Isolation and characterization of alkane degrading bacteria from petroleum reservoir waste water in Iran (Kerman and Tehran provenances)

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Isolation and characterization of alkane degrading bacteria frompetroleum reservoir waste water in Iran (Kerman and Tehranprovenances)

Mehdi Hassanshahian a, Mohammad Ahmadinejad b,⇑, Hamid Tebyanian c, Ashraf Kariminik d

a Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iranb Dermatology and Leishmaniasis Research Center, Department of Parasitology, School of Medicine, Kerman University of Medical Sciences, End of 22nd Bahman BLVD,Kerman 7616714115, Iranc Department of Microbiology, Science and Research Branch, Islamic Azad University, Kerman, Irand Department of Microbiology, Islamic Azad University, Kerman, Iran

a r t i c l e i n f o

Keywords:AlkaneBiodegradationBacteriaIran

a b s t r a c t

Petroleum products spill and leakage have become two major environmental challenges in Iran. Samplingwas performed in the petroleum reservoir waste water of Tehran and Kerman Provinces of Iran. Alkanedegrading bacteria were isolated by enrichment in a Bushnel–Hass medium, with hexadecane as solesource of carbon and energy. The isolated strains were identified by amplification of 16S rDNA geneand sequencing. Specific primers were used for identification of alkane hydroxylase gene. Fifteen alkanedegrading bacteria were isolated and 8 strains were selected as powerful degradative bacteria. These 8strains relate to Rhodococcus jostii, Stenotrophomonas maltophilia, Achromobacter piechaudii, Tsukamurellatyrosinosolvens, Pseudomonas fluorescens, Rhodococcus erythropolis, Stenotrophomonas maltophilia, Pseudo-monas aeruginosa genera. The optimum concentration of hexadecane that allowed high growth was 2.5%.Gas chromatography results show that all strains can degrade approximately half of hexadecane in oneweek of incubation. All of the strains have alkane hydroxylase gene which are important for biodegrada-tion. As a result, this study indicates that there is a high diversity of degradative bacteria in petroleumreservoir waste water in Iran.

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1. Introduction

Petroleum hydrocarbons are the most common environmentalpollutants and oil spills pose a great hazard to terrestrial and mar-ine ecosystem. 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 as they severely damage the surrounding ecosystem(Head et al., 2006).

The traditional methods to cope with oil spills are confined tophysical containment. Biological methods can have an edge overthe physicochemical treatment in removing spills as they offer abiodegradation of oil fraction by microorganisms (Chaillan et al.,2004; Hanson et al., 1997).

Microbiological decontamination of oil derivatives in pollutedenvironments is claimed to represent an efficient, economic andversatile alternative to physicochemical treatment (Emtiazi et al.,

2009). The rate of biodegradation depends on oil concentration, al-kanes length, biosurfactant and type of microorganisms (Cappelloet al., 2012a). It has been observed that the saturated componentsof crude oil (alkanes) are particularly the alkanes of intermediatelength (C10–C20) which are biodegraded more readily (Subarnaet al., 2002). The rate of uptake and mineralization of many organiccompounds depend on the concentration of the compound, highconcentration of hydrocarbon which causes inhibition of biodegra-dation. It is done by nutrient or oxygen limitation or through toxiceffects exerted by volatile hydrocarbons (Luis et al., 2000; Chaneauet al., 2005; Emtiazi et al., 2005).

The growth of microorganisms on hydrocarbons is often accom-panied by the emulsification of the insoluble carbon source in theculture medium. In most cases, this has been due to the productionof extra cellular emulsifying agents, during the breakdown ofhydrocarbons. These processes aid microorganisms to grow onand metabolize crude oil (Cappello et al., 2012b).

Alkane hydroxylase is a key enzyme in alkane degradation. Thisenzyme which introduces oxygen atom is derived from molecularoxygen which is in the alkane substrate and plays an importantrole in crude oil bioremediation. Alkane hydroxylase genes are

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⇑ Corresponding author. Tel.: +98 9131410143.E-mail address: [email protected] (M. Ahmadinejad).

