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Biodegradation of Baleym Mix CrudeOil in Soil Microcosm by Some LocallyIsolated Egyptian Bacterial StrainsLaila A. Farahat a & Nour Sh. El-Gendy aa Egyptian Petroleum Research Institute (EPRI) , EgyptPublished online: 25 Feb 2008.

To cite this article: Laila A. Farahat & Nour Sh. El-Gendy (2008) Biodegradation of Baleym MixCrude Oil in Soil Microcosm by Some Locally Isolated Egyptian Bacterial Strains, Soil and SedimentContamination: An International Journal, 17:2, 150-162

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Soil & Sediment Contamination, 17:150–162, 2008Copyright © Taylor & Francis Group, LLCISSN: 1532-0383 print / 1549-7887 onlineDOI: 10.1080/15320380701872886

Biodegradation of Baleym Mix Crude Oil in SoilMicrocosm by Some Locally Isolated Egyptian

Bacterial Strains

LAILA A. FARAHAT AND NOUR SH. EL-GENDY

Egyptian Petroleum Research Institute (EPRI), Egypt

Petroleum refining, transportation and storage are sources of soil contamination. InEgypt, a large number of contaminated sites exist as a result of different crude oil ac-tivities. In this work, biodegradation experiments in artificially oil contaminated soilmicrocosms (30 cm × 26 cm × 8 cm) of shallow polypropylene plastic trays were car-ried out with special emphasis on biodegradation of polycyclic aromatic hydrocarbons(PAHs). Two promising isolates, Pseudomonas aeruginosa I.1.1.6 and Brevibacteriumcasei I.2.1.7, previously isolated from Egyptian hydrocarbon contaminated soil, wereused. The microcosms were inoculated at 30◦C for 60 days with a water content of60%wt. TPH were quantitatively monitored, gas chromatographic (GC) and high per-formance liquid chromatographic (HPLC) analysis were preformed to follow up thebiodegradation process. The results obtained indicate that after 60 days of incubation;Pseudomonas aeruginosa I.1.1.6 showed higher TPH biodegradation efficiency (64%)than that of Brevibacterium casei I.2.1.7 (58%). Bacterial isolates I.1.1.6 and I.2.1.7showed total normal alkanes degradation of approximately 74% and 90%, respectively.Generally they are capable of degrading high molecular weight PAHs (five and six mem-bered ring compounds, benzo[b]fluoranthene; BbF and indeno[1,2,3-cd]Pyrene, IP, andhave broad versatility in their action on the16PAH compounds currently regulated bythe US Environmental Protection Agency (EPA) as priority pollutants, and could beuseful in bioremediation of soil contaminated with crude oil.

Keywords Bioremediation, soil, microcosm, crude oil, polycyclic aromatic hydrocar-bons, Pseudomonas aeruginosa, Brevibacterium casei

Introduction

Environmental contamination by petroleum and its products is widespread. Petroleumrefining, transportation, and storage are sources of soil contamination. In some cases, it isdue to accidental events (oil spills), but in others it is a consequence of negligent disposal ofsludge residues that accumulate in oil storage tanks. Soil contamination with hydrocarbonscauses extensive damage to local ecosystem since accumulation of such pollutants in tissuesof animals and plants may cause death or mutation (Labud et al., 2007).

In Egypt, a large number of contaminated sites exist as a result of different petroleum oilactivities. Recently, this problem has motivated researchers to remediate these contaminatedsites. Mechanical and chemical techniques are the commonly applied methods in Egyptfor getting rid of oil spills or the diverse by-products obtained from the petroleum industry.

Address correspondence to Nour Sh. El-Gendy, Egyptian Petroleum Research Institute, NasrCity, Cairo, Egypt. E-mail: nourepri@yahoo.com

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Biodegradation of Baleym Mix Crude Oil in Soil Microcosm 151

Actually, these tools are not adequate for complete removal of such oil contaminants. Toconfront and resolve this enormous problem, remediation must be simple and cost-effectivefor both industrialized nations and under-developed countries. Therefore, bioremediationis considered as a viable alternative for this purpose. The Egyptian oil industry viewsbioremediation of drilling mud, tank bottoms and contaminated refinery soils as a toppriority. It is well known that it can be used successfully to remediate many hydrocarbon(HC) polluted sites, converting the toxic compounds of oil to non-toxic products withoutfurther disruption of the local environment, and can be applied as a polishing step afterconventional mechanical and/or chemical clean-up options have been applied (Van-Hammeet al., 2003; Chaineau et al., 2005).

