Appl Microbiol Biotechnol (2001) 55:311–316 DOI 10.1007/s002530000488

O R I G I N A L PA P E R

S. Manohar · C.K. Kim · T.B. Karegoudar

Enhanced degradation of naphthalene by immobilization of Pseudomonas sp. strain NGK1 in polyurethane foam

Received: 25 April 2000 / Received revision: 8 August 2000 / Accepted: 13 August 2000 / Published online: 13 March 2001 © Springer-Verlag 2001

Abstract A Pseudomonas sp. strain NGK1 (NCIM 5120) capable of degrading naphthalene was immobilized in polyurethane foam. The naphthalene-degrading activity of the freely suspended cells was compared with that of immobilized cells in batches in shaken culture and in a continuous culture system in a packed-bed reactor. Increasing concentrations of naphthalene were better tolerated and more quickly degraded by immobilized cell cultures than by free cells. An initial naphthalene concentration of 25 mM was completely degraded by freely suspended cells (4×1010 cfu ml–1) and polyurethane-foamimmobilized cells (0.8–1×1012 cfu g–1 foam cubes) after 4 days and 2 days of incubation, respectively. Free cells degraded a maximum of 30 mM naphthalene after 4 days of incubation with 50 mM naphthalene, and no further degradation was observed even after 15 days of incubation, whereas foam-immobilized cells brought about the complete degradation of 50 mM initial naphthalene after 6 days of incubation. Furthermore, with 25 mM naphthalene, the polyurethane-foam-immobilized cells were reused 45 times over a period of 90 days without losing naphthalene-degrading activity. By contrast, with the same amount of naphthalene, alginate-, agar-, and polyacrylamide-entrapped cells could be reused for 18, 12, and 23 times over a period of 44, 28, and 50 days, respectively. During continuous degradation in a packedbed reactor, foam-immobilized cells degraded 80 mM naphthalene at a rate of 150 ml–1 h–1. With the same flow rate and 40 mM naphthalene, this system operated efficiently and continuously for about 120 days, whereas the packed-bed reactor with alginate-, agar-, and polyacrylamide-entrapped cells could be operated only for 45, 40, S. Manohar · T.B. Karegoudar (✉) Department of Biochemistry, Gulbarga University, Gulbarga-585 106, India e-mail: [email protected] Tel.: +91-8472-45781, Fax: +91-8472-45632 C.K. Kim Department of Microbiology and Research Institute of Genetic Engineering, Chungbuk, National University, Cheongju, 361-763, Korea

and 60 days respectively. Thus, more efficient degradation of naphthalene could be achieved by immobilizing cells of Pseudomonas sp. strain NGK1 in polyurethane foam, rather than in the other matrices tested.

Introduction Polycyclic aromatic hydrocarbons (PAHs) are a class of compounds regarded as ubiquitous pollutants. Many PAHs exhibit carcinogenic, teratogenic or mutagenic properties (Gundlach et al. 1983; Vandermeulen 1981). Naphthalene is a PAH that is frequently involved in contamination of the environment by transport, accidental discharge, the disposal of petroleum products or through industrial effluents. Naphthalene is considered to be a primary irritant and the US-Environmental Protection Agency (EPA) has classified it as a “priority toxic pollutant” (US-EPA 1980, 1986). Due to its potential toxicity, the behavior and fate of naphthalene in the environment is of great concern for public health monitoring and environmental toxicology (Anderson et al. 1974; Strubble and Hormon 1983; Darville and Wilhm 1984). Furthermore, naphthalene, which is the simplest homologue in the polycyclic series, has received considerable interest as a model compound for understanding the microbial metabolic pathway of more complex PAHs. The biodegradation of naphthalene by free microbial cells has been extensively studied (Gibson and Subramanian 1984; Grund et al. 1992; Eaton and Chapman 1992; Atlas and Cerniglia 1995; Ashok and Saxena 1995; Manohar and Karegoudar 1995). To avoid the problems of handling and cell separation, which are associated with the use of free bacteria, cell immobilization is one of the most attractive alternatives. The main advantages of immobilized whole microorganisms are their higher operational stability, high cell density and their use in continuous reactors (Hulst et al. 1985). Various matrices, such as k-carrageenan, alginate, agar, polyacrylamide-hydrazide and polyurethane foam, have been successfully used for immobilization of microorganisms

