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Immobilization of Rhodococcus erythropolis B4 on radiation crosslinked poly(vinylpyrrolidone) hydrogel: Application to the degradation of polycyclic aromatic hydrocarbons
Available online at www.sciencedirect.com
NIM B Beam Interactions with Materials & Atoms
Nuclear Instruments and Methods in Physics Research B 265 (2007) 370–374 www.elsevier.com/locate/nimb
Immobilization of Rhodococcus erythropolis B4 on radiation crosslinked poly(vinylpyrrolidone) hydrogel: Application to the degradation of polycyclic aromatic hydrocarbons A. Djefal-Kerrar a
a,*
, S. Gais a, K. Ouallouche a, A. Nacer Khodja a, M. Mahlous a, H. Hace`ne b
Division of Nuclear Applications, Nuclear Research Centre of Algiers, 2 Bd Frantz Fanon BP-399 Alger-gare, Algiers, Algeria b Biological Sciences Institute, Science and Technology University Houari Boumediene, Algiers, Algeria Available online 8 September 2007
Abstract A poly(vinylpyrrolidone) (PVP) hydrogel crosslinked by gamma radiation was used to immobilize, by adsorption, Rhodococcus erythropolis B4 strain. Immobilized cells were tested for their capacity to degrade naphthalene and anthracene, under aerobic conditions. The results showed that, the strain fixed is capable of growing in the presence of naphthalene or anthracene as a unique source of carbon. It was also shown that, the fixed strain can be preserved by freeze-drying for further use. The biodegradation capacity was improved during the second use. 2007 Elsevier B.V. All rights reserved. PACS: 61.82.Pv; 87.68+z; 89.20.Bb; 92.20.jb Keywords: Cell immobilization; Poly(vinylpyrrolidone); Radiation crosslinking; Anthracene; Naphthalene; Biodegradation
1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental pollutants. They are generated from petroleum and many pyrolysis processes. They cause concern because of their potentially deleterious effects on human health and many can be recalcitrant in the environment [1]. Among them, naphthalene, a bicycle PAH, is released into the environment as coal tar and coal tar products [2]. Whereas anthracene, a tricycle PAH, is found in high amounts in PAH contaminated sediments, surface soils and waste sites. This hydrophobic contaminant is toxic to aquatic life [3]. Various existing clean-up technologies for hydrocarbon treatment can be categorised in three general schemes: chemical, physical and biological. The degradation by *
Corresponding author. Tel.: +213 21 43 44 44; fax: +213 21 43 42 80. E-mail address: [email protected] (A. Djefal-Kerrar).
0168-583X/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2007.09.005
microorganisms is the subject of many works [4–7]. A wide range of different bacteria is able to completely assimilate a defined range of compound, or exhibit just partial metabolism [8]. Therefore, the use of pure cultures of microorganisms, specially adapted to metabolize the contaminant, is envisaged as an attractive alternative. Rhodococcus erythropolis is well known microorganism containing a large set of enzymes that allows carrying out an enormous number of bioconversion and degradation [9,10]. Feasibility and performance of recently developed immobilized bacteria on inert support for biodegradation of recalcitrant compounds has attracted considerable and increasing interest since this strategy allows obtaining much more profit from this process. The main advantages in the use of immobilized cells in comparison with suspended ones include the retention in the reactor of higher concentration of microorganisms, easy removal of bacteria after use from the reaction mixture, providing
A. Djefal-Kerrar et al. / Nucl. Instr. and Meth. in Phys. Res. B 265 (2007) 370–374
the ability to control reaction time, reuse of cells for many reaction cycles, lowering the total production cost of cells-mediated reactions, provide pure products [11–13]. Microbial cells can be immobilized on various polymeric entrapment matrices. The most promising materials and preparation technique for such a purpose are hydrogels and radiation crosslinking, respectively [11,14]. Among hydrophilic polymers, poly(vinylpyrrolidone) PVP, poly(vinyl alcohol) PVA, poly(hydroxyethylmetacrylate) PHEMA, poly(ethylene oxide) PEO and others, are the most often used for hydrogel’s synthesis [15]. In this work PVP was used for the preparation of the immobilization matrix. The objectives of the study are, on one hand, to investigate the immobilization of R. erythropolis B4 pure strain on the PVP matrix crosslinked by gamma radiation, and on the other hand, to evaluate the ability of immobilized cells to degrade PAHs, such as naphthalene and anthracene. 2. Materials and methods 2.1. Chemicals Anthracene and naphtalene were obtained from SigmaAldrich. Poly(vinylpyrrolidone) K90 with an average molecular weight of 360,000 Da was purchased from Fluka-Chemica. Solvents were of analytical grade.