Microbiologists have known since the 1940s (ZoBell, 1946) that microorganisms un-der favorable conditions can utilize petroleum hydrocarbons as a metabolic carbon source.Microbial interactions in the soil are very complex and play an important role in the trans-formation or decomposition of hazardous waste components (JRB Associates, Inc., 1984).Some members of a microbial community might be able to provide important degradingenzymes, whereas others may supply surfactants (Wiesel et al., 1993). Brevibacteriumstrains were investigated in different bioremediation studies of PAHs and polyaromaticsulfur heterocyclic compounds (Trenz et al., 1994; Chaillan et al., 2004). Pseudomonassp. is often isolated from hydrocarbon-contaminated sites and hydrocarbon-degrading cul-tures. This genus has a broad affinity for hydrocarbons and can degrade selected alkanes,alicyclics, thiophenes, and aromatics (Van-Hamme et al., 2003). It has potential for appli-cation in treatment systems for Egyptian industrial effluents rich in aromatic compounds(El-Tayeb et al., 1998; Abd-El-Haleem et al., 2002; El-Masry et al., 2004). It also producesa range of biosurfactants, e.g. rhamnolipids (Sim et al., 1997; Desai and Banat, 1999).Different Pseudomonas aeruginosa strains that have the ability to degrade different com-ponents of crude oils have also been isolated (Hwang and Cutright, 2002; Jacques et al.,2005).

PAHs are among the most common pollutants found at contaminated industrial sites.Although they are naturally occurring compounds and essentially ubiquitous at low concen-trations in the terrestrial environment, high concentrations of these compounds are found incontaminated soils near petroleum processing operations. There are wide ranges of PAHs,from the simple two-ring compounds naphthalene (Naph.) to the large multi-ring com-pounds. Compounds with two or three rings are arbitrarily defined as low molecular weight(LMW) species, while those with four or more rings as high molecular weight (HMW)species. Particular attention was paid to polycyclic aromatic hydrocarbons (PAHs) as theyare a class of ubiquitous persistent environmental contaminants with high toxic, mutagenic,and carcinogenic properties (Aichberger et al., 2006). The US Environmental ProtectionAgency (EPA) currently regulates 16 PAH compounds as priority pollutants for remediation(King et al., 2004; Abbondanzi et al., 2006).

Measuring the success of oil spills removal is based on several parameters, amongthem the degradation of PAHs present. Lab-scale microcosms serve as an important stagein the study of the biodegradation efficiencies of microorganisms and can also serve asstandard test systems that can be adapted to different environmental conditions. Results ofmicrocosm studies are often used to develop remedial pilot test specifications (Lepo andCripe, 1999).

In a previous work two bacterial strains, Pseudomonas aeruginosa I.1.1.6 and Bre-vibacterium casei I.2.1.7, were isolated from Egyptian hydrocarbon polluted soil for theirability to degrade Baleym Mix crude oil in two liquid phase laboratory experiments (Farahatet al., 2006).

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This work aims to study the efficiency of these two bacterial isolates to bioremediateartificially contaminated soil with Baleym Mix crude oil on microcosm levels with specialemphasis on biodegradation of PAHs. The use of artificially sterilized polluted soil is afirst step to study the biodegradation effect of each tested bacteria separately without theinfluence of any other factors or organisms.

Materials and Methods

Medium

Luria-Bertani medium (LB) used for obtaining biomass was prepared according to Kirimuraet al. (2001) and consists of 10 g/l Tryptone, 5 g/l Yeast extract and 10 g/l NaCl, dissolvedin 1liter distilled water and adjusted to pH 7 with 10% NaOH before sterilization.