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(Chibata 1978; Cheetham 1980; Mattiasson 1983; Hall and Rao 1989). Immobilized cells have been used extensively in the production of useful chemicals, the degradation of wastewaters (Chibata et al. 1986; Bisping and Rehm 1988; Hall and Rao 1989),and the bioremediation of numerous toxic chemicals including phenol (Bettmann and Rehm 1984, 1985), 4-chlorobenzoate (Sahasrabudhe et al. 1988), 4-chlorophenol (Balfanz and Rehm 1991), pyridine (Lee et al. 1994), and 2,4-dichlorophenoxyacetic acid (Kochar and Kahlon 1995). Recently, we demonstrated the degradation of naphthalene (Manohar and Karegoudar 1998) by cells of Pseudomonas sp. strain NGK1 immobilized in alginate, agar, and polyacrylamide. In the present study, Pseudomonas sp. strain NGK1 immobilized in polyurethane foam was compared with freely suspended cells and the same cells entrapped in alginate, agar, polyacrylamide with regard to naphthalenedegrading capacity at various initial naphthalene concentrations. The longevity and operational stability of naphthalene-degrading capacity by immobilized cells in semicontinuous and continuous culture systems were also studied and compared. Furthermore, the efficiency of polyurethane-foam-immobilized cells over alginate-, agar-, and polyacrylamide-entrapped cells is reported and discussed.

Immobilization Pseudomonas sp. strain NGK1 (NCIM 5120) was grown in precultivation medium. The cells were harvested during the exponential growth phase by centrifugation at 5,000 g for 10 min at 15 °C and then immobilized in either alginate, agar, polyacrylamide or polyurethane foam. Alginate entrapment of cells The alginate entrapment of cells was carried out according to the method of Bettmann and Rehm (1984). A 50-ml bacterial cell suspension (15 g wet weight/50 ml sterilized alginate solution) was added to 200 ml sterilized alginate solution (4% w/v) and mixed by stirring on a magnetic stirrer. This alginate/cell mixture was extruded drop by drop into a cold, sterile 0.2 M CaCl2 solution. Gel beads of 2 mm diameter were obtained. The beads were hardened by resuspending into a fresh CaCl2 solution for 2 h with gentle agitation. Agar entrapment of cells The procedure was carried out according to the method described by Nilson et al. (1983). The bacterial cell suspension (15 g wet weight/50 ml saline) was added to 200 ml sterilized agar solution (agar 4% w/v in physiological saline) at 50 °C and mixed by stirring on a magnetic stirrer. This agar/cell mixture was poured into vigorously stirring, prewarmed paraffin oil (40 °C, 1,000 ml oil/50 ml agar/cell mixture). This mixture was allowed to cool by itself, while stirring continued, and the emulsion slowly solidified. The cell-entrapped agar beads of 1–2 mm thus obtained were washed with sterilized saline and distilled water.

Materials and methods Polyacrylamide entrapment of cells Chemicals Naphthalene was purchased from SD Fine Chemicals, India. Polyurethane foam was obtained from a local supplier, Mukesh Foam Products, Bangalore, India. The foam was elastic and its low density (16 kg m3) provided a large surface area. All other chemicals used in this study were of analytical grade.

The entrapment procedure was carried out according to the methods of Chibata et al. (1978) and Starostina et al. (1987). Bacterial cells (15 g wet weight in saline) were preincubated with MgSO4 to a final concentration of 1 M. The cell suspension (50 ml) was added into 200 ml acrylamide/bis-acrylamide solution and mixed by stirring on a magnetic stirrer. Heat evolved during polymerization was eliminated using an ice bath. The cell-entrapped polymer sheet was cut and ground to give fine granules 1 mm in diameter.

