Effect of Adsorption and Bead Size of Immobilized Biomass on the

6820

Ind. Eng. Chem. Res. 2007, 46, 6820-6824

Effect of Adsorption and Bead Size of Immobilized Biomass on the Rate of Biodegradation of Phenol at High Concentration Levels Nedal Massalha, Sobhi Basheer, and Isam Sabbah* The Regional Research & DeVelopment Center, The Galilee Society, Shefa-Amr, 20200, Israel

The main objective of this research is to examine the modification of a well-known immobilization technique of biomass to enhance the aerobic biodegradation of phenol at high initial concentrations. This work focused on testing the effect of entrapping different low-cost mineral additives (clay and activated carbon) at the immobilization matrix, while considering the diffusive internal mass-transfer limitations. Aerobic biodegradation of phenol was performed using isolated microorganism cells from a compost pile of agricultural waste. The influence of a different initial concentration of phenol (400-2000 mg/L) on the rate of biodegradation was tested in systems that were based on free and immobilized cells. The results show that immobilized cells in modified immobilizers tolerated and completely degraded phenol at initial concentrations of 2000 mg/L and higher. A bead size of 4 mm (diameter) and a composition of alginate with clay and activated carbon was determined to be the optimal and most-effective bead size and composition for the immobilized biomass to degrade phenol at an initial concentration of ∼2000 mg/L. The bead size significantly affected the biodegradation rate of phenol, where the highest biodegradation rate was obtained for the 1-mm beads and the lowest value was obtained for the 6-mm beads. Immobilized cells in 1-mm beads of alginate with clay and activated carbon could not tolerate an initial phenol concentration of 2000 mg/L at the first run. However, increasing the initial phenol concentration gradually enables the cells to utilize phenol at initial concentration of 2600 mg/L in the third run, using the same beads. Introduction Phenol is an organic, aromatic compound that occurs naturally in the environment. However, it appears in high concentrations from a wide spectrum of industrial1 and agricultural wastewaters, including petroleum refineries, pulp and paper, plastic, dye, polymeric resins, the pharmaceutical industry,2 and olive mill wastewater.3 Phenol is water-soluble and highly mobile. It can penetrate water resources very quickly, even at very low concentrations, and it can cause odor and taste problems. Phenols generally are considered to be priority pollutants, because they are harmful to organisms, even at relatively low concentrations. The common methods to treat phenols in water and wastewater include biodegradation, adsorption on activated carbon, oxidation, solvent extraction, etc.4 Among the different mentioned treatment methods, microbial degradation is considered the most cost-effective; however, the biological treatment is limited, because of the toxicity and slow biodegradation process, especially at high concentrations.2,5,6 Wastewater with a low concentration level of phenol (up to 500 mg/L) could be treated biologically in a reactor that is based on free cells; however, many constraints are associated with this method for the biodegradation of phenol, such as the maintenance of optimal initial concentration, optimal cell concentration, and removal of produced cells (sludge). These disadvantages could be eliminated by immobilizing cells in a porous solid (gel) matrix, usually calcium alginate, where the immobilization techniques have exhibited little sludge production.7 Entrapping bacteria protects them from predation, water, and nutrient stress and allows for a larger bacterial population * To whom correspondence should be addressed. Tel: (+)972-49504523. Fax: (+)972-4-9504525. E-mail address: [email protected].

over a longer time interval.8,9 In addition, the immobilizing material is inert, nontoxic to cells, inexpensive, and practical.10-12 More importantly, the immobilization technique of biomass provides a high cell concentration at the startup of the bioreactor, which allows for cell reuse, and prevents cell and biomass washout in continuous processes.10 In 2003, Chung et al. reported that phenol at an initial concentration up to 600 mg/L was degraded in a free-cell system, while immobilized biomass could tolerate a higher level of initial concentration of up to 1000 mg/L.14 The phenol inhibition effect was observed at initial concentrations of 100-150 mg/L, using a free-cell-based reactor.10,13,14 On the other hand, the diffusive mass transfer within the solid matrix is considered a limiting factor that reduces the productivity of the process.14 The “beads” act like a slow-release delivery system, where the substrate (contaminant) is slowly released to the cells for microbial mineralization without a significant impact of the surrounding environment. This protection is necessary for industrial effluents with high toxicant concentration. Many types of solid matrixes, such as calcium alginate, activated carbon, polyurethane foams, k-carrageenan, and polymer matrixes, have been used for the immobilization of cells, which are suitable for the degradation of phenol.15 Alginate is the most popular material for gel preparation,16 because of the low cost and mild conditions of immobilization. The objective of the present study is to test the modification of an encapsulation procedure with mineral additives for phenol biodegradation at high concentrations that are inhibitory to microbial growth and biodegradation. The biodegradation of phenol using isolated bacteria from compost, and immobilized in various matrixes amended with bentonite clay and powdered activated carbon, was investigated. The rate of degradation of phenol at high concentrations by immobilized and freely suspended cells was evaluated.

