- Email: [email protected]
Isolation and characterization of biosurfactant producing bacteria from Persian Gulf (Bushehr provenance)
Marine Pollution Bulletin 86 (2014) 361–366
Contents lists available at ScienceDirect
Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Isolation and characterization of biosurfactant producing bacteria from Persian Gulf (Bushehr provenance) Mehdi Hassanshahian ⇑ Department of Biology, Faculty of Science, Shahid Bahonar University of Kerman, Kerman, Iran
a r t i c l e
i n f o
Article history: Available online 15 July 2014 Keywords: Biodegradation Biosurfactant Persian Gulf Marine environment
a b s t r a c t Biosurfactants are surface active materials that are produced by some microorganisms. These molecules increase biodegradation of insoluble pollutants. In this study sediments and seawater samples were collected from the coastline of Bushehr provenance in the Persian Gulf and their biosurfactant producing bacteria were isolated. Biosurfactant producing bacteria were isolated by using an enrichment method in Bushnell-Hass medium with diesel oil as the sole carbon source. Five screening tests were used for selection of Biosurfactant producing bacteria: hemolysis in blood agar, oil spreading, drop collapse, emulsification activity and Bacterial Adhesion to Hydrocarbon test (BATH). These bacteria were identified using biochemical and molecular methods. Eighty different colonies were isolated from the collected samples. The most biosurfactant producing isolates related to petrochemical plants of Khark Island. Fourteen biosurfactant producing bacteria were selected between these isolates and 7 isolates were screened as these were predominant producers that belong to Shewanella alga, Shewanella upenei, Vibrio furnissii, Gallaecimonas pentaromativorans, Brevibacterium epidermidis, Psychrobacter namhaensis and Pseudomonas fluorescens. The largest clear zone diameters in oil spreading were observed for G. pentaromativorans strain O15. Also, this strain has the best emulsification activity and reduction of surface tension, suggesting it is the best of thee isolated strains. The results of this study confirmed that there is high diversity of biosurfactant producing bacteria in marine ecosystem of Iran and by application of these bacteria in petrochemical waste water environmental problems can be assisted. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Microbial compounds which exhibit pronounced surface activity are classified as biosurfactants (Cappello et al., 2012b; Bodour and Maier 2002). They decrease surface tension at the air–water interface and between immiscible liquids, or at the solid–liquid interface. These molecules chemically belong to various categories such as glycolipids, lipopeptides, polysaccharide–protein complexes, phospholipids, fatty acids and neutral lipids (Cappello et al., 2012a,c). Excellent detergency, emulsification, dispersing traits, penetrating, thickening, microbial growth enhancement, metal sequestering and resource recovering (oil) are important characteristics of biosurfactants which make them suitable to replace with chemical surfactants. Therefore, they have wide applications in cosmetics, oil recovery and bioremediation (Emtiazi et al., 2005; Ghanavati et al., 2008). The marine environment represents the major component of the Earth’s biosphere. It covers a majority (70%) part of earth’s ⇑ Tel.: +98 9132906971; fax: +98 3222032. E-mail address: [email protected] http://dx.doi.org/10.1016/j.marpolbul.2014.06.043 0025-326X/Ó 2014 Elsevier Ltd. All rights reserved.
surface and makes 90% of the volume of its crust. Oceans represent a vast and exhaustive source of natural products in the globe, harboring the most diverse groups of flora and fauna (Hasanshahian and Emtiazi, 2008). Marine microorganisms have developed unique metabolic and physiological capabilities to thrive in extreme habitats and produce novel metabolites which are not often present in the microbes of terrestrial origin (Fenical, 1993; Hassanshahian et al., 2013). Therefore, this rich marine habitat provides a magnificent opportunity to discover newer compounds such as antibiotics, enzymes, vitamins, drugs, biosurfactant (BS), bioemulsifier (BE) and other valuable compounds of commercial importance (Jensen and Fenical, 1994; Austin, 1989; Romanenko et al., 2001; Lang and Wagner, 1993). Biosurfactant producing microorganisms are ubiquitous, inhabiting both water (sea, fresh water, and groundwater) and land (soil, sediment, and sludge) as well as extreme environments (e.g. hypersaline sites, oil reservoirs), and thriving at a wide range of temperatures, pH values and salinity. Microorganisms produce BS/BE to mediate solubilisation of hydrophobic compounds in their environment to be able to utilize them as substrates, however, this fact may not be always true. Few microbes produce BS/BE on water
362
M. Hassanshahian / Marine Pollution Bulletin 86 (2014) 361–366
soluble substrates. It has been suggested that the presence of surface active molecules on the microbial cell surface increases the hydrophobicity of the cell and helps it to survive in hydrophobic environment (Hassanshahian et al., 2014b; Das et al., 2009). Despite the fact that marine environment represents a wealthy basin of diverse microorganisms, it is important to know that at the same time they are suffering from anthropogenic pollution with domestic and industrial wastes (Hassan et al., 1996; Hassanshahian et al., 2014c). Large quantities of crude oil, hydrocarbons, petroleum oil products and halogenated compounds finds their way into the marine ecosystem through accidental spillage (Satpute et al., 2005). To treat, emulsify or simply overcome such spills, on the one hand, many petroleum based synthetic chemical surfactants often get used (Hassanshahian et al., 2014a). Such synthetic compounds on the other hand, however often have detrimental ecological effects (Hassanshahian et al., 2012a). The use of biosurfactants and bioemulsifiers therefore may represent a better alternative to overcome the toxicity of synthetic compounds. The environmental roles of the biosurfactants produced by marine microorganisms have been reported earlier (Poremba et al., 1991; Schulz et al., 1991; Abraham et al., 1998). Persian Gulf is a marine environment that was polluted with crude oil during the 1991 Gulf war. The pollution impact of this episode has been evaluated in several studies, all indicated that crude oil accumulated and remained for long time in coastal area (Hassanshahian et al., 2010; Emtiazi et al., 2009). The oil pollution problem is particularly acute in an oil producing area of this marine environment such as Bushehr provenance. The aims of this study was to understanding the diversity of biosurfactant producing bacteria in Persian Gulf and especially at Bushehr provenance, also isolation and characterization of these important bacteria is another purpose of this research. 2. Materials and methods 2.1. Sampling Sediments and seawater samples were collected from five stations in the Persian Gulf at Bushehr provenance (Khark Island (KI), Ganaveh Port (GP), Siraf Port (SP), Halileh Shoreline (HS) and Asalooye Shoreline (AS) 26°150 N; 54°150 E). From each station five sediments and seawater samples were taken. These stations located near to petrochemical plants and oil refinery. Sediment samples were taken from 1–12 cm below the surface of coastal using a sterile knife. Seawater samples were collected from a depth of 15 cm in sterile 100 ml bottles and transported on ice to the laboratory for isolation. 2.2. Isolation and selection of biosurfactant producing bacteria ONR7a medium was used for isolation of biosurfactant producing bacteria. ONR7a contained (per liter of distilled water) 22.79 g of NaCl, 11.18 g of MgCl26H2O, 3.98 g of Na2SO4, 1.46 g of CaC12 2H2O, 1.3 g of TAPS0 {3-[N-tris(hydroxymethyl)methylamino]-2hydroxypropanesulfonica cid}, 0.72 g of KCl, 0.27 g of NH4Cl, 89 mg of Na2HP047H2O, 83 mg of NaBr, 31 mg of NaHCO3, 27 mg of H3BO3, 24 mg of SrCl26H2O, 2.6 mg of NaF, and 2 mg of FeCl24H2O. For solid media, Bacto Agar (Difco) (15 g/l) was added to the solution (Dyksterhouse et al., 1995). ONR7a medium were supplemented with 1% (v/v) diesel oil as sole carbon source and energy. Portion of sediment (10 g) or condensed seawater (10 ml) were added to Erlenmeyer flasks containing 100 ml of medium and the flasks were incubated for 10 days at 30 °C on rotary shaker (180 rpm, INFORS AG). Then