Bacterial strain and culture media The naphthalene-degrading Pseudomonas sp. strain NGK1 used in this study was isolated by enrichment culture technique on naphthalene (Manohar and Karegoudar 1995). This culture is deposited in the National Collection of Industrial Microorganisms (NCIM), National Chemical Laboratory, Pune, India, under accession number NCIM 5120. The medium used for pre-cultivation of the bacterium contained (g l–1) K2HPO4 0.38, Mg SO4·7H2O 0.2, NH4Cl 1.0, FeCl3 0.05, yeast extract 1.0, and sodium salicylate 0.25. The pH of the medium was adjusted to 7.0. One hundred ml aliquots of this medium were transferred into 250-ml conical flasks and sterilized by autoclaving at 121 °C for 15 min. The sterilized medium was then supplemented with naphthalene (0.1% w/v). For naphthalene degradation studies, the mineral salts medium contained (g l–1) K2HPO4 0.38, MgSO4·7H2O 0.2, NH4Cl 1.0, and FeCl3 0.05. The pH of the medium was adjusted to 7 and sterilized by autoclaving. Naphthalene at various concentrations (20–80 mM) was dissolved in 3.8 ml of N, N′-dimethylformamide (DMF) and transferred into mineral salts medium containing Tween-80 (approximately 80× critical micelle concentration). This medium was subjected to ultrasonication prior to use.

Polyurethane foam immobilization of cells Cells were immobilized in polyurethane foam cubes according to the method of Hall and Rao (1989). The polyurethane foam was cut into approximately 5 mm cubes, washed twice with distilled water and dried. The freshly grown bacterial cell suspension (100 ml, 8×1010 cfu ml–1) was added to a 500-ml conical flask containing sterilized foam cubes (4 g). The contents of the flask were mixed by stirring on a magnetic stirrer for 2 h. The flask was kept on a rotary shaker for 1 h at 140 rpm and left undisturbed for another 2 h. The medium was removed and foam cubes containing the immobilized bacteria were washed with saline. The decanted bacterial suspension and the saline wash were combined and the bacterial population in the mixture was counted by the spreadplate method. Fermentation conditions Batch fermentations Batch fermentations for the degradation of naphthalene were performed for both freely suspended cells and cells immobilized in polyurethane foam cubes. For freely suspended cell cultures, exponentially growing cells were added to 250-ml conical flasks

313 containing 100 ml mineral salts medium. The cell concentration was adjusted (4×1010 cfu ml–1) and various amounts of naphthalene (25, 50, and 75 mM) were added. The degradation process was carried out at 30 °C on a rotary shaker at 150 rpm for the desired incubation period. Samples of the culture broth were taken at the indicated times for the analysis of naphthalene. For immobilized cells, 4 g of polyurethane-foam-immobilized bacteria were added to a 250-ml conical flask containing 100 ml mineral salts medium with various amounts of naphthalene as indicated. The initial cell concentration was in the range of 0.8×1012–1×1012 cfu g–1 foam cubes. The degradation of naphthalene was also carried out using alginate-, agar-, and polyacrylamide-entrapped cells (Manohar and Karegoudar 1998). Like the freely suspended cell culture, the immobilized cultures were incubated at 30 °C on the rotary shaker under identical fermentation conditions. The control experiments for the evaporation of naphthalene were performed in sterile medium at the previously indicated naphthalene loadings. The evaporation of naphthalene from the sterile control was found to be about 0.15 mM day–1, which was negligible and thus not used to correct the naphthalene degradation values. Repeated batch fermentation Repeated batch fermentations were carried out in order to establish the long-term stability and operational stability of naphthalene degradation by polyurethane-foam-immobilized cells. After every 2-day incubation period, the used medium was decanted and fresh medium containing naphthalene (25 and 50 mM) was added to the flasks. The degradation process was carried out under identical fermentation conditions. Continuous fermentations The continuous fermentations were carried out in a packed-bed reactor. A cylindrical glass column (4×50 cm, volume 650 ml) with an outlet facility every 5 cm was used as the reactor. The bottom of the column was packed with a circular foam pad (diameter 4 cm) followed by a porous glass-frit. The reactor was packed with 60 g of foam cubes, containing immobilized bacteria, to a height of 45 cm; the working volume was 260 ml. Aeration was maintained at 0.5 bar throughout the entire system, including through the bottom of the column; this guaranteed that the culture medium was well-mixed. The column was attached to a reservoir of naphthalene/mineral salts medium and kept on a magnetic stirrer for proper mixing of naphthalene into the medium. The degradation process was carried out by the continuous supply of medium, through a side arm present at the bottom of the column, with various concentrations of naphthalene added at different flow rates (100–200 ml h–1) via a peristaltic pump (Miclins, India). Residual naphthalene was measured in the effluent for each set of experiments.

