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International Biodeterioration & Biodegradation 89 (2014) 58e66

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier .com/locate/ ibiod

Characterization of hydrocarbon-degrading bacteria isolated fromoil-contaminated sediments in the Sultanate of Oman and evaluationof bioaugmentation and biostimulation approaches in microcosmexperiments

Raeid M.M. Abed a,*, Jamal Al-Sabahi b, Fatema Al-Maqrashi a, Amal Al-Habsi a,Manal Al-Hinai a

aBiology Department, College of Science, Sultan Qaboos University, P. O. Box 36, PC 123 Al Khoud, OmanbCentral Instrument Laboratory, College of Agricultural and Marine Sciences, Sultan Qaboos University, P. O. Box 34, PC 123 Al Khoud, Oman

a r t i c l e i n f o

Article history:Received 16 April 2013Received in revised form16 July 2013Accepted 13 January 2014Available online 11 February 2014

Keywords:BioremediationBioaugmentation16S rRNANutrientsAlkanesBacterial consortia

* Corresponding author. Tel.: þ968 92867442; fax:E-mail address: rabed@mpi-bremen.de (R.M.M. Ab

0964-8305/$ e see front matter � 2014 Elsevier Ltd.http://dx.doi.org/10.1016/j.ibiod.2014.01.006

a b s t r a c t

Two oil-polluted sediments (PD and KH) were sampled from a coastal region in Oman for the isolation ofhydrocarbon-degrading bacteria and for testing different bioremediation approaches. Fourty strains wereisolated, eighteen were affiliated to Marinobacter whereas the rest belonged to Pseudomonas, Halomonas,Hahella and Alcanivorax. All strains grew well at 2e7% salinity and between 20 and 60 �C. The strainsexhibited a better growth on long chain than on short chain alkanes. Biostimulation and bio-augmentation were compared in both sediments and oil biodegradation was followed by measuring CO2

evolution and by gas chromatography (GC). The evolved CO2 reached 0.45 � 0.02 and2.23 � 0.07 mg CO2 g�1 sediment after 88 days in the untreated PD and KH sediments, respectively.While the addition of inorganic nutrients resulted in 1.2e3.7 fold increase in CO2 evolution in bothsediments, the addition of the bacterial consortiumwas only effective in the PD sediment. The maximumCO2 evolution was measured when both nutrients and bacteria were added and this corresponded to atotal oil mineralization of 2.6 � 0.12 and 1.49 � 0.04% of the initial oil after 88 days in the PD and KHsediments, respectively. GC analysis confirmed the CO2 data and showed that most of the degradedcompounds belonged to alkanes. We conclude that the Omani polluted sediments contain halotolerantand thermotolerant bacteria and biostimulation is more efficient than bioaugmentation for their cleanup.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Oil production is the backbone of the economy in the Sultanateof Oman, and more than 60% of the oil transported from the Gulfregion passes through its waters (Petrov, 2008). Enormous effortsare exerted to prevent oil contamination, however, small and largescale discrete spills can never be completely excluded. Contami-nation can occur by tanker accidents or tankers dumping ballastwater along the coasts as well as during oil exploration and pro-duction. Oil spills, regardless of their cause, pose a serious threat forterrestrial and marine ecosystems and urgent remediationmethods are always needed (Swannell et al., 1996).

Oil spill incidents have prompted the development of differentphysical, chemical, as well as biological techniques for dealing with

þ968 24141437.ed).

All rights reserved.

oil pollution (Swannell et al., 1996; Smith, 1996). Bioremediationhas been widely applied as a successful and cost-effective tech-nique to treat different oil spills around the globe (Swannell et al.,1996; Vogel, 1996; Lee and Merlin, 1999; Timmis and Pieper,1999). Two main bioremediation approaches are known; bio-stimulation and bioaugmentation. Biostimulation involves theaddition of inorganic (mainly nitrogen and phosphorus) andorganic fertilizers and was found to stimulate the growth ofindigenous oil degraders and to increase biodegradation rates(Ward and Brock, 1978; Rivet et al., 1993; Bragg et al., 1994; Lee andMerlin, 1999). Bioaugmentation involves the addition of oil-degrading bacteria to supplement the existing microbial commu-nities. This approach showed considerable success in bio-remediating several polluted sites, although in some cases, bacteriaisolated from other contaminated sites could not survive in the newenvironment (Vogel, 1996; Ko and Lebeault, 1999; Lee and Merlin,1999; Timmis and Pieper, 1999). Bioaugmentation was beneficialin shortening the period required for the degradation of pollutants

Table 1Characteristics of the two studied sediments.

Parameter Unit PD KH

TPH mg g�1 22 � 0.7 55 � 0.6MPN on hexadecane 2.7 � 106 5.6 � 106

pH 7.46 7.43Salinity % 2 3.5Soil textureSand % 30 43Clay % 8 6Silt % 62 51

Soil type Silt loam Silt loamNitrate mg l�1 21.13 2.18Phosphate mg l�1 0.46 NDFluoride mg l�1 0.048 0.034Bromide mg l�1 12.15 3.45Sulfate mg l�1 451 27

TPH: Total petroleum hydrocarbons; ND: not detected.MPN: Most probable number.

