample was analyzed using a (HPLC) instrument model Waters600E, equipped with dual UV absorbance detector Waters 2487and auto sampler Waters 717 plus attached to a computerized sys-tem with Millennium 3.2 software. PAHs standards were obtainedfrom Supelco. The conditions of separation (Chen et al., 2006) areas follows: Column: Supelcosil. LC-PAH, 5 lm particles, 15 cmlength and 4.6 mm ID, Mobile phase: gradient acetonitrile: water60–100% acetonitrile (v/v) over 45 min. Flow rate: 0–2 min.0.2 ml/min., 2–45 min. 1.0 ml/min. Detector set at 254 nm.

3. Results and discussion

3.1. Physicochemical properties of water sample

The different physicochemical parameters of the water sampleobtained from Gemsa Bay, Red Sea, Egypt was determined andlisted in Table 1.

The results obtained from Table 1 indicated that high electricalconductivity of tested water sample than the recommended value,also high total dissolved solid, density and salinity.

Table 1Physicochemical characteristics of water sample.

Properties Sea water c

pH value at 25 �C 8.2 ± 0.1Electrical conductivity at mohS/cm2 4.9 �C 0.10 � 10�6

Total dissolved solids TDS, mg/l 34481.6Density, g/ml at 60 F 1.020–1.02Specific gravity 1.0264Salinity value, mg/l (PPM) 35,000


3.2. Oil content of water sample

Oil spills cause extensive damage to marine life, terrestrial life,human health and natural resources. Therefore, to unambiguouslycharacterize spilled oil and to link them to the known source isextremely important for environmental damage assessment. Thefirst step in assessing the seriousness of oil pollution in the marineenvironment is the determination the magnitude of its concentra-tion (Produced Water Society., 2011 <http://70.86.131.22/’pro-ducedpage.php?page_name=produced_water_facts> Sin.; Khalil,2007).

The result obtained from the quantitative determinations of oilcontent in the studied water collected as far one meter fromonshore sample, is equal (496.4 ppm). This indicates relatively highoil content than the recommended standard = 15 ppm according tothe environmental law (Environment Law promulgated No. 4 of1994, EGYPT).

3.3. Evaluation of the chemical composition of oil extracted from waterand crude oil samples

3.3.1. Gas chromatographic analysisThe gas chromatograms for crude oil sample and the extracted

oil from sea water are shown in (Fig. 2a and b). The Fig. 2(a and b)indicated that carbon number of n-alkane of crude oil range fromnC11–nC38 while in water sample range from nC12–nC45 and thereconcentrations were 5390.94 (lg/g) and 72164 (lg/g) respectivelywhile concentration of total aliphatic and alicyclic compoundswere 46524.9 lg/g and 250568 lg/g respectively.

The identification of the homologous n-alkanes allows thedetermination of Carbon Preference Index (CPI) and Cmax for eachsample, indicates the relative contributions of n-alkanes frompetroleum pollution (Huault et al., 2009; Liu et al., 2012b).

In the two samples CPI (1.4 � 1 and 1.32 � 1) the resultsrevealed petrogenic pollution which is confirmed by Cmax at nC38

and nC45.

The presence of a prominent UCM is normally taken to indicatethe presence of petroleum hydrocarbons. Also the UCM high per-cent (88.41% and 71.20%) as shown in Fig. 2(a and b), confirmthe widespread of petroleum origin in this area (Gemsa Bay)(Zaghden et al., 2007)

The relative importance of the UCM, expressed as the ratio ofunresolved to resolved compounds (U/R), is used as diagnostic cri-teria of pollutant inputs (Readman et al., 2002).

Significant contamination by petroleum products exists whenthe value of U/R is P2 according to (Simoneit, 1986) and >4 accord-ing to (Yan et al., 2005). So in the two samples under study a (U/R = 7.6 and �3) which is P2 show a significant contamination bypetroleum products exist (Zaghden et al., 2007).

The isoprenoid hydrocarbons pristane and phytane are com-monly present in crude oils (Volkman et al., 1992) usually as majorconstituents among a much wider distribution of isoprenoidalkanes. Thus they are often considered as good indicators of petro-leum contamination (Volkman et al., 1992; Zaghden et al., 2007).

omposition Gemsa Sea water (extracted) sample

7.946.70 � 10�2

47365.09 1.03563

1.0366636052.5



5 10 15 20 25 30 35 40 45 50 55 60 65 70

V

b

a

α-Fe2O3

Zn5(OH)8Cl2

Fig. 2. (a) Chromatogram for crude oil sample and (b) chromatogram for the extracted oil from sea water.