Results Degradation of naphthalene by freely suspended and immobilized cells Batch fermentations for the degradation of naphthalene were carried out by freely suspended cells and compared with that by cells immobilized in polyurethane foam. Degradation of naphthalene by freely suspended cells (4×1010 cfu ml–1) and polyurethane-foam-immobilized cells (0.8–1×1012 cfu g–1 foam cubes) with three different initial concentrations of naphthalene (25, 50, and 75 mM) was carried out at different incubation periods. The results of these studies are given in Fig. 1. With freely suspended cells at the above cell concentrations, it was shown that the initial 25 mM of naphthalene was completely degraded within 3–4 days of incubation. At an initial naphthalene concentration of 50 mM, the same cell concentration showed a maximum of 30 mM naphthalene degradation after 4 days of incubation. No further degradation of naphthalene was observed even at 15 days of incubation. When the initial amount of naphthalene was increased to 75 mM, only 30 mM was degraded after 4 days of incubation, and no further degradation was observed with the same cell concentration even after 15 days of incubation. The results obtained with immobilized cells in batch culture are given in Fig. 1. It is evident that complete degradation of 25 mM and 50 mM initial naphthalene occurred after 2 and 6 days of incubation, respectively, with polyurethane-foam-immobilized cells (cell concentration 0.8–1×1012 cfu g–1 foam cubes). Furthermore, it was observed that, with an initial naphthalene concentration of 75 mM, a maximum of 65 mM naphthalene could be degraded by polyurethane-foam-immobilized cells (with the same cell concentration) after 15 days of incubation.

Analysis of naphthalene Analysis of naphthalene was carried out by a reversed-phase HPLC (model Shimadzu LC-6A Liquid Chromatograph) with a UV (276 nm) detector (Shimadzu SPD-6AV, UV/visible spectrophotometer detector). A Shim Pack CLC-C8 (M) octadecylsilane (ODS) column (4.6×150 mm) with methanol:water (9:1) at a flow rate of 1 ml min–1 was employed. The peak area was calculated with a peak analyzer (Shimadzu, C-R6A Chromatopack) attached to the HPLC system. Naphthalene was also quantified in the spent medium by using a UV/vis light spectrophotometer (Shimadzu, model 160A) as described previously (Manohar and Karegoudar 1998).

Fig. 1 Degradation of naphthalene by freely suspended cells of Pseudomonas sp. strain NGK1 (4×1010 cfu ml–1) and polyurethane-foam-immobilized cells (1×1012 cfu g–1 foam cubes) in shaken cultures. Open triangles, diamonds and squares represent degradation by freely suspended cells, filled triangles, diamonds and squares degradation by immobilized cells

314 Table 1 Degradation of naphthalene by cells of Pseudomonas sp. strain NGK1 immobilized in different matrices in a batch culture system. Cell numbers are in the range of 1.8×1011 cfug–1 for cells immobilized on alginate-, agar-, and polyacrylamide gel beads, and 0.8×1012 cfu g–1 for cells immobilized in polyurethane foam cubes. The final cell population in all of the above fermentation systems are nearly same

Matrix

Initial naphthalene (mM)

Naphthalene degraded (mM)

Incubation period (days)

Number of times immobilized matrices reused

Alginate

25 50 25 50 25 50 25 50

25 50 25 50 25 50 25 50

3.5 7.0 2.5 6.0 3.0 6.5 2.0 6.0

18 03 12 03 23 05 45 05

Agar Polyacrylamide Polyurethane foam

Fig. 2 Repetitive batch fermentations for semi-continuous degradation of naphthalene by polyurethane-foam-immobilized cells with 25 mM naphthalene loadings. Cell concentration 0.8 –1×1012 cfu g–1 polyurethane foam cubes