Microorganisms

The bacterial strains Pseudomonas aeruginosa I.1.1.6 and Brevibacterium casei I.2.1.7 usedin this study were previously isolated from Egyptian hydrocarbon contaminated soil (Fara-hat et al., 2006). They were characterized by their morphological and biochemical propertiesaccording to the Bergey’s Manual of Systematic Bacteriology (Sneath et al., 1986; Hensyl,1994) and Bergey’s Manual of Determinative Bacteriology (Holt et al., 1994). Identifica-tion of bacterial strains was confirmed using Biolog system model, Biolog/Microlog 3420program (Egyptian Plant Disease Research Institute).

Analytical Methods

GC-FID Analysis. Gas chromatography (GC) model Agilent 6890 was used to follow upthe degradation of aliphatic hydrocarbons, using the following system:

Column Capillary HP-1 (polymethylsiloxane)30 m × 0.35 mm (I. D.) × 0.25µm (Film)

Injector temperature 320◦CDetector FIDOven temperature From 100◦C to 320◦C at fixed rate 5◦C/minCarrier gas Nitrogen with flow rate; 2 ml/minSample size 0.5 µl

High Performance Liquid Chromatographic Analysis (HPLC). PAHs identification andquantification in the extracted oil of all samples were performed using HPLC modelWaters 600E equipped with auto sampler Waters 717 and dual wavelength absorbancedetector Waters 2487 set at 254 nm. PAHs standards (HPLC grade) were obtainedfrom Supelco. Standard curves were established. The conditions of separation were asfollows:

Column Supelcosil LC-PAH, 15 cm × 4.6 mm ID 5 µm particles sizeMobile phase Acetonitrile/H2O HPLC grades, gradient from 50:50 to 100% AcetonitrileFlow rate Gradient program; 0–2 min; 0.2 ml/min then 2–45 min; 1.0 ml/min

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Biodegradation of Baleym Mix Crude Oil in Soil Microcosm 153

Preparation of the Soil and Supplementation of Crude Oil. In this work the laboratory testwas conducted in shallow polypropylene plastic trays of approximately 30 cm × 26 cm ×8 cm (width, length, height).

Sandy soil obtained from El-Soukhna Beach, Egypt, was ground in a mortar to a finegranular powder and sieved through standard electrical sieves (<6 mm) to simulate uniformsoil composition.

500 g of the sieved dry soil were placed in each of the plastic trays. 5 g of Baleym Mix(BX) crude oil were added, uniformly mixed and homogenized well.

The soils were sterilized three times by autoclaving at 121◦C for 30 min (twice beforethe addition of the crude oil and once after its addition). These steps were carried out to killindigenous bacteria in the soil.

Addition of Nutrients and Surfactant. Due to the high carbon content of oil and the lowcontents of the other essential nutrients necessary for microbial growth, supplementationof contaminated soil with phosphorous and nitrogen is generally required to enhance thegrowth of HC degrading bacteria and to stimulate oil degradation (Swannel et al., 1995).

Nitrogen as NH4Cl and potassium as K2HPO4 were added to give a final concentrationequivalent to a C: N: P mass ratio of 100:10:1; the nutrients used are of GR grade.

30 µl of the surfactant; Tween 80 in 20 ml of distilled water was autoclaved at 121◦Cfor 30 min, then added to each tray and the soil was homogenized well. Tween80 has beenused in several studies and recommended as an efficient emulsifier (Sabate et al., 2004).

Inocula Preparation. Each of the bacterial isolates was grown separately in LB mediumfor 48 hours at 30◦C, pH 7 on a shaking incubator of 150 rpm.

Each isolate was harvested by centrifugation at 5000 rpm for 5 min., washed twicewith sterile saline (8.5 g NaCl per one liter distilled water), suspended and then added tothe trays of contaminated soil, yielding a final concentration of approximately 108 TCFU/gsoil at zero time, the most recommended inoculum size in most of the microcosm studies(Mishra et al., 2004; Sabate et al., 2004).

One tray was kept without the addition of microorganisms under the same conditionsof the inoculated trays as a negative control.

Incubation

The microcosms were incubated at 30◦C for 60 days. During this period of time sterilizeddistilled water was added regularly to keep the water content 60% wt. This moisture contenthas been used in several studies and lies within the range recommended by Dibble and Bartha(1979) and Sabate et al. (2004).