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The poly(vinylpyrrolidone) (PVP) at a concentration of 7% w/v was dissolved in distilled water at 70 C overnight, and poured into plastic syringes, sealed with parafilm then irradiated by c rays at a dose of 25 kGy. Gel fraction was determined according to the formula: Gf ¼ W dg =W ip 100%;
ð1Þ
where Wdg is the weight of the dry gel and Wip is the initial weight of the polymer. The equilibrium swelling ratio of the PVP hydrogel in water was measured gravimetrically as the ratio of the swollen gel weight at the equilibrium to that of the dry gel. Cross-linked gels were cut into discs of about 0.5 cm thickness and 1.2 cm diameter and maintained in sterile conditions until their use for cell immobilization. 2.5. Immobilization of the cells Hydrogel discs were dropped into 50 ml of sterile vitamin-supplemented MSM, and then 2 ml of the suspension cell containing 108 CFU (Colony Forming Unit)/ml were added. In order to allow a full contact of the hydrogel discs with suspended cells without sticking each to other, a maximum of 30 discs were used. The mixture was incubated at 30 C under shaking. After 24 h, the discs were removed from the culture medium, and then washed with distilled water to remove non adherent cells. The immobilized cells are ready to be tested for their PAHs degradation ability.
2.2. Microorganisms 2.6. Scanning electron microscopy R. erythropolis B4 strain was obtained from the microbiology laboratory collection, Science & Technology University of Algiers. It was maintained on nutrient agar (Difco) slants at 4 C. 2.3. Culture conditions First, bacterial cells were reactivated in Tryptic Soy broth at 30 C. For their adaptation to the PAH, the cells were pre-grown in vitamin-supplemented mineral salt medium (MSM) containing per liter of distilled water: Na2HPO4, 4.5 g; NH4NO3, 1 g; KH2PO4, 0.68 g; MgSO4 7H2O, 0.1 g; Fe2SO4 7H2O, 0.001 g, trace elements and vitamin solution [16], and two different carbon sources: glucose and naphthalene (1/3:2/3) or glucose and anthracene (1/3:2/3). After an incubation of about 72 h at 30 C in an orbital shaker incubator at 120 rpm, the cells were harvested by centrifugation (3000 rpm, 20 min) and then resuspended in distilled water. 2.4. Hydrogel preparation and characterisation Polymeric hydrogel was synthesised by gamma radiation induced crosslinking and sterilisation, according to the method reported previously [17].
R. erythropolis B4 cells immobilized on radiation crosslinked and sterilised poly(vinylpyrrolidone) hydrogel matrix were fixed with 2.5% (v/v) glutaraldehyde in saline solution for 1 h, rinsed in water and then dehydrated by sequential immersion in increasing concentration of ethanol (10%, 50%, 80%, 100%) for 10 min each. Subsequently the specimens were freeze dried and then coated with gold to be finally observed with a JEOL JSM 6360 LV scanning electron microscope at an acceleration voltage of 10 kV. 2.7. Biodegradation test The capacity of the bacterial cells to degrade PAH was tested by inoculating 200 ml of MSM containing anthracene or naphthalene as a sole carbon source in N,Ndimethylformamide and ethanol, respectively, with either, 2 ml of the free cell suspension, or with 30 hydrogel discs containing fixed cells. The PAH concentrations were 0.7 and 1 g/l for anthracene and, 1 and 1.5 g/l for naphthalene [18–22]. Incubation was made at 30 C under continuous shaking. Periodically, samples were taken aseptically from the culture flask for HPLC analysis.
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2.8. Reusability of the immobilized cells After the first test, the discs with immobilized bacteria were freeze dried and stored at room temperature. After 15 days of storage, the discs were reused to test the PAHs degradation ability of immobilized cells, in the same conditions as in the first test. 2.9. High performance liquid chromatography (HPLC) analysis Identification and quantification of remaining naphthalene and anthracene in the culture media were performed by HPLC (Waters 2487) with UV detection at a wavelength of 254 nm. Remaining PAH, were extracted from the culture probe after centrifugation (3000 rpm, 20 min), with an equal weighted volume of cyclohexane for 2 h at 160 rpm. An aliquot (20 ll) was analysed using a thermo hypersil BDS-C18 column (375 · 125 mm) and elution flow rate 1.5 ml/min (water/acetonitril: 25/75 v/v).
mean that the irradiation dose delivered to sterilise the hydrogel, at the same time produced a high degree of crosslinking, which render 86% of it insoluble in water. The swelling property of the hydrogel will assure a maximum contact of the fixed bacteria with the PAH contained in water. 3.2. Cell immobilization on PVP matrix As shown in SEM microphotographs, the surface of the hydrogel matrix unexposed to bacterial suspension (Fig. 1(A)) was covered by Rhodococcal cells after its immersion into the bacterial suspension (Fig. 1(B)). Higher magnification SEM image (Fig. 1(C)) revealed the ovalshaped bacterium growth, in the presence of naphthalene, invading uniformly the entire hydrogel surface. Bacterial adhesion and growth was also shown, in presence of anthracene, onto the hydrogel surface by forming heaps of cells (Fig. 1(D)). PVP hydrogel matrix has a high adsorption capacity [23]. The immobilization rate calculated as the ratio of the total number of immobilized cells to the total surface of the hydrogel discs was 1.6 · 105 cell/mm2.