Our previous studies (Manohar and Karegoudar 1998) on the degradation of naphthalene by the same bacteria showed that the initial 25 mM of naphthalene was completely degraded after 3.5, 2.5and about 2.5–3 days of incubation by alginate-, agar-, and polyacrylamide entrapped cells, respectively. With the same cell concentration, the cells entrapped in alginate, agar, and polyacrylamide completely degraded 50 mM naphthalene after about 7 days of incubation (Table 1). Semi-continuous degradation of naphthalene by immobilized cells The repetitive batch fermentations for the semi-continuous degradation of naphthalene by polyurethane-foamimmobilized cells was carried out at two different naphthalene loadings (25 mM and 50 mM). It was observed that, for an initial 25 mM naphthalene, the polyurethanefoam-immobilized cells could be reused more than 45 times without losing naphthalene-degrading capacity over a period of 90 days, after which the cells continued naphthalene degradation but at a decreased rate. The results of these studies are shown in Fig. 2. By contrast, with 25 mM initial naphthalene, alginate-entrapped cells could be reused 18 times, agar-entrapped cells 12 times and polyacrylamide-entrapped cells 23 times over a period of 44, 28, and 48 days respectively (Manohar and Karegoudar 1998). However when the initial concentration of naphthalene was increased to 50 mM, the foamimmobilized cells could be reused five times, whereas alginate- and agar-entrapped cells could be reused three

times and polyacrylamide-entrapped cells five times, with a decreased naphthalene-degrading rate (Table 1). Continuous degradation of naphthalene by immobilized cells The continuous degradation of naphthalene by Pseudomonas sp. strain NGK1 immobilized in polyurethane foam cubes was performed in a packed-bed reactor (bed height 45 cm, 60 g foam-immobilized bacteria-) by allowing the naphthalene to pass through the reactor at different flow rates (150, 175, and 200 ml h–1). The results showed that foam-immobilized bacteria (0.8×1012 cfu g–1 foam cubes) completely degraded 40 mM of naphthalene at the above flow rates. With the same cell concentration and an initial loading of 60 mM naphthalene, 59, 56 and 54 mM of naphthalene was degraded at the above flow rates. Furthermore, when the concentration of naphthalene was increased to 80 mM, the foam-immobilized cells degraded 78, 70, and 64 mM naphthalene at the above respective flow rates (Table 2). Our previous studies showed the complete degradation of 40 mM naphthalene by the cells entrapped in alginate, agar, and polyacrylamide (cells: 1.8×1011 cfu g–1 foam cubes) at the above flow rates. With 80 mM naphthalene loading, the alginate-entrapped cells degraded 58 mM naphthalene, agar-entrapped cells 69 mM naphthalene, and polyacrylamide-entrapped cells 64 mM naphthalene at a flow rate of 175 ml h–1 (Table 3). It was observed that, for an initial 40 mM naphthalene loading with a flow rate 150 ml h–1, and for 60 mM naphthalene

315 Table 2 Degradation of naphthalene by foam-immobilized Pseudomonas sp. strain NGK1 in a packed-bed reactor. Cell numbers were 0.8×1012 cfu g–1 foam cubes for cells in polyurethane foam matrix Flow rate Residence time (ml h–1) (min)

Initial naphthalene Naphthalene (mM) degraded (mM)

150

104

175

89

200

78

40 60 80 40 60 80 40 60 80

40.0 59.0 78.0 40.0 56.0 70.0 38.0 54.0 64.0

Table 3 Degradation of naphthalene by cells of Pseudomonas sp. strain NGK1 immobilized in different matrices in a packed-bed reactor with 80 mM initial naphthalene concentration at different flow rates. Cell numbers were 1.8×1011 cfu g–1 for cells on beads and 0.8×1012 cfu g–1 for cells in polyurethane foam cubes Matrix

Alginate

Flow rate Residence Naphthalene Degradation (ml h–1) time degraded efficiency (min) (mM) (%)

175 200 Agar 175 200 Polyacrylamide 175 200 Polyurethane 175 foam 200

48 42 48 42 41 36 89 78

58 50 69 63 64 58 70 64

72.4 62.4 86.2 77.5 80.0 70.2 87.4 80.0

with a flow rate of 100 ml h–1, the packed-bed reactor with polyurethane-foam-immobilized cells operated efficiently and continuously for more than 120 days. By contrast, the packed-bed reactor with alginate-, agar-, and polyacrylamide-entrapped cells could be operated continuously for only 45, 40 and 60 days respectively, under identical fermentation conditions.