The ratio of pristane to phytane (Pri/Phy = 0.22 and 0.20) in thetwo samples can be used as, fingerprint of oil contamination, alsothe occurrence of petroleum hydrocarbons is confirmed frompresence of phytane > pristane in agreement with previousinvestigators (Powell, 1988; Waseda and Nishita, 1998). AlsoC17/Pri) (n-Heptadecane/Pristine ratio), C18/Phy (nOctadecane/Phytaneratio), that showed that C17/Pri > 1(=38.07and 36.19) and C18/Phy > 1(=6.81and 8.15), indicating a fresh input of petroleum oilsin the studied area (Deng et al., 2013).

The absence of high peaks (n-C25 and C23) in the control studiedsample, revealed an absence of biogenic origin pollution (Leahyand Colwell, 1990).

3.4. Characterization of nanoparticles

Fig. 3 showed the XRD patterns obtained for a-Fe2O3 andZn5(OH)8Cl2 samples. The XRD patterns for a-Fe2O3 (Fig. 3a) indi-cated that, high intensity reflections at 2h = 33.18, 35.68 that ischaracteristic to a-Fe2O3 phase. None of the diffraction lines attrib-uted to any other phases was observed in the iron oxide sample.Fig. 3(b) showed clear sharp peaks which are characteristic forthe presence of Zn5(OH)8Cl2 material. Fig. 4 revealed, themicrographs of the a-Fe2O3 and Zn5(OH)8Cl2 nanoparticles. TheZn5(OH)8Cl2 particles (Fig. 4a) were highly agglomerated with

Fig. 3. The XRD pattern for the as prepar


roughly the average particle size of 60 mm. Fig. 4(b) illustratedthat, the iron oxide prepared by the thermal decompositionmethod has a relatively uniform particle size distribution ofapproximately 57 nm. Figure showed also that the prepared ironoxide has a hexagonal shape with slightly agglomerated particles.

3.5. Enumeration of halophilic bacteria in contaminated sea water

The isolation of total halophilic bacterial isolate from contami-nated sea water was enumerated on Luria broth (LB) plates med-ium. The halophilic bacterial count collected from the sea watersample was found to be 7 � 10�6 CFU/ml.

3.6. Isolation and identification of crude oil degrading bacteria

Hydrocarbon degrading bacteria were first isolated almost acentury ago. And a recent review lists 79 bacterial genera thatcan use hydrocarbons as the sole source of carbon and energy, aswell as, 9 cyanobacterial genera, 103 fungal genera and 14 algalgenera that are known to degrade or transform hydrocarbons(Head et al., 2006).

Fifteen bacterial strains were isolated from BHMS medium thatwas established at 30 �C for 4 weeks. Two of the isolated strainsthat showed a higher growth rate on crude oil were selected from

ed (a) Zn5(OH)8Cl2 and (b) a-Fe2O3.



Fig. 4. TEM micrographs of (a) a-Fe2O3 and (b) Zn5(OH)8Cl2.


the fifteen isolates for further study. During the growth of two bac-terial isolates, various changes of color, turbidity and dispersion ofcrude oil were observed within one day of incubation. Thesechanges indicated the ability of the different microorganisms inthe culture media to utilize petroleum hydrocarbons as a carbonand energy source. These strains (I1 and I2) were first identifiedusing molecular identification performed by amplifying andsequencing the 16S rRNA gene sequences. The results of the iden-tification procedure showed that the two isolated bacteria belongto the Pseudomonas xanthomarina KMM 1447 and Pseudomonasstutzeri ATCC 17588. Literature search revealed that various speciesof Pseudomonas and Bacillus are common inhabitants of petroleum-polluted ecosystems. Furthermore, these organisms are wellknown for their capacity to degrade a range of petroleum hydro-carbons (Radwan et al., 2005; Zhang et al., 2010).