R.M.M. Abed et al. / International Biodeterioration & Biodegradation 89 (2014) 58e66 59

and in the degradation of compounds that are relatively recalcitrantto degradation such as chlorinated hydrocarbons (Vogel, 1996).

Although there is a substantial database concerning the types ofbacteria capable of degrading oil components and ways to accel-erate their degradation rates (Mearns, 1997; Jones, 1998; Head andSwannell, 1999), this knowledge cannot be simply extrapolated topolluted sites in Oman without keeping in mind the significantdifferences in environmental conditions. The soils of Oman arecharacterized by temperatures that can reach more than 55 �C. Thecoastal sediments experience salinities up to 10% due to highevaporation rates. Such features influence the diversity and activityof soil microorganisms as well as rates of natural degradation ofpollutants, which are typically low at such extreme conditions(Margesin and Schinner, 2001). Bacteria isolated from elsewhereare unlikely to survive the harsh conditions of the Omani envi-ronment. Therefore, bioremediation strategies that rely on indige-nous oil-degrading bacteria and their stimulation under fieldenvironmental conditions are required.

The present study was undertaken 1) to find out whether bac-terial isolates from sediments of Oman with the ability to grow onpetroleum compounds are similar or different than those isolatedfrom other ecosystems and 2) to evaluate the effectiveness of bio-stimulation and bioaugmentation approaches in enhancing oilbiodegradability in two heavily contaminated sediments. Indige-nous bacteria were isolated from oil-polluted sediments, identifiedusing 16S rRNA-based phylogeny and characterized for theirgrowth at different salinities and temperatures, as well as ondifferent alkanes. While biostimulation was achieved by the addi-tion of nutrients (N and P), bioaugmentation was achieved by theaddition of a bacterial consortium designed from our isolates. Theextent of oil-degradation was studied by following the CO2 evolu-tion and was further verified by gas chromatography (GC) analysisof the residual oil fraction.

2. Materials and method

2.1. Physical and chemical characterization of the samples

Two sediment samples (PD and KH) were collected from the oil-contaminated land at Mina Al-Fahal (lat. 23� 370 49.9200N; long. 58�

310 23.8400E), a coastal area in Muscat, Oman. Oil contamination ofthe KH sediment was relatively more recent (six months ago) thanthat of the PD sediment (2 years ago). The shade temperature at thetime of sampling was 32.1 �C. For salinity measurements, 10 g ofsediment weremixedwith 50ml of deionizedwater and allowed torest for 20 min. The pH and salinity of the filtrate were measuredusing calibrated YSI instruments. Total petroleum hydrocarbons(TPH) in the sediments were measured using GC as describedbefore (Weisman, 1998). A standard soil hydrometer method wasused to determine the percent sand, silt and clay in both samples(Klute, 1986), and this composition was used to identify the soiltexture from a soil triangle (Brady, 1984). Nutrients were extractedand analyzed using Ion Chromatography (Jackson, 2000).

2.2. Isolation and characterization of hydrocarbon-degradingbacteria

2.2.1. Enrichment and isolationThe abundance of alkane-degrading bacteria in both samples

was compared by estimating the most probable number (MPN)counts using hexadecane as a sole carbon source (Abed et al., 2007).Bacteria from both sediments were enriched on a defined artificialseawater medium supplemented with 1% (w/v) Omani crude oil asthe sole carbon source as previously described (Abed et al., 2007).The medium contained MgCl2.6H2O (5.6 gl�1), MgSO4.7H2O

(6.8 gl�1), CaCl2.2H2O (1.47 gl�1), KCl (0.66 gl�1), KBr (0.09 gl�1),KH2PO4 (0.15 gl�1) and NH4Cl (0.2 gl�1) and was supplementedwith trace elements mixtures (Widdel and Bak, 1992) and vitamins(Heijthuijsen and Hansen, 1986). Enrichments were performedusing media of 2% and 7% salinity (w/v NaCl). All enrichments wereperformed under aerobic conditions at 30 �C. Axenic strains wereobtained by plating on agar medium containing 20 mM acetate asthe sole carbon source, and then re-tested for growth on oil.

2.2.2. Identification of the isolatesNucleic acids from the isolates were extracted and 16S rRNA

genes were amplified using polymerase chain reaction (PCR) aspreviously described (Muyzer et al., 1995; Abed and Garcia-Pichel,2001). The PCR products were purified using the QIAquick PCRpurification kit (Qiagen, Düsseldorf, Germany) and then sequencedwith an ABI PRISM 3100 genetic analyzer (Applied Biosystems,Foster city, Calif.). Our sequences were aligned to sequences in theARB database using the alignment tool of the ARB software package(Ludwig et al., 1998). These sequences were inserted into amaximum likelihood pre-established tree using the parsimony ARBtool. The 16S rRNA sequences of our isolates were submitted to theGenBank under the accession numbers JQ284260eJQ284299.

2.2.3. Growth of the isolates at different salinities and temperaturesand on different alkanes

The growth of the obtained isolates at different salinities andtemperatures was tested. The isolates were inoculated in tubesfilled with 10 ml artificial seawater media with the salinities 2, 3, 5,7, 10, 12 and 16% (w/v NaCl), amended with 20 mM acetate. Thetubes were incubated on a rotary shaker at 100 rpm at 30 �C. Thegrowth of the isolates was also measured at the temperatures 5, 20,30, 40, 50 and 60 �C. Growth was monitored by following thechanges of the optical densities of the cultures at 660 nm over aperiod of 4 weeks.