3. Results and discussion 3.3. Application to PAH biodegradation 3.1. Characterisation of the PVP matrix PVP is soluble in water, once crosslinked by irradiation it acquires a permanent three-dimensional network of polymer chains, which render it insoluble gel. The measured values of the gel fraction and the equilibrium swelling rate of the hydrogel were: 86% and 2293%, respectively. These results
3.3.1. PAH degradation by free cells The strain R. erythropolis B4 grows with naphthalene or anthracene as a sole source of carbon and energy. The ability to degrade naphthalene and anthracene by the free suspended bacteria was observed as shown in Figs. 2 and 3. Maximal naphthalene degradation (99.46%) was reached
Fig. 1. SEM microphotographs of radiation cross-linked poly(vinylpyrrolidone) matrix prior to inoculation (A), after 5 days of R. erythropolis growth in naphthalene (B, C) and in anthracene (D).
A. Djefal-Kerrar et al. / Nucl. Instr. and Meth. in Phys. Res. B 265 (2007) 370–374
after an incubation time of 5 days with a naphthalene concentration of 1 g/l, while it was of 82.02% with a naphthalene concentration of 1.5 g/l. Maximal anthracene degradation (87%) was reached after incubation of 11 days with an anthracene concentration of 0.7 g/l while it was of 69% with an anthracene concentration of 1 g/l. The strain degradation ability seems to be more efficient on naphthalene than on anthracene. For both hydrocarbons the degradation efficiency is concentration dependent. The lower is the PAH concentration, the higher is the degradation rate.
Naphtalene degradation (%)
100
80
60
373
1 g/l 1.5 g/l
40
20
0 0
2
4
6
8
10
12
Time (days) Fig. 2. Naphthalene degradation by free Rhodococcus erythropolis B4.
Anthracene degradation (%)
100
80
60
40
0.7 g/l 1 g/l
20
0 0
2
4
6
8
10
12
Time (days) Fig. 3. Anthracene degradation by free Rhodococcus erythropolis B4.
3.3.2. PAH degradation by immobilized R. erythropolis B4 Through our results, we have put in evidence that R. erythropolis B4 has been fixed onto the PVP matrix. This was verified by its positive growth further to many rinsing of the matrix. The bioadhesion to the matrix was realized and bacteria fixed to the support remained well active. The matrix does not affect the microbial activity, no loss of activity was observed and the so fixed bacteria were able to reactivate even after a period of storage [24]. Furthermore, PVP hydrogel seems to be biocompatible with biological systems [23]. We observed a high capability of the reused immobilized strain to degrade PAHs. The degradation rates as a function of the PAH concentration and culture time are given in Tables 1 and 2. We can notice from the results of the Tables 1 and 2, that the reused immobilized cells have a better capability to degrade PAHs. The degradation rate obtained with reused cells after 2 days is higher than that obtained even after 10 days with the first use. This has been reported by several studies [25–27]. This was most likely due to the increase availability of the substrates for the cells and a better interaction between the substrates and the immobilized cells [26,28]. The prolonged acclimation and proliferation at the expense of the chemicals of concern, also improves degradative activity of microorganisms towards the contaminants [27]. The maximum degradation is observed at the lowest concentration for both PAHs.