5 ml aliquots were removed to fresh medium. After a series of four further subcultures, inoculums from the flask were streaked out and phenotypically different colonies purified on ONR7a agar medium. The procedure was repeated and isolates only exhibiting pronounced growth on diesel oil were stored in stock media with glycerol at 20 °C for further characterization (Chaillan et al., 2004; Tebyanian et al., 2013). 2.3. Screening of biosurfactant producing bacteria Three tests were used for screening and selection of prevailing biosurfactant producing bacteria. These tests were described below. 2.3.1. Hemolytic test Hemolytic activity was carried out as described by Carrillo et al. (1996). Isolated strains were screened on blood agar plates containing 5% (v/v) blood and incubated at 30 °C for 24–48 h. Hemolytic activity was detected as the presence of a clear zone around a colony. 2.3.2. Drop collapse method The drop-collapse technique was carried out in the polystyrene lid of a 96-microwell plate (Biolog, Harward, CA, USA) as described by Jain et al. (1991) and 100 ll culture supernatant was added to wells of a 96-well microtiter plate lid, and then 5 ll of crude oil was added to the surface of the culture supernatant. Biosurfactant-producing culture gave flat drops. Aliquots from a culture of each strain were analyzed on two separate microtiter plates. 2.3.3. Oil spreading method Oil spread technique was carried out according to Morikova et al. (2000) and Youssef et al. (2004). 50 ml of distilled water was added to Petri dishes followed by addition of 100 ll of crude oil to the surface of the water. Then, 10 ll of the culture filtrates was put on the crude oil surface. The diameter of the clear zone on the oil surface was measured. 2.4. Liquid surface tension The surface tension (ST) of the culture supernatants was measured with a digital surface tensiometer (DCAT, DataPhysics Instruments GmbH, Filderstadt, Germany) working on the principles of Wilhelmy plate method (Fernandes et al., 2007). The validity of the surface tension readings was checked with pure water (70.78 ± 0.02 mN/m) before each reading. All surface tension readings were taken in triplicate. 2.5. Emulsification activity and Bacterial Adhesion To hydrocarbon (BATH test) The emulsification activity (E24) was determined by the addition 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 emulsification activity was determined as the percentage of height of the emulsified layer (mm) divided by the total height of the liquid column (mm) (Emtiazi et al., 2009). Bacterial adhesion to hydrocarbon was carried out according to Pruthi and Cameotra, 1997. 2.6. Identification of the isolates 2.6.1. Biochemical identification In order to identify and characterize the bacteria isolates, some biochemical tests was carried out such as: Gram staining, oxidation/fermentation, production of acid from carbohydrates,
M. Hassanshahian / Marine Pollution Bulletin 86 (2014) 361–366
hydrolysis of gelatin and citrate. These tests done according to the Bergey’s manual for identification taxonomy (Holt et al., 1998). 2.6.2. Molecular identification Analysis of 16S rRNA was performed for the taxonomic characterization of isolated strains. Total DNA extraction of bacterial strains was performed with the CTAB method. The bacterial 16S rRNA loci were amplified using the forward domain specific bacteria primer, Bac27_F (50 -AGAGTTTGATCCTGGCTCAG-30 ) and universal reverse primer Uni_1492R (50 -TACGYTACCTTGTTACGACTT-30 ). The amplification reaction was performed in the total volume of 25 ll consisting, 2 mM MgCl2 (1 ll), 10X PCR reaction buffer (200 mM Tris; 500 mM KCl) (2.5 ll), 2 mM each dNTP (2 ll), 0.15 mM each primer (1 ll), 1U (0.5 ll) Taq DNA polymerase (Qiagen, Hilden, Germany) and 2 ll of template DNA (50 pm). The distill water was added for remaining of reaction (15 ll). Amplification was performed in a thermocycler GeneAmp 5700 (PE Applied Biosystem, Foster City, CA, USA). The temperature profile for PCR was kept, 94 °C for 5 min, 94 °C for 1 min, 54 °C for 1 min, 72 °C for 1 min, 30 cycles; then 72 °C for 10 min and finally storage at 4 °C (Troussellier et al., 2005). The 16S rRNA amplified was sequenced with a Big Dye terminator V3.1 cycle sequencing kit on an automated capillary sequencer (model 3100 Avant Genetic Analyzer, Applied Biosystems). Similarity rank from the Ribosomal Database Project RDP) (Maidak et al., 1997) and FASTA Nucleotide Database. Query were used to determine partial 16S rRNA sequences to estimate the degree of similarity to other 16S rRNA gene sequences. Analysis and phylogenetic affiliates of sequences was performed as previously described protocols (Yakimov et al., 2006). 3. Results 3.1. Isolation and screening of biosurfactant producing bacteria from marine samples Eighty morphologically distinct microbial colonies were isolated from collected sea water and sediment samples. These isolates belong to these geographic zones: 21 were isolated from the KI samples, 15 from the GP samples, 17 from the SP samples, 12 from the HS samples and 15 from the AS samples. Ninety percent of the bacterial isolates (72 of 80) were Gram-negative. Forty-six isolates were determined as biosurfactant producing bacteria using three screening tests. However the responses of these isolates to screening methods were different. As shown in Fig. 1 drop collapse method has the most positive response and
363
hemolysis on blood agar has the lowest positive response while the positive and negative response of oil spreading method were equal (Fig. 1). 3.2. Biosurfactant production by isolated strains Fourteen isolated strains that show high growth rate on diesel oil were selected between 46 isolates for further study. Table 1 illustrates the results of screening tests for these isolated strains. As shown in this table the best isolates that have sufficient response to all screening tests were: I36, O15, N4, U15, E14, T26, N3 and H4. However some isolated strains such as K1, P1, and B16 have positive response to one or two screening tests (Table 1). 3.3. Biochemical properties of the isolated strains Some preliminary identification tests were done to discriminate between isolated strains. Table 2 shows the results of these diagnostic tests. As shown in this table the most isolated strains were Gram negative, oxidase positive and motility positive. However there are some differences between isolated bacteria (Table 2). 3.4. Cell surface hydrophobicity, emulsification index and decrease of surface tension by isolated strains Emulsification activity, bacterial adhesion to hydrocarbon (BATH) and reduction of surface tension assay were analyzed for each strain separately. Data obtained from these tests were shown in Table 3. Strains I36, O15, N4, U15, E14, T26, and N3 have the highest values of emulsification activity and cell surface hydrophobicity. These strains also produce more biosurfactant and dramatically decrease surface tension than other strains. The results indicated that there are a direct relationship between cell surface hydrophobicity and biosurfactant production with emulsification activity (Table 3). 3.5. Molecular identification and phylogenetic analysis of isolated strains Molecular identification of the isolates were perform for I36, O15, N4, U15, E14, T26, and N3 strains by amplification and sequencing the 16S rRNA gene sequencing and comparing them to the database of known 16S rRNA sequences. The molecular identification of these strains show that these strains belongs to: Shewanella alga (Strain N4), Shewanella upenei (Strain E14), Vibrio furnissii (Strain U15), Gallaecimonas pentaromativorans (Strain O15), Brevibacterium epidermidis (Strain N3), Psychrobacter namhaensis (Strain I36) and Pseudomonas fluorescens (Strain T26). The phylogeny tree of these isolated strains was shown in the Fig. 2. All sequences of seven bacteria were submitted to Genetic sequence database at the National Center for Biotechnical Information (NCBI). Gene Bank ID of these strains in NCBI is: HF968433 to HF968439. 4. Discussion
Fig. 1. The response of total isolates to the three biosurfactant production assays.
Biosurfactants can fulfill various physiological roles and provide ecological advantages to the producing strains (Ron and Rosenberg, 2001). For instance, they improve the bioavailability of water insoluble substrates by emulsification, thus enhancing biodegradation of several environmental pollutants. Biosurfactant-producing microbes can be found in various ecosystems, although the environments that are impacted with hydrophobic contaminants such as refinery wastes and petroleum are more yielding than undisturbed ones (Batista et al., 2006).
364
M. Hassanshahian / Marine Pollution Bulletin 86 (2014) 361–366
Table 1 Biosurfactant production assays for isolated strains. Isolate
Hemolytic test
Drop collapse (shape of drop in comparison to blank)
Oil spreading (area of oil displaced in mm) (mm)
I36 O15 N4 U15 B16 E14 T26 N3 P1 H4 W35 K1 K3 W25
+ + + + + + + + +
+++ +++ +++ +++ +++ +++ +++ ++ +++ + + ++ ++
10 14 8 13 10 10 10 8.5 7 10 6 6 8 9
Abbreviations: +, positive response; , negative response (for hemolytic test); +, low flat; ++, middle flat, +++, completely flat (for drop collapse test).
Table 2 Some biochemical characteristics of selected biosurfactant producing strains. Isolate
Origin
Gram staining
Oxidase
Motility
Acid from glucose
I36 O15 N4 U15 B16 E14 T26 N3 P1 H4 W35 K1 K3 W25
KI-sea water GP-sea water SP-sea water HS-sea water AS-sea water KI-sediment GP-sediment SP-sediment HS-sediment AS-sediment KI-sediment AS-sea water KI-sea water HS-sea water
N N N N N N N P N N P N N N
+ + + + + + +
+ + + + + + + +
+ + + + + +
Abbreviations: N, gram negative; P, gram positive; +, positive response; , negative response.