Twenty three strains, representing closely related species indifferent generawere tested for their growth on particular alkanes(Table 2), as a sole carbon source (Abed et al., 2007). The strainswere inoculated in test tubes, each containing 10 ml of artificialseawater medium and 100 ml of a single alkane. The alkanes werefilter-sterilized using solvent-resistant cellulose filters (0.2 mmpore size) prior to use. All incubations were done in triplicates at30 �C in the dark with continuous shaking at 150 rpm. Growth wasmeasured by following changes in optical density at 660 nmagainst biotic (without a substrate) and sterile (without bacteria)controls.

Table 2Growth of twenty three representative strains on selected alkanes as monitored by changes in the optical density at 660 nm against biotic (without substrate) and sterile (nobacteria) controls.

Strain ID Alkane

Pentane Hexane Heptane Tridecane Tetradecane Pentadecane Hexadecane Heptadecane Octadecane Nonadecane

C5H12 C6H14 C7H16 C13H28 C14H30 C15H32 C16H34 C17H36 C18H38 C19H40

MH1 � � � � � � � � � þMH3 � þ þ þþ � � þþ � þ þMH4 � þ þ þ � þ þ þ þþ �MH5 þ þ � þ þ þ þ � þ þMH6 � þ � þ þ � � � þ þMH7 � � þ þ þ þ þ � þ þþMH9 þ þ � þ � � þ þ � þMH12 � � � þ þ þ þ � þ þMH13 � � � þþ þ þ þ � þ þMH16 þ þ � þ þ � þ � þ �MH19 þ þ � þþ � þ þ � � �MH21 þ þ þ þ þ þ þ þ þ þMH22 � � � þ þ þ þ þ þ þAH2 � � � þþ þþ þ þ þ þ þAH3 � � � � þ þ þþ þþ þ þAH4 � � � þþ þ þþ þþ þþ þþ �AH5 � � � þþ þþ þ þþ þþ þþ þAH6 � � � þ þ � þþ þþ þ þAH8 � � � þþ þþ þþ þþ þ þþ þAH9 � þ þ þþ þþ þþ þþ þ þ þAH14 � � � þþ þ þþ þ þþ þþ �AH18 � � � þþ þþ þþ þþ þþ þþ �AH23 � � � þþ þ þþ þþ þ þþ þ

(þþ): fast growth; (þ): growth and (e): no or very slow growth.

R.M.M. Abed et al. / International Biodeterioration & Biodegradation 89 (2014) 58e6660

2.2.4. Fatty acids detection in the isolatesFatty acids were detected in twenty three representative strains

using the MIDI Sherlock Microbial Identification System (www.midi-inc.com, Technical Note #101). 40e50 mg of the bacterialcells were harvested at the stationary phase. The cells were sub-jected to saponification in dilute NaOH/methanol solution followedby derivatization with dilute HCl/methanol solution to give therespective fatty acid methyl esters (FAMEs). The FAMEs were thenextracted from the aqueous phase by the use of hexane/methyl tert-butyl ether and the resulting extract was analyzed by using GC(Perkin Elmer, Model Clarus 600). Calibration was done using anexternal calibration standard (Microbial ID, MIDI Inc.). The obtainedpeaks were identified and quantified by comparing them to theexternal standard.

2.3. Bioremediation experiments

2.3.1. Preparation of bioaugmentation consortiumThe consortium for bioaugmentation experiments was prepared

by mixing equal proportions of pure cultures of the followingbacterial strains: MH2, MH3, MH21, MH22, AH2, AH3, AH6, AH8and AH23, pre-grown in acetate-containing artificial seawatermedium. The strains of the consortium were morphologically andbiochemically characterized (Table 1S). The selection of thesestrains was based on their growth patterns on the tested alkanes(Table 3) and to represent different genera. The cells wereconcentrated by centrifugation, washed three times with auto-claved medium in order to remove traces of acetate in the originalmedium, and then suspended in 20 ml artificial seawater mediumdeprived of KH2PO4 and NH4Cl.

2.3.2. Biostimulation and bioaugmentation experimentsTen grams of each polluted sediment were mixed with 20 ml of

carbon- and nutrient-free (i.e. without KH2PO4 and NH4Cl) artificialseawater medium (see above 2.2.1) in a 165 ml serum bottle. Thesesediments were subjected to the following treatments, each in

triplicate: 1) biostimulation (BS), performed by adding NH4Cl andNaH2PO4 as N and P sources at a final concentration of 2 and0.32 gl�1, respectively; 2) bioaugmentation (BA), performed byadding 1ml of the bacterial consortium in nutrient-free medium, 3)bioaugmentation and biostimulation (BA þ BS) were both thebacterial consortium and NH4Cl and NaH2PO4 were added and 4) acontrol sediment where neither bacteria nor nutrients were added.The bottles were then closed and sealed immediately with thick,black rubber stoppers, which were sealed with glue and screw capsto ensure no gas leakage. The bottles were incubated at 30 �Cwithout shaking in order to mimic the situation that occurs innature following an oil spill. CO2 measurements were performedusing GC every 2e3 weeks for 88 days. The oxygen concentration inthe bottles was monitored throughout the experiments, throughthe resolved GC air peaks, to verify that it did not become thelimiting factor.