Table 1 Naphthalene degradation rates (%) by immobilized R. erythropolis B4 Naphthalene 0.1%
Naphthalene 0.15%
Fixed cells (5 days)
Fixed cells (10 days)
Reused fixed cells (2 days)
Fixed cells (5 days)
Fixed cells (10 days)
Reused fixed cells (2 days)
58.67
70.97
82.23
42.97
53.4
67.23
Table 2 Anthracene degradation rates (%) by immobilized R. erythropolis B4 Anthracene 0.07%
Anthracene 0.1%
Fixed cells (5 days)
Fixed cells (10 days)
Reused fixed cells (2 days)
Fixed cells (5 days)
Fixed cells (10 days)
Reused fixed cells (2 days)
54.67
76.65
84.77
38.43
69.7
77.72
374
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Further investigation is required to evaluate the degradation performance of immobilized cells for many times repeated use in a bioreactor system. 4. Conclusion In this work the use of a pure strain of R. erythropolis B4 immobilized on PVP matrix crosslinked by gamma radiation was investigated for an application to the degradation of polycyclic aromatic hydrocarbons such as naphthalene and anthracene. The cells have been successfully immobilized onto the hydrogel discs, which appears to not affect microbial activity. The immobilized strain causes high degradation rates of naphthalene and anthracene. The higher degradation rates obtained with reused fixed bacteria reflect the capability of the PVP matrix to retain immobilized cells and to form a stable system that can be used for several degradation processes. Acknowledgements The authors thank Ms Belaroussi from the Centre of Development of Advanced Technologies, Algeria for her kind assistance in taking SEM microphotographs. References [1] E.J. Baum, in: H.V. Gelboin, P.O.P. Ts’o (Eds.), Polycyclic Hydrocarbons and Cancer, Environment Chemistry and Metabolism, Vol. 1, Academic Press, New York, San Francisco, London, 1978, pp. 45–46. [2] M.J. Larkin, C.C.R. Allen, L.A. Kulakov, D.A. Lipscomb, J. Bacteriol. 181 (1999) 6200. [3] D. Dean-Ross, J.D. Moody, J.P. Freeman, D.R. Doerge, C.E. Cerniglia, FEMS Microbiol. Lett. 204 (2001) 205. [4] C.E. Cerniglia, Adv. Appl. Microbiol. 30 (1984) 31. [5] C.E. Cerniglia, Biodegradation 3 (1992) 351. [6] C.E. Cerniglia, M.A. Heitkamp, in: U. Varanasi (Ed.), Metabolism of Polycyclic Aromatic Hydrocarbons in the Aquatic Environment, CRC Press, Inc., Boca Raton, FL, 1989, pp. 41–68. [7] C.C.C.R. Carvalho, M.M.R. da Fonseca, Appl. Microbiol. Biotechnol. 67 (2005) 715.
[8] K.L. Shuttleworth, C.E. Cerniglia, Appl. Biochem. Biotechnol. 54 (1995) 291. [9] P. Wattiau, in: S.N. Agathos, W. Reineke (Eds.), Biotechnology for the Environment: Strategy and Fundamentals, Kluwer Academic Publishers., Netherlands, 2002, pp. 69–89. [10] M.J. Larkin, L.A. Kulakov, C.C.R. Allen, Environ. Biotechnol. 16 (2005) 282. [11] A. Abd El-hady, H.A. Abd El-Rehim, J. Biomed. Biotechnol. 4 (2004) 219. [12] S.F. Karel, B. Libik, C.R. Roberstson, Chem. Eng. Sci. 40 (1985) 1321. [13] M.S. Kuyukina, I.B. Ivshina, A. Yu Gavrin, E.A. Podorozhko, V.I. Lozinsky, C.E. Jeffree, J.C. Philp, J. Microbiol. Meth. 65 (2005) 596. [14] C.F. Degiorgi, R.A. Pizarro, E.E. Smolko, S. Lora, M. Carenza, Radiat. Phys. Chem. 63 (2002) 109. [15] J.M. Rosiak, I. Janik, S. Kadlubowski, M. Kozicki, Nucl. Instr. and Meth. B 208 (2003) 325. [16] M. Bouchez, D. Blanchet, J.P. Vandecasteele, Appl. Microbiol. Biotechnol. 43 (1995) 156. [17] S. Benamer, M. Mahlous, A. Boukrif, B. Mansouri, S. Larbi Youcef, Nucl. Instr. and Meth. B 248 (2006) 284. [18] E. Grund, B. Denecke, R. Eichenlaub, Appl. Environ. Microbiol. 58 (1992) 1874. [19] B. Boldrin, A. Tiehm, C. Fritzsche, Appl. Environ. Microbiol. 59 (1993) 1927. [20] S. Meyer, R. Moser, A. Neef, U. Stahl, P. Kampfer, Microbiology 145 (1999) 1731. [21] M. Bouchez, D. Blanchet, B. Besnainou, J.-Y. Leveau, J.-P. Vandecasteele, J. Appl. Microbiol. 82 (1997) 310. [22] K. Malatova, A thesis submitted of the requirement for the Degree of Master of Science in Chemistry, Department of Chemistry Rochester. Institute of Technology Rochester, 2005, p. 108. [23] H. Kaplan Can, B.K. Denizli, S. Kavlak, A. Guner, Radiat. Phys. Chem. 72 (2005) 483. [24] Y. Kourkoutas, M. Kanellaki, A.A. Koutinas, C. Tzia, J. Food Eng. 4 (2006) 217. [25] S. Manohar, C.K. Kim, T.K. Karegoudar, Appl. Microbiol. Biot. 55 (2001) 311. [26] M.P. Diaz, K.G. Boyd, S.J.W. Grigson, J.G. Burgess, Biotechnol. Bioeng. 79 (2002) 145. [27] A. Kermanshahi pour, D. Karamanev, A. Margaritis, Water Res. 39 (2005) 3704. [28] K. Na, Y. Lee, W. Lee, Y. Huh, J. Lee, M. Kubo, S. Chung, J. Biosci. Bioeng. 90 (2000) 368.