10.1021/ie070057v CCC: $37.00 © 2007 American Chemical Society Published on Web 06/19/2007

Ind. Eng. Chem. Res., Vol. 46, No. 21, 2007 6821

Description of the Biodegradation Rate The Haldane equation (eq 1) was used to describe the substrate inhibition effect:17

µ)

µmaxC C2 Ks + C + Ki

(1)

where µ is the growth rate (in units of h-1), µmax the maximum growth rate (in units of h-1), Ks the substrate affinity constant (in units of mg/L), Ki the substrate inbibition constant (in units of mg/L), and C the phenol concentration in the biodegradation medium (in units of mg/L). Materials and Methods (1) Reagents. Phenol (CAS No. 108-95-2) and all reagents were purchased from Riedel-de Haen Fine Chemicals. The sodium alginate salt (C6H8O6)n for microorganism immobilization (CAS No. 950-32-7) from Fluka and powdered activated charcoal (PAC) (CAS No. 64365-11-3) were purchased from Sigma. Technical-grade clay (sodium bentonite) was purchased from a local supplier. Double-distilled water was filtered through a Millipore membrane filter before use. (2) Microorganism-Culture Enrichment Medium. An enrichment culture was developed from compost, which included a mixture of olive mill and piggery solid waste. Samples were taken from this source, based on the assumption that it contains phenol-degrading microorganisms. Samples of 2 g each were initially grown on a phenol-rich medium as a sole source of carbon. Each 2-g sample was added to a 300-mL Erlenmeyer flask containing 100 mL of the growth medium (see below) and 0.5 mM phenol as a carbon source. The microorganisms were grown and maintained in a nutrient medium containing KH2PO4 (0.5 mg/L), K2HPO4 (1 mg/L), (NH4)2SO4 (1 mg/L), MgSO4 (0.2 mg/L), MnCl2‚4H2O (0.1 mg/L), CaCl2‚2H2O (0.1 mg/L), and FeSO4‚7H2O (0.005 mg/L), with a final pH of 6.9. The flasks were incubated at 35 °C and 200 rpm for one week. A volume of 2 mL of the suspension was taken from each Erlenmeyer flask and transferred to a fresh nutrient medium, under the same incubation conditions, for 48 h. Four subsequent iterations (i.e., identical sampling and transferring to fresh medium) were performed, and cells were grown under the same conditions. Optical density (OD) analysis was used to indicate the cell concentration. (3) Kinetic Study with Free Cells. To determine the specific growth rate, a kinetic study of phenol biodegradation was conducted. Phenol biodegrading microorganisms were grown in the mineral media with a phenol concentration of 500 mg/L. The cells collected by centrifugation (4500 rpm for 10 min) were resuspended in the mineral medium and recentrifuged. Subsequent cleaning, resuspension, and recentrifugation were conducted until a final cell concentration (dry weight) of ∼0.057 g/mL was attained. A volume of 1 mL of the concentrated cells were inoculated into the mineral medium (100 mL) in 300-mL bottles. The bottles were capped with cotton plugs to allow air circulation and placed in a shaker at 25-27 °C and 150 rpm. The biodegradation kinetic experiments were identically conducted at different initial phenol concentrations in the range of 100-1600 mg/L. (4) Preparation of Immobilized Cells Beads. A stock of sodium alginate was prepared by adding 2% (w/v) of alginate to the medium. Three different matrixes were prepared by adding calcium bentonite clay (2%, w/v), PAC (1%, w/v), or