2.3.3. Chemical analyses and quantification of oil mineralizationCO2 in the headspace was measured by withdrawing 250 ml at

different time intervals using a gas-tight glass syringe and injectingit manually into GC (GC, Agilent model 6890N). The GC wasequipped with thermal conductivity detector and a 30 m � 250 mmcapillary column (HP-PLOT Q). Heliumwas used as a carrier gas at aflow rate of 4 ml min�1 and the injector and detector temperatureswere maintained at 200 �C and 210 �C, respectively. The oventemperature was programmed from 50 �C to 80 �C (final hold time3min) at a rate of 20 �C min�1. CO2 evolution data were statisticallyanalyzed by one-way ANOVA using the SPSS software (10th edition,Chicago, USA). P-values were adjusted using the sequential Bon-ferroni (Quinn and Keough, 2002) and Tukey’s test was used todetermine differences between individual means. The extent of oilmineralization was calculated from the CO2 evolution data bycomparing the experimentally quantified CO2 with the theoreticalamount of CO2 that would be formed by complete oxidation of thepresent oil after subtraction of the CO2 evolved from an oil-freesediment (Musat F, personal communication).

Table 3Percentage distribution of fatty acids in twenty three representative strains as detected by GC and identified by comparison to a library of fatty acid compounds.

Strain ID Fatty acid

DO HDO TD 13-MTD 12-MTD PD MPD C-HXD HD 15-MHD C-MHD HPD C-ODDE C-ODE T-ODE OD C-MOD

C12:0 3-OH C12:0 C14:0 i-C15:0 a-C15:0 C15:0 i-C16:0 C16:1 C16:0 i-C17:0 C17:0 C17:0 C18:2 C18:1 C18:1 C18:0 C19:0

MH1 6.0 e 1.7 e 7.9 e e 8.1 38.3 e 2.3 e e e 21.5 e 14.2MH3 6.5 e 1.3 3.7 20.9 e 10.4 3.2 24.6 0.9 2.7 1.0 e e 10.8 1.4 12.6MH4 e e 6.3 1.4 21.7 e 13.6 3.3 9.6 e 1.6 e 5.7 13.1 15.8 8.0 e

MH5 e 3.4 e e 12.2 e 24.8 e 25.9 1.4 6.2 e 1.3 1.0 6.0 4.4 13.3MH6 e e 3.9 e 1.0 e e 6.5 41.5 e e e e 33.4 e 11.7 1.8MH7 e e 4.4 e 2.9 e 14.6 1.4 26.3 2.4 2.9 1.5 e 2.5 8.9 7.3 24.7MH9 e e e 8.4 69.2 e 5.3 e 8.7 3.3 e e e e e 5.1 e

MH12 e e 2.9 e 7.8 e e 6.0 40.0 e 2.0 e e e 34.4 4.4 2.6MH13 9.9 1.8 1.5 e 5.4 e 7.0 11.2 33.8 e 3.7 e e e 23.9 1.8 e

MH16 e e 2.1 3.2 18.4 e 6.7 6.0 30.7 e 2.9 e e e 23.9 2.2 3.9MH19 10.0 1.4 2.3 e e e 1.5 6.7 35.9 e 7.5 e e 2.3 12.8 5.4 14.4MH21 e e 2.6 e 10.1 e 27.1 e 26.1 e e e e 19.7 e 12.2 2.3MH22 e e 1.5 1.2 e 2.0 7.6 2.2 29.5 e 1.7 1.5 e 5.0 3.1 5.0 39.7AH2 7.4 e e e 2.1 e e 8.2 33.6 e 5.4 e e e 25.5 1.6 16.3AH3 6.4 e 1.9 e 8.2 e e 7.3 36.3 e 4.0 e e e 21.5 e 14.4AH4 4.5 e 1.4 e e e e 7.0 41.1 e 6.3 e e e 18.2 e 21.6AH5 3.2 e e e e e e 7.7 39.3 e 3.5 e e e 24.0 e 22.4AH6 9.7 e e e e e e 9.3 43.0 e 5.4 e e e 15.1 e 17.5AH8 5.3 e 1.0 e e e e 7.3 40.0 e 4.6 e e e 22.3 1.1 18.5AH9 6.7 e e e e e e 9.4 43.3 e 4.4 e e e 20.3 e 15.8AH14 16.7 e e e e e e 7.1 35.6 e 3.0 e e e 20.5 e 17.1AH18 6.0 e e e e e e 12.4 39.2 e 3.5 e e e 17.4 e 21.6AH23 5.2 e e e e e e 7.6 40.5 e 6.0 e e e 16.2 e 24.6

DO: Dodecanoate; HDO: 3-hydroxydodecanoate; TD: tetradecanoate; 13-MTD: 13-methyltetradecanoate; 12-MTD: 12-methyltetradecanoate; PD: pentadecanoate; MPD: 14-methylpentadecanoate; C-HXD: cis-9-hexadecenoate HD: hexadecanoate; 15-MHD: 15-methylhexadecanoate; C-MHD: cis-9,10-methylenehexadecanoate; HPD: heptade-canoate; C-ODDE: cis-9,12-octadecadienoate; C-ODE: cis-9-octadecenoate; T-ODE:trans-9-octadecenoate OD: octadecanoate; C-MOD: cis-9,10-methyleneoctadecanoate.

