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Adaptation Mechanisms to Toxic Solvents in Shewanella Putrefaciens
Available online at www.sciencedirect.com
ScienceDirect Agriculture and Agricultural Science Procedia 6 (2015) 601 – 607
“ST26733”, International Conference "Agriculture for Life, Life for Agriculture"
Adaptation Mechanisms to Toxic Solvents in Shewanella putrefaciens Mihaela Marilena Stancu* Institute of Biology Bucharest of Romanian Academy, 296 Splaiul Independentei, 060031 Bucharest, P.O. Box 56-53, Romania
Abstract Shewanella putrefaciens is a Gram-negative bacterium which is widespread in various environments, including in sites polluted with petroleum and petroleum products. S. putrefaciens IBBPo6 used organic solvents with log POW (logarithm of the partition coefficient of the solvent in an octanol-water mixture) values above 4 (i.e., n-decane) and below 4 (i.e., cyclohexane, n-hexane, toluene, o-xylene, ethylbenzene) as growth substrates. More-robust biofilms were developed when S. putrefaciens IBBPo6 cells were grown in the presence of cyclohexane, n-hexane, toluene, o-xylene or ethylbenzene, as compared with those formed in the presence of n-decane. Four biosurfactants were detected in the cell-free culture broths of S. putrefaciens IBBPo6 cells grown in the presence of tested organic solvents. There were observed differences between the PCR pattern of S. putrefaciens IBBPo6 cells grown in the presence of n-decane, and those of cells grown in the presence other tested organic solvents (i.e., cyclohexane, nhexane, toluene, o-xylene, ethylbenzene).
© Published by byElsevier ElsevierB.V. B.V. This is an open access article under the CC BY-NC-ND license Authors. Published © 2015 2015 The The Authors. (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the University of Agronomic Sciences and Veterinary Medicine Bucharest. Peer-review under responsibility of the University of Agronomic Sciences and Veterinary Medicine Bucharest Keywords: adaptation; mechanisms; Shewanella; solvents
1. Introduction The genus Shewanella is a member of the class Gammaproteobacteria and includes a group of Gram-negative, motile, rod-shaped, oxidase-positive, non-fermentative and facultative anaerobic bacteria (Park et al., 2009). Shewanella species are widely distributed in diverse environments due to their flexible respiration. These bacteria can use a wide range of electron acceptors and are classified in the group of dissimilatory metal-reducing bacteria
* Corresponding author. Tel.: +40212219202; fax: +40212219071. E-mail address: [email protected]
2210-7843 © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the University of Agronomic Sciences and Veterinary Medicine Bucharest doi:10.1016/j.aaspro.2015.08.097
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(Rodrigues et al., 2011; Wu et al., 2013). Their respiratory versatility allows them to play an important role in the geochemical cycling of iron, manganese and carbon sources, as well as in bioremediation and bio-engineering applications (Wu et al., 2013). Shewanella putrefaciens strains are able to use metals for respiration and they are also able to degrade several pollutants (e.g., petroleum, chlorinated solvents) which makes these microorganisms useful tools for bioremediation (Martín-Gil et al., 2004; Gao et al., 2006). The mechanisms underlying organic solvents tolerance in bacteria are not yet fully understood, although a number of factors involved in the process have been characterized over the last years. These studies have revealed a number of mechanisms, including: changes in membrane protein pattern; alteration in phospholipids composition; organic solvents removal through active efflux systems (efflux pumps); changes in energy metabolism; changes in cell morphology; changes in the cell wall; vesicle formation; transformation or degradation of the organic solvents (Heipieper et al., 2007; Schrewe et al., 2013). The aim of this study was to investigate the possible cell adaptation mechanisms pursued by S. putrefaciens IBBPo6 cells grown in the presence of 0.5% (v/v) organic solvents. Changes in the cell growth rate, biofilm formation, biosurfactant production, and in PCR pattern were observed in S. putrefaciens IBBPo6 cells grown in the presence of 0.5% (v/v) organic solvents. 2. Materials and Methods 2.1 Solvent tolerance in S. putrefaciens IBBPo6 Bacterial cells were cultivated on liquid LB-Mg medium (Stancu and Grifoll, 2011) containing 15 Pg ml-1 ampicillin and incubated at 28°C on a rotary shaker (200 rpm) until they reached the exponential growth phase. Bacterial cells were harvested by centrifugation, washed and finally resuspended (105 cells ml-1) in minimal medium (Stancu and Grifoll, 2011). 0.5% (w/v) yeast extract and 0.5% (v/v) organic solvents (alkanes: cyclohexane, nhexane, n-decane; aromatics: toluene, o-xylene, ethylbenzene) were supplied to the cell suspensions as the carbon source. Flasks were sealed and incubated for 24 hours at 28°C on a rotary shaker (200 rpm). 2.1.1. Cell growth Cell growth in the presence of organic solvents was determined by measuring optical density (OD) at 660 nm using a SPECORD 200 UV-visible spectrophotometer. 2.1.2. Biofilm formation Samples were fixed on SEM holder and gold-coated with a JEOL JFC-1300 auto fine coater, in a deep vacuum. The samples were examined with a JEOL JSM-6610LV scanning electron microscope (SEM). 2.1.3. Biosurfactant production. Biosurfactants were extracted from the cell-free culture broths with chloroform-methanol (2:1 v/v), using the method described by Stancu (2014). For thin layer chromatography (TLC) analysis, the samples were spotted with a Linomat 5 sample applicator (CAMAG), on a 10×10 cm precoated silica gel 60 TLC aluminium sheets (Merck). The separation was performed using the chloroform-methanol (90:10 v/v) mixture as mobile phase. After development, a densitometric scan at 254 nm in a TLC Scanner 4 (CAMAG) was performed for detection and quantification of the biosurfactants. 