Int. J. Environ. Sci. Technol. (2015) 12:2801–2808 DOI 10.1007/s13762-015-0779-0 ORIGINAL PAPER Evaluation of bioremediation potential and biopolymer production of pseudomonads isolated from petroleum hydrocarbon-contaminated areas A. Goudarztalejerdi • M. Tabatabaei • M. H. Eskandari • D. Mowla • A. Iraji Received: 17 October 2014 / Revised: 6 January 2015 / Accepted: 14 February 2015 / Published online: 4 March 2015 Ó Islamic Azad University (IAU) 2015 Abstract Bacteria are diverse and abundant in soils, but polyhydroxyalkanoate has a functional role in bacterial only a few bacteria have known to grow on hydrocarbon- survival and stress tolerance in the toxic environments and contaminated areas and utilize complex carbon source such poor nutrient availability. as crude oil for the synthesis of polyhydroxyalkanoate (bioremediation potential and the ability to produce impor- Keywords Biopolymers Á Bioremediation Á tant biopolymers). Among 32 samples collected from sev- Polyhydroxyalkanoate Á Pseudomonads eral sites of petroleum refinery soil and oily sludge of Iranian southwestern refineries, 45 oil-degrading pseudomonads were identified, and 33 % of the isolated Pseudomonas Introduction strains were able to produce polyhydroxyalkanoate using Gachsaran crude oil (2 % v/v) as carbon source. The re- Soil contamination with petroleum and petroleum-based peated monomer composition of the copolymer produced hydrocarbons has caused critical environmental and health from Gachsaran crude oil was determined by gas chro- defects, and increasing attention has been paid for devel- matography/mass spectrometry. The produced monomers oping and implementing innovative technology for clean- composites contained: C8 (3-hydroxyoctanoate), C10 (3- ing up this contaminant. Bioremediation methods are hydroxydecanoate), C12 (2-hydroxydodecanoate), C14 (3- currently receiving favorable publicity as promising envi- hydroxytetradecanoate), and C16 (3-hydroxydecahex- ronmental-friendly, efficient, and cheap treatment tech- anoate), which are known as biopolymers. This study nologies for the remediation of hydrocarbons and can be indicates oil-contaminated areas can be important sources described as the conversion of chemical compounds by for polyhydroxyalkanoate producers which can be used living organisms, especially microorganisms, into energy, for the bioremediation of crude-oil-polluted sites; also cell mass, and biological waste products (Rahman et al. 2002; Minai-Tehrani et al. 2015; Barin et al. 2014). Some A. Goudarztalejerdi Á M. Tabatabaei (&) of the most important of these bioremediation products are Department of Pathobiology, School of Veterinary Medicine, polyhydroxyalkanoic acids (PHAs), which are family of Shiraz University, Shiraz, Iran biopolymers formed by the biological condensation of e-mail: [email protected] hydroxyalkanoic acids, produced by bacteria and archaea. M. H. Eskandari PHAs are deposited as water-insoluble inclusions in the Department of Food Science and Technology, cells (Rehm and Steinbuchel 1999; Anderson and Dawes College of Agriculture, Shiraz University, Shiraz, Iran 1990). These natural polyesters are considered for several D. Mowla applications in the packaging, medical, pharmaceutical, Environmental Research Center in Petroleum and Petrochemical agricultural, and food industries or as raw materials for the Industry, Shiraz University, Shiraz, Iran synthesis of enantiomerically pure chemicals and the pro- duction of paints due to its biodegradability (Rehm and A. Iraji Central Research Laboratory, Shiraz University of Medical Steinbuchel 1999; Zinn et al. 2001; Luengo et al. 2003; Sciences, Shiraz, Iran Reddy et al. 2008). PHAs are produced by bacteria under 123 2802 Int. J. Environ. Sci. Technol. (2015) 12:2801–2808 unbalanced growth conditions when a carbon source is Isolation and identification of Pseudomonas strains available in excess, and other nutrients are growth-limiting and act as a mechanism to store excess carbon and energy Pseudomonas strains were isolated from contaminated soil (Zinn et al. 2001;Rehm2003; Ivanov et al. 2015). and oily sludge samples collected from Iranian south- Polyester synthases are the key enzymes of polyester western refineries (Table 1). A total of 32 samples were biosynthesis and catalyze the conversion of (R)-hydrox- collected during the sampling period. Samples were seri- yacyl-CoA thioesters to polyesters with the concomitant ally diluted using sterile normal saline (10-1–10-5) and release of CoA (Zinn et al. 2001; Rehm 2003). enriched by culturing aerobically at 30 °C in tryptic soy PHAs are produced by many different bacterial cultures, broth (TSB) (Merck, Germany) overnight. The enriched the first report of PHA formation by a Pseudomonas spe- samples were