Discussion The degradation of naphthalene by polyurethane-foamimmobilized Pseudomonas sp. strain NGK1 (NCIM 5120) was compared with alginate-, agar-, and polyacrylamide-entrapped cells and freely suspended cells. In freely suspended cell cultures, the complete degradation of naphthalene was observed only at lower initial loadings (25 mM). The data obtained from the degradation of naphthalene by polyurethane-foam-immobilized cells in batch culture suggest that the rate of degradation of naphthalene even at higher loadings (50 mM and 75 mM) was much higher than that with free cells, alginate- entrapped cells, and polyacrylamide-entrapped cells. The incomplete degradation of naphthalene in free

cell cultures may be due to swelling of the cells caused by the accumulation of phenolic compounds (Klousmeier and Strawinski 1957). The results obtained from the continuous fermentation studies suggest that degradation of naphthalene was effective even at high concentrations. Polyurethane-foam-immobilized cells degraded 70 mM naphthalene at a rate of 175 ml–1 h–1 from an initial 80 mM naphthalene load, whereas at the same rate 58 mM, 69 mM, and 64 mM naphthalene were degraded by alginate-, agar- and polyacrylamide-entrapped cells. Polyurethane-foam-immobilized cells and freely suspended cells degraded naphthalene without an initial lag period. The initially low rate of naphthalene degradation in other immobilized systems may be attributed to slow diffusion of the compound into the gel beads. The complete degradation of 25 mM and 50 mM naphthalene occurred after 2 and 6 days of incubation with foam-immobilized cells in batch cultures. Alginateentrapped cells degraded 25 mM and 50 mM naphthalene after 3.5 days and 7 days, agar-entrapped cells after 2.5 and 6.5 days and polyacrylamide-entrapped cells after 3 and about 7 days of incubation, respectively. Polyurethane-foam-immobilized cells in batch cultures with 25 mM naphthalene could be reused 45 times, while alginate-, agar-, and polyacrylamide-entrapped cells were reused 18, 12, and 23 times, respectively. Polyurethanefoam-immobilized cells in a packed-bed reactor degraded 40 mM naphthalene continuously at a rate of 150 ml–1 h–1 and 60 mM at a rate of 100 ml–1 h–1 for more than 120 days, whereas the packed-bed reactor with alginate-, agar- and polyacrylamide-entrapped cells operated for 45, 40 and 60 days, respectively, under identical fermentation conditions (Manohar and Karegouder 1998). The very slow degradation of naphthalene in batch culture systems may be due to the limited air supply. The increased degradation by immobilized cells is due to the accelerated reaction rates caused by the high local cell density in or on the immobilized matrix. Immobilization also provides a kind of membrane stabilization and increases cell permeability, which is assumed to be responsible for the cell protection and better degradation rates in immobilized cells. Such observations were made by Hall and Rao (1989), who studied the production of fuels and chemicals. Bisping and Rehm (1988) reported multistep reactions with immobilized cells in the production of alcohol and glycerol. Lee et al. (1994) reported increased degradation of pyridine by immobilized Pimelobacter sp. This study reveals that more efficient and long-term degradation of naphthalene was achieved by Pseudomonas sp. strain NGK1 cells immobilized in polyurethane foam than by cells immobilized in alginate, agar and polyacrylamide or their free cell counterparts. Polyurethane foam is readily available and easy to handle for immobilization. Furthermore, the operational stability and longevity of cells immobilized in polyurethane foam is significantly better than with the other materials tested. Moreover, the immobilization of bacterial cells in polyurethane is extremely versatile and cost-effective.

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With this approach, there is a potential for the development of microbial technology for detoxification of effluents containing naphthalene. Acknowledgements The financial assistance from the Council of Scientific and Industrial Research and All India Council for Technical Education, New Delhi, India, in the form of research projects to T.B.K. is gratefully acknowledged.

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