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classified into three groups through phylogenetic analysis. The al-kane hydroxylase group (I) is encoded as alk-B gene which cata-lyzes medium chain length (C6–C12) n-alkanes. The geneclassified group (II) is encoded as alk-M which catalyzes long chainalkanes >C12 and are possessed by Acinetobacter. The gene classi-fied group (III) is encoded as alk-B which is unknown for substratespecificity, alkane oxidation pathway and oxidation system (Kohnoet al., 2002; Heissblanquet et al., 2005).

Oil reservoirs have a complex mixture of microbial communitywhich is related to type of hydrocarbons. Waste water producedfrom these oil reservoirs makes many environmental problems.The elimination of pollutant from this waste water is importantfor quality control of the oil reservoirs (Ghanavati et al., 2008).

The aims of this study were to isolate and characterize hexadec-ane degrading bacteria from petroleum reservoirs waste water intwo different regions of Iran. In this study, we report some bacte-rial strains capable of efficiently growing on hexadecane.

2. Materials and methods

2.1. Sampling

To isolate of alkane hydrocarbon degrading bacteria from con-taminated soils and petroleum reservoirs waste water, sampleswere collected from 16 different sites in Tehran and Kerman petro-leum reservoirs regions (36�15, N; 44�15, E). Sixteen samples hadbeen collected into sampling, 5 samples have collected from Ker-man Petroleum reservoirs waste water, the other 5 samples fromTehran petroleum reservoirs and the remaining 6 samples hadbeen collected from soil, contaminated to hydrocarbons. The soilsamples were collected from 1 to 12 cm below the surface of soilusing a sterile knife. Waste water samples were collected from adepth of 3 cm, in sterile 100 ml bottles and had been transportedon ice to the laboratory for isolation (Hassanshahian et al., 2012b).

2.2. Isolation and selection of alkane degrading bacteria

A synthetic Bushnell Haas Mineral Salts medium (BHMS) wasused for the isolation of alkane degrading bacteria (Kohno et al.,2002). BHMS medium contain (g l�1) KH2PO4, 1; K2HPO4, 0.2;MgSO4�7H2O, 0.2; CaCl2, 0.02; NH4NO3, 1; and 2 droplet of FeCl3

60%. The pH adjusted to 7 (Cappello et al., 2012b). BHMS mediumwas supplemented with 1% (v/v) hexadecane as sole carbon sourceand energy. Portion of soil (1 g) or waste water (1 ml) sampleswere added to 250 ml Erlenmeyer flasks containing 100 ml BHMSmedium and the flask incubated for 10 days at 30 �C on rotary sha-ker (INFORS AG, Switzerland) operation at 180 rpm. Then 5 ml ali-quots were removed to a fresh BHMS medium. After a series of fourfurther subcultures, inoculums from the flask were streaked outand phenotypically different colonies were purified on BHMS agarmedium. Phenotypically different colonies obtained from theplates were transferred to a fresh BHMS medium with and withouthexadecane to eliminate autotrophs and agar-utilizing bacteria.The procedure was repeated and isolated, only exhibiting pro-nounced growth on hexadecane were stored for further character-ization (Chaillan et al., 2004; Hassanshahian et al., 2012a).

2.3. Identification of isolates

2.3.1. Biochemical characterizationTo identify and characterize the isolated bacteria, biochemical

tests such as gram staining, oxidation/fermentation, productionof acid from carbohydrates, hydrolysis of gelatin and citrate werecarried out according to the Bergey’s manual of Systematic Bacte-riology (Holt et al., 1998).