R.M.M. Abed et al. / International Biodeterioration & Biodegradation 89 (2014) 58e66 61

Oil biodegradation (mainly C14eC23 alkanes) was furtherconfirmed using GC analysis of the sediments at the end of theexperiment. Two and half g of each sediment were extracted 4e5times in 15 ml hexane. The pooled extract was filtered with non-absorbent cotton to remove solid particles and the filtrate wasevaporated using a rotary evaporator. Hydrocarbons were analyzedusing a GC equipped with a flame ionization detector and30 m � 250 mm capillary column (HP-5MS) and were quantifiedusing an internal standard. Helium was used as a carrier gas at aflow rate of 1 ml min�1 and the injector and detector were main-tained at 290 �C. The oven temperature was programmed from80 �C (initial hold time 2min) to 290 �C (final hold time 30min) at arate of 10 �C min�1.

3. Results

3.1. Physical and chemical characteristics of the studied sediments

The TPH content of the PD and KH sediments were 22� 0.7 and55 � 0.6 mg g�1 sediment and the MPN counts on hexadecane asthe sole carbon source reached 2.7 � 106 and 5.6 � 106, respec-tively (Table 1). Both soils contained more silt (51e62%) than sandand clay and thus had a silt loam texture. The pH of both sampleswas around 7.4 and the salinity was between 2% and 3.5%. Whilethe concentrations of phosphate and nitrate in the PD soil reached21.13 and 0.46 mg l�1, respectively, these concentrations weremuch lower in the KH sediment (Table 1). Sulfate was alsodetected at a much higher concentration in the PD than in the KHsediment.

3.2. Characterization of the isolates

3.2.1. Phylogenetic affiliationA total of fourty bacterial strains were isolated, nineteen at 2%

salinity (AH strains) and twenty one at 7% salinity (MH strains). All

strains were Gram negative and rod in shape (Table 1S). Thirty fourstrains belonged to Gammaproteobacteria and six to Alphaproteo-bacteria (Fig. 1). The gammaproteobacterial strains were phyloge-netically affiliated to sequences from the genera Pseudomonas,Halomonas, Marinobacter, Hahella and Alcanivorax (Fig. 1). Eighteensequences of the gammaproteobacterial strains grouped withspecies of the genus Marinobacter. These strains shared 92e98%sequence similarity to the known oil-degrading strains M. hydro-carbonoclasticus and M. alkaliphilus. Four strains (i.e. MH8, 16, 17and 18) were related to the sequences of Pseudomonas stutzeriisolated from oil pipelines and Pseudomonas sp. 3CB6, which is ahalobenzoate-degrading denitrifier with >97% sequence similarity.Only one strain (i.e. AH14) was related to the halophilic Halomonasspp. The strains AH1 and MH3 were related to the known poly-aromatic hydrocarbon (PAH)-degrading bacteria Alcanivoraxvenustensis with 96% sequence similarity. Of the alphaproteo-bacterial strains, MH21 strain had >98% sequence similarity withthe marine Parvibaculum spp., whereas the strain AH2 had 92%similarity to the Azospirillum sp. 5C isolated from fuel contaminatedsoils. The remaining four strains (i.e. AH7, AH8, AH18 and AH23)were related to sequences of unknown bacteria.

3.2.2. Growth propertiesAll isolates grew well at salinities between 2 and 7% (Fig. 2).

The strains AH8, MH2, MH6, MH9 and MH13 showed detectablegrowth at salinities up to 10%. The most favorable salinity forgrowth was 7% for twenty six of the fourty strains, whereas therest had their best growth at 3% salinity. Nearly all strains grew attemperatures between 20 and 60 �C but with variations in theirtemperature optima (Fig. 2). While the optimum temperature oftwenty four strains was 30 �C, it was 40 �C for the remainingsixteen strains.

Growth spectra on different alkanes showed a unique substrateutilization pattern for each species (Table 2). Growth was morepronounced on long chain compared to short chain alkanes. While

Fig. 1. Unrooted phylogenetic tree showing the affiliations based on 16S rRNA genes of the 40 strains obtained from oil-contaminated sediments in Oman and their closest relatives.All strains were affiliated to the groups Gammaproteobacteria and Alphaproteobacteria. The tree was simplified for clarity by omitting all sequences between clusters. The barindicates 10% sequence divergence.

R.M.M. Abed et al. / International Biodeterioration & Biodegradation 89 (2014) 58e6662

only few strains grew on pentane (five out of twenty three), hexane(nine strains) and heptane (five strains), most strains grew on tri-decane, tetradecane, pentadecane, hexadecane and octadecane. AHstrains exhibited relatively better growth on C13eC18 alkanescompared to MH strains. None of the AH strains grew on pentaneand only AH9 grew on hexane and pentane. While MH21 straingrew on all tested alkanes, MH1 could not grow on any of themexcept nonadecane.