3.7. Evaluation of growth rate and some surface properties ofbiosurfactant after biodegradation process

Biosurfactants, a group of surface active molecules synthesizedby diverse microorganisms, can reduce both surface and intersur-face tension, making these molecules useful in emulsification pro-cesses. The use of biosurfactants has been found to enhance the

Table 2Growth count, surface properties of biosurfactant and biodegradation percentage after tre

Sample Incubation periods (days)

After 3 days

Logcount

Emulsificationpower (E24)

Surfacetension (m

Control crude oil 0 0 65Crude oil + I1 8.3 30 40Crude oil + I1 + biosurfactant 8.8 58 42Crude oil + I2 7.8 0 40Crude oil + I2 + biosurfactant 8.8 0 40Crude oil + I1 + I2 7.8 70 38Crude oil + I1 + I2 + biosurfactant 8.8 68 40Crude oil + I1 + Fe2O3(NP) 7.0 65 45Crude oil + I1 + bio. + Fe2O3 (NP) 6.9 0 45Crude oil + I2 + Fe2O3 (NP) 8.3 60 46Crude oil + I2 + bio. + Fe2O3 (NP) 8.5 0 48Crude oil + I1 + I2 + Fe2O3 (NP) 8.0 0 40Crude oil + I1 + I2 + Fe2O3 (NP) + bio. 8.7 55 38Crude oil + I1 + Zn5(OH)8Cl2 (NP) 6 60 40Crude oil + I1 + Zn5(OH)8Cl2(NP) + bio. 8.3 75 40Crude oil + I2 + Zn5(OH)8Cl2 (NP) 8 0 45Crude oil + I1 + I2 + Zn5(OH)8Cl2 (NP) 6 0 45Crude oil + I1 + I2 + bio. + Zn5(OH)8Cl2(NP) 6.9 57 48

I1: Bacterial isolate no. 1, I2: Bacterial isolate no. 2, c: Crude oil, bio: Biosurfactant, (NP)a Percentage biodegradation = Weight of original oil – wt. of residual/wt. of original o


degradation of crude oil and other hydrocarbons (Batista et al.,2006).

Production of biosurfactants is known among crude oil andhydrocarbon-degrading bacteria (Ward, 2010; Franzetti et al.,2010; Mnif et al., 2011). In contrast to the proposed bacterialadherence to the oil droplets which was inferred from the observedfluctuations in culture density, the production of biosurfactantswas confirmed by the observed reduction in surface tension andinduction of emulsification power in crude oil culture. Moreover,dispersion of crude oil in most of the cultures was observed afew hours after the incubation. It is also worth mentioning thatthe presence of oils in the growth medium usually interferes withsurface tension measurements (Walter et al., 2010). Finally, therole of biosurfactants in biodegradation of hydrophobic com-pounds is still controversial (Ward, 2010).

In the present study, two bacterial strains (I1 and I2) were growntogether or separately on MSM medium. This medium contains 1%crude oil concentration with/without biosurfactants and two dif-ferent types of nanoparticles. Some factors related to biodegrada-tion were assayed after 3 days and 1 week. Data are shown in(Table 2) the growth capacity of the I1and I2 bacterial strainstogether with biosurfactants was greater than that of two bacterialstrains separately with/without biosurfactants. Moreover, Addition

atment by two different bacterial isolates.

Percentage ofbiodegradationa (%)

After 7 days

N/m)Logcount

Emulsificationpower (E24)

Surfacetension (mN/m)

0 0 65 –8.6 40 39 309.2 75 37 408.0 0 36 309.1 0 37 308.8 75 36 309.0 75 34 507.5 60 40 508.3 50 38 408.6 64 39 308.8 0 40 408.5 55 36 509.3 75 35 508.5 68 36 509.3 80 34 508.6 0 40 508.2 0 40 309.3 80 36 50

: nanoparticlesil � 100.




of a definite nanoparticle to the previous media enhanced the bac-terial growth rate and the biosurfactant efficiency. Furthermore,the microcosms (experimental ecosystem) containing crude oiland nanoparticles only enhanced the bacterial strain to producebiosurfactant. This achievement proved that the nanoparticleswere capable of assisting the microbial activities which agreeswith several other studies (Banat, 1995; Banat et al., 2000). Thehigher growth rate, lowest surface tension and the highest emulsi-fication power were obtained by the bacterial strain in microcosmscontaining the crude oil, biosurfactant and two different types ofnanoparticles (Fe2O3 and Zn5(OH)8Cl2). Additionally, there weredirect relationships between both the emulsification activity (E24)and the decrease in surface tension with increasing growth rateon hydrocarbons (Shahian et al., 2012; Ismail et al., 2013a,b). Bio-surfactant production by the bacterial isolate was associated withhigher values of growth, (Vater, 1986) which proved that the bio-surfactant production by Bacillus sp. was associated with cellulargrowth.