Table 3 Measurement of emulsification activity (%, E24), cell surface hydrophobicity (%, BATH) and decrease of surface tension (mN/m) by strains in this study. All data that reported in this table is the mean of three values and cited value in the table is standard deviation.
a
Isolate
Emulsification activity (E24%)
Cell surface hydrophobicity (BATH%)
Surface tensiona (mN m1)
I36 O15 N4 U15 B16 E14 T26 N3 P1 H4 W35 K1 K3 W25
34 65 45 44 18 34 44 43 10 25 40 20 25 30
27 59 27 19 5.2 38.8 38 39 20 8.2 2.3 3.7 1.7 3.7
42.1 34.5 43.4 44.6 43 44.6 41.5 40.3 45.3 45.5 46.8 47.8 50.2 49.8
Surface tension of ONR medium without bacteria as blank is 53.6.
In this study, bacteria that dominated in the enrichment media were selected due to their ability to utilize the hydrophobic substrates. This is in line with the fact that hydrophobic pollutants in collected seawater and sediment samples contain several of the organic substrates added to the enrichments. Therefore, microorganisms in those samples are adapted to live with hydrophobic compounds as carbon source. These cultures yielded 80 isolates that were also differentiated based on colony morphology on ONR7a-agar plates.
Are there any biosurfactant producers among the isolated bacteria? To answer this question, we adopted a variety of screening assays as suggested by Walter et al. (2010). The isolated bacteria were searched for potential biosurfactant producers via three screening phases of ascending certainty. To get a preliminary indication, these isolated bacteria were grown on ONR7a containing diesel oil as a sole carbon source and inducer of biosurfactant production (initial screening phase). Emulsification of the diesel oil in the growth medium indicated biosurfactant production by
M. Hassanshahian / Marine Pollution Bulletin 86 (2014) 361–366
365
Fig. 2. Phylogenetic tree of 16S rRNA sequences of the isolated strains obtained from Persian Gulf. The tree was constructed using sequences of comparable region of the 16S rRNA gene sequences available in public databases. Neighbour-joining analysis using 1000 bootstrap replicates was used to infer tree topology. The bar represents 0.02% sequence divergence. Sequenced data showing the location of isolated strains.
tested bacteria. The cultures that exhibited weak emulsification showed scarce or no turbidity in the aqueous phase. In those cultures the cells might be adhering to the emulsified oil slicks (Bredholt et al., 1998). Emulsification of the diesel oil in water is a prerequisite that paves the way for biodegradation of this environmental pollutant by many bacteria. It enhances the bioavailability of the oil and thus increases the biodegradation rate (Bredholt et al., 1998; Minf et al., 2011; Hassanshahian et al., 2012b). However, visual inspection of the diesel oil cultures for biosurfactant production is an indirect screening method as the growth with hydrophobic compounds indicates the production of biosurfactants, but does not always correlate with this trait (Willumsen and Karlson, 1997). Second screening phase to further investigate biosurfactant production contain three tests: oil spreading, drop collapse and hemolytic activity assays. The clear zone diameters were larger for cultures of the strains I36, O15, N4, U15, E14, T26, N3 and H4 however the largest diameters observed for strain O15. Thus, indicating the presence of higher concentrations of biosurfactant. This is in good agreement with the drop collapse and hemolytic activity results. Microbial production of the surface-active compounds on diesel oil and other hydrophobic substrates has frequently been reported (Iqbal et al., 1995; Kumara et al., 2006). The data of the third screening phase (surface tension reduction and emulsification activity) confirmed unequivocally the production of biosurfactants by the mentioned strains. Taken together, the data of the screening assays in the three phases are coherent and support each other. Since all the screening assays were performed with cell-free culture supernatants, it can be concluded that the cells produce biosurfactant and secrete them to the extracellular medium. Similar results have been reported in many cases (Batista et al., 2006; Cappello et al., 2012b). Isolation of biosurfactant producing from marine environment was reported by other researcher in the world. For example Coelho et al. (2003) reported production of biosurfactant by quinoline degrading marine Pseudomonas sp. strain GU 104. Species of Alcanivorax produces a potent glucose–lipid surfactant (Abraham et al., 1998). In this research six different genus of biosurfactant producing bacteria were isolated from Persian Gulf at Bushehr provenance. These results are in agreement with the results that reported by other researcher. Pruthi and Cameotra (1997) screened biosurfactant producing bacteria by assay the cell surface hydrophobicity. They found a direct relationship between cell surface hydrophobicity and biosurfactant production. Batista et al. (2006) isolated 17 biosurfactant producing bacteria from oil contaminated beaches in Brazil. They found that six strains can reduce surface tension below than 40 mN m1.
5. Conclusion Marine microorganisms that produced biosurfactant have high diversity and ability in Persian Gulf. In this survey from 14 biosurfactant producing bacteria, 7 strains can produce high biosurfactant and reduce surface tension. Also we found a direct relationship between cell surface hydrophobicity and biosurfactant production with emulsification activity as the strain O15 that had high biosurfactant production also had efficient emulsification activity. Acknowledgment This work was supported financially by Shahid Bahonar University of Kerman. References Abraham, W.R., Meyer, H., Yakimov, M., 1998. Novel glycine containing glucolipids from the alkane using bacterium Alcanivorax borkumensis. Biochem. Biophys. Acta 1393, 57–62. Austin, B., 1989. Novel pharmaceutical compounds from marine bacteria. J. Appl. Bacteriol. 67, 461–470. Batista, S., Mounteer, A., Amorim, F., 2006. Isolation and characterization of biosurfactant/bioemulsifier producing bacteria from petroleum contaminated sites. Bioresour. Technol. 97, 868–875. Bodour, A.A., Maier, R.M., 2002. Biosurfactants: types, screening methods and application. Encycl. Environ. Microbio, vol. 2. Springer, New York, pp. 750–769. Bredholt, H., Josefen, K., Vatland, A., Bruhbeim, P., EImhjellen, K., 1998. Emulsification of crude oil by an alkane-oxidizing Rhodococcus species isolated from seawater. Can. J. Microbiol. 44, 330–340. Cappello, S., Genovese, M., Della Torre, C., Crisari, A., Hassanshahian, M., Santisi, S., Calogero, R., Yakimov, M.M., 2012a. Effect of bioemulsificant exopolysaccharide EPS2003 on microbial community dynamics during as says of oil spill bioremediation: a microcosm study. Mar. Pollut. Bull. 64 (12), 2820–2828. Cappello, S., Crisari, A., Hassanshahian, M., Genovese, M., Santisi, S., Yakimov, M.M., 2012b. Effect of a bioemulsificant exopolysaccharide (EPS2003) on abundance and vitality of marine bacteria. Water Air Soil Pollut.. http://dx.doi.org/10.1007/ s11270-012-1159-8. Cappello, S., Santisi, S., Calogero, R., Hassanshahian, M., Yakimov, M.M., 2012c. Characterization of oil-degrading bacteria isolated from bilge water. Water Air Soil Pollut.. http://dx.doi.org/10.1007/s11270-012-1103-. Carrillo, P.G., Mardaraz, C., Pitta-Alvarez, S.J., Giulietti, A.M., 1996. Isolation and selection of biosurfactant-producing bacteria. World J. Microbiol. Biotechnol. 12, 82–84. Chaillan, F., Le, F.A., Bury, E., Phantavong, Y.H., Grimont, P., Saliot, A., Oudot, J., 2004. Identification and biodegradation potential of tropical aerobic hydrocarbondegrading microorganisms. Res. Microbiol. 155 (7), 587–595. Coelho, J., Rivonkar, C.U., Bhavesh, N.S., Jothi, M., Sagodkar, U.M., 2003. Biosurfactant production by the quinoline degrading marine bacterium Pseudomonas sp. Strain GU 104, and its effect on the metabolism of green mussel Perna viridis L. Indian J. Mar. Sci. 32, 202–207. Das, P., Mukherjee, S., Sen, R., 2009. Biosurfactant of marine origin exhibiting heavy metal remediation properties. Biores. Technol. 100 (20), 4887–4890. Dyksterhouse, S.E., Gray, J.P., Herwig, R.P., Lara, J.C., Staley, J.T., 1995. Cycloclasticus pugetii gen. nov., sp. nov., an aromatic hydrocarbon degrading bacterium from marine sediments. Int. J. Syst. Bacteriol. 45, 116–123.