3.2.3. Bacterial fatty acidsSeventeen different fatty acids were detected, and their percent

distribution was different among different strains (Table 3). Hex-adecanoate was the most abundant fatty acid (24e43% of the totalamount) in twenty out of the twenty three strains, whereas 12-methyltetradecanoate and 14-methylpentadecanoate were themost abundant in the remaining three strains, namely MH4, MH9and MH21. The fatty acids 3-hydroxydodecanoate, 14-

Fig. 2. Growth of the strains at different salinities and temperatures as monitored byfollowing changes in the optical density at 660 nm against biotic (without substrate)and sterile (no bacteria) controls. The white dots represent the most favorable salinityor temperature of growth.

R.M.M. Abed et al. / International Biodeterioration & Biodegradation 89 (2014) 58e66 63

methylpentadecanoate, heptadecanoate, cis-9,12-octadecadienoateand cis-9-octadecenoate were detected in some MH strains but notin any of the AH strains. All AH strains produced dodecanoate, cis-9-hexadecanoate, cis-9,10-methylenehexadecanoate, trans-9-octadecanoate and cis-9,10-methyleneoctadecanoate. The fattyacids tetradecanoate, 12-methyltetradecanoate and octadecanoatewere present in few AH strains but not in others.

3.3. Bioremediation experiments

Clear differences were observed in the cumulative amounts ofCO2 evolved by the two studied sediments upon the addition of thebacterial consortium and/or inorganic nutrients (Table 4, Fig. 3).The total amount of CO2 produced after 88 days was greater in theoriginal KH sediment than in the PD sediment, with values reaching2.23 � 0.07 and 0.45 � 0.02 mg CO2 g�1 sediment, respectively(Table 4). While CO2 production did not significantly change whenthe bacterial consortium was added without inorganic nutrients(KH2PO4 and NH4Cl) to the KH sediment (P ¼ 0.992), it showed asignificant increase in the PD sediment from 0.45 � 0.02 to0.67 � 0.03 mg CO2 g�1 sediment (P ¼ 0.05). The addition ofinorganic nutrients to both sediments caused a remarkable in-crease in the CO2 production (Fig. 3). The total amount of CO2

evolved after 88 days upon the addition of inorganic nutrientsreached 2.74 � 0.14 and 1.67 � 0.18 mg CO2 g�1 soil in the KH andPD sediments, respectively. When both the bacterial consortiumand inorganic nutrients were added together, a significant increasein the CO2 production was observed in the PD sediment (P ¼ 0.05),whereas the produced amounts of CO2 were comparable in thepresence of nutrients with or without the bacterial consortium inthe KH sediment.

The extent of oil mineralization, calculated from the evolvedCO2, was 0.6 and 1.2% of the initial oil after 88 days in the originalPD and KH sediments without any additions, respectively (Table 4).In the KH sediment, oil mineralization rate increased from 1.2% to1.49% only when inorganic nutrients were added, with a totalamount of 0.82 mg oil degraded per g sediment. The maximum oilmineralization rate in the PD sediment was obtained when bothnutrients and bacteria were added and reached around 2.6% of theinitial oil in the sediment. This value indicates that only 0.6 mg ofoil was degraded g�1 of PD sediment. GC analysis showed that mostof the degraded fraction of oil belonged to C14eC23 alkanes (Table 4,Fig. 1S). The concentrations of C14eC23 before and after applyingbioremediation showed that these compounds were degraded inboth PD and KH sediments, however to different levels. Whiledegradation was between 46 and 67% of the initial concentrationsin the PD sediment, it was only between 28 and 42% in the KHsediment. In both sediments, the degradation was apparentlyhigher in the presence of inorganic nutrients with or without thebacterial consortium compared to the treatment with bacterialconsortium alone. The degradation patterns of these alkanes atdifferent bioremediation treatments confirmed the results basedon the CO2 measurements.

4. Discussion

The enriched strains belonged mainly to genera previouslyknown to contain hydrocarbon-degrading species (Gauthier et al.,1992; Yakimov et al., 1998; Huu et al., 1999; Chang et al., 2000;Syutsubo et al., 2001). The dominance of Marinobacter-relatedstrains in our culture collection (eighteen out of thirty two) sug-gests the abundance of this group in the studied sediments as oneof easily culturable bacteria. Marinobacter species such as M.hydrocarbonoclasticus, M. aquaeolei and M. alkaliphilus were shownto utilize various alkanes and polyaromatic hydrocarbons (PAHs) asthe sole source of carbon and energy (Gauthier et al., 1992; Yakimovet al., 1998; Huu et al., 1999). Alcanivorax species are regarded asmain degraders of aliphatic hydrocarbons in the marine environ-ment, and Pseudomonas species are renowned for their metabolicversatility (Sikorski et al., 2005; Yakimov et al., 2007). Alcanivoraxand Marinobacter species seem to have a global distribution, astheir sequences have been retrieved from sediments of differentorigins (Yakimov et al., 2007). They were detected in the Japan Seaafter the Nakhodka spill (Kasai et al., 2001), in sediments fromSpain after the oil spill from the tanker Prestige in 2002 (Vila et al.,2010), in the oil-polluted sediments of the Arabian Gulf (Abed et al.,2007; Al-Awadhi et al., 2007) and after the spill in the Ria de Vigo(Alonso-Gutierrez et al., 2008). Alkane-degrading bacteria fromthese two genera have even been isolated from deep-sea sediments(Tapilatu et al., 2009). Oil contamination of marine sediments wasshown to result in bacterial community changes in the favor ofspecies belonging toMarinobacter and Alcanivorax species (Jimenezet al., 2011).