3.8. Biodegradation of crude oil by different bacterial strains

3.8.1. Gravimetric analysisThe percentage biodegradation of crude oil by the two bacterial

isolates (I1 and I2) was estimated by gravimetric analysis (Fig. 5)and listed in (Table 2). Table and figure showed that the bacterialstrains degraded in the range from 30% to 50% of the crude oil.The medium containing biosurfactant with two types of nanopar-ticles separately and two bacterial strains together gave higherpercentage of degradation. El-Sheshtawy (2011) the productionof biosurfactants by the activated bacterial strain (Bacillus subtilis)doubled its capacity to degrade crude petroleum oil from 12.2% to31.3% after incubation for 168 h under the optimum cultural con-ditions. Shahian et al. (2012) found a relationship between the lev-els of biosurfactant production and crude oil biodegradation; thestrains that produce high levels of biosurfactant can better degradecrude oil.

Table 3Distribution of carbon numbers in gas chromatogram of residual crude oil after

3.8.2. Gas chromatographic analysisThe first step in the aerobic degradation of alkanes by bacteria is

catalyzed by oxygenases. These enzymes, which introduce oxygenatoms derived from molecular oxygen into the alkane substrate,

0

5

10

15

20

25

30

35

40

45

50

55log count (%) biodegrada�on

log

coun

t and

(%)b

iode

grad

atio

n

Fig. 5. Relation between growth count and biodegradation percentage of crude oilby two different bacterial isolates after 7 days incubation period. Control crude oil,S1: Crude oil + I1, S2: Crude oil + I1 + biosurfactant, S3: Crude oil + I2, S4: Crudeoil + I2 + biosurfactant, S5: Crude oil + I1 + I2, S6: Crude oil + I1 + I2 + biosurfactant,S7: Crude oil + I1 + Fe2O3(NP), S8: Crude oil + I1+ bio. + Fe2O3 (NP), S9: Crudeoil + I2 + Fe2O3 (NP), S10: Crude oil + I2 + bio. + Fe2O3 (NP), S11: Crude oil + I1 + I2 +Fe2O3 (NP), S12: Crude oil + I1 + I2 + Fe2O3 (NP) + bio., S13: Crude oil + I1 + Zn5

(OH)8Cl2 (NP), S14: Crude oil + I1 + Zn5(OH)8Cl2(NP)+bio., S15: Crude oil + I2 + Zn5

(OH)8Cl2 (NP), S16: Crude oil + I1 + I2 + Zn5(OH)8Cl2 (NP), S17: Crudeoil + I1 + I2 + bio. + Zn5(OH)8Cl2(NP).


play an important role in oil bioremediation and in the co-meta-bolic degradation of compounds (Vomberg and Klinner, 2000).

In this study, the biodegradation of crude oil was analyzed after7 days of incubation period using the GC for aliphatic compounds.Table 3 showed the results of Gas Chromatographic analysis (GC)of the residual crude oil samples of different microcosms, andthe control sample (crude oil sample without treatment bymicroorganisms).

The percentage of residual n-paraffins and iso-paraffins presentin crude oil after biodegradation was calculated by comparing withthe undegraded control. The obtained data showed better degrada-tion of n-paraffins than iso-paraffins in microcosms containing bio-surfactants. While in microcosms containing Fe2O3 nanoparticlewith biosurfactants, better degradation of iso-paraffins than n-par-affins took place. The best biodegradation of iso-paraffins wasobserved in microcosm containing biosurfactant, Fe2O3 nanoparti-cle and (I1) bacterial isolate. On the other hand, the higher percent-age degradation of iso-paraffins was also obtained in culture mediacontaining biosurfactant, Zn5(OH)8Cl2 nanoparticles and (I2) bacte-rial isolate. These results indicate that, the biosurfactants assistedthe bacterial isolates to consume the n-paraffins over the iso-par-affins. On the contrary, the presence of biosurfactant with differenttypes of nanoparticles helps the bacterial isolates to consume theiso-paraffins more than n-paraffins which seems to be newly andvaluable biodegradation trend.