To each microcosm, cheesecloth-covered cotton batting about 3cm thick was applied.By this means microbiological control of the microcosms over the entire test period wasestablished as much as it could be.

The soil was mixed well daily under aseptic conditions with a sterile spatula to keepthe humidity homogenous and to guarantee good aeration. All of the above steps were doneunder aseptic conditions.

Monitoring of Biodegradation and Bacterial Growth. 10 g of the artificially contaminatedsoil were taken at different prescribed time intervals for TPH, GC and HPLC analysisto follow up the biodegradation process and to estimate the total viable bacterial count(TCFU/g soil). Briefly, 1 g soil samples were added to 10 ml of sterile saline (8.5 g NaCl

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per one liter of distilled water), vortexed for 30 min, and then serially diluted to 10−10.Serial dilutions of these suspensions were inoculated on LB/agar plates and incubated at30◦C for 48 hours to enumerate TCFU.

Hydrocarbon Assessment of Oily Soil. Extraction and analysis of the total petroleum hy-drocarbons (TPH) of the oily soil (OS) were carried out as follows:

� Soil dry mass (DM) and moisture content were calculated from the weight loss afterheat treatment for 20h at 105◦C (Margesin et al., 2003).

� The TPH concentration in the extract was determined gravimetrically. Extraction ofdried soil sample was carried out in a Soxhlet extractor apparatus using a mixture of n-hexane and dichloromethane (1:1 v/v) (Viguri et al., 2002). Percentage of degradationwas calculated according to Bento et al. (2004) by the following expression:Percentage degradation = [(TPHinitial– TPHtreated) /TPHinitial] × 100

� The extracted oil samples were analyzed employing GC/FID to determine the degreeof biodegradation of aliphatic hydrocarbons (Mac Naughton et al., 1999). The %loss of biodegraded n- and iso-paraffins was calculated by comparing the areas ofthe peaks obtained in case of treated samples with those of crude oil used (at zerotime), taking into consideration that the amounts of injected samples were exactlythe same in all cases.

� The total concentration of the US-EPA 16PAHs in the extracted crude oil from themicrocosms before and after treatment with the two bacterial isolates were monitoredat each prescribed time using HPLC and recorded.

Results and Discussion

Laboratory-contaminated microcosms serve as an important function in the study ofbiodegradation efficiency of natural strains or genetically modified organisms (GMOs)and can also serve as standard test systems that can be adapted to a variety of environmentalconditions (Mishra et al., 2004).

Monitoring the survival of the introduced highly efficient microbial degraders at thecontaminated site is an essential attribute in the reduction of contamination levels by theintroduced organisms (Mac Naughton et al., 1999; Margesin and Schimer, 2001; Margesinet al., 2003; Mishra et al., 2004).

No culturable microorganisms were found in the negative control microcosm through-out the incubation period.

There was a general depletion in total viable count (TCFU/g soil) observed after 10 daysof incubation, as listed in Table 1. Similar observation was reported by Sabate et al. (2004).According to Bento et al. (2004), initially bacterial isolates were stimulated by labilehydrocarbon sources that induce a high rate of biodegradation. As those sources decrease,microbial population uses the more recalcitrant hydrocarbons less efficiently. With the de-crease in labile carbon sources, nutrients become most likely limited in supporting microbialgrowth. The decrease of total viable count could be attributed to the subsequent depletionof nutrients and the presence of toxic metabolites resulting from degradation (Boopathy,2000).

Degradation efficiency for TPH of the bacterial isolates was estimated and illustratedin Table 1. Pseudomonas aeruginosa I.1.1.6 showed higher TPH biodegradation efficiency(64%) than that of Brevibacterium casei I.2.1.7 (58%).