both at the same percentage to the sodium alginate stock. A small amount (0.057 g) of dry cells were resuspended within a suspension of 10 g of the three different mixtures. Each mixture was dropped into a 2% CaCl2 solution, using a 10-mL syringe connected to different tips to produce beads with diameters of 1, 2, 4, and 6 mm. The beads were stored in 2% CaCl2 solution for 2 h at room temperature to ensure more stable hydrogels. The beads then were rinsed within a mineral medium and immediately soaked in a fresh mineral medium for 30 min to filter all CaCl2 residues. (5) Biodegradation Rate Experiments. Biodegradation rate experiments were performed to evaluate the effect of initial phenol concentration on the biodegradation rate of phenol by free and immobilized cells, as well as to examine the tolerance threshold of both free and immobilized cells. The experiments were performed in 300-mL bottles containing 100 mL of solution at 27-29 °C and 150 rpm, where 0.057 g of dried cells (free or immobilized) were inoculated at time zero. Samples were taken out at different time intervals for biomass and phenol analysis. All experiments were performed in triplicate, and average values were determined, as outlined in the results. (6) Abiotic System. To distinguish between the decrease of phenol as a result of biodegradation or of adsorption, a set of experiments similar to the biodegradation rate experiments were conducted using beads of 1 mm and 4 mm of the different composites without biomass (abiotic systems). Here (and later in this manuscript), we refer to this part as “blank” experiments. A set of duplicate of (blank) experiments were performed for each initial phenol concentration and all matrixes of the beads. Similarly to the biodegradation rate experiments, the blank systems were conducted in 300-mL bottles at 27-29 °C and shaken at 150 rpm. (7) Measurement of Cell and Phenol Concentrations. Samples of 0.3 mL each were taken at different time intervals. The samples were centrifuged at 14 000 rpm for 10 min before diluting the samples with a culture medium for phenol measurement by the spectrophotometer at 270 nm. The phenol concentration was determined using an ultraviolet (UV) spectrophotometer (Jasco UV-550). The UV absorbance method is effective at phenol detection of concentrations of 50 mg/L. Absorbencies were measured at 270 nm, using a 1-cm-path-length square quartz cuvette with a culture medium as a reference. As for cells, the turbidity was measured as OD at 600 nm. For proving full mineralization of phenol in the biodegradation experiments, a set of total organic carbon (TOC) analyses was conducted using a TOC analyzer (Shimadzu, model 5000A). Results and Discussion (1) Growth and Degradation Rate of Free Cells. The results of phenol degradation in a mineral medium at pH 6.9 and a temperature of 27 °C for different initial phenol concentrations are shown in Figure 1a. The phenol was almost completely degraded within 5 h for phenol at initial concentrations of 100 and 200 mg/L, and within 20 h for phenol at initial concentrations of 400-1000 mg/L. A longer time interval was necessary for complete degradation of phenol at an initial concentration of 1300 mg/L, and almost no degradation was observed for phenol at an initial concentration of 1600 mg/L. From this figure and the cell growth (are not shown here), it is clear that substrate inhibition is significant at an initial phenol concentration of ∼500 mg/L. This result can be seen in Figure 1b, which shows the specific growth of phenol as a function of initial phenol concentration. The line represents the best fit to the Haldane model (eq 1)17 used to describe substrate inhibition kinetics. In

6822

Ind. Eng. Chem. Res., Vol. 46, No. 21, 2007

Figure 3. Effect of matrix composition on the biodegradation of the phenol using beads of 4 mm at pH 6.9, T ) 25-27 °C. Legend: Alg ) alginate; AC ) activated carbon.

Figure 1. (a) Kinetics of phenol biodegradation with free cells at various initial phenol concentrations (pH 6.9, T ) 25-27 °C). (b) Specific growth rate of free cells under different initial phenol concentrations (25-27 °C, pH 6.9).

Figure 2. Normalized phenol concentration versus time for the different matrices without cells for beads of (a) 1 mm and (b) 4 mm (pH 6.9, T ) 25-27 °C). Legend: Alg ) alginate; AC ) activated carbon.