2.3.2. Molecular identification of bacteria and detection of alkanehydroxylase gene (alk-B) in the isolated bacteria

Analysis of 16S rDNA was performed to the taxonomic charac-terization of isolated strains. Also, the purified DNA extracts weresubsequently screened by PCR to detect catabolic alkB gene thatencodes enzymes involved in alkane degradation pathways. TotalDNA extraction of bacterial strains was performed with the CTABmethod (Winnepenninckx et al., 1993). The bacterial 16S rDNA lociwas amplified using the forward domain specific bacteria primer,Bac27_F (50-AGAGTTTGATCCTGGCTCAG-30) and universal reverseprimer Uni_1492R (50-TACGYTACCTTGTTACGACTT-30). Alkanehydroxylase gene was detected by alk-3F (50-TCGAGCA-CATCCGCGGCCACCA-30) and alk-3R (50-CCG TAG TGCTCGACG-TAGTT-30) primers (Kohno et al., 2002; Hassanshahian et al.,2010). The amplification reaction was performed in a total volumeof 50 ll consisting, 1X solution Q (Qiagen, Hilden, Germany), 1XQiagen reaction buffer, 1 lM of each forward and reverse primer,10 lM dNTPs (Gobco, Invitrogen Co., Carlsbad, CA), and 2 U of Qia-gen Taq polymerase (Qiagen). Amplification for 35 cycles was per-formed in a thermacycler GeneAmp 5700 (PE Applied Biosystem,Foster City, CA, USA). The temperature profile for PCR was kept,95 �C for 5 min (1 cycle); 94 �C for 1 min and 72 �C for 2 min (35cycles), followed by 72 �C for 10 min at the end of final cycle(Troussellier et al., 2005). PCR products were visualized by gel elec-trophoresis using a horizontal 2% agarose gel (Sigma, St. Louis, MO)with 1X TBE buffer. Gels were stained in a solution of ethidium bro-mide and visualized with a UVP UV transilluminator (UVP Inc., SanGabriel, CA) (Kohno et al., 2002; Sei et al., 2003).

The 16S rDNA amplified was sequenced with a Big Dye termina-tor V3.1 cycle sequencing kit on an automated capillary sequencer(model 3100 Avant Genetic Analyzer, Applied Biosystems). Analy-sis and phylogenetic affiliates of sequences was performed as pre-viously described (Yakimov et al., 2006; Maidak et al., 1997).

2.4. Growth and hexadecane removal assay

Growth curves of bacteria in the study were routinely assessedindirectly by turbidity measurement (O.D600 nm) in a UV–visiblespectrophotometer (Shimadzu UV-160, Japan). The hexadecane re-moval assay was carried out by Gas Chromatography Flame Ioniza-tion Detector (GC-FID) analysis (Hassanshahian et al., 2012a).

2.5. Extraction of hexadecane and gas chromatography analysis

Residual hexadecane in liquid was extracted using a liquid–li-quid extraction technique with acetone/hexane (1:1) and was ana-lyzed by GC-FID for residual hexadecane (Chaneau et al., 2005).First, liquid was placed in the refrigerator until the hexadecane be-comes a solid. After 4 h, 5 ml of hexane and 5 ml of acetone wasadded and was stirred vigorously for 2 min. The liquid obtainedwas centrifuged for 10 min, 5000 rpm. The upper liquid was thenpassed through a PFTE filter (0.22 lm). Then, hexadecane concen-trations were determined by GC-FID. In order to avoid losses ofhexadecane during extraction, the whole sample was extracted di-rectly in the experimental flasks. Then, hexadecane concentrationswere determined by GC-FID. AHP-5MS (Agilent, USA) column (5%phenyl 95% methylpolysiloxane; 30 m length � 0.025 mmid � 0.25 lm film thickness) was used at a temperature programof 120 �C for 1 min, increased to 180 �C at 20 �C/min, and held at180 �C for 5 min. Nitrogen was used as a carrier gas at a constantflow of 1.5 ml/min. Injector and detector temperatures were 250and 270 �C, respectively. The injected volume was 2 ll and dode-cane (C12H26) was used as internal standard (Chaneau et al., 2005).

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2.6. Emulsification activity and bacterial adherence to hydrocarbons(BATH) test

The emulsification activity (E24) was determined by the addi-tion of hexadecane, to the same volume of cell free culture broth,mixed with a vortex for 2 min and left to stand for 24 h. The emul-sification activity was determined as the percentage of height ofthe emulsified layer (mm) divided by the total height of the liquidcolumn (mm) (Hassanshahian et al., 2012b). Bacterial adhesion tohydrocarbon was carried out according to Pruthi and Cameotra(1997).