366
M. Hassanshahian / Marine Pollution Bulletin 86 (2014) 361–366
Emtiazi, G., Hassanshahyan, M., Golbang, N., 2005. Development of a microtiter plate method for determination of phenol utilization, biofilm formation and respiratory activity by environmental bacterial isolates. Int. Biodeterior. Biodegrad. 56, 231–235. Emtiazi, G., Saleh, T., Hassanshahian, M., 2009. The effect of bacterial glutathione S-transferase on morpholine degradation. Biotechnol. J. 4, 202–205. Fenical, W., 1993. Chemical studies of marine bacteria: developing a new resource. Chem. Rev. 93, 1673–1683. Fernandes, P.A.V., Arruda, I.R., Santos, A.F.A.B., Araujo, A.A., Maiors, A.M.S., Ximenes, F.A., 2007. Antimicrobial activity of surfactants produced by Bacillus subtilis R14 against multidrug-resistant bacteria. Braz. J. Microbiol. 38, 704–709. Ghanavati, H., Emtiazi, G., Hassanshahian, M., 2008. Synergism effects of phenol degrading yeast and ammonia oxidizing bacteria for nitrification in coke wastewater of Esfahan steel company. Waste Manage. Res. 26 (2), 203–208. Hasanshahian, M., Emtiazi, G., 2008. Investigation of alkane biodegradation using the microtiter plate method and correlation between biofilm formation, biosurfactant production and crude oil biodegradation. Int. Biodeterior. Biodegrad. 62, 170–178. Hassan, E.S., Banat, I.M., El-Shahawi, M.S., Abu-Hilal, A.H., 1996. Bacterial, nutrients and heavy metals pollution assessment along the eastern coastal area of the United Arab Emirates. J. Aquat. Ecosys. Health 5, 73–81. Hassanshahian, M., Emtiazi, G., Kermanshahi, R., Cappello, S., 2010. Comparison of oil degrading microbial communities in sediments from the Persian Gulf and Caspian Sea. Soil Sediment Contaminat. 19 (3), 277–291. Hassanshahian, M., Tebyanian, H., Cappello, S., 2012a. Isolation and characterization of two crude-oil degrading yeast strains, Yarrowia lipolytica PG-20 and PG-32 from Persian Gulf. Mar. Pollut. Bull. 64, 1386–1391. Hassanshahian, M., Emtiazi, G., Cappello, S., 2012b. Isolation and characterization of crude-oil-degrading bacteria from the Persian Gulf and the Caspian Sea. Mar. Pollut. Bull. 64, 7–12. Hassanshahian, M., Ahmadinejad, M., Tebyanian, H., Kariminik, A., 2013. Isolation and characterization of alkane degrading bacteria from petroleum reservoir waste water in Iran (Kerman and Tehran provenances). Mar. Pollut. Bull. 73, 300–305. Hassanshahian, M., Emtiazi, G., Caruso, G., Cappello, S., 2014a. Bioremediation (bioaugmentation/biostimulation) trials of oil polluted seawater: a mesocosm simulation study. Mar. Environ. Res. 95, 28–38. Hassanshahian, M., Zeynalipour, M.S., Hosseinzadeh Musa, F., 2014b. Isolation and characterization of crude oil degrading bacteria from the Persian Gulf (Khorramshahr provenance). Mar. Pollut. Bull. 82, 39–44. Hassanshahian, M., Yakimov, M.M., Denaro, R., Genovese, M., Cappello, S., 2014c. Using Real-Time PCR to assess changes in the crude oil degrading microbial community in contaminated seawater mesocosms. Inte. Biodeterior. Biodegrad. 93, 241–248. Holt, S.G., Kriey, N.R., Sneath, P.H.A., Staley, J.T., Williams, S.T., 1998. Bergy’s manual of determinative for bacteriology. Williams and Wilkins, New York. Iqbal, S., Khalid, Z.M., Malik, K.A., 1995. Enhanced biodegradation and emulsification of crude oil and hyperproduction of biosurfactants by a gamma ray-induced mutant of Pseudomonas aeruginosa. Lett. Appl. Microbiol. 21 (3), 176–179. Jain, D.K., Collins-Thompson, D.L., Lee, H., Trevors, J.T., 1991. A drop-collapsing test for screening surfactant-producing microorganisms. J. Microbiol. Method 13, 271–279. Jensen, P.R., Fenical, W., 1994. Strategies for the discovery of secondary metabolites from marine bacteria: ecological perspectives. Annu. Rev. Microbiol. 48, 559– 584.
Kumara, M., Leon, V., Materano, A., Ilzins, O.A., Galindo-Castro, I., Fuenmayor, S.L., 2006. Polycyclic aromatic hydrocarbon degradation by biosurfactant-producing Pseudomonas sp. IR1. Z. Naturforsch. C. J. Biosci. 61, 203–212. Lang, S., Wagner, F., 1993. Biosurfactants from marine microorganisms. In: Kosaric, N. (Ed.), Biosurfactants: Production Properties Applications (Surfactant Science Series), vol. 48. Marcel Dekker, New York, pp. 391–417. Maidak, B.L., Olsen, G.J., Larsen, N., 1997. The RDP ribosomal database project. Nucleic Acids Res. 25 (109–1), 11. Minf, S., Chamkha, M., Labat, M., Sayadi, S., 2011. Simultaneous hydrocarbon biodegradation and biosurfactant production by oil field-selected bacteria. J. Appl. Microbiol. 111, 525–536. Morikova, M., Hirata, Y., Imanaka, T., 2000. A study on the structure–function relationship of the lipopeptide biosurfactants. Biochim. Biophys. Acta 1488, 211–218. Poremba, K., Gunkel, W., Lang, S., Wagner, F., 1991. Toxicity testing of synthetic and biogenic surfactants on marine microorganisms. Environ. Toxicol. Water Qual. 6, 157–163. Pruthi, V., Cameotra, S.S., 1997. Rapid identification of biosurfactant-producing bacterial strains using a cell surface hydrophobicity technique. Biotechnol. Tech. 11, 671–674. Romanenko, L.A., Kalinovskaya, N.I., Mikhailov, V.V., 2001. Taxonomic composition and biological activity of microorganisms associated with a marine Ascidian Halocynthia aurantium. Russ. J. Mar. Biol. 27, 291–295. Ron, E., Rosenberg, E., 2001. Biosurfactants and oil bioremediation. Curr. Opin. Biotechnol. 13, 249–252. Satpute, S. K., Dhakephalkar, P. K., Chopade, B. A., 2005. Biosurfactants and bioemulsifiers in hydrocarbon biodegradation and spilled oil bioremediation. Indo-Italian brain storming workshop on technology transfer for industrial applications of novel methods and materials for environmental problem, vol. 2, pp. 1–18. Schulz, D., Passeri, A., Schmidt, M., Lang, S., Wagner, F., Wray, V., 1991. Screening for biosurfactants among crude oil degrading marine microorganisms from the North Sea. Naturforschung 46 (3–4), 197–203. Tebyanian, H., Hassanshahian, M., Kariminik, A., 2013. hexadecane-degradation by Teskumurella and Stenotrophomonas strains iso lated from hydrocarbon contaminated soils. Jundishapur. J. Microbiol. 26 (7), e9182. Troussellier, M., Got, P., Mboup, M., Corbin, D., Giuliano, L., Cappello, S., Bouvy, M., 2005. Daily bacterio plankton in a sub-Saharan estuary (Senegal River, West Africa): a mesocosm study. Aquat. Microb. Ecol. 40, 13–24. Walter, V., Syldatk, C., Hausmann, R., 2010. Biosurfactants: screening concepts for the isolation of biosurfactant producing microorganisms. Adv. Exp. Med. Biol. 672, 1–13. Willumsen, P., Karlson, U., 1997. Screening of bacteria isolated from PAH contaminated soils for production of biosurfactants and bioemulsifiers. Biodegradation 7, 415–423. Yakimov, M.M., Cappello, S., Crisafi, E., Tursi, A., et al., 2006. Phylogenetic survey of metabolically active microbial communities associated with the deep-sea coral Lophelia pertusa from the Apulian Plateau, Central Mediterranean Sea. Deep Sea Res. Part I 53, 62–75. Youssef, N.H., Duncan, K.E., Nagle, D.P., Savager, K.N., Knapp, R.M., McInerney, M.J., 2004. Comparison of methods to detect biosurfactant production by diverse microorganisms. J. Microbiol. Methods 56, 339–346.