Hydrocarbons differ in their susceptibility to microbial attackand in the past they had generally been ranked in the followingorder of decreasing susceptibility: n-alkanes > branched alkane-s > low molecular weight aromatics > cycloalkanes (Mohamedet al., 2006; Paudyn et al., 2008). From the above results, it canbe concluded that the presence of nanoparticles can enhance theability of the bacterial strain in terms of improving the biosurfac-tant properties. Also, in microcosms containing different nanopar-ticles without adding biosurfactant, the bacterial strains were ableto produce biosurfactants which provide efficient surface proper-ties. However, so far, very limited studies have been reported onnanoparticles effect on the microbiological reaction rates (Zhanget al., 2011).

treatment by bacterial strain using biosurfactant and two different types ofnanoparticles.

Sample % Paraffins

n-Paraffins

Iso-paraffins

Negative control 55.93 44.07Crude oil + I1 60.05 39.95Crude oil + I1 + biosurfactant 50.98 49.02Crude oil + I2 46.45 53.55Crude oil + I2 + biosurfactant 54.46 45.54Crude oil + I1 + I2 56.44 43.56Crude oil + I1 + I2 + biosurfactant 47.81 52.19

Different types of nanoparticles (NP)Crude oil + I1 + F2eO3 nanoparticles (NP) 80.67 19.33Crude oil + I1 + biosurfactant + Fe2O3(NP) 83.85 16.15Crude oil + I2 + Fe2O3 nanoparticles (NP) 74.27 25.73Crude oil + I2 + biosurfactant + Fe2O3(NP) 81.24 18.76Crude oil + I1++ I2 + Fe2O3 (NP) 69.68 30.32Crude oil + I1++ I2 + Fe2O3 (NP) + biosurfactant 65.14 34.86Crude oil + I1 + Zn5(OH)8Cl2 (NP) 71.64 28.36Crude oil + I1 + Zn5(OH)8Cl2 (NP) + biosurfactant 74.08 25.92Crude oil + I2 + Zn5(OH)8Cl2 (NP) 70.8 29.2Crude oil + I2 + Zn5(OH)8Cl2 (NP) + biosurfactant 82.8 17.2Crude oil + I1 + I2 + Zn5(OH)8Cl2 (NP) 66.31 33.69Crude oil + I1 + I2 + biosurfactant + Zn5(OH)8Cl2

(NP)76.54 23.46

I1: Bacterial isolate no. 1, I2: Bacterial isolate no. 2.




3.8.3. High performance liquid chromatographic analysisThe biodegradation potential of the bacterial strains on polyaro-

matics was detected by HPLC analysis as given in Tables 4–6. Thedata in these tables show that, the different membered rings ofpolyaromatics were fully utilized in all microcosms of bacterial iso-late (I1) including (2- and 5- membered rings polyaromatics)except in microcosm containing zinc nanoparticles (NP). In addi-tion, the microcosm containing biosurfactant and Fe2O3 (NP)enhanced the strains to fully utilize 4- member rings polyaromat-ics. Also, in microcosms containing biosurfactant and different (NP)separately increased the percentage biodegradation of 2-, 3- and 6-membered rings polyaromatics.

The microcosms containing bacterial isolate (I2), different (NP)separately and crude oil, the 2-, 3- and 5- membered rings polyaro-matics were fully utilized Tables 5 and 6. Meanwhile, the

Table 4Polyaromatics distribution in crude petroleum oil after treatment by two bacterial isolate

Aromatic ring Compound of polyaromatics Negative control I1 + c

2 Naphthalene 35.55 0Total concentration (%) 35.55 0

3 Acenaphthylene 27.56 47.8Acenaphthene 2.51 4.1Fluorine 2.77 15.6Phenanthrene 0.14 1.5Anthracene 19.28 19.51Total concentration (%) 52.26 88.51

4 Fluoranthene 13.25 7.1Pyrene 6.21 3.8Benzo (a) anthracene 0 0Chrysene 0.07 0.09Total concentration (%) 19.53 10.99

5 Bezno (b) fluoranthene 0 0.07Bezno (k) fluoranthene 0 0Dibenzo(a,h)anthracene 0.25 0Total concentration (%) 0.25 0.07

6 Benzo(g,h,i) perylene 0 0Indeno(1,2,3-cd)pyrene 2.12 0.43Total concentration (%) 2.12 0.43

I1: Bacterial isolate no. 1, I2: Bacterial isolate no. 2, c: Crude oil, bio: Biosurfactant.