3. Results

3.1. Isolation and identification of bacteria

Fifteen alkane-degrading bacterial strains were isolated fromenrichment cultures that established at 30 �C for 2 weeks. Sevenstrains were isolated from Tehran petroleum reservoir wastewater, 5 strains were isolated from Kerman Petroleum reservoirwaste water and 3 strains were isolated from soil contaminated

hydrocarbons. Eight isolated strains that show higher growth rateon hexadecane were selected between the fifteen isolated bacteriafor further study. Strains isolated were identified by classical bio-chemical tests. Molecular identification of the isolated bacteriawere perform by amplification and sequencing the 16S rDNA genesequencing and comparing them to the database of known 16SrDNA sequences (Fig. 1 and Table 1). The results of identificationwere showed in Table 1. The phylogenic trees of these eight iso-lated strains were illustrated in Fig. 1. Sequences in study weresubmitted to EMBL database. Accession numbers of these strainsin EMBL are: HE862281–HE862288.

3.2. Growth rate and hexadecane removal by strains

All bacterial strains were grown in hexadecane (1%) for 1 weekwith shaking (160 rpm). during this week, ever day optical densitywas read for each strain until the end of week and O.D that was re-lated to exponential phase was reported as growth rate. After1 week, hexadecane biodegradation was analyzed by GC-FID meth-od. The results of the growth and degradation were presented inTable 2. As reported in this table, the strains Achromobacter pie-chaudii strain O1 and Rhodococcus erythropolis strain G2 have highpercentage of hexadecane biodegradation and growth rate, respec-tively of 93% and 84%. On the other hand, the Pseudomonas aerugin-osa strain B has the lowest percentage of hexadecane removal(38%) and low growth rate between all the isolated bacteria.

3.3. Effect of various concentrations of hexadecane on bacterial growth

For determining the effect of various concentrations of hexadec-ane on bacterial growth, selected the isolated bacteria were grownin different concentration of hexadecane (1%, 2.5%, 4%, 5.5% and 7%)then optical density was read at 600 nm ever day for each strainthat incubated at 30 �C. The result of this experiment was pre-sented in Fig. 2. As shown in this figure, when the concentrationsof hexadecane were increased, the growth rate of degradative bac-

Fig. 1. Phylogenetic tree of 16S rDNA sequences of the bacterial isolates obtained from petroleum reservoirs waste water. The tree was constructed using sequences ofcomparable region of the 16S rDNA gene sequences available in public databases. Neighbour-joining analysis using 1000 bootstrap replicates was used to infer tree topology.The bar represents 0.1% sequence divergence. Sequenced data showing the location of selected isolated strain.

Table 1Closest relatives of 16rDNA gene sequences of bacteria isolated in this study.

Isolate Closest hit Accessionnumber

ID(%)

G2 Rhodococcus erythropolis isolate PhyCEm-1365

HE862281 98

M2 Stenotrophomonas maltophilia strain MHFENV 20

HE862282 98

B Pseudomonas aeruginosa strain MSSRFD43 HE862283 99L1 Rhodococcus jostii RHA1 HE862284 100Q1 Stenotrophomonas maltophilia strain Ags-9 HE862285 98O1 Achromobacter piechaudii strain Shan11 HE862286 98Q3 Tsukamurella tyrosinosolvens IFM 10623 HE862287 100L2 Pseudomonas fluorescens strain NBRC

15832HE862288 99

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teria were decreased. For example, Stenotrophomonas maltophiliastrain M2 has optical density of 1.5 in 1% of hexadecane, however,this value decreases to 0.3 in 7% concentration of hexadecane. Thebest concentration of hexadecane that allowed to high growth ratein all bacterial strains was 2.5%. Between the bacterial strains A.piechaudii strain O1 and R. erythropolis strain G2 can adapt and de-grade high concentration of hexadecane better, as these strainshave the value of O.D 1.5 and 1.4 in 5% concentration of hexadec-ane. The lowest growth in different concentration of hexadecane inall bacterial strains belongs to P. aeruginosa strain B that has only0.3 O.D at 7% concentration of hexadecane.