2.1.4. Polymerase chain reaction (PCR) For PCR amplification, 1 ȝl of DNA extract was added to a final volume of 25 ȝl reaction mixture, containing: 5×GoTaq flexi buffer, MgCl2, dNTP mix, specific primers for catabolic (alkB, alkM, alkB1, alkM1, todM, xylM, ndoM, C23DO) genes (ALK1-f and ALK1-r, ALK2-f and ALK2-r, ALK3-f and ALK3-r, Kohno et al., 2002; alkM-f and alkM-r, todM-f and todM-r, xylM-f and xylM-r, ndoM-f and ndoM-r, Márquez-Rocha et al., 2005; 23CAT-f and 23CAT-r, Mesarch et al., 2000), specific primers for hydrophobic/amphiphilic efflux 1 (HAE1) transporter gene (A24f2 and A577r2, Meguro et al., 2005) or specific primers for trehalose-phosphate synthase (otsA1) gene (otsA-f and otsA-r, Tischler et al., 2013), and GoTaq G2 hot start polymerase (Promega). PCR was performed with a Mastercycler pro S (Eppendorf). PCR assays were performed according to Meguro et al. (2005) protocol: initial denaturation for 10 min at 94°C, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 50°C or 62°C
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for 30 sec, extension at 72°C for 2 min, and a final extension at 72°C for 10 min. After electrophoretic separation on 1.5% (w/v) TBE agarose gel (Sambrook et al., 1989) and staining with fast blast DNA stain (Bio-Rad) the PCR products were analyzed. Reagents used during this study were procured from Merck, Sigma-Aldrich, Promega, Invitrogen, Applied Biosystems or Bio-Rad Laboratories. The PCR primers were purchased from Biosearch Technologies, Integrated DNA Technologies and Invitrogen. 3. Results and Discussions The strain used in the present study was S. putrefaciens IBBPo6 (KM405339). This Gram-negative bacterium was isolated (Stancu, 2015) from Poeni oily sludge (Teleorman County, Romania). Their taxonomic affiliation was determined on the basis of phenotypic characteristics and of 16S rRNA gene sequence (Stancu, 2015). 3.1. Solvent tolerance in S. putrefaciens IBBPo6 S. putrefaciens isolates are widespread in various environments, including in sites polluted with petroleum and petroleum products. Nevertheless, the mechanisms of solvent tolerance in this bacterium have not been investigated so far. 3.1.1. Cell growth Bacterial cells susceptibility to toxic organic solvents depends on the logarithm of the partition coefficient of the solvent in an octanol-water mixture (log POW) (Sikkema et al., 1995; Heipieper et al., 2007). The growth of S. putrefaciens IBBPo6 cells on minimal medium supplemented with 0.5% yeast extract was higher (OD = 0.905), compared with the growth of the cells on the same medium in the presence of 0.5% organic solvents (OD = 0.4130.762). As expected, the growth was higher (OD = 0.762) when S. putrefaciens IBBPo6 cells were grown in the presence of n-decane with log POW value over 4, as compared with the growth of cells (OD = 0.413-0.637) in the presence of other tested organic solvents (i.e., cyclohexane, n-hexane, toluene, o-xylene, ethylbenzene) with log POW values under 4 (Table 1). Table 1. Growth of S. putrefaciens IBBPo6 cells in the presence of organic solvents Assay Cell growtha
Control 0.905
Cyclohexane (3.35) 0.523
n-Hexane (3.86) 0.637
Organic solvents (log POWb) n-Decane Toluene (5.98) (2.64) 0.762 0.413
o-Xylene (3.09) 0.468
Ethylbenzene (3.17) 0.497
Legend: a = the absorbance of the culture broths was measured at 660 nm; b = logarithm of the partition coefficient of the solvent in an octanolwater mixture.
According to Schrewe et al. (2013), organic solvents with a log POW between 1 and 4 are considered to be toxic to bacterial cells. However, some bacteria (e.g., Pseudomonas strains) are tolerant to organic solvents with a log POW under 4 and can utilize such compounds as growth substrates (Schrewe et al., 2013). These solvent-tolerant organisms are able to adapt to the presence of toxic organic solvents. More hydrophobic, larger compounds (log POW over 4) are considered non-toxic and do not accumulate in cellular membranes, as they are virtually unable to pass the outer membrane due to their size and hydrophobicity (Schrewe et al., 2013). 3.1.2. Biofilm formation Like other bacteria, Shewanella species can form biofilms under diverse environments (Wu et al. 2013). Microbial biofilms are surface-associated, dynamic communities that respond to changing external and internal environments, allowing continued acclimation of the biofilm population (Saville et al., 2011). Scanning electron micrographs (Figures 1a-1g) showed biofilms formation when S. putrefaciens IBBPo6 cells were grown 24 hours on minimal medium supplemented with 0.5% yeast extract and on the same medium in the presence of 0.5% organic solvents. When S. putrefaciens IBBPo6 cells were grown in the presence of cyclohexane, nhexane, toluene, o-xylene or ethylbenzene more-robust biofilms were developed, as compared with those formed in the presence of n-decane or yeast extract. In biofilms, cells are embedded in a self-produced matrix composed of extracellular polymeric substances (EPS), including proteins, polysaccharides, lipids, and extracellular DNA (Ding et al., 2014). Extensive studies have demonstrated a wide range of advantages of the presence of an EPS matrix for
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the biofilm mode of life (Ding et al., 2014). For example, compared with planktonically grown cells, biofilms have been reported to be more tolerant to harsh physicochemical conditions generated by various toxic contaminants (Ding et al., 2014). Studies of Shewanella species showed that the type of carbon source and the nutrient concentration influence the development and architecture of the biofilms. Therefore, more-robust biofilms with enhanced microbe-surface interactions were observed in poor nutrient conditions (Thormann et al., 2004). a.
b.
c.
d.
e.
f.
g.