Table 5Polyaromatics distribution in crude petroleum oil after treatment by two bacterial strains

Number of rings Compound of polyaromatics Negative control I1 + Fe + c I1

2 Naphthalene 35.55 0 10Total concentration (%) 35.55 0 10

3 Acenaphthylene 27.56 48.48 0Acenaphthene 2.51 4.0 0Fluorine 2.77 12.0 0Phenanthrene 0.14 1.5 0Anthracene 19.28 20.87 0Total concentration (%) 52.26 86.85 0

4 Fluoranthene 13.25 7.3 0Pyrene 6.21 4.8 0Benzo (a) anthracene 0 0 0Chrysene 0.07 0.84 0Total concentration (%) 19.53 12.94 0

5 Bezno (b) fluoranthene 0 0 0Bezno (k) fluoranthene 0 0 0Dibenzo(a,h)anthracene 0.25 0.10 0Total concentration (%) 0.25 0.10 0

6 Benzo(g,h,i) perylene 0 0 19Indeno(1,2,3-cd)pyrene 2.12 0.11 69Total concentration (%) 2.12 0.11 89

I1: Bacterial isolate no. 1, I2: Bacterial isolate no. 2, c: Crude oil, bio: Biosurfactant, Fe: F


microcosms containing the bacterial isolates together (I1 and I2),biosurfactants and Fe2O3, Zn5(OH)8Cl2 (NP) separately enhancedthe isolates for increased degradation of 2-, 3- and 5- memberedrings polyaromatics Tables 5 and 6.

It is necessary to mention that, the increased degradation per-centage of any compound may be attributed either to a realincrease due to the formation of additional amounts of this polyar-omatic hydrocarbon (this is only accepted in the case of lowerpolyaromatics) or the other probability is the enhanced consump-tion of other compounds leading to a relative accumulation of suchployaromatic hydrocarbon (El-Bastawissy et al., 2004).

From the results obtained in this study, it can be concluded that,the complete degradation of some different membered rings ofpolyaromatics and the percentage biodegradation of other polyaro-matics increased in microcosms containing two different types of

with biosurfactant using HPLC analysis.

I1 + c + bio I2 + c I2 + c + bio I1 + I2 + c I1 + I2 + c + bio

0.49 31.28 0 0 00.49 31.28 0 0 0

19.43 22.14 8.3 8.9 20.310 1.49 0.94 0.73 5.772.31 9.67 0 0.5 23.300.49 1.72 3.2 0.2 2.192.3 17.58 0 1.1 29.46

24.53 52.6 12.44 11.43 81.03

0.73 7.65 0.94 0.1 10.886.07 6.72 0 0.1 6.210 0 0.1 0 00 0.09 0 0.1 0.586.8 14.46 1.04 0.3 17.67

0 0 0.09 0.12 00 0 0 0.05 00 0 0 0.5 00 0 0.09 0.67 0

0 0 44.79 85.90 068.08 1.66 41.64 1.7 1.368.08 1.66 86.43 87.6 1.3

with Fe2O3 nanoparticles using HPLC analysis.

+ Fe + c + bio I2 + Fe + c I2 + Fe + c + bio I1 + I2 + Fe + c I1 + I2 + Fe + c + bio

.1 32 0 23.97 0

.1 32 0 23.97 0

0 51.95 0 48.41.9 3.32 2.48 3.90 11.05 0 9

.4 1.3 1.19 1.16 1.50 10.67 0 20.59

.4 3.2 78.18 3.64 83.39

0.9 0 1.65 6.70 14.22 0 4.40 0 0 00 0 0 0.40.9 14.22 1.65 11.50

0 0 0.33 00 0 0.17 0.110 0 0 00 0 0.5 0.11

.2 0 0 0 0

.8 63.9 7.6 70.24 5.0

.00 63.9 7.6 70.24 5.0

2eO3 nanoparticles (NP).



Table 6Polyaromatics distribution in crude petroleum oil after treatment by two bacterial isolate with Zn5(OH)8Cl2 nanoparticles using HPLC analysis.