inoculated on cetrimide agar (Merck, Ger- cies; P. oleovorans, described the production of a polymer many) containing: Peptone from gelatin 20.0 (g/l); mag- containing 3-OH-octanoate by assimilation of medium- nesium chloride 1.4 (g/l); potassium sulfate 10.0 (g/l); N- and long-chain length fatty acids (Desmet et al. 1983) and cetyl-N,N,N-trimethylammoniumbromide (cetrimide) later reported that variety of pseudomonads, such as 0.3 (g/l); agar–agar 13.6 (g/l); and 10 ml/l glycerol (Mer- P. putida, P. oleovorans, and P. aeruginosa can produce ck, Germany) and then incubated at 30 °C, overnight. PHAs with many different structures as energy and carbon Single colonies were achieved by repeated streaking storage materials when the cells are cultivated in the method on cetrimide agar plates. After gram staining and presence of various carbon sources (Huisman et al. 1989). determination of catalase and oxidase activities, isolates Most of the carbon substrates supplied for PHA pro- were identified by physiological and biochemical tests in- duction are pure alkanes, fatty acids or carbohydrates (Kim cluding: methyl red (MR), Voges–Proskauer (VP) test, et al. 1997; Ashby et al. 2002), complex substrates, such as gelatin hydrolysis, motility test, MacConkey growth, pig- castor or euphorbia oil, resulted in PHA of (C6–C14) with ment production, urease test, indole test, nitrate reduction P. aeruginosa (Eggink et al. 1995); however, the use of test, and lactose, glucose, and maltose utilization tests. subproducts or wastes has hardly been explored at all, These tests were selected to provide high discrimination which may well be due to the complexity of their com- among Pseudomonas spp., based on a high probability of a position. P. resinovorans accumulated 15 % of the cellular positive or negative result. Different phenotypic charac- dry weight of PHA from tallow (Cromwick et al. 1996). teristics were evaluated as outlined in Bergey’s manual of Recently PHA productions by bacteria have been stud- systematic bacteriology (Breed et al. 1975). ied and their PHAs properties well-characterized. Howev- er, our finding about PHA-producing bacteria in Screening of PHA-producing microorganism hydrocarbon-contaminated regions and properties of these PHA remains limited. So far two PHA producers Pseu- The pseudomonad-isolated strains from contaminated area domonas spp. from hydrocarbon-contaminated area have were assayed for PHA production by using an initial Sudan been known: P. stutzeri 1317 (He et al. 1998) and Black B staining (Burdon 1946). Positively stained isolates P. pseudoalcaligenes strain YS1 isolated from oil-con- were induced to accumulate PHA. These strains were in- taminated soils (Hang et al. 2002). cubated in 100 ml PHA production medium (6.0 g The main objective of this study was to investigate Na2HPO4, 3.0 g KH2PO4, 0.5 g NH4Cl, 0.5 g NaCl, bioremediation potential and biopolymer production of 1.0 mM MgSO4, and 0.1 mM CaCl2 per liter of medium) pseudomonads isolated from petroleum hydrocarbon-con- (Goh and Tan 2012) containing 2 % (v/v) sterilized Gach- taminated areas of different regions of Iranian southwest- saran crude oil (with identified compounds; Table 2) as sole ern refineries. This study was carried out between October carbon source and then checked for PHA production by 2012 and February 2014. specific Nile red A dye staining method (Spiekermann et al. 1999). The PHA-accumulating colonies, after Nile red A staining, showed bright orange fluorescence on irradiation Materials and methods with UV light, and their fluorescence intensity increased with increase in PHA content of the bacterial cells. Reference strains Quantitative analysis of PHAs In this study, the following reference strains were used: P. aeruginosa ATCC 15442, P. aeruginosa ATCC 9027, The isolates showing bright orange fluorescence on irra- P. putida ATCC 12633, P. putida ATCC 47054, Al- diation with UV light after Nile red staining were selected caligenes eutrophus ATCC 17699, and Escherichia coli as PHA accumulators. These isolates were first grown in ATCC 25922. PHA production medium in 100-ml flasks and incubated at 123 Int. J. Environ. Sci. Technol. (2015) 12:2801–2808 2803 Table 1 Geographic location and type of contamination prevalent in different sampling sites Isolation sites Geographic location Type of sample Hydrocarbon-contaminated soil Oily sludge Gachsaran refinery 3559287N 479844.88E 5 2 Shiraz Oil Refining Company 3290352N 660341.84E 2 2 Farrashband gas refinery 3192389N 607274E 2 3 Asalouyeh refineries 3043170N 660111E 6 4 Shiraz petrochemical company 3307721.23N 668001E 2 – Khonj gasoline-contaminated soil 3087454N 739653.26E 2 – Shiraz (control-negative samples) 3290352N 660341.84E 2 – Table 2 Gachsaran oil analysis using GC/MS