Figure 1. Scanning electron micrographs of S. putrefaciens IBBPo6 cells grown in the presence of organic solvents Control (panel a), cyclohexane (panel b), n-hexane (panel c), n-decane (panel d), toluene (panel e), o-xylene (panel f), ethylbenzene (panel g).
3.1.3. Biosurfactant production Biosurfactants are surface active materials that are produced by some bacteria, including by different Shewanella strains (Hassanshahian, 2014). According to literature (Davey et al., 2003), biosurfactants produced by some bacteria (e.g., rhamnolipids synthesized by Pseudomonas aeruginosa PAO1) are thought to maintain biofilm architecture by influencing cell-cell interactions and bacterial attachment to surfaces (Davey et al., 2003). Furthermore, the biosurfactant molecules increase biodegradation of insoluble pollutants (Hassanshahian, 2014). In the cell-free culture broths of S. putrefaciens IBBPo6 cells grown on minimal medium supplemented with 0.5% yeast extract were detected four biosurfactants (Figures 2a, 2b): the BS1 with a Rf (retardation factor) of 0.43, BS2 with a Rf of 0.53, BS3 with a Rf of 0.59, and BS4 with a Rf of 0.68. The same biosurfactants were detected in the extract of S. putrefaciens IBBPo6 cells grown in the presence of 0.5% organic solvents. The Rf values were between 0.36-0.41 for BS1, 0.46-0.51 for BS2, 0.53-0.59 for BS3, and 0.65-0.69 for BS4. As evident from TLC analysis (Figures 2a, 2b), BS3 and BS4 were detected in barely quantities in the extracts of S. putrefaciens IBBPo6 cells, as compared with other two biosurfactants (BS1, BS2). To recover crystalline biosurfactant, the cell-free culture broths
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of S. putrefaciens IBBPo6 cells were acidified to pH 2 + 0.5 with HCl. Biosurfactant crystals produced by S. putrefaciens IBBPo6 cells grown on minimal medium supplemented with 0.5% yeast extract appeared as frost flowers-like crystals (data not shown). Similar data were obtained by us (Stancu, 2015) when S. putrefaciens IBBPo6 cells were grown on LB medium, as well as when the cells were exposed to 5% organic solvents. a.
b.
All tracks at 254 nm
BS4 BS3 BS2
1000.0 [AU] 800.0 700.0 600.0 500.0 400.0 300.0 200.0 100.0 0.0 0.00 0.10
BS1
R T
1000.0 [AU] 800.0 700.0 600.0 500.0 400.0 300.0 200.0 100.0 0.0 100.0 S [mm] 80.0 70.0 0.20 0.30 60.0 0.40 50.0 0.50 40.0 0.60 30.0 0.70 20.0 0.80 [Rf] 10.0 1.00 0.0
1
1
2
3
4
5
7
6
SS
23
4
5
67
S
Figure 2. Biosurfactants of S. putrefaciens IBBPo6 cells grown in the presence of organic solvents Control (lane 1), cyclohexane (lane 2), n-hexane (lane 3), n-decane (lane 4), toluene (lane 5), o-xylene (lane 6), ethylbenzene (lane 7); sugars standards (SS), trehalose (T), L-rhamnose (R); biosurfactants (BS1, BS2, BS3, BS4). Panels a, b. The TLC plate was visualized and scanned under a 254 nm ultraviolet light. The biosurfactants spots and their corresponding peak have been marked by arrows.
3.1.4. Polymerase chain reaction (PCR) Genomic DNA extracted from S. putrefaciens IBBPo6 cells grown on minimal medium supplemented with 0.5% yeast extract and from the cells grown in the presence of 0.5% organic solvents was used as template for PCR amplification (Table 2) of some catabolic (i.e., alkB, alkM, alkB1, alkM1, todM, xylM, ndoM, C23DO), transporter (i.e., HAE1) and trehalose-phosphate synthase (i.e., otsA1) genes. Table 2. Detection of catabolic, transporter and trehalose-phosphate synthase genes in S. putrefaciens IBBPo6 cells grown in the presence of organic solvents by PCR assay Organic solvents (log POWc)
PCR assay Gene
Primers ALK1-f ALK1-r ALK2-f ALK2-r ALK3-f ALK3-r alkM-f alkM-r todM-f todM-r xylM-f xylM-r ndoM-f ndoM-r 23CAT-f 23CAT-r A24f2 A577r2 otsA-f otsA-r
alkB alkM alkB1 alkM1 todM xylM ndoM C23DO HAE1 otsA1 a
ATa (°C)
EFSb (bp)
Control
Cyclohexane (3.35)
n-Hexane (3.86)
n-Decane (5.98)
Toluene (2.64)
o-Xylene (3.09)
Ethylbenzene (3.17)
50
185
-
-
-
-
-
-
-
50
271
-
-
-
-
-
-
-
50
330
+
+
+
+
+
-
+
62
870
+
-
-
+
-
-
-
62
560
-
-
-
-
-
-
-
62
834
-
-
-
-
-
-
-
62
642
-
-
-
-
-
-
-
50
238
-
-
-
-
-
-
-
50
550
+
+
+
+
+
-
+
50
760
+
+
+
+
+
-
+
b
c
Legend: AT = annealing temperature; EFS = expected fragment size; log POW = logarithm of the partition coefficient of the solvent in an octanol-water mixture; f = forward PCR primer; r = reverse PCR primer; + = positive reaction; - = negative reaction.