NIM B Beam Interactions with Materials & Atoms
Nuclear Instruments and Methods in Physics Research B 265 (2007) 370–374 www.elsevier.com/locate/nimb
Immobilization of Rhodococcus erythropolis B4 on radiation crosslinked poly(vinylpyrrolidone) hydrogel: Application to the degradation of polycyclic aromatic hydrocarbons A. Djefal-Kerrar a
a,*
, S. Gais a, K. Ouallouche a, A. Nacer Khodja a, M. Mahlous a, H. Hace`ne b
Division of Nuclear Applications, Nuclear Research Centre of Algiers, 2 Bd Frantz Fanon BP-399 Alger-gare, Algiers, Algeria b Biological Sciences Institute, Science and Technology University Houari Boumediene, Algiers, Algeria Available online 8 September 2007
Abstract A poly(vinylpyrrolidone) (PVP) hydrogel crosslinked by gamma radiation was used to immobilize, by adsorption, Rhodococcus erythropolis B4 strain. Immobilized cells were tested for their capacity to degrade naphthalene and anthracene, under aerobic conditions. The results showed that, the strain fixed is capable of growing in the presence of naphthalene or anthracene as a unique source of carbon. It was also shown that, the fixed strain can be preserved by freeze-drying for further use. The biodegradation capacity was improved during the second use. 2007 Elsevier B.V. All rights reserved. PACS: 61.82.Pv; 87.68+z; 89.20.Bb; 92.20.jb Keywords: Cell immobilization; Poly(vinylpyrrolidone); Radiation crosslinking; Anthracene; Naphthalene; Biodegradation
1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental pollutants. They are generated from petroleum and many pyrolysis processes. They cause concern because of their potentially deleterious effects on human health and many can be recalcitrant in the environment [1]. Among them, naphthalene, a bicycle PAH, is released into the environment as coal tar and coal tar products [2]. Whereas anthracene, a tricycle PAH, is found in high amounts in PAH contaminated sediments, surface soils and waste sites. This hydrophobic contaminant is toxic to aquatic life [3]. Various existing clean-up technologies for hydrocarbon treatment can be categorised in three general schemes: chemical, physical and biological. The degradation by *
Corresponding author. Tel.: +213 21 43 44 44; fax: +213 21 43 42 80. E-mail address: [email protected] (A. Djefal-Kerrar).
0168-583X/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2007.09.005
microorganisms is the subject of many works [4–7]. A wide range of different bacteria is able to completely assimilate a defined range of compound, or exhibit just partial metabolism [8]. Therefore, the use of pure cultures of microorganisms, specially adapted to metabolize the contaminant, is envisaged as an attractive alternative. Rhodococcus erythropolis is well known microorganism containing a large set of enzymes that allows carrying out an enormous number of bioconversion and degradation [9,10]. Feasibility and performance of recently developed immobilized bacteria on inert support for biodegradation of recalcitrant compounds has attracted considerable and increasing interest since this strategy allows obtaining much more profit from this process. The main advantages in the use of immobilized cells in comparison with suspended ones include the retention in the reactor of higher concentration of microorganisms, easy removal of bacteria after use from the reaction mixture, providing
A. Djefal-Kerrar et al. / Nucl. Instr. and Meth. in Phys. Res. B 265 (2007) 370–374
the ability to control reaction time, reuse of cells for many reaction cycles, lowering the total production cost of cells-mediated reactions, provide pure products [11–13]. Microbial cells can be immobilized on various polymeric entrapment matrices. The most promising materials and preparation technique for such a purpose are hydrogels and radiation crosslinking, respectively [11,14]. Among hydrophilic polymers, poly(vinylpyrrolidone) PVP, poly(vinyl alcohol) PVA, poly(hydroxyethylmetacrylate) PHEMA, poly(ethylene oxide) PEO and others, are the most often used for hydrogel’s synthesis [15]. In this work PVP was used for the preparation of the immobilization matrix. The objectives of the study are, on one hand, to investigate the immobilization of R. erythropolis B4 pure strain on the PVP matrix crosslinked by gamma radiation, and on the other hand, to evaluate the ability of immobilized cells to degrade PAHs, such as naphthalene and anthracene. 2. Materials and methods 2.1. Chemicals Anthracene and naphtalene were obtained from SigmaAldrich. Poly(vinylpyrrolidone) K90 with an average molecular weight of 360,000 Da was purchased from Fluka-Chemica. Solvents were of analytical grade.