Contents lists available at ScienceDirect
Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Isolation and characterization of biosurfactant producing bacteria from Persian Gulf (Bushehr provenance) Mehdi Hassanshahian ⇑ Department of Biology, Faculty of Science, Shahid Bahonar University of Kerman, Kerman, Iran
a r t i c l e
i n f o
Article history: Available online 15 July 2014 Keywords: Biodegradation Biosurfactant Persian Gulf Marine environment
a b s t r a c t Biosurfactants are surface active materials that are produced by some microorganisms. These molecules increase biodegradation of insoluble pollutants. In this study sediments and seawater samples were collected from the coastline of Bushehr provenance in the Persian Gulf and their biosurfactant producing bacteria were isolated. Biosurfactant producing bacteria were isolated by using an enrichment method in Bushnell-Hass medium with diesel oil as the sole carbon source. Five screening tests were used for selection of Biosurfactant producing bacteria: hemolysis in blood agar, oil spreading, drop collapse, emulsification activity and Bacterial Adhesion to Hydrocarbon test (BATH). These bacteria were identified using biochemical and molecular methods. Eighty different colonies were isolated from the collected samples. The most biosurfactant producing isolates related to petrochemical plants of Khark Island. Fourteen biosurfactant producing bacteria were selected between these isolates and 7 isolates were screened as these were predominant producers that belong to Shewanella alga, Shewanella upenei, Vibrio furnissii, Gallaecimonas pentaromativorans, Brevibacterium epidermidis, Psychrobacter namhaensis and Pseudomonas fluorescens. The largest clear zone diameters in oil spreading were observed for G. pentaromativorans strain O15. Also, this strain has the best emulsification activity and reduction of surface tension, suggesting it is the best of thee isolated strains. The results of this study confirmed that there is high diversity of biosurfactant producing bacteria in marine ecosystem of Iran and by application of these bacteria in petrochemical waste water environmental problems can be assisted. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Microbial compounds which exhibit pronounced surface activity are classified as biosurfactants (Cappello et al., 2012b; Bodour and Maier 2002). They decrease surface tension at the air–water interface and between immiscible liquids, or at the solid–liquid interface. These molecules chemically belong to various categories such as glycolipids, lipopeptides, polysaccharide–protein complexes, phospholipids, fatty acids and neutral lipids (Cappello et al., 2012a,c). Excellent detergency, emulsification, dispersing traits, penetrating, thickening, microbial growth enhancement, metal sequestering and resource recovering (oil) are important characteristics of biosurfactants which make them suitable to replace with chemical surfactants. Therefore, they have wide applications in cosmetics, oil recovery and bioremediation (Emtiazi et al., 2005; Ghanavati et al., 2008). The marine environment represents the major component of the Earth’s biosphere. It covers a majority (70%) part of earth’s ⇑ Tel.: +98 9132906971; fax: +98 3222032. E-mail address: [email protected] http://dx.doi.org/10.1016/j.marpolbul.2014.06.043 0025-326X/Ó 2014 Elsevier Ltd. All rights reserved.
surface and makes 90% of the volume of its crust. Oceans represent a vast and exhaustive source of natural products in the globe, harboring the most diverse groups of flora and fauna (Hasanshahian and Emtiazi, 2008). Marine microorganisms have developed unique metabolic and physiological capabilities to thrive in extreme habitats and produce novel metabolites which are not often present in the microbes of terrestrial origin (Fenical, 1993; Hassanshahian et al., 2013). Therefore, this rich marine habitat provides a magnificent opportunity to discover newer compounds such as antibiotics, enzymes, vitamins, drugs, biosurfactant (BS), bioemulsifier (BE) and other valuable compounds of commercial importance (Jensen and Fenical, 1994; Austin, 1989; Romanenko et al., 2001; Lang and Wagner, 1993). Biosurfactant producing microorganisms are ubiquitous, inhabiting both water (sea, fresh water, and groundwater) and land (soil, sediment, and sludge) as well as extreme environments (e.g. hypersaline sites, oil reservoirs), and thriving at a wide range of temperatures, pH values and salinity. Microorganisms produce BS/BE to mediate solubilisation of hydrophobic compounds in their environment to be able to utilize them as substrates, however, this fact may not be always true. Few microbes produce BS/BE on water
362
M. Hassanshahian / Marine Pollution Bulletin 86 (2014) 361–366
soluble substrates. It has been suggested that the presence of surface active molecules on the microbial cell surface increases the hydrophobicity of the cell and helps it to survive in hydrophobic environment (Hassanshahian et al., 2014b; Das et al., 2009). Despite the fact that marine environment represents a wealthy basin of diverse microorganisms, it is important to know that at the same time they are suffering from anthropogenic pollution with domestic and industrial wastes (Hassan et al., 1996; Hassanshahian et al., 2014c). Large quantities of crude oil, hydrocarbons, petroleum oil products and halogenated compounds finds their way into the marine ecosystem through accidental spillage (Satpute et al., 2005). To treat, emulsify or simply overcome such spills, on the one hand, many petroleum based synthetic chemical surfactants often get used (Hassanshahian et al., 2014a). Such synthetic compounds on the other hand, however often have detrimental ecological effects (Hassanshahian et al., 2012a). The use of biosurfactants and bioemulsifiers therefore may represent a better alternative to overcome the toxicity of synthetic compounds. The environmental roles of the biosurfactants produced by marine microorganisms have been reported earlier (Poremba et al., 1991; Schulz et al., 1991; Abraham et al., 1998). Persian Gulf is a marine environment that was polluted with crude oil during the 1991 Gulf war. The pollution impact of this episode has been evaluated in several studies, all indicated that crude oil accumulated and remained for long time in coastal area (Hassanshahian et al., 2010; Emtiazi et al., 2009). The oil pollution problem is particularly acute in an oil producing area of this marine environment such as Bushehr provenance. The aims of this study was to understanding the diversity of biosurfactant producing bacteria in Persian Gulf and especially at Bushehr provenance, also isolation and characterization of these important bacteria is another purpose of this research. 2. Materials and methods 2.1. Sampling Sediments and seawater samples were collected from five stations in the Persian Gulf at Bushehr provenance (Khark Island (KI), Ganaveh Port (GP), Siraf Port (SP), Halileh Shoreline (HS) and Asalooye Shoreline (AS) 26°150 N; 54°150 E). From each station five sediments and seawater samples were taken. These stations located near to petrochemical plants and oil refinery. Sediment samples were taken from 1–12 cm below the surface of coastal using a sterile knife. Seawater samples were collected from a depth of 15 cm in sterile 100 ml bottles and transported on ice to the laboratory for isolation. 2.2. Isolation and selection of biosurfactant producing bacteria ONR7a medium was used for isolation of biosurfactant producing bacteria. ONR7a contained (per liter of distilled water) 22.79 g of NaCl, 11.18 g of MgCl26H2O, 3.98 g of Na2SO4, 1.46 g of CaC12 2H2O, 1.3 g of TAPS0 {3-[N-tris(hydroxymethyl)methylamino]-2hydroxypropanesulfonica cid}, 0.72 g of KCl, 0.27 g of NH4Cl, 89 mg of Na2HP047H2O, 83 mg of NaBr, 31 mg of NaHCO3, 27 mg of H3BO3, 24 mg of SrCl26H2O, 2.6 mg of NaF, and 2 mg of FeCl24H2O. For solid media, Bacto Agar (Difco) (15 g/l) was added to the solution (Dyksterhouse et al., 1995). ONR7a medium were supplemented with 1% (v/v) diesel oil as sole carbon source and energy. Portion of sediment (10 g) or condensed seawater (10 ml) were added to Erlenmeyer flasks containing 100 ml of medium and the flasks were incubated for 10 days at 30 °C on rotary shaker (180 rpm, INFORS AG). Then
5 ml aliquots were removed to fresh medium. After a series of four further subcultures, inoculums from the flask were streaked out and phenotypically different colonies purified on ONR7a agar medium. The procedure was repeated and isolates only exhibiting pronounced growth on diesel oil were stored in stock media with glycerol at 20 °C for further characterization (Chaillan et al., 2004; Tebyanian et al., 2013). 2.3. Screening of biosurfactant producing bacteria Three tests were used for screening and selection of prevailing biosurfactant producing bacteria. These tests were described below. 2.3.1. Hemolytic test Hemolytic activity was carried out as described by Carrillo et al. (1996). Isolated strains were screened on blood agar plates containing 5% (v/v) blood and incubated at 30 °C for 24–48 h. Hemolytic activity was detected as the presence of a clear zone around a colony. 2.3.2. Drop collapse method The drop-collapse technique was carried out in the polystyrene lid of a 96-microwell plate (Biolog, Harward, CA, USA) as described by Jain et al. (1991) and 100 ll culture supernatant was added to wells of a 96-well microtiter plate lid, and then 5 ll of crude oil was added to the surface of the culture supernatant. Biosurfactant-producing culture gave flat drops. Aliquots from a culture of each strain were analyzed on two separate microtiter plates. 2.3.3. Oil spreading method Oil spread technique was carried out according to Morikova et al. (2000) and Youssef et al. (2004). 50 ml of distilled water was added to Petri dishes followed by addition of 100 ll of crude oil to the surface of the water. Then, 10 ll of the culture filtrates was put on the crude oil surface. The diameter of the clear zone on the oil surface was measured. 2.4. Liquid surface tension The surface tension (ST) of the culture supernatants was measured with a digital surface tensiometer (DCAT, DataPhysics Instruments GmbH, Filderstadt, Germany) working on the principles of Wilhelmy plate method (Fernandes et al., 2007). The validity of the surface tension readings was checked with pure water (70.78 ± 0.02 mN/m) before each reading. All surface tension readings were taken in triplicate. 2.5. Emulsification activity and Bacterial Adhesion To hydrocarbon (BATH test) The emulsification activity (E24) was determined by the addition 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 emulsification activity was determined as the percentage of height of the emulsified layer (mm) divided by the total height of the liquid column (mm) (Emtiazi et al., 2009). Bacterial adhesion to hydrocarbon was carried out according to Pruthi and Cameotra, 1997. 2.6. Identification of the isolates 2.6.1. Biochemical identification In order to identify and characterize the bacteria isolates, some biochemical tests was carried out such as: Gram staining, oxidation/fermentation, production of acid from carbohydrates,
M. Hassanshahian / Marine Pollution Bulletin 86 (2014) 361–366
hydrolysis of gelatin and citrate. These tests done according to the Bergey’s manual for identification taxonomy (Holt et al., 1998). 2.6.2. Molecular identification Analysis of 16S rRNA was performed for the taxonomic characterization of isolated strains. Total DNA extraction of bacterial strains was performed with the CTAB method. The bacterial 16S rRNA loci were amplified using the forward domain specific bacteria primer, Bac27_F (50 -AGAGTTTGATCCTGGCTCAG-30 ) and universal reverse primer Uni_1492R (50 -TACGYTACCTTGTTACGACTT-30 ). The amplification reaction was performed in the total volume of 25 ll consisting, 2 mM MgCl2 (1 ll), 10X PCR reaction buffer (200 mM Tris; 500 mM KCl) (2.5 ll), 2 mM each dNTP (2 ll), 0.15 mM each primer (1 ll), 1U (0.5 ll) Taq DNA polymerase (Qiagen, Hilden, Germany) and 2 ll of template DNA (50 pm). The distill water was added for remaining of reaction (15 ll). Amplification was performed in a thermocycler GeneAmp 5700 (PE Applied Biosystem, Foster City, CA, USA). The temperature profile for PCR was kept, 94 °C for 5 min, 94 °C for 1 min, 54 °C for 1 min, 72 °C for 1 min, 30 cycles; then 72 °C for 10 min and finally storage at 4 °C (Troussellier et al., 2005). The 16S rRNA amplified was sequenced with a Big Dye terminator V3.1 cycle sequencing kit on an automated capillary sequencer (model 3100 Avant Genetic Analyzer, Applied Biosystems). Similarity rank from the Ribosomal Database Project RDP) (Maidak et al., 1997) and FASTA Nucleotide Database. Query were used to determine partial 16S rRNA sequences to estimate the degree of similarity to other 16S rRNA gene sequences. Analysis and phylogenetic affiliates of sequences was performed as previously described protocols (Yakimov et al., 2006). 3. Results 3.1. Isolation and screening of biosurfactant producing bacteria from marine samples Eighty morphologically distinct microbial colonies were isolated from collected sea water and sediment samples. These isolates belong to these geographic zones: 21 were isolated from the KI samples, 15 from the GP samples, 17 from the SP samples, 12 from the HS samples and 15 from the AS samples. Ninety percent of the bacterial isolates (72 of 80) were Gram-negative. Forty-six isolates were determined as biosurfactant producing bacteria using three screening tests. However the responses of these isolates to screening methods were different. As shown in Fig. 1 drop collapse method has the most positive response and
363
hemolysis on blood agar has the lowest positive response while the positive and negative response of oil spreading method were equal (Fig. 1). 3.2. Biosurfactant production by isolated strains Fourteen isolated strains that show high growth rate on diesel oil were selected between 46 isolates for further study. Table 1 illustrates the results of screening tests for these isolated strains. As shown in this table the best isolates that have sufficient response to all screening tests were: I36, O15, N4, U15, E14, T26, N3 and H4. However some isolated strains such as K1, P1, and B16 have positive response to one or two screening tests (Table 1). 3.3. Biochemical properties of the isolated strains Some preliminary identification tests were done to discriminate between isolated strains. Table 2 shows the results of these diagnostic tests. As shown in this table the most isolated strains were Gram negative, oxidase positive and motility positive. However there are some differences between isolated bacteria (Table 2). 3.4. Cell surface hydrophobicity, emulsification index and decrease of surface tension by isolated strains Emulsification activity, bacterial adhesion to hydrocarbon (BATH) and reduction of surface tension assay were analyzed for each strain separately. Data obtained from these tests were shown in Table 3. Strains I36, O15, N4, U15, E14, T26, and N3 have the highest values of emulsification activity and cell surface hydrophobicity. These strains also produce more biosurfactant and dramatically decrease surface tension than other strains. The results indicated that there are a direct relationship between cell surface hydrophobicity and biosurfactant production with emulsification activity (Table 3). 3.5. Molecular identification and phylogenetic analysis of isolated strains Molecular identification of the isolates were perform for I36, O15, N4, U15, E14, T26, and N3 strains by amplification and sequencing the 16S rRNA gene sequencing and comparing them to the database of known 16S rRNA sequences. The molecular identification of these strains show that these strains belongs to: Shewanella alga (Strain N4), Shewanella upenei (Strain E14), Vibrio furnissii (Strain U15), Gallaecimonas pentaromativorans (Strain O15), Brevibacterium epidermidis (Strain N3), Psychrobacter namhaensis (Strain I36) and Pseudomonas fluorescens (Strain T26). The phylogeny tree of these isolated strains was shown in the Fig. 2. All sequences of seven bacteria were submitted to Genetic sequence database at the National Center for Biotechnical Information (NCBI). Gene Bank ID of these strains in NCBI is: HF968433 to HF968439. 4. Discussion
Fig. 1. The response of total isolates to the three biosurfactant production assays.
Biosurfactants can fulfill various physiological roles and provide ecological advantages to the producing strains (Ron and Rosenberg, 2001). For instance, they improve the bioavailability of water insoluble substrates by emulsification, thus enhancing biodegradation of several environmental pollutants. Biosurfactant-producing microbes can be found in various ecosystems, although the environments that are impacted with hydrophobic contaminants such as refinery wastes and petroleum are more yielding than undisturbed ones (Batista et al., 2006).
364
M. Hassanshahian / Marine Pollution Bulletin 86 (2014) 361–366
Table 1 Biosurfactant production assays for isolated strains. Isolate
Hemolytic test
Drop collapse (shape of drop in comparison to blank)
Oil spreading (area of oil displaced in mm) (mm)
I36 O15 N4 U15 B16 E14 T26 N3 P1 H4 W35 K1 K3 W25
+ + + + + + + + +
+++ +++ +++ +++ +++ +++ +++ ++ +++ + + ++ ++
10 14 8 13 10 10 10 8.5 7 10 6 6 8 9
Abbreviations: +, positive response; , negative response (for hemolytic test); +, low flat; ++, middle flat, +++, completely flat (for drop collapse test).
Table 2 Some biochemical characteristics of selected biosurfactant producing strains. Isolate
Origin
Gram staining
Oxidase
Motility
Acid from glucose
I36 O15 N4 U15 B16 E14 T26 N3 P1 H4 W35 K1 K3 W25
KI-sea water GP-sea water SP-sea water HS-sea water AS-sea water KI-sediment GP-sediment SP-sediment HS-sediment AS-sediment KI-sediment AS-sea water KI-sea water HS-sea water
N N N N N N N P N N P N N N
+ + + + + + +
+ + + + + + + +
+ + + + + +
Abbreviations: N, gram negative; P, gram positive; +, positive response; , negative response.
Table 3 Measurement of emulsification activity (%, E24), cell surface hydrophobicity (%, BATH) and decrease of surface tension (mN/m) by strains in this study. All data that reported in this table is the mean of three values and cited value in the table is standard deviation.
a
Isolate
Emulsification activity (E24%)
Cell surface hydrophobicity (BATH%)
Surface tensiona (mN m1)
I36 O15 N4 U15 B16 E14 T26 N3 P1 H4 W35 K1 K3 W25
34 65 45 44 18 34 44 43 10 25 40 20 25 30
27 59 27 19 5.2 38.8 38 39 20 8.2 2.3 3.7 1.7 3.7
42.1 34.5 43.4 44.6 43 44.6 41.5 40.3 45.3 45.5 46.8 47.8 50.2 49.8
Surface tension of ONR medium without bacteria as blank is 53.6.
In this study, bacteria that dominated in the enrichment media were selected due to their ability to utilize the hydrophobic substrates. This is in line with the fact that hydrophobic pollutants in collected seawater and sediment samples contain several of the organic substrates added to the enrichments. Therefore, microorganisms in those samples are adapted to live with hydrophobic compounds as carbon source. These cultures yielded 80 isolates that were also differentiated based on colony morphology on ONR7a-agar plates.
Are there any biosurfactant producers among the isolated bacteria? To answer this question, we adopted a variety of screening assays as suggested by Walter et al. (2010). The isolated bacteria were searched for potential biosurfactant producers via three screening phases of ascending certainty. To get a preliminary indication, these isolated bacteria were grown on ONR7a containing diesel oil as a sole carbon source and inducer of biosurfactant production (initial screening phase). Emulsification of the diesel oil in the growth medium indicated biosurfactant production by
M. Hassanshahian / Marine Pollution Bulletin 86 (2014) 361–366
365
Fig. 2. Phylogenetic tree of 16S rRNA sequences of the isolated strains obtained from Persian Gulf. The tree was constructed using sequences of comparable region of the 16S rRNA gene sequences available in public databases. Neighbour-joining analysis using 1000 bootstrap replicates was used to infer tree topology. The bar represents 0.02% sequence divergence. Sequenced data showing the location of isolated strains.
tested bacteria. The cultures that exhibited weak emulsification showed scarce or no turbidity in the aqueous phase. In those cultures the cells might be adhering to the emulsified oil slicks (Bredholt et al., 1998). Emulsification of the diesel oil in water is a prerequisite that paves the way for biodegradation of this environmental pollutant by many bacteria. It enhances the bioavailability of the oil and thus increases the biodegradation rate (Bredholt et al., 1998; Minf et al., 2011; Hassanshahian et al., 2012b). However, visual inspection of the diesel oil cultures for biosurfactant production is an indirect screening method as the growth with hydrophobic compounds indicates the production of biosurfactants, but does not always correlate with this trait (Willumsen and Karlson, 1997). Second screening phase to further investigate biosurfactant production contain three tests: oil spreading, drop collapse and hemolytic activity assays. The clear zone diameters were larger for cultures of the strains I36, O15, N4, U15, E14, T26, N3 and H4 however the largest diameters observed for strain O15. Thus, indicating the presence of higher concentrations of biosurfactant. This is in good agreement with the drop collapse and hemolytic activity results. Microbial production of the surface-active compounds on diesel oil and other hydrophobic substrates has frequently been reported (Iqbal et al., 1995; Kumara et al., 2006). The data of the third screening phase (surface tension reduction and emulsification activity) confirmed unequivocally the production of biosurfactants by the mentioned strains. Taken together, the data of the screening assays in the three phases are coherent and support each other. Since all the screening assays were performed with cell-free culture supernatants, it can be concluded that the cells produce biosurfactant and secrete them to the extracellular medium. Similar results have been reported in many cases (Batista et al., 2006; Cappello et al., 2012b). Isolation of biosurfactant producing from marine environment was reported by other researcher in the world. For example Coelho et al. (2003) reported production of biosurfactant by quinoline degrading marine Pseudomonas sp. strain GU 104. Species of Alcanivorax produces a potent glucose–lipid surfactant (Abraham et al., 1998). In this research six different genus of biosurfactant producing bacteria were isolated from Persian Gulf at Bushehr provenance. These results are in agreement with the results that reported by other researcher. Pruthi and Cameotra (1997) screened biosurfactant producing bacteria by assay the cell surface hydrophobicity. They found a direct relationship between cell surface hydrophobicity and biosurfactant production. Batista et al. (2006) isolated 17 biosurfactant producing bacteria from oil contaminated beaches in Brazil. They found that six strains can reduce surface tension below than 40 mN m1.
5. Conclusion Marine microorganisms that produced biosurfactant have high diversity and ability in Persian Gulf. In this survey from 14 biosurfactant producing bacteria, 7 strains can produce high biosurfactant and reduce surface tension. Also we found a direct relationship between cell surface hydrophobicity and biosurfactant production with emulsification activity as the strain O15 that had high biosurfactant production also had efficient emulsification activity. Acknowledgment This work was supported financially by Shahid Bahonar University of Kerman. References Abraham, W.R., Meyer, H., Yakimov, M., 1998. Novel glycine containing glucolipids from the alkane using bacterium Alcanivorax borkumensis. Biochem. Biophys. Acta 1393, 57–62. Austin, B., 1989. Novel pharmaceutical compounds from marine bacteria. J. Appl. Bacteriol. 67, 461–470. Batista, S., Mounteer, A., Amorim, F., 2006. Isolation and characterization of biosurfactant/bioemulsifier producing bacteria from petroleum contaminated sites. Bioresour. Technol. 97, 868–875. Bodour, A.A., Maier, R.M., 2002. Biosurfactants: types, screening methods and application. Encycl. Environ. Microbio, vol. 2. Springer, New York, pp. 750–769. Bredholt, H., Josefen, K., Vatland, A., Bruhbeim, P., EImhjellen, K., 1998. Emulsification of crude oil by an alkane-oxidizing Rhodococcus species isolated from seawater. Can. J. Microbiol. 44, 330–340. Cappello, S., Genovese, M., Della Torre, C., Crisari, A., Hassanshahian, M., Santisi, S., Calogero, R., Yakimov, M.M., 2012a. Effect of bioemulsificant exopolysaccharide EPS2003 on microbial community dynamics during as says of oil spill bioremediation: a microcosm study. Mar. Pollut. Bull. 64 (12), 2820–2828. Cappello, S., Crisari, A., Hassanshahian, M., Genovese, M., Santisi, S., Yakimov, M.M., 2012b. Effect of a bioemulsificant exopolysaccharide (EPS2003) on abundance and vitality of marine bacteria. Water Air Soil Pollut.. http://dx.doi.org/10.1007/ s11270-012-1159-8. Cappello, S., Santisi, S., Calogero, R., Hassanshahian, M., Yakimov, M.M., 2012c. Characterization of oil-degrading bacteria isolated from bilge water. Water Air Soil Pollut.. http://dx.doi.org/10.1007/s11270-012-1103-. Carrillo, P.G., Mardaraz, C., Pitta-Alvarez, S.J., Giulietti, A.M., 1996. Isolation and selection of biosurfactant-producing bacteria. World J. Microbiol. Biotechnol. 12, 82–84. Chaillan, F., Le, F.A., Bury, E., Phantavong, Y.H., Grimont, P., Saliot, A., Oudot, J., 2004. Identification and biodegradation potential of tropical aerobic hydrocarbondegrading microorganisms. Res. Microbiol. 155 (7), 587–595. Coelho, J., Rivonkar, C.U., Bhavesh, N.S., Jothi, M., Sagodkar, U.M., 2003. Biosurfactant production by the quinoline degrading marine bacterium Pseudomonas sp. Strain GU 104, and its effect on the metabolism of green mussel Perna viridis L. Indian J. Mar. Sci. 32, 202–207. Das, P., Mukherjee, S., Sen, R., 2009. Biosurfactant of marine origin exhibiting heavy metal remediation properties. Biores. Technol. 100 (20), 4887–4890. Dyksterhouse, S.E., Gray, J.P., Herwig, R.P., Lara, J.C., Staley, J.T., 1995. Cycloclasticus pugetii gen. nov., sp. nov., an aromatic hydrocarbon degrading bacterium from marine sediments. Int. J. Syst. Bacteriol. 45, 116–123.
366
M. Hassanshahian / Marine Pollution Bulletin 86 (2014) 361–366
Emtiazi, G., Hassanshahyan, M., Golbang, N., 2005. Development of a microtiter plate method for determination of phenol utilization, biofilm formation and respiratory activity by environmental bacterial isolates. Int. Biodeterior. Biodegrad. 56, 231–235. Emtiazi, G., Saleh, T., Hassanshahian, M., 2009. The effect of bacterial glutathione S-transferase on morpholine degradation. Biotechnol. J. 4, 202–205. Fenical, W., 1993. Chemical studies of marine bacteria: developing a new resource. Chem. Rev. 93, 1673–1683. Fernandes, P.A.V., Arruda, I.R., Santos, A.F.A.B., Araujo, A.A., Maiors, A.M.S., Ximenes, F.A., 2007. Antimicrobial activity of surfactants produced by Bacillus subtilis R14 against multidrug-resistant bacteria. Braz. J. Microbiol. 38, 704–709. Ghanavati, H., Emtiazi, G., Hassanshahian, M., 2008. Synergism effects of phenol degrading yeast and ammonia oxidizing bacteria for nitrification in coke wastewater of Esfahan steel company. Waste Manage. Res. 26 (2), 203–208. Hasanshahian, M., Emtiazi, G., 2008. Investigation of alkane biodegradation using the microtiter plate method and correlation between biofilm formation, biosurfactant production and crude oil biodegradation. Int. Biodeterior. Biodegrad. 62, 170–178. Hassan, E.S., Banat, I.M., El-Shahawi, M.S., Abu-Hilal, A.H., 1996. Bacterial, nutrients and heavy metals pollution assessment along the eastern coastal area of the United Arab Emirates. J. Aquat. Ecosys. Health 5, 73–81. Hassanshahian, M., Emtiazi, G., Kermanshahi, R., Cappello, S., 2010. Comparison of oil degrading microbial communities in sediments from the Persian Gulf and Caspian Sea. Soil Sediment Contaminat. 19 (3), 277–291. Hassanshahian, M., Tebyanian, H., Cappello, S., 2012a. Isolation and characterization of two crude-oil degrading yeast strains, Yarrowia lipolytica PG-20 and PG-32 from Persian Gulf. Mar. Pollut. Bull. 64, 1386–1391. Hassanshahian, M., Emtiazi, G., Cappello, S., 2012b. Isolation and characterization of crude-oil-degrading bacteria from the Persian Gulf and the Caspian Sea. Mar. Pollut. Bull. 64, 7–12. Hassanshahian, M., Ahmadinejad, M., Tebyanian, H., Kariminik, A., 2013. Isolation and characterization of alkane degrading bacteria from petroleum reservoir waste water in Iran (Kerman and Tehran provenances). Mar. Pollut. Bull. 73, 300–305. Hassanshahian, M., Emtiazi, G., Caruso, G., Cappello, S., 2014a. Bioremediation (bioaugmentation/biostimulation) trials of oil polluted seawater: a mesocosm simulation study. Mar. Environ. Res. 95, 28–38. Hassanshahian, M., Zeynalipour, M.S., Hosseinzadeh Musa, F., 2014b. Isolation and characterization of crude oil degrading bacteria from the Persian Gulf (Khorramshahr provenance). Mar. Pollut. Bull. 82, 39–44. Hassanshahian, M., Yakimov, M.M., Denaro, R., Genovese, M., Cappello, S., 2014c. Using Real-Time PCR to assess changes in the crude oil degrading microbial community in contaminated seawater mesocosms. Inte. Biodeterior. Biodegrad. 93, 241–248. Holt, S.G., Kriey, N.R., Sneath, P.H.A., Staley, J.T., Williams, S.T., 1998. Bergy’s manual of determinative for bacteriology. Williams and Wilkins, New York. Iqbal, S., Khalid, Z.M., Malik, K.A., 1995. Enhanced biodegradation and emulsification of crude oil and hyperproduction of biosurfactants by a gamma ray-induced mutant of Pseudomonas aeruginosa. Lett. Appl. Microbiol. 21 (3), 176–179. Jain, D.K., Collins-Thompson, D.L., Lee, H., Trevors, J.T., 1991. A drop-collapsing test for screening surfactant-producing microorganisms. J. Microbiol. Method 13, 271–279. Jensen, P.R., Fenical, W., 1994. Strategies for the discovery of secondary metabolites from marine bacteria: ecological perspectives. Annu. Rev. Microbiol. 48, 559– 584.
Kumara, M., Leon, V., Materano, A., Ilzins, O.A., Galindo-Castro, I., Fuenmayor, S.L., 2006. Polycyclic aromatic hydrocarbon degradation by biosurfactant-producing Pseudomonas sp. IR1. Z. Naturforsch. C. J. Biosci. 61, 203–212. Lang, S., Wagner, F., 1993. Biosurfactants from marine microorganisms. In: Kosaric, N. (Ed.), Biosurfactants: Production Properties Applications (Surfactant Science Series), vol. 48. Marcel Dekker, New York, pp. 391–417. Maidak, B.L., Olsen, G.J., Larsen, N., 1997. The RDP ribosomal database project. Nucleic Acids Res. 25 (109–1), 11. Minf, S., Chamkha, M., Labat, M., Sayadi, S., 2011. Simultaneous hydrocarbon biodegradation and biosurfactant production by oil field-selected bacteria. J. Appl. Microbiol. 111, 525–536. Morikova, M., Hirata, Y., Imanaka, T., 2000. A study on the structure–function relationship of the lipopeptide biosurfactants. Biochim. Biophys. Acta 1488, 211–218. Poremba, K., Gunkel, W., Lang, S., Wagner, F., 1991. Toxicity testing of synthetic and biogenic surfactants on marine microorganisms. Environ. Toxicol. Water Qual. 6, 157–163. Pruthi, V., Cameotra, S.S., 1997. Rapid identification of biosurfactant-producing bacterial strains using a cell surface hydrophobicity technique. Biotechnol. Tech. 11, 671–674. Romanenko, L.A., Kalinovskaya, N.I., Mikhailov, V.V., 2001. Taxonomic composition and biological activity of microorganisms associated with a marine Ascidian Halocynthia aurantium. Russ. J. Mar. Biol. 27, 291–295. Ron, E., Rosenberg, E., 2001. Biosurfactants and oil bioremediation. Curr. Opin. Biotechnol. 13, 249–252. Satpute, S. K., Dhakephalkar, P. K., Chopade, B. A., 2005. Biosurfactants and bioemulsifiers in hydrocarbon biodegradation and spilled oil bioremediation. Indo-Italian brain storming workshop on technology transfer for industrial applications of novel methods and materials for environmental problem, vol. 2, pp. 1–18. Schulz, D., Passeri, A., Schmidt, M., Lang, S., Wagner, F., Wray, V., 1991. Screening for biosurfactants among crude oil degrading marine microorganisms from the North Sea. Naturforschung 46 (3–4), 197–203. Tebyanian, H., Hassanshahian, M., Kariminik, A., 2013. hexadecane-degradation by Teskumurella and Stenotrophomonas strains iso lated from hydrocarbon contaminated soils. Jundishapur. J. Microbiol. 26 (7), e9182. Troussellier, M., Got, P., Mboup, M., Corbin, D., Giuliano, L., Cappello, S., Bouvy, M., 2005. Daily bacterio plankton in a sub-Saharan estuary (Senegal River, West Africa): a mesocosm study. Aquat. Microb. Ecol. 40, 13–24. Walter, V., Syldatk, C., Hausmann, R., 2010. Biosurfactants: screening concepts for the isolation of biosurfactant producing microorganisms. Adv. Exp. Med. Biol. 672, 1–13. Willumsen, P., Karlson, U., 1997. Screening of bacteria isolated from PAH contaminated soils for production of biosurfactants and bioemulsifiers. Biodegradation 7, 415–423. Yakimov, M.M., Cappello, S., Crisafi, E., Tursi, A., et al., 2006. Phylogenetic survey of metabolically active microbial communities associated with the deep-sea coral Lophelia pertusa from the Apulian Plateau, Central Mediterranean Sea. Deep Sea Res. Part I 53, 62–75. Youssef, N.H., Duncan, K.E., Nagle, D.P., Savager, K.N., Knapp, R.M., McInerney, M.J., 2004. Comparison of methods to detect biosurfactant production by diverse microorganisms. J. Microbiol. Methods 56, 339–346.