The optimum growth of our strains was detected at salinitiesbetween 2 and 7% and temperatures between 30 and 40 �C, sug-gesting that they are halotolerant and mesophilic. The salinity andtemperature tolerance of the strains is in accordance with theprevailing conditions in the field where the original sediments

Table 4The initial evolution rates of CO2, the total evolved CO2 during 88 days of incubation, the percentage of oil mineralization calculated from the evolved CO2 and the percentage ofbiodegradation of hydrocarbons C14eC23 as detected by GC analysis for the different treatments.

Sediment Type of treatment Initial CO2 evolution rate(mg-CO2 g�1 sediment d�1)a

Total evolved CO2 after 88 days(mg CO2 g�1 sediment)

% of oil mineralizationcalculated fromevolved CO2

% of biodegradation of C14eC23calculated from GC

PD Untreated 0.007 0.45 � 0.02 0.60 � 0.02 46.2 � 5.7Bioaugmentation (BA) 0.011 0.67 � 0.03 0.90 � 0.04 44.9 � 2.8Biostimulation (BS) 0.025 1.67 � 0.18 2.20 � 0.24 54.8 � 3.9BA þ BS 0.029 1.94 � 0.09 2.6 � 0.12 67.2 � 1.6

KH Untreated 0.032 2.23 � 0.07 1.20 � 0.04 28.5 � 2.8Bioaugmentation (BA) 0.033 2.26 � 0.09 1.22 � 0.05 24.0 � 3.9Biostimulation (BS) 0.047 2.74 � 0.14 1.48 � 0.08 41.4 � 3.2BA þ BS 0.048 2.76 � 0.08 1.49 � 0.04 41.9 � 5.0

a Initial maximum evolution rate of CO2 was calculated from the linear regression of the increase in the period between 10 and 30 days.

R.M.M. Abed et al. / International Biodeterioration & Biodegradation 89 (2014) 58e6664

were collected. Although the sediments had a salinity between 2and 3.5% at the time of sampling, their salinity could increase up to10% during hot summers due to high evaporation rates, thusexplaining the halotolerance of enriched strains. Previous isolatesfrom the genera Marinobacter, Alcanivorax and Halomonas werealso found to grow at similar ranges of salinity and temperature(Gauthier et al., 1992; Fernandez-Martinez et al., 2003; Takai et al.,2005). For example, the optimum salinity and temperature for thegrowth of the strains M. hydrocarbonoclasticus and M. alkaliphiluswere 3.5% and 2.5e3.2% and 32 �C and 30e35 �C, respectively(Gauthier et al., 1992; Takai et al., 2005). The detected fatty acids inour strains were typically found in species belonging to similargenera (Rainey et al., 1994; Doumenq et al., 2001; Fernandez-Martinez et al., 2003). For instance, the fatty acids C16:0 and C18:0and their derivatives were found abundant in our strains as well asin other alkane-degrading bacteria such as A. venustensis and M.hydrocarbonoclasticus (Doumenq et al., 2001; Fernandez-Martinezet al., 2003). Besides their use as biomarkers in the identificationof bacteria, fatty acids are produced by some of the oil-degradingmicroorganisms in order to improve hydrocarbon uptake throughemulsification (de Vasconcellos et al., 2009). Indeed, the fatty acidcomposition of alkane-degrading bacteria was shown to be influ-enced by the type of hydrocarbons provided to them as a carbonsource (Doumenq et al., 2001).

Our strains exhibited good growth on hydrocarbons so theycould potentially contribute to the bioremediation of oil-pollutedsediments. Similar compounds were shown to be utilized by Mar-inobacter, Alcanivorax and Pseudomonas species as the sole carbonand energy source (Gauthier et al., 1992; Fernandez-Martinez et al.,2003; Takai et al., 2005). The strains ability to degrade alkanes and

Fig. 3. The cumulative amount of CO2 (in mg g�1 sediment) evolved as a result of minerabacterial consortium in the PD and KH sediments. Common alphabetic superscripts indicat

to grow at temperatures around 40 �C and salinities up to 7% makethem suitable for the cleanup of Omani polluted sediments, whereenvironmental conditions are often extreme. The notion thatdifferent strains degrade different alkanes underlines the need tocombine several strains together in order to achieve maximum oildegradation. A consortium composed of several bacteria wasshown to degrade complex oil components more efficiently thansingle strains, probably because of a wider range of degradationpathways (Rahman et al., 2002).

The evolution of much higher CO2 in the untreated KH and PDsediments than the oil-free sediments from the same region (notshown) indicates that the indigenous microorganisms within thesesediments were capable of degrading hydrocarbons and thus couldcontribute to natural attenuation of pollutants in these sites. MPNcounts and isolation results further confirmed the presence ofhydrocarbon-degrading bacteria in these sediments. Numerouslaboratory and field studies have shown that hydrocarbon-degrading microorganisms are ubiquitous in contaminated sedi-ments and natural attenuation is effective in the cleanup of petro-leum contaminants (Bento et al., 2005; Couto et al., 2012; Tang et al.,2012). Indigenous microorganisms have a distinct advantage overintroduced microorganisms to enhance biodegradation becausethey are well adapted to the physical and chemical conditions. TheKH sediment exhibited higher rates of CO2 evolution and oilmineralization than the PD sediment. This could be attributed to theageing effect of hydrocarbons in the PD sediment, which reduces thebioavailability of contaminants due to their sequestration in the soilmatrix (Tang et al., 2012). Moreover, differences in the soil texture,particle size, salinity as well as nutrient content of the two sedi-ments (Table 1) may have differently affected degradation rates.

lization of crude oil hydrocarbons during the addition of inorganic nutrients and/or ae no significant difference using Tukey’s test within each time point.