371
The poly(vinylpyrrolidone) (PVP) at a concentration of 7% w/v was dissolved in distilled water at 70 C overnight, and poured into plastic syringes, sealed with parafilm then irradiated by c rays at a dose of 25 kGy. Gel fraction was determined according to the formula: Gf ¼ W dg =W ip 100%;
ð1Þ
where Wdg is the weight of the dry gel and Wip is the initial weight of the polymer. The equilibrium swelling ratio of the PVP hydrogel in water was measured gravimetrically as the ratio of the swollen gel weight at the equilibrium to that of the dry gel. Cross-linked gels were cut into discs of about 0.5 cm thickness and 1.2 cm diameter and maintained in sterile conditions until their use for cell immobilization. 2.5. Immobilization of the cells Hydrogel discs were dropped into 50 ml of sterile vitamin-supplemented MSM, and then 2 ml of the suspension cell containing 108 CFU (Colony Forming Unit)/ml were added. In order to allow a full contact of the hydrogel discs with suspended cells without sticking each to other, a maximum of 30 discs were used. The mixture was incubated at 30 C under shaking. After 24 h, the discs were removed from the culture medium, and then washed with distilled water to remove non adherent cells. The immobilized cells are ready to be tested for their PAHs degradation ability.
2.2. Microorganisms 2.6. Scanning electron microscopy R. erythropolis B4 strain was obtained from the microbiology laboratory collection, Science & Technology University of Algiers. It was maintained on nutrient agar (Difco) slants at 4 C. 2.3. Culture conditions First, bacterial cells were reactivated in Tryptic Soy broth at 30 C. For their adaptation to the PAH, the cells were pre-grown in vitamin-supplemented mineral salt medium (MSM) containing per liter of distilled water: Na2HPO4, 4.5 g; NH4NO3, 1 g; KH2PO4, 0.68 g; MgSO4 7H2O, 0.1 g; Fe2SO4 7H2O, 0.001 g, trace elements and vitamin solution [16], and two different carbon sources: glucose and naphthalene (1/3:2/3) or glucose and anthracene (1/3:2/3). After an incubation of about 72 h at 30 C in an orbital shaker incubator at 120 rpm, the cells were harvested by centrifugation (3000 rpm, 20 min) and then resuspended in distilled water. 2.4. Hydrogel preparation and characterisation Polymeric hydrogel was synthesised by gamma radiation induced crosslinking and sterilisation, according to the method reported previously [17].
R. erythropolis B4 cells immobilized on radiation crosslinked and sterilised poly(vinylpyrrolidone) hydrogel matrix were fixed with 2.5% (v/v) glutaraldehyde in saline solution for 1 h, rinsed in water and then dehydrated by sequential immersion in increasing concentration of ethanol (10%, 50%, 80%, 100%) for 10 min each. Subsequently the specimens were freeze dried and then coated with gold to be finally observed with a JEOL JSM 6360 LV scanning electron microscope at an acceleration voltage of 10 kV. 2.7. Biodegradation test The capacity of the bacterial cells to degrade PAH was tested by inoculating 200 ml of MSM containing anthracene or naphthalene as a sole carbon source in N,Ndimethylformamide and ethanol, respectively, with either, 2 ml of the free cell suspension, or with 30 hydrogel discs containing fixed cells. The PAH concentrations were 0.7 and 1 g/l for anthracene and, 1 and 1.5 g/l for naphthalene [18–22]. Incubation was made at 30 C under continuous shaking. Periodically, samples were taken aseptically from the culture flask for HPLC analysis.
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2.8. Reusability of the immobilized cells After the first test, the discs with immobilized bacteria were freeze dried and stored at room temperature. After 15 days of storage, the discs were reused to test the PAHs degradation ability of immobilized cells, in the same conditions as in the first test. 2.9. High performance liquid chromatography (HPLC) analysis Identification and quantification of remaining naphthalene and anthracene in the culture media were performed by HPLC (Waters 2487) with UV detection at a wavelength of 254 nm. Remaining PAH, were extracted from the culture probe after centrifugation (3000 rpm, 20 min), with an equal weighted volume of cyclohexane for 2 h at 160 rpm. An aliquot (20 ll) was analysed using a thermo hypersil BDS-C18 column (375 · 125 mm) and elution flow rate 1.5 ml/min (water/acetonitril: 25/75 v/v).
mean that the irradiation dose delivered to sterilise the hydrogel, at the same time produced a high degree of crosslinking, which render 86% of it insoluble in water. The swelling property of the hydrogel will assure a maximum contact of the fixed bacteria with the PAH contained in water. 3.2. Cell immobilization on PVP matrix As shown in SEM microphotographs, the surface of the hydrogel matrix unexposed to bacterial suspension (Fig. 1(A)) was covered by Rhodococcal cells after its immersion into the bacterial suspension (Fig. 1(B)). Higher magnification SEM image (Fig. 1(C)) revealed the ovalshaped bacterium growth, in the presence of naphthalene, invading uniformly the entire hydrogel surface. Bacterial adhesion and growth was also shown, in presence of anthracene, onto the hydrogel surface by forming heaps of cells (Fig. 1(D)). PVP hydrogel matrix has a high adsorption capacity [23]. The immobilization rate calculated as the ratio of the total number of immobilized cells to the total surface of the hydrogel discs was 1.6 · 105 cell/mm2.