Number of rings Compound of polyaromatics Negative control I1 + Zn + c I1 + Zn + c + bio I2 + Zn + c I2 + Zn + c + bio I1 + I2 + Zn + c I1 + I2 + Zn + c + bio

2 Naphthalene 35.55 0 32.22 43.07 0 0 20.92Total concentration (%) 35.55 0 32.22 43.07 0 0 20.92

3 Acenaphthylene 27.56 0 13.44 0 22.38 3.41 0Acenaphthene 2.51 5.89 2.67 2.5 5.12 0.90 5.77Fluorine 2.77 21.60 0 6.6 0 14.02 12.98Phenanthrene 0.14 2.22 1.6 1.6 0.33 3.25 5.77Anthracene 19.28 34.27 31.53 0 62.65 32.65 0Total concentration (%) 52.26 63.98 49.24 10.7 90.48 54.23 24.52

4 Fluoranthene 13.25 21.60 0 7.5 0 12.35 3.35Pyrene 6.21 8.62 17.33 29.8 5.75 29.57 2.88Benzo (a) anthracene 0 0 0 0 0 0.08 0.03Chrysene 0.07 1.62 0 0 0 0.39 0Total concentration (%) 19.53 31.84 17.33 37.3 5.75 42.39 6.26

5 Bezno (b) fluoranthene 0 0 0.30 0.23 0 0.48 0Bezno (k) fluoranthene 0 0.17 0.04 0 0 0.21 0Dibenzo(a,h)anthracene 0.25 0 0 0 0.1 0 0Total concentration (%) 0.25 0.17 0.34 0.23 0.1 0.69 0

6 Benzo(g,h,i) perylene 0 0 0 0 2.58 0.82 0Indeno(1,2,3-cd)pyrene 2.12 4.01 0.87 8.7 1.09 1.87 48.30Total concentration (%) 2.12 4.01 0.87 8.7 3.67 2.69 48.30

I1: Bacterial isolate no. 1, I2: Bacterial isolate no. 2, c: Crude oil, bio: Biosurfactant, Zn: Zn5(OH)8Cl2 nanoparticles (NP).


nanoparticles with biosurfactant at 7 days. However, so far, verylimited studies have been reported on nanoparticles effect on thebiodegradation of crude oil contaminated sites in the presence ofbiosurfactants. Thus, there are plenty of aspects required to bestudied. Each variety of nanoparticles has their own characters.Meanwhile, the selection of the optimal band of nanoparticlesand microorganisms is considered a great help of the reactionrates. Proper nanoparticle concentration should be explored refer-ring to that the excessive concentration can be toxic to microor-ganism, thus, it may reduce the reaction rate. The reactionconditions in the presence of nanoparticles and microorganismsshould be studied to find out the optimal conditions for reaction.The combination pattern, coated on the microorganism or inde-pendently dispersed with microorganisms in the solution, is alsoan important aspect of the reaction rate effect (Zhang et al., 2011).

4. Conclusions

In this study, two crude oil-degrading bacterial strains were iso-lated from the Gemsa Bay, in the Red Sea, Egypt. There is a directrelationship between both the emulsification activity (E24) andthe decrease in surface tension with increasing the growth rateon hydrocarbons. The presence of biosurfactant with differenttypes of nanoparticles helps the bacterial isolates to consume theiso-paraffins more than n-paraffins. This result seems to be newlyand valuable biodegradation trend. The complete degradation ofsome different membered rings of polyaromatics and the percent-age biodegradation of other polyaromatics increased in micro-cosms containing two different types of nanoparticles withbiosurfactant at 7 days. Thus, these bacterial isolates have a poten-tial to be applied in the bioremediation of petroleum contaminatedsites using biosurfactant and specific concentration of Fe2O3 andZn5(OH)8Cl2 nanoparticles.

Acknowledgments

This work was sponsored by the Egyptian Petroleum ResearchInstitute (EPRI), Cairo, Egypt. The authors would also like to thankProf. Dr. Ahmed Hashem faculty of science, Ain Shams University.


Also the authors would like to thank Mr. Walid Gad Kamal (bio-technology lab in EPRI) for his assistance and laboratory support.

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http://dx.doi.org/10.4172/2157-7463.1000163


































http://dx.doi.org/10.1038/333604b0




































http://dx.doi.org/10.1016/j.marchem.2006.12.016

http://dx.doi.org/10.1016/j.marchem.2006.12.016










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