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The expected PCR products sizes were 185, 271, 330, 870 bp for alkane hydroxylase genes (alkB, alkM, alkB1, alkM1), 560 bp for toluene dioxygenase (todM), 834 bp for xylene monooxygenase (xylM), 642 bp for naphthalene dioxygenase (ndoM), 238 bp for catechol 2,3-dioxygenase, 550 bp for HAE1 gene, and 760 bp for otsA1 gene. The annealing temperature was 50°C when primers for alkB, alkM, alkB1, C23DO, HAE1 and otsA1 genes were used, while in the case of the primers for alkM1, todM, xylM and ndoM genes the annealing temperature was 62°C. S. putrefaciens IBBPo6 cells grown on minimal medium supplemented with 0.5% yeast extract possesses alkB1, alkM1, HAE1 and otsA1 genes (Table 2). The other catabolic genes (i.e., alkB, alkM, todM, xylM, ndoM and C23DO) were not detected in this bacterium. Similar data were obtained by us when S. putrefaciens IBBPo6 cells were grown on LB medium (Stancu, 2015). As observed in Table 2, alkB1, HAE1 and otsA1 genes were also detected in S. putrefaciens IBBPo6 cells grown in the presence of 0.5% cyclohexane, n-hexane, n-decane, toluene or ethylbenzene; when the cells were grown in the presence of 0.5% o-xylene, the alkB1, HAE1 and otsA1 genes were not detected. The alkM1gene was detected in S. putrefaciens IBBPo6 cells grown in the presence of 0.5% n-decane; in DNA extracted from cells grown in the presence of 0.5% cyclohexane, n-hexane, toluene, o-xylene or ethylbenzene the alkM1gene was not detected. 4. Conclusions The acquired results showed that S. putrefaciens IBBPo6 cells used organic solvents with log POW values under 4, such as cyclohexane, n-hexane, toluene, o-xylene or ethylbenzene as growth substrates. As expected, n-decane with log POW value over 4 was also used as growth substrate by S. putrefaciens IBBPo6 cells. When S. putrefaciens IBBPo6 cells were grown in the presence of cyclohexane, n-hexane, toluene, o-xylene or ethylbenzene more-robust biofilms were formed, as compared with those developed in the presence of n-decane. Biosurfactants detected in the cell-free culture broths of S. putrefaciens IBBPo6 cells grown in the presence of organic solvents could have an important role in their biofilm architecture. S. putrefaciens IBBPo6 cells grown in the presence of n-decane possesses alkB1, alkM1, HAE1 and otsA1 genes, while the cells grown in the presence of cyclohexane, n-hexane, toluene or ethylbenzene possesses only alkB1, HAE1 and otsA1. In the DNA extracted from cells grown in the presence of o-xylene, the alkB1, alkM1, HAE1 and otsA1 genes were not detected. There were observed some differences between the PCR pattern of S. putrefaciens IBBPo6 cells grown in the presence of n-decane with log POW value over 4, and those of cells grown in the presence other organic solvents with log POW values under 4. Acknowledgements The study was funded by project no. RO1567-IBB05/2015 from the Institute of Biology Bucharest of Romanian Academy. The author is grateful to Ana Dinu and Alexandru Brînzan for technical support. References Davey, M.E., Caiazza, N.C., O'Toole, G.A., 2003. Rhamnolipid surfactant production affects biofilm architecture in Pseudomonas aeruginosa PAO1. Journal of Bacteriology 185, 1027–1036. Ding, Y., Peng, N., Du, Y., Ji, L., Cao, B., 2014. Disruption of putrescine biosynthesis in Shewanella oneidensis enhances biofilm cohesiveness and performance in Cr(VI) immobilization. Applied and Environmental Microbiology 80, 1498–1506. Gao, H.A., Obraztova, A., Stewart, N., Popa, R., Fredrickson, J.K., Tiedje, J.M., Nealson, K.H., Zhou, J., 2006. Shewanella loihica sp. nov., isolated from iron-rich microbial mats in the Pacific Ocean. International Journal of Systematic and Evolutionary Microbiology 56, 1911– 1916. Hassanshahian, M., 2014. Isolation and characterization of biosurfactant producing bacteria from Persian Gulf (Bushehr provenance). Marine Pollution Bulletin 86, 361–366. Heipieper, H.J., Neumann, G., Cornelissen, S., Meinhardt, F., 2007. Solvent-tolerant bacteria for biotransformations in two-phase fermentation systems. Applied Microbiology and Biotechnology 74, 961–973. Kohno, T., Sugimoto, Y., Sei, K., Mori, K., 2002. Design of PCR primers and gene probes for general detection of alkane-degrading bacteria. Microbes and Environments 17, 114–121. Márquez-Rocha, F.J., Olmos-Soto, J., Rosano-Hernández, M.C., Muriel-Garcia, M., 2005. Determination of the hydrocarbon-degrading metabolic capabilities of tropical bacterial isolates. International Biodeterioration and Biodegradation 55, 17–23.