3. Results and discussion 3.3. Application to PAH biodegradation 3.1. Characterisation of the PVP matrix PVP is soluble in water, once crosslinked by irradiation it acquires a permanent three-dimensional network of polymer chains, which render it insoluble gel. The measured values of the gel fraction and the equilibrium swelling rate of the hydrogel were: 86% and 2293%, respectively. These results
3.3.1. PAH degradation by free cells The strain R. erythropolis B4 grows with naphthalene or anthracene as a sole source of carbon and energy. The ability to degrade naphthalene and anthracene by the free suspended bacteria was observed as shown in Figs. 2 and 3. Maximal naphthalene degradation (99.46%) was reached
Fig. 1. SEM microphotographs of radiation cross-linked poly(vinylpyrrolidone) matrix prior to inoculation (A), after 5 days of R. erythropolis growth in naphthalene (B, C) and in anthracene (D).
A. Djefal-Kerrar et al. / Nucl. Instr. and Meth. in Phys. Res. B 265 (2007) 370–374
after an incubation time of 5 days with a naphthalene concentration of 1 g/l, while it was of 82.02% with a naphthalene concentration of 1.5 g/l. Maximal anthracene degradation (87%) was reached after incubation of 11 days with an anthracene concentration of 0.7 g/l while it was of 69% with an anthracene concentration of 1 g/l. The strain degradation ability seems to be more efficient on naphthalene than on anthracene. For both hydrocarbons the degradation efficiency is concentration dependent. The lower is the PAH concentration, the higher is the degradation rate.
Naphtalene degradation (%)
100
80
60
373
1 g/l 1.5 g/l
40
20
0 0
2
4
6
8
10
12
Time (days) Fig. 2. Naphthalene degradation by free Rhodococcus erythropolis B4.
Anthracene degradation (%)
100
80
60
40
0.7 g/l 1 g/l
20
0 0
2
4
6
8
10
12
Time (days) Fig. 3. Anthracene degradation by free Rhodococcus erythropolis B4.
3.3.2. PAH degradation by immobilized R. erythropolis B4 Through our results, we have put in evidence that R. erythropolis B4 has been fixed onto the PVP matrix. This was verified by its positive growth further to many rinsing of the matrix. The bioadhesion to the matrix was realized and bacteria fixed to the support remained well active. The matrix does not affect the microbial activity, no loss of activity was observed and the so fixed bacteria were able to reactivate even after a period of storage [24]. Furthermore, PVP hydrogel seems to be biocompatible with biological systems [23]. We observed a high capability of the reused immobilized strain to degrade PAHs. The degradation rates as a function of the PAH concentration and culture time are given in Tables 1 and 2. We can notice from the results of the Tables 1 and 2, that the reused immobilized cells have a better capability to degrade PAHs. The degradation rate obtained with reused cells after 2 days is higher than that obtained even after 10 days with the first use. This has been reported by several studies [25–27]. This was most likely due to the increase availability of the substrates for the cells and a better interaction between the substrates and the immobilized cells [26,28]. The prolonged acclimation and proliferation at the expense of the chemicals of concern, also improves degradative activity of microorganisms towards the contaminants [27]. The maximum degradation is observed at the lowest concentration for both PAHs.