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Biodegradation of Baleym Mix Crude Oil in Soil Microcosm 155

Table 1Growth (TCFU/g soil) and biodegradation potential (%BD) of bacterial isolates on oily soil

microcosm

Pseudomonas aeruginosa Brevibacterium caseiI.1.1.6 I.2.1.7

Time (days) TCFU %BD TCFU %BD

Zero 3 × 108 — 3 × 108 —10 2 × 1010 39 2.5 × 1010 4520 2 × 109 43 4 × 109 5230 9 × 104 59 3 × 106 5445 3 × 104 60 2 × 105 5860 4 × 103 64 3 × 104 58

There was no significant loss in TPH concentration in the negative control microcosmrecorded after 60 days of incubation.

The rate of biodegradation of TPH was high during the first 20 days of incubationand then decreased. Song et al. (1990) and Sabate et al. (2004) reported similar resultswith a residual fraction in soil after a bioremediation experiment. As described in otherstudies, the degradation pattern of organic chemicals in soil usually shows a rapid initialphase of degradation followed by a period of little or no change in concentration. Thiskinetic is known as the “hockey stick” phenomenon (Alexander, 1999). A depletion ofnutrients, a decrease of microbial populations, lower bioavailability and higher recalcitranceof residual contaminants explain this kind of dynamic (Alexander, 1999; Sabate et al., 2004).Formation of toxic metabolites has been suggested in many instances, but the search fortoxic metabolites has not yet been successful.

It was evident by GC/FID analysis that the aliphatic hydrocarbons were degradedextensively in the studied soil microcosms, as shown in Figure 1 and recorded in Table 2.For example; total normal alkanes were degraded by approximately 74% and 90% after 60days of incubation using Pseudomonas aeruginosa I.1.1.6 and Brevibacterium casei I.2.1.7,respectively. nC17 was degraded by approximately 93% and 91% and nC18 was degradedby approximately 62% and 98% after 60 days of incubation using I.1.1.6 and I.2.1.7,respectively. It was reported by Lepo and Cripe (1999) that the addition of nitrogen andphosphorous nutrients stimulate the alkane-degrading microbes and result in a substantialdepletion of the n-alkane fraction. All the n-alkanes groups, as listed in Table 2, weredegraded at approximately the same rate, regardless of the chain length of n-alkanes. Thesame observation was reported by De Jonge et al. (1997).

There was no significant loss observed in the aliphatic hydrocarbons of the negativecontrol microcosm after 60 days of incubation, except in the relatively light fraction C10-C15,where there was a loss of approximately 10%.

Although the branched alkanes, pristane Pr (2, 6, 10, 14-tetramethylpentadecane) andphytane Ph (2, 6, 10, 14-tetramethylhexadecane), have been used as conservative biomarkersin oil bioremediation studies, their recalcitrance to biodegradation has been examined inseveral reports (Pritchard, 1993; Huang et al., 2004; Chaineau et al., 2005). In spite of therecalcitrance of Pr and Ph, there was evidence that both bacterial isolates were able to attackthese hydrocarbons. I.1.1.6 and I.2.1.7 degraded Pr by approximately 99% and 98% after 60days of incubation, respectively, while Ph was degraded by approximately 96% and 99%,

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Figure 1. Biodegradation of aliphatic hydrocarbon over time.

respectively, after the same incubation period. In a 105-day field experiment on petroleumhydrocarbons contaminated sediment conducted by Xu et al. (2004), approximately 97%of the branched alkanes were lost. In addition, Xu et al. (2004) reported that pristaneand phytane are not reliable to serve as conservative biomarkers for monitoring of oilbiodegradation in beach sediments.

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Biodegradation of Baleym Mix Crude Oil in Soil Microcosm 157

Table 2Monitoring of n- and iso-alkanes of crude oil over time

Pseudomonas aeruginosa Brevibacterium caseiI.1.1.6 I.2.1.7

Sample Zero 10 30 60 Zero 10 30 60parameter time days days days time days days days

Pr/Ph 1.1 1 1.7 0.3 1.1 0.4 5 2.5nC17/Pr 2.4 4.3 4.8 16.9 2.4 13 4.9 18.3nC18/Ph 2.6 6.3 10.1 22.1 2.6 12 68.5 54.3