R.M.M. Abed et al. / International Biodeterioration & Biodegradation 89 (2014) 58e66 65

The bioremediation experiments suggested that biostimulationthrough the addition of inorganic nutrients (BS treatment)increased the rates of CO2 evolution and oil mineralization byindigenous microorganisms in both sediments, however todifferent extents. The linear and continuous increase in CO2 evo-lution over the period of 88 days indicates that oxygen was notlimited in the bottles and aerobic respiration was still possible.Moreover, the resolved GC peaks indicated that oxygen levels wasstill present in excess at the end of the experiment. The success ofnutrients supplementation in speeding up the degradation processhas been previously demonstrated (Xu and Lu, 2010; Kauppi et al.,2011). This is attributed to the fact that marine environments aregenerally limited with nutrients (see Table 1) and/or the addition oflarge amounts of oil results in a high C:N ratio that is unfavorable tomicrobial activity (Ward and Brock, 1978). The addition of nutrientsresulted in themineralization of about 2.2% of the initially added oilafter 88 days incubation in the PD sediment, but only 1.48% in theKH sediment. Although the percentage of oil mineralization in theKH sediment is lower than in the PD sediment, the total amount ofdegraded TPH would be around 0.8 mg g�1 of the KH sediment and0.5 mg g�1 of the PD sediment. This is due to the higher concen-trations of TPH in the KH than in the PD sediment.

While biostimulation increased biodegradation rates in bothsediments, bioaugmentation (i.e. the addition of bacteria) was onlysuccessful in the case of the PD sediment. Perhaps the inoculatedmicroorganisms were outcompeted by the indigenous bacteria inthe KH sediment, but grew better in the PD sediment. Althoughalkanes are relatively easy to degrade, the introduction of alkane-degrading bacteria was mainly to shorten the period required forthe degradation of these compounds. Bioaugmentation was oftensuccessful in cases where the number and activity of indigenousmicroorganisms were low and where pollutants were toxic (Vogel,1996). For example, the degradation of the recalcitrant pollutants2,4,6-trinitrotoluene, pentachlorophenol and carbon tetrachloridewas enhanced by bioaugmenting the soils with microorganismscapable of degrading these compounds (Edgehill, 1995; Shin andCrawford, 1995; Witt et al., 1995). In spite of the success of bio-augmentation in the PD sediment, biostimulation still seems to bemore effective. The success of either approach is site-specific anddepends on many parameters such as the nature of pollutants(concentration, toxicity and bioavailability), soil physico-chemicalcharacteristics and indigenous microorganisms (number, activity,sensitivity and interspecies competition).

The use of CO2 evolution as an indication of oil mineralizationprovided an easier and faster alternative than the use of GC anal-ysis. This approach was successfully used to follow oil biodegra-dation in several studies (Atlas and Bartha, 1972a,b; Kim et al.,2005; Morais and Tauk-Tornisielo, 2009). Using this approach, itwas shown that 42% of a paraffinic petroleum was mineralizedwithin 32 days (Atlas and Bartha, 1972a,b). In our experiments, weestimated that between 1.49 and 2.6% of the initial oil weremineralized within 88 days of bioremediation treatments.Although these values are relatively low, it should be kept in mindthat the used sediments were heavily polluted with oil and thesevalues corresponded to the degradation of approximately1mg oil g�1 sediment. Furthermore, this methodmeasures only thecompounds that are completely mineralized to CO2 but not thosethat are partially degraded. This might be the reason why thedegradation of C14eC23 alkanes by GC was much higher, since thismethod measures the degradation of compounds, regardlesswhether they were completely or partially degraded. Thebiodegradation-resistant fraction of oil may include the polycyclicaromatic hydrocarbons with more than 3 rings and the polarfraction of crude oil. Some of these compounds may be partiallyoxidized, leading to consumption of oxygen but not to CO2

production. CO2 method also fails to measure the carbon fractionthat could be assimilated in biomass. It has been demonstrated thatisolates of bacteria and yeasts assimilate as much as 50% of thesupplied carbon (Linton and Stephenson, 1978).

In conclusion, the isolated strains from Omani oil-contaminatedsediments belong to the common genera found in other pollutedsediments around the world such as Marinobacter, Pseudomonas,Alcanivorax and Holomonas. The bioaugmentation of contaminatedsediments with a mixture of these strains can accelerate thebiodegradation processes, however biostimulation through theaddition of inorganic nutrients still seems to be a more efficientapproach.

Acknowledgment

We would like to thank Michael Barry, Gary Brown and MichaelRobinson, Stjepko Golubic and Reginald Victor for reviewing themanuscript and for their suggestions. This research was financiallysupported by The Research Council (TRC) of Oman (grant RC/SCI/BIOL/11/01).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ibiod.2014.01.006.

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