the study of Chung et al.,14 using P. putida to degrade phenols, free cells could tolerate initial phenol concentrations up to 600 mg/L, whereas the same biomass was totally inhibited under an initial phenol concentration of 800 mg/L. (2) Rate of Phenol Decrease in Abiotic Systems. Figures 2a and 2b describe the normalized phenol concentration (initial concentration of 1600-1700 mg/L) versus time for the different matrixes of beads (alginate; alginate + PAC; alginate + clay;

and alginate + PAC + clay) with diameters of 1 and 4 mm, respectively. The decrease in phenol, which was attributed to adsorption processes, was observed only for the matrixes that contain PAC (Figures 2a and 2b). However, no significant decrease of phenol concentration was observed for the same initial concentration of the matrixes without PAC. These results indicate that the reduction of phenol attributed to adsorption is 2.5% for beads that contain alginate and clay; 12.5% for beads that contain alginate, clay, and activated carbon; and 15% for alginate and activated carbon. This reduction of 12%-15% of the initial concentration was fully consistent with the sorption capacity of phenol to activated carbon obtained from the adsorption isotherm of phenol with PAC (results are not shown here). (3) Rate of Degradation of Immobilized Cells. (A) Effect of Matrix Composition. The biodegradation of phenol was identical for all the different matrixes of immobilization (alginate; alginate + PAC; alginate + clay; and alginate + PAC + clay) at initial phenol concentrations of 400-1600 mg/L, using beads with diameters of 4 mm (result not shown here). However, a slower degradation rate was observed using immobilized cells rather than free cells in this range of initial phenol concentration. A significant effect of the composition of the matrix was obtained for higher initial phenol concentrations (∼2000 mg/L). Figure 3 shows the degradation kinetics of phenol at an initial concentration of 2000 mg/L, using immobilized cells in the three studied matrixes with beads 4 mm in diameter at 27 °C and pH 6.9. As observed in this figure, the addition of both PAC and bentonite clay to the alginate matrix significantly enhances the phenol degradation, where complete degradation was achieved after 75 h of contact time. The addition of clay and activated carbon (this work) increases the toxicity threshold of biomass to initial phenol concentrations of up to 2000 mg/L, where the total removal of phenol was achieved at ∼100 h. However, Chung et al.14 reported that the immobilized cells (biomass) could tolerate initial phenol concentrations of up to 1000 mg/L with 70% removal of residual phenol after ∼150 h. This comparison shows that the matrix composition has a significant effect on reducing the toxicity threshold value of phenols to immobilized microorganisms. (B) Effect of Bead Size. To assess the most effective immobilizing matrix (alginate, clay, and PAC), four bead sizes (1, 2, 4, and 6 mm) were studied. Figure 4a presents the degradation rate of phenol at an initial concentration of 1730 mg/L, using free and immobilized cells in beads with diameters of 1, 2, 4, and 6 mm. The larger the bead size, the slower the

Ind. Eng. Chem. Res., Vol. 46, No. 21, 2007 6823

Figure 5. Phenol degradation kinetics with repeated use of immobilized cells in one run for an initial concentration of 1960 mg/L and three consecutive cycles of initial concentrations of 1700, 2000, and 2600 mg/L, respectively.

Figure 4. Effect of bead size on the kinetics of biodegradation of phenol (pH 6.9, T ) 25-27 °C), beads matrix consist of (Alg + clay + AC) at an initial phenol concentration of (a) 1730 mg/L and (b) 2000 mg/L. (Alg ) alginate; AC ) activated carbon).

phenol degradation rate. This result can be attributed to diffusive mass-transfer limitations. This size can be critical when cells are exposed to higher initial concentrations (Figure 4b). The biodegradation of phenol clearly was not observed, even with the best matrix of the two additives at the optimal percentage, when the bead size was 1 mm, where complete degradation was reached under the same conditions using immobilized cells in a bead size of 4 mm. In 1999, Aksu and Bulbul10 reported that the effect of diffusion resistance on the biodegradation rate is very significant and should not be ignored in any engineering consideration, when using immobilized cells in an alginate matrix. They reported that diffusion resistance increases as the bead size increases. A similar concept was obtained by Fan et al. in 1990, where the diffusion coefficient of phenol, within biofilm attached to activated carbon particles, was reduced by increasing the biofilm density (equivalent to particle size in this study).18 (C) Reuse of Beads. One of the advantages of using immobilized biomass is the easy reuse of beads. To evaluate the repeated use of immobilized biomass, two sets of biodegradation kinetics were conducted. The first set was conducted at an initial phenol concentration of 2000 mg/L with immobilized beads 1 mm in diameter, while, in the second set, a graduated increase of the initial phenol concentration was evaluated with beads 1 mm in diameter. Figure 5 shows a comparison between the kinetic of phenol degradation when beads 1 mm in diameter are exposed to an initial phenol concentration of 2000 mg/L at the first run and with fresh beads, where the concentration of phenol was gradually increased from 1700