Loss of each n-alkane group (%)C10–C15 — 21.4 42.1 75.3 — 52.4 77.8 87.3C16–C20 — 35.4 56.4 67.6 — 64 85.3 92.1C21–C25 — 56.5 65.7 79.4 — 52.7 87.6 91.2C26–C30 — 28.1 54.8 77.2 — 33.4 79.9 90.7total — 34.2 53.3 74 — 53.5 81.9 90.1Pristane (Pr) — 75 82 99 — 95 96 98Phytane (Ph) — 73 88 96 — 84 98 99

Changes in the ratios of nC17/Pr, nC18/Ph and Pr/Ph have been long recognized andused as indicators of biodegradation (Wang and Fingas, 2003; Zytner et al., 2006). Ratiosof Pr/Ph, nC17/Pr and nC18/Ph, calculated over the prescribed time intervals, were recordedin Table 2 and illustrated in Figure 2. Generally, there is an increase in nC17/Pr and nC18/Phratios after 60 days of incubation. By plotting nC17/Pr vs. nC18/Ph it is clear that degradationratios generally increase linearly with time in the case of microcosms treated with I.1.1.6.But they fluctuated throughout the incubation period and didn’t follow a certain trend inmicrocosms treated with I.2.1.7. The observed degradation of pristane and phytane in thisstudy gave an explanation for the general increase of nC17/Pr and nC18/Ph ratios. Generally,the nC17/Pr ratio was lower than that of nC18/Ph for both treatments with I.1.1.6 and I.2.1.7.Similar observations were reported by Diaz et al. (2000) and this was thought to be anindication of the preferential biodegradation of phytane. On the other hand, the Pr/Phratio fluctuated throughout the incubation period, sometimes lower than zero time value,suggesting selective degradation of pristane, especially after 60 days of incubation in thecase of I.1.1.6 and after 10 days of incubation in the case of I.2.1.7. The high value of Pr/Phratio after 30 days of incubation in the case of I.2.1.7 confirms the selective and preferentialbiodegradation of phytane. Although it decreased again after 60 days of incubation, it isstill higher than the ratio at zero time.

The concentrations of the 16-PAHs in the extracted crude oil from the microcosmsbefore and after treatment with the two bacterial isolates were monitored at the pre-scribed time intervals using HPLC. Biodegradation percentages of the PAHs are recorded inTable 3.

HPLC qualitative analysis of the individual PAHs for the extracted sample of BX-crudeoil at zero time shows that some compounds of the16-PAHs listed by EPA were not detected.These are acenapthylene, benzo[k]fluoranthene, benzo[a]Pyrene, dibenzo[a,h]anthracene,benzo[g,h,i]perylene. Generally, petrogenic origin of PAHs is characterized by the domi-nance of LPAHs over the HPAHs (Moustafa, 2004).

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Figure 2. Biodegradation effect on ratio of nC17/Pr, nC18/Ph and Pr/Ph.

In the negative control microcosm after 60 days of incubation, the amount of PAHsis slightly affected, showing slight loss in naphthalene (Naph.) of about 15%, triaromaticring compounds of about 5%, four aromatic ring compounds of 1%. No loss occurred forthe five and six membered ring compounds. This indicates that the abiotic loss of PAHswas generally negligible and no or slight biodegradation occurred as bacteria had beeninactivated or killed by autoclaving.

It is obvious from the results listed in Table 3 that the two bacterial isolates, Pseu-domonas aeruginosa I.1.1.6 and Brevibacterium casei I.2.1.7, have broad versatility in theiraction on PAHs. Generally, the rate of biodegradation of PAHs using I.1.1.6 was higher thanthat of I.2.1.7. They showed good utilization of naphthalene (diaromatic ring compound),so that no remaining naphthalene was observed after 60 days of incubation. This resultis in agreement with that of Brakstand et al. (2004), who showed that biodegradation ofnaphthalene can reach 99–100% after 21 days.