Table 1 Naphthalene degradation rates (%) by immobilized R. erythropolis B4 Naphthalene 0.1%
Naphthalene 0.15%
Fixed cells (5 days)
Fixed cells (10 days)
Reused fixed cells (2 days)
Fixed cells (5 days)
Fixed cells (10 days)
Reused fixed cells (2 days)
58.67
70.97
82.23
42.97
53.4
67.23
Table 2 Anthracene degradation rates (%) by immobilized R. erythropolis B4 Anthracene 0.07%
Anthracene 0.1%
Fixed cells (5 days)
Fixed cells (10 days)
Reused fixed cells (2 days)
Fixed cells (5 days)
Fixed cells (10 days)
Reused fixed cells (2 days)
54.67
76.65
84.77
38.43
69.7
77.72
374
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Further investigation is required to evaluate the degradation performance of immobilized cells for many times repeated use in a bioreactor system. 4. Conclusion In this work the use of a pure strain of R. erythropolis B4 immobilized on PVP matrix crosslinked by gamma radiation was investigated for an application to the degradation of polycyclic aromatic hydrocarbons such as naphthalene and anthracene. The cells have been successfully immobilized onto the hydrogel discs, which appears to not affect microbial activity. The immobilized strain causes high degradation rates of naphthalene and anthracene. The higher degradation rates obtained with reused fixed bacteria reflect the capability of the PVP matrix to retain immobilized cells and to form a stable system that can be used for several degradation processes. Acknowledgements The authors thank Ms Belaroussi from the Centre of Development of Advanced Technologies, Algeria for her kind assistance in taking SEM microphotographs. References [1] E.J. Baum, in: H.V. Gelboin, P.O.P. Ts’o (Eds.), Polycyclic Hydrocarbons and Cancer, Environment Chemistry and Metabolism, Vol. 1, Academic Press, New York, San Francisco, London, 1978, pp. 45–46. [2] M.J. Larkin, C.C.R. Allen, L.A. Kulakov, D.A. Lipscomb, J. Bacteriol. 181 (1999) 6200. [3] D. Dean-Ross, J.D. Moody, J.P. Freeman, D.R. Doerge, C.E. Cerniglia, FEMS Microbiol. Lett. 204 (2001) 205. [4] C.E. Cerniglia, Adv. Appl. Microbiol. 30 (1984) 31. [5] C.E. Cerniglia, Biodegradation 3 (1992) 351. [6] C.E. Cerniglia, M.A. Heitkamp, in: U. Varanasi (Ed.), Metabolism of Polycyclic Aromatic Hydrocarbons in the Aquatic Environment, CRC Press, Inc., Boca Raton, FL, 1989, pp. 41–68. [7] C.C.C.R. Carvalho, M.M.R. da Fonseca, Appl. Microbiol. Biotechnol. 67 (2005) 715.
[8] K.L. Shuttleworth, C.E. Cerniglia, Appl. Biochem. Biotechnol. 54 (1995) 291. [9] P. Wattiau, in: S.N. Agathos, W. Reineke (Eds.), Biotechnology for the Environment: Strategy and Fundamentals, Kluwer Academic Publishers., Netherlands, 2002, pp. 69–89. [10] M.J. Larkin, L.A. Kulakov, C.C.R. Allen, Environ. Biotechnol. 16 (2005) 282. [11] A. Abd El-hady, H.A. Abd El-Rehim, J. Biomed. Biotechnol. 4 (2004) 219. [12] S.F. Karel, B. Libik, C.R. Roberstson, Chem. Eng. Sci. 40 (1985) 1321. [13] M.S. Kuyukina, I.B. Ivshina, A. Yu Gavrin, E.A. Podorozhko, V.I. Lozinsky, C.E. Jeffree, J.C. Philp, J. Microbiol. Meth. 65 (2005) 596. [14] C.F. Degiorgi, R.A. Pizarro, E.E. Smolko, S. Lora, M. Carenza, Radiat. Phys. Chem. 63 (2002) 109. [15] J.M. Rosiak, I. Janik, S. Kadlubowski, M. Kozicki, Nucl. Instr. and Meth. B 208 (2003) 325. [16] M. Bouchez, D. Blanchet, J.P. Vandecasteele, Appl. Microbiol. Biotechnol. 43 (1995) 156. [17] S. Benamer, M. Mahlous, A. Boukrif, B. Mansouri, S. Larbi Youcef, Nucl. Instr. and Meth. B 248 (2006) 284. [18] E. Grund, B. Denecke, R. Eichenlaub, Appl. Environ. Microbiol. 58 (1992) 1874. [19] B. Boldrin, A. Tiehm, C. Fritzsche, Appl. Environ. Microbiol. 59 (1993) 1927. [20] S. Meyer, R. Moser, A. Neef, U. Stahl, P. Kampfer, Microbiology 145 (1999) 1731. [21] M. Bouchez, D. Blanchet, B. Besnainou, J.-Y. Leveau, J.-P. Vandecasteele, J. Appl. Microbiol. 82 (1997) 310. [22] K. Malatova, A thesis submitted of the requirement for the Degree of Master of Science in Chemistry, Department of Chemistry Rochester. Institute of Technology Rochester, 2005, p. 108. [23] H. Kaplan Can, B.K. Denizli, S. Kavlak, A. Guner, Radiat. Phys. Chem. 72 (2005) 483. [24] Y. Kourkoutas, M. Kanellaki, A.A. Koutinas, C. Tzia, J. Food Eng. 4 (2006) 217. [25] S. Manohar, C.K. Kim, T.K. Karegoudar, Appl. Microbiol. Biot. 55 (2001) 311. [26] M.P. Diaz, K.G. Boyd, S.J.W. Grigson, J.G. Burgess, Biotechnol. Bioeng. 79 (2002) 145. [27] A. Kermanshahi pour, D. Karamanev, A. Margaritis, Water Res. 39 (2005) 3704. [28] K. Na, Y. Lee, W. Lee, Y. Huh, J. Lee, M. Kubo, S. Chung, J. Biosci. Bioeng. 90 (2000) 368.