3.4. Adherence to organic phase and emulsification activity by theisolated bacteria

Cell surface hydrophobicity and emulsification activity of theisolated bacteria were studied by emulsification (E24%) and bacte-rial adhesion to hydrocarbon tests (BATH). The results were pre-sented in Table 3. R. erythropolis strain G2 and A. piechaudii strainO1 have high cell surface hydrophobicity (27, 32 BATH%) andemulsification activity (30.8, 35.7 E24%), however, P. aeruginosastrain B has the lowest hydrophobicity and emulsification betweenall the isolated bacteria (4, 1.42).

3.5. Detection of catabolic gene by PCR

The presence of catabolic gene encoding alkane hydroxylasesenzymes alkB gene between isolated strains was determined byPCR using specific primers. Alk-B primers successfully amplified

PCR products of expected size from all isolated strains. Fig. 3 showsthe results of PCR analysis which verified alkane hydroxylase genewhich existed in all isolated strains.

4. Discussion

In lots of researches, alkane degrading bacteria have been iso-lated from different environment such as waste water and oil con-taminated soils (Cappello et al., 2012b). Jurelevicius et al. (2012)described alkane degrading bacteria in soils taken from KingGeorge Island in Maritime Antarctic. They found that there is ahigh diversity of alkane degrading bacteria in this environment.Quatrini et al. (2008) isolated some gram positive n-alkane degrad-ers from a hydrocarbon contaminated Mediterranean shoreline.One major difference between our study and other reports wasthe isolation of hexadecane degrading bacteria from petroleumreservoirs waste water in Iran. There are many reports about theisolation of hydrocarbon degrading bacteria from contaminatedsoil, sediment, sludge and water which are provided but no reporton the isolation of hexadecane degrading bacteria from petroleumreservoirs waste water has been published.

Hexadecane is a persistent pollutant in the environment be-cause of low water solubility of this hydrophobic contaminantwhich makes it unavailable for microorganisms. In this study, fif-teen bacterial strains capable of hexadecane degradation were iso-lated and eight strains that have high growth rate and degradationpotential were selected and identified by 16S rDNA sequence anal-

Fig. 2. Effect of various concentrations of hexadecane (1–7%) on bacterial growth (as Optical Density [O.D] at 600 nm) of eight bacterial strains. All strains were grown for7 day at 30 �C.

Table 2Growth and hexadecane removal by strains. All strains were cultured in BHMSmedium with hexadecane (1%) with shaking (160 rpm) for 1 weak at 30 �C.

Isolate Growth rate(O.D600/h)

Percentageof hexadecaneremoval

Rhodococcus erythropolis strain G2 1.9 93Stenotrophomonas maltophilia strain M2 1.32 74Pseudomonas aeruginosa strain B 0.53 38Rhodococcus jostii strain L1 1.52 51Stenotrophomonas maltophilia strain Q1 1.4 68Achromobacter piechaudii strain O1 1.85 84Tsukamurella tyrosinosolvens strain Q3 1.12 76Pseudomonas fluorescens strain L2 0.7 61

Table 3Measurement of emulsification activity (E24%) and cell surface hydrophobicity(BATH%) in isolated strain.

Strain Emulsification activity(E24%)

Cell surfacehydrophobicity(BATH%)

Rhodococcus erythropolis strain G2 30.8 27Stenotrophomonas maltophilia

strain M229 6

Pseudomonas aeruginosa strain B 1.42 4Rhodococcus jostii strain L1 28.7 22Stenotrophomonas maltophilia

strain Q16.12 24

Achromobacter piechaudii strain O1 35.7 32Tsukamurella tyrosinosolvens strain

Q37.14 29

Pseudomonas fluorescens strain L2 28.6 31

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ysis. These strains belong to the genus Rhodococcus, Stenotropho-monas, Pseudomonas, Achromobacter and Tsukamurella. Someresearchers described these genera as biodegradative bacteria.Song et al. (2011) isolated Rhodococcus which is capable of degrad-ing aliphatic hydrocarbons, Hua et al. (2007) described the genus ofPseudomonas capable of degrading of hexadecane.

Biodegradation is based on the use of microbial populationsthat possess the ability to modify or decompose certain pollutants.In this process, pollutants are mineralized completely (Atlas, 1994;Cunha and Leite, 2000; Watanabe and Hamamura, 2003). In thepresent study, the removals of hexadecane by selected isolatedbacterial strains were studied. After 1 week at 32 �C, hexadecaneconcentration showed 70% decrease approximately in all strains,however, two strains have more degradation capacity (more that80%). Other investigators, over a period of 10 days of culture, haveachieved 40% (Roy and Greer, 2000) and 80% (Dashti et al., 2008).

Most researchers have studied biodegradation of aliphatichydrocarbons in concentration less than 4% in liquid system (Qua-trini et al., 2008). In this study, increased concentration of hexadec-ane from 1% to 7% on growth of isolated strains was studied. Wefound that when the concentration of hexadecane increases thegrowth rate of bacteria decreases, however, this decrease is notthe same for all strains. The optimum concentration of hexadecaneto grow was 2.5%.

The solubility of hexadecane in water is less than0.9 � 10�6 mg/L (Bernardez, 2009). Therefore, it is supposed thatbacteria must have some mechanisms to uptake and use thishydrophobic substrate. The cell surface hydrophobicity and pro-duction of emulsifier may be the two mechanisms for better uptak-ing and degradating of hexadecane in liquid solution. The results ofthis study indicated that there are a direct relationship betweencell surface hydrophobicity and emulsification activity with alkanebiodegradation. Since strains G2 and O1 have high cell surfacehydrophobicity and emulsification activity and can degrade alkane

in more quantity and higher concentration than other strains.These results demonstrate that when a bacterial strain has highcell surface hydrophobicity, It can produce more emulsifier andthis will enhance alkane biodegradation.

In our previous study (Hasanshahian and Emtiazi, 2008) we re-ported a clear correlation between emulsification activity, cell-adherence to hydrocarbon and growth rate of the crude oil degrad-ing bacteria in crude oil media.

In this study, it is confirmed that bacterial strains that have highhydrophobicity can produce more bio emulsifier and these mecha-nisms lead to better biodegradation of hexadecane.

The first step in the aerobic degradation of alkanes by bacteria iscatalyzed by oxygenases. These enzymes, which introduce oxygenatoms, are derived from molecular oxygen taken from the alkanesubstrate, play an important role in oil bioremediation and in thecometabolic degradation of compounds. Vomberg and Klinner(2000) isolated 45 alkane degrading bacteria belonging to 37 bac-teria species from soil, contaminated by crude-oil in Germany.They studied distribution of alk-B between isolates by PCR-hybrid-ization method. They concluded that group (III) alkane hydroxylaseis predominant between isolates. The genes alkBs have been foundin various environments, such as Alaskan sediments, contaminatedsoil, cold ecosystems, a fuel oil-contaminated site, a shallow aqui-fer, bulk soil, a land treatment unit, Arctic and Antarctic soil andSea water (Van Beilen et al., 2003). The gene alkB could possiblybe used as a marker to predict the potential of different environ-ments for oil degradation (Van Beilen et al., 2003). Heissblanquetet al. (2005) showed that there are clear differences in the predom-inance of the two alk-B genotypes in freshwater and soil micro-cosms. However, both types of alk-B gene were increased in themost polluted soils (Heissblanquet et al., 2005).

In this study distribution of alk-B is almost the same between 8bacterial isolates. This result indicated that alkane hydroxylase en-zyme encoded by alk-B gene is essential for hydrocarbonbiodegradation.

5. Conclusion

The results described in this paper show that hexadecanedegrading bacteria have more diversity and ability for biodegrada-tion in waste water petroleum reservoirs in Iran. By applying thesebacteria for bioremediation purpose, we can manage waste whichare produced in petroleum reservoirs better.

Acknowledgment

This work was financially supported by Shahid Bahonar Univer-sity of Kerman.

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