For the three membered rings compounds, the isolates are superior in completelyremoving acenaphthene at different rates, taking into consideration that the sample beforetreatment is characterized by a relatively high concentration of acenaphthene. They have the

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Table 3Biodegradation potential of bacterial isolates on PAHs

%Biodegradation through out the incubation period (days)

Pseudomonas aeruginosa Brevibacterium CaseiI.1.1.6 I.2.1.7

Isolates 20 30 60 20 30 60PAHs days days days days days days

Naphthalene 97.47 100 100 4.57 73.95 100Acenaphthene 100 100 100 99.91 99.94 100Fluorene 100 100 100 73.32 100 100Phenanthrene 100 100 100 100 100 100Anthracene 90.20 90.20 100 100 100 100Fluoranthene 100 100 100 100 100 100Pyrene 96.64 100 100 51.83 98.51 100Benzo[a]anthracene 94.87 100 100 91.93 93.76 97.99Chrysene 82.14 82.14 100 47.32 82.14 100Benzo[b]fluoranthene 60 60 100 18 100 100Indeno[1,2,3-cd]pyrene 100 100 100 100 100 100

ability to completely degrade phenanthrene (Phe) and anthracene (Ant) at different intervaltimes. Results obtained reveal that isolate I.1.1.6 was superior in degrading fluorene (F)completely after 20 days of incubation, while isolate I.2.1.7 needed more than 20 days forits complete degradation.

The two individual bacterial cultures are able to degrade the four membered ringscompounds fluoranthene (Flu), pyrene (Pyr), benzo[a]anthracene (BaA) and chrysene (Chr)at different rates.

Generally, the two bacterial isolates are capable of degrading high molecularweight PAHs (five and six membered rings), including benzo[b]fluoranthene (BbF) andindeno[1,2,3-cd]pyrene (IP).

Aitken et al. (2005) reported the mineralization of benzo[a]anthracene, chrysene andbenzo[a]pyrene by pseudomonas saccharaphila p15. As none of the three compounds areused as growth substrates by the organism, this suggests that the metabolism of high molec-ular (H) PAHs using P. saccharaphila is linked to the metabolism of low molecular weight(L) PAHs. This might explain the complete degradation of the HPAHs in the microcosmsof the bacterial isolates throughout the 60 days incubation period, although the bacterialgrowth was inhibited as shown in Table 1.

Moustafa et al. (2003) studied biodegradation potentials of bacterial strain (ICM) iso-lated from Ismailya Canal, Egypt, for Baleym middle base oil and showed complete re-moval of pyrene, benzo[a]anthracene, benzo[k]fluoranthene and benzo[g,h,i]perylene after21 days of incubation. They attributed this to the chronic exposure of the isolated bacterialstrain to different hydrocarbon pollutants, mainly aromatics and alicyclic compounds in thecollection site, which may direct bacterial actions towards similar compounds.

The inhibition of growth, after 10 days of incubation, and decrease of TPH degradationrate, after 20 days of incubation, could be due to the accumulation of toxic intermediatesformed from the biodegradation of the four-ring compounds and other LPAHs. Many PAH

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intermediates, e.g. phenolic and benzoic acid intermediates, affect the survival and activityof microorganisms. These compounds are considered as biocidal at elevated concentrationsthat can rapidly limit biodegradation process (Kazunga and Aitken, 2000).

According to Supaka et al. (2001), the complete degradation of high molecular weightPAHs of four rings, five and six membered rings, indeno[1,2,3-cd]pyrene (IP), in absenceof growth may be attributed to co-metabolism, where the biodegradation of more labile lowmolecular weight PAHs of di- and tri-rings enhances and improves the biodegradation ofhigh molecular weight PAHs, in particular those with four or more aromatic rings.

Conclusion

From this study it can be concluded that the two bacterial isolates, Pseudomonas aeruginosaI.1.1.6 and Brevibacterium casei I.2.1.7, can be used in the bioremediation of soil systemscontaminated with crude oil. They have a broad versatility in biodegradation of differentconstituents of Baleym Mix crude oil n- and iso-alkanes, especially pristane and phytane.Although Pr and Ph are known to be used as biomarkers due to their recalcitrance forbiodegradation compared to nC17 and nC18. I.1.1.6 and I.2.1.7 have high biodegradingeffect on the 16 PAH compounds currently regulated by the US Environmental ProtectionAgency (EPA) as priority pollutants. It is always difficult to bridge the gap between basicand applied research and further study will be needed on a chronically oil-contaminatedsoil.

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