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ScienceDirect Agriculture and Agricultural Science Procedia 6 (2015) 601 – 607
“ST26733”, International Conference "Agriculture for Life, Life for Agriculture"
Adaptation Mechanisms to Toxic Solvents in Shewanella putrefaciens Mihaela Marilena Stancu* Institute of Biology Bucharest of Romanian Academy, 296 Splaiul Independentei, 060031 Bucharest, P.O. Box 56-53, Romania
Abstract Shewanella putrefaciens is a Gram-negative bacterium which is widespread in various environments, including in sites polluted with petroleum and petroleum products. S. putrefaciens IBBPo6 used organic solvents with log POW (logarithm of the partition coefficient of the solvent in an octanol-water mixture) values above 4 (i.e., n-decane) and below 4 (i.e., cyclohexane, n-hexane, toluene, o-xylene, ethylbenzene) as growth substrates. More-robust biofilms were developed when S. putrefaciens IBBPo6 cells were grown in the presence of cyclohexane, n-hexane, toluene, o-xylene or ethylbenzene, as compared with those formed in the presence of n-decane. Four biosurfactants were detected in the cell-free culture broths of S. putrefaciens IBBPo6 cells grown in the presence of tested organic solvents. There were observed differences between the PCR pattern of S. putrefaciens IBBPo6 cells grown in the presence of n-decane, and those of cells grown in the presence other tested organic solvents (i.e., cyclohexane, nhexane, toluene, o-xylene, ethylbenzene).
© Published by byElsevier ElsevierB.V. B.V. This is an open access article under the CC BY-NC-ND license Authors. Published © 2015 2015 The The Authors. (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the University of Agronomic Sciences and Veterinary Medicine Bucharest. Peer-review under responsibility of the University of Agronomic Sciences and Veterinary Medicine Bucharest Keywords: adaptation; mechanisms; Shewanella; solvents
1. Introduction The genus Shewanella is a member of the class Gammaproteobacteria and includes a group of Gram-negative, motile, rod-shaped, oxidase-positive, non-fermentative and facultative anaerobic bacteria (Park et al., 2009). Shewanella species are widely distributed in diverse environments due to their flexible respiration. These bacteria can use a wide range of electron acceptors and are classified in the group of dissimilatory metal-reducing bacteria
* Corresponding author. Tel.: +40212219202; fax: +40212219071. E-mail address: [email protected]
2210-7843 © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the University of Agronomic Sciences and Veterinary Medicine Bucharest doi:10.1016/j.aaspro.2015.08.097
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(Rodrigues et al., 2011; Wu et al., 2013). Their respiratory versatility allows them to play an important role in the geochemical cycling of iron, manganese and carbon sources, as well as in bioremediation and bio-engineering applications (Wu et al., 2013). Shewanella putrefaciens strains are able to use metals for respiration and they are also able to degrade several pollutants (e.g., petroleum, chlorinated solvents) which makes these microorganisms useful tools for bioremediation (Martín-Gil et al., 2004; Gao et al., 2006). The mechanisms underlying organic solvents tolerance in bacteria are not yet fully understood, although a number of factors involved in the process have been characterized over the last years. These studies have revealed a number of mechanisms, including: changes in membrane protein pattern; alteration in phospholipids composition; organic solvents removal through active efflux systems (efflux pumps); changes in energy metabolism; changes in cell morphology; changes in the cell wall; vesicle formation; transformation or degradation of the organic solvents (Heipieper et al., 2007; Schrewe et al., 2013). The aim of this study was to investigate the possible cell adaptation mechanisms pursued by S. putrefaciens IBBPo6 cells grown in the presence of 0.5% (v/v) organic solvents. Changes in the cell growth rate, biofilm formation, biosurfactant production, and in PCR pattern were observed in S. putrefaciens IBBPo6 cells grown in the presence of 0.5% (v/v) organic solvents. 2. Materials and Methods 2.1 Solvent tolerance in S. putrefaciens IBBPo6 Bacterial cells were cultivated on liquid LB-Mg medium (Stancu and Grifoll, 2011) containing 15 Pg ml-1 ampicillin and incubated at 28°C on a rotary shaker (200 rpm) until they reached the exponential growth phase. Bacterial cells were harvested by centrifugation, washed and finally resuspended (105 cells ml-1) in minimal medium (Stancu and Grifoll, 2011). 0.5% (w/v) yeast extract and 0.5% (v/v) organic solvents (alkanes: cyclohexane, nhexane, n-decane; aromatics: toluene, o-xylene, ethylbenzene) were supplied to the cell suspensions as the carbon source. Flasks were sealed and incubated for 24 hours at 28°C on a rotary shaker (200 rpm). 2.1.1. Cell growth Cell growth in the presence of organic solvents was determined by measuring optical density (OD) at 660 nm using a SPECORD 200 UV-visible spectrophotometer. 2.1.2. Biofilm formation Samples were fixed on SEM holder and gold-coated with a JEOL JFC-1300 auto fine coater, in a deep vacuum. The samples were examined with a JEOL JSM-6610LV scanning electron microscope (SEM). 2.1.3. Biosurfactant production. Biosurfactants were extracted from the cell-free culture broths with chloroform-methanol (2:1 v/v), using the method described by Stancu (2014). For thin layer chromatography (TLC) analysis, the samples were spotted with a Linomat 5 sample applicator (CAMAG), on a 10×10 cm precoated silica gel 60 TLC aluminium sheets (Merck). The separation was performed using the chloroform-methanol (90:10 v/v) mixture as mobile phase. After development, a densitometric scan at 254 nm in a TLC Scanner 4 (CAMAG) was performed for detection and quantification of the biosurfactants. 2.1.4. Polymerase chain reaction (PCR) For PCR amplification, 1 ȝl of DNA extract was added to a final volume of 25 ȝl reaction mixture, containing: 5×GoTaq flexi buffer, MgCl2, dNTP mix, specific primers for catabolic (alkB, alkM, alkB1, alkM1, todM, xylM, ndoM, C23DO) genes (ALK1-f and ALK1-r, ALK2-f and ALK2-r, ALK3-f and ALK3-r, Kohno et al., 2002; alkM-f and alkM-r, todM-f and todM-r, xylM-f and xylM-r, ndoM-f and ndoM-r, Márquez-Rocha et al., 2005; 23CAT-f and 23CAT-r, Mesarch et al., 2000), specific primers for hydrophobic/amphiphilic efflux 1 (HAE1) transporter gene (A24f2 and A577r2, Meguro et al., 2005) or specific primers for trehalose-phosphate synthase (otsA1) gene (otsA-f and otsA-r, Tischler et al., 2013), and GoTaq G2 hot start polymerase (Promega). PCR was performed with a Mastercycler pro S (Eppendorf). PCR assays were performed according to Meguro et al. (2005) protocol: initial denaturation for 10 min at 94°C, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 50°C or 62°C
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for 30 sec, extension at 72°C for 2 min, and a final extension at 72°C for 10 min. After electrophoretic separation on 1.5% (w/v) TBE agarose gel (Sambrook et al., 1989) and staining with fast blast DNA stain (Bio-Rad) the PCR products were analyzed. Reagents used during this study were procured from Merck, Sigma-Aldrich, Promega, Invitrogen, Applied Biosystems or Bio-Rad Laboratories. The PCR primers were purchased from Biosearch Technologies, Integrated DNA Technologies and Invitrogen. 3. Results and Discussions The strain used in the present study was S. putrefaciens IBBPo6 (KM405339). This Gram-negative bacterium was isolated (Stancu, 2015) from Poeni oily sludge (Teleorman County, Romania). Their taxonomic affiliation was determined on the basis of phenotypic characteristics and of 16S rRNA gene sequence (Stancu, 2015). 3.1. Solvent tolerance in S. putrefaciens IBBPo6 S. putrefaciens isolates are widespread in various environments, including in sites polluted with petroleum and petroleum products. Nevertheless, the mechanisms of solvent tolerance in this bacterium have not been investigated so far. 3.1.1. Cell growth Bacterial cells susceptibility to toxic organic solvents depends on the logarithm of the partition coefficient of the solvent in an octanol-water mixture (log POW) (Sikkema et al., 1995; Heipieper et al., 2007). The growth of S. putrefaciens IBBPo6 cells on minimal medium supplemented with 0.5% yeast extract was higher (OD = 0.905), compared with the growth of the cells on the same medium in the presence of 0.5% organic solvents (OD = 0.4130.762). As expected, the growth was higher (OD = 0.762) when S. putrefaciens IBBPo6 cells were grown in the presence of n-decane with log POW value over 4, as compared with the growth of cells (OD = 0.413-0.637) in the presence of other tested organic solvents (i.e., cyclohexane, n-hexane, toluene, o-xylene, ethylbenzene) with log POW values under 4 (Table 1). Table 1. Growth of S. putrefaciens IBBPo6 cells in the presence of organic solvents Assay Cell growtha
Control 0.905
Cyclohexane (3.35) 0.523
n-Hexane (3.86) 0.637
Organic solvents (log POWb) n-Decane Toluene (5.98) (2.64) 0.762 0.413
o-Xylene (3.09) 0.468
Ethylbenzene (3.17) 0.497
Legend: a = the absorbance of the culture broths was measured at 660 nm; b = logarithm of the partition coefficient of the solvent in an octanolwater mixture.
According to Schrewe et al. (2013), organic solvents with a log POW between 1 and 4 are considered to be toxic to bacterial cells. However, some bacteria (e.g., Pseudomonas strains) are tolerant to organic solvents with a log POW under 4 and can utilize such compounds as growth substrates (Schrewe et al., 2013). These solvent-tolerant organisms are able to adapt to the presence of toxic organic solvents. More hydrophobic, larger compounds (log POW over 4) are considered non-toxic and do not accumulate in cellular membranes, as they are virtually unable to pass the outer membrane due to their size and hydrophobicity (Schrewe et al., 2013). 3.1.2. Biofilm formation Like other bacteria, Shewanella species can form biofilms under diverse environments (Wu et al. 2013). Microbial biofilms are surface-associated, dynamic communities that respond to changing external and internal environments, allowing continued acclimation of the biofilm population (Saville et al., 2011). Scanning electron micrographs (Figures 1a-1g) showed biofilms formation when S. putrefaciens IBBPo6 cells were grown 24 hours on minimal medium supplemented with 0.5% yeast extract and on the same medium in the presence of 0.5% organic solvents. When S. putrefaciens IBBPo6 cells were grown in the presence of cyclohexane, nhexane, toluene, o-xylene or ethylbenzene more-robust biofilms were developed, as compared with those formed in the presence of n-decane or yeast extract. In biofilms, cells are embedded in a self-produced matrix composed of extracellular polymeric substances (EPS), including proteins, polysaccharides, lipids, and extracellular DNA (Ding et al., 2014). Extensive studies have demonstrated a wide range of advantages of the presence of an EPS matrix for
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the biofilm mode of life (Ding et al., 2014). For example, compared with planktonically grown cells, biofilms have been reported to be more tolerant to harsh physicochemical conditions generated by various toxic contaminants (Ding et al., 2014). Studies of Shewanella species showed that the type of carbon source and the nutrient concentration influence the development and architecture of the biofilms. Therefore, more-robust biofilms with enhanced microbe-surface interactions were observed in poor nutrient conditions (Thormann et al., 2004). a.
b.
c.
d.
e.
f.
g.
Figure 1. Scanning electron micrographs of S. putrefaciens IBBPo6 cells grown in the presence of organic solvents Control (panel a), cyclohexane (panel b), n-hexane (panel c), n-decane (panel d), toluene (panel e), o-xylene (panel f), ethylbenzene (panel g).
3.1.3. Biosurfactant production Biosurfactants are surface active materials that are produced by some bacteria, including by different Shewanella strains (Hassanshahian, 2014). According to literature (Davey et al., 2003), biosurfactants produced by some bacteria (e.g., rhamnolipids synthesized by Pseudomonas aeruginosa PAO1) are thought to maintain biofilm architecture by influencing cell-cell interactions and bacterial attachment to surfaces (Davey et al., 2003). Furthermore, the biosurfactant molecules increase biodegradation of insoluble pollutants (Hassanshahian, 2014). In the cell-free culture broths of S. putrefaciens IBBPo6 cells grown on minimal medium supplemented with 0.5% yeast extract were detected four biosurfactants (Figures 2a, 2b): the BS1 with a Rf (retardation factor) of 0.43, BS2 with a Rf of 0.53, BS3 with a Rf of 0.59, and BS4 with a Rf of 0.68. The same biosurfactants were detected in the extract of S. putrefaciens IBBPo6 cells grown in the presence of 0.5% organic solvents. The Rf values were between 0.36-0.41 for BS1, 0.46-0.51 for BS2, 0.53-0.59 for BS3, and 0.65-0.69 for BS4. As evident from TLC analysis (Figures 2a, 2b), BS3 and BS4 were detected in barely quantities in the extracts of S. putrefaciens IBBPo6 cells, as compared with other two biosurfactants (BS1, BS2). To recover crystalline biosurfactant, the cell-free culture broths
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of S. putrefaciens IBBPo6 cells were acidified to pH 2 + 0.5 with HCl. Biosurfactant crystals produced by S. putrefaciens IBBPo6 cells grown on minimal medium supplemented with 0.5% yeast extract appeared as frost flowers-like crystals (data not shown). Similar data were obtained by us (Stancu, 2015) when S. putrefaciens IBBPo6 cells were grown on LB medium, as well as when the cells were exposed to 5% organic solvents. a.
b.
All tracks at 254 nm
BS4 BS3 BS2
1000.0 [AU] 800.0 700.0 600.0 500.0 400.0 300.0 200.0 100.0 0.0 0.00 0.10
BS1
R T
1000.0 [AU] 800.0 700.0 600.0 500.0 400.0 300.0 200.0 100.0 0.0 100.0 S [mm] 80.0 70.0 0.20 0.30 60.0 0.40 50.0 0.50 40.0 0.60 30.0 0.70 20.0 0.80 [Rf] 10.0 1.00 0.0
1
1
2
3
4
5
7
6
SS
23
4
5
67
S
Figure 2. Biosurfactants of S. putrefaciens IBBPo6 cells grown in the presence of organic solvents Control (lane 1), cyclohexane (lane 2), n-hexane (lane 3), n-decane (lane 4), toluene (lane 5), o-xylene (lane 6), ethylbenzene (lane 7); sugars standards (SS), trehalose (T), L-rhamnose (R); biosurfactants (BS1, BS2, BS3, BS4). Panels a, b. The TLC plate was visualized and scanned under a 254 nm ultraviolet light. The biosurfactants spots and their corresponding peak have been marked by arrows.
3.1.4. Polymerase chain reaction (PCR) Genomic DNA extracted from S. putrefaciens IBBPo6 cells grown on minimal medium supplemented with 0.5% yeast extract and from the cells grown in the presence of 0.5% organic solvents was used as template for PCR amplification (Table 2) of some catabolic (i.e., alkB, alkM, alkB1, alkM1, todM, xylM, ndoM, C23DO), transporter (i.e., HAE1) and trehalose-phosphate synthase (i.e., otsA1) genes. Table 2. Detection of catabolic, transporter and trehalose-phosphate synthase genes in S. putrefaciens IBBPo6 cells grown in the presence of organic solvents by PCR assay Organic solvents (log POWc)
PCR assay Gene
Primers ALK1-f ALK1-r ALK2-f ALK2-r ALK3-f ALK3-r alkM-f alkM-r todM-f todM-r xylM-f xylM-r ndoM-f ndoM-r 23CAT-f 23CAT-r A24f2 A577r2 otsA-f otsA-r
alkB alkM alkB1 alkM1 todM xylM ndoM C23DO HAE1 otsA1 a
ATa (°C)
EFSb (bp)
Control
Cyclohexane (3.35)
n-Hexane (3.86)
n-Decane (5.98)
Toluene (2.64)
o-Xylene (3.09)
Ethylbenzene (3.17)
50
185
-
-
-
-
-
-
-
50
271
-
-
-
-
-
-
-
50
330
+
+
+
+
+
-
+
62
870
+
-
-
+
-
-
-
62
560
-
-
-
-
-
-
-
62
834
-
-
-
-
-
-
-
62
642
-
-
-
-
-
-
-
50
238
-
-
-
-
-
-
-
50
550
+
+
+
+
+
-
+
50
760
+
+
+
+
+
-
+
b
c
Legend: AT = annealing temperature; EFS = expected fragment size; log POW = logarithm of the partition coefficient of the solvent in an octanol-water mixture; f = forward PCR primer; r = reverse PCR primer; + = positive reaction; - = negative reaction.
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The expected PCR products sizes were 185, 271, 330, 870 bp for alkane hydroxylase genes (alkB, alkM, alkB1, alkM1), 560 bp for toluene dioxygenase (todM), 834 bp for xylene monooxygenase (xylM), 642 bp for naphthalene dioxygenase (ndoM), 238 bp for catechol 2,3-dioxygenase, 550 bp for HAE1 gene, and 760 bp for otsA1 gene. The annealing temperature was 50°C when primers for alkB, alkM, alkB1, C23DO, HAE1 and otsA1 genes were used, while in the case of the primers for alkM1, todM, xylM and ndoM genes the annealing temperature was 62°C. S. putrefaciens IBBPo6 cells grown on minimal medium supplemented with 0.5% yeast extract possesses alkB1, alkM1, HAE1 and otsA1 genes (Table 2). The other catabolic genes (i.e., alkB, alkM, todM, xylM, ndoM and C23DO) were not detected in this bacterium. Similar data were obtained by us when S. putrefaciens IBBPo6 cells were grown on LB medium (Stancu, 2015). As observed in Table 2, alkB1, HAE1 and otsA1 genes were also detected in S. putrefaciens IBBPo6 cells grown in the presence of 0.5% cyclohexane, n-hexane, n-decane, toluene or ethylbenzene; when the cells were grown in the presence of 0.5% o-xylene, the alkB1, HAE1 and otsA1 genes were not detected. The alkM1gene was detected in S. putrefaciens IBBPo6 cells grown in the presence of 0.5% n-decane; in DNA extracted from cells grown in the presence of 0.5% cyclohexane, n-hexane, toluene, o-xylene or ethylbenzene the alkM1gene was not detected. 4. Conclusions The acquired results showed that S. putrefaciens IBBPo6 cells used organic solvents with log POW values under 4, such as cyclohexane, n-hexane, toluene, o-xylene or ethylbenzene as growth substrates. As expected, n-decane with log POW value over 4 was also used as growth substrate by S. putrefaciens IBBPo6 cells. When S. putrefaciens IBBPo6 cells were grown in the presence of cyclohexane, n-hexane, toluene, o-xylene or ethylbenzene more-robust biofilms were formed, as compared with those developed in the presence of n-decane. Biosurfactants detected in the cell-free culture broths of S. putrefaciens IBBPo6 cells grown in the presence of organic solvents could have an important role in their biofilm architecture. S. putrefaciens IBBPo6 cells grown in the presence of n-decane possesses alkB1, alkM1, HAE1 and otsA1 genes, while the cells grown in the presence of cyclohexane, n-hexane, toluene or ethylbenzene possesses only alkB1, HAE1 and otsA1. In the DNA extracted from cells grown in the presence of o-xylene, the alkB1, alkM1, HAE1 and otsA1 genes were not detected. There were observed some differences between the PCR pattern of S. putrefaciens IBBPo6 cells grown in the presence of n-decane with log POW value over 4, and those of cells grown in the presence other organic solvents with log POW values under 4. Acknowledgements The study was funded by project no. RO1567-IBB05/2015 from the Institute of Biology Bucharest of Romanian Academy. The author is grateful to Ana Dinu and Alexandru Brînzan for technical support. References Davey, M.E., Caiazza, N.C., O'Toole, G.A., 2003. Rhamnolipid surfactant production affects biofilm architecture in Pseudomonas aeruginosa PAO1. Journal of Bacteriology 185, 1027–1036. Ding, Y., Peng, N., Du, Y., Ji, L., Cao, B., 2014. Disruption of putrescine biosynthesis in Shewanella oneidensis enhances biofilm cohesiveness and performance in Cr(VI) immobilization. Applied and Environmental Microbiology 80, 1498–1506. Gao, H.A., Obraztova, A., Stewart, N., Popa, R., Fredrickson, J.K., Tiedje, J.M., Nealson, K.H., Zhou, J., 2006. Shewanella loihica sp. nov., isolated from iron-rich microbial mats in the Pacific Ocean. International Journal of Systematic and Evolutionary Microbiology 56, 1911– 1916. Hassanshahian, M., 2014. Isolation and characterization of biosurfactant producing bacteria from Persian Gulf (Bushehr provenance). Marine Pollution Bulletin 86, 361–366. Heipieper, H.J., Neumann, G., Cornelissen, S., Meinhardt, F., 2007. Solvent-tolerant bacteria for biotransformations in two-phase fermentation systems. Applied Microbiology and Biotechnology 74, 961–973. Kohno, T., Sugimoto, Y., Sei, K., Mori, K., 2002. Design of PCR primers and gene probes for general detection of alkane-degrading bacteria. Microbes and Environments 17, 114–121. Márquez-Rocha, F.J., Olmos-Soto, J., Rosano-Hernández, M.C., Muriel-Garcia, M., 2005. Determination of the hydrocarbon-degrading metabolic capabilities of tropical bacterial isolates. International Biodeterioration and Biodegradation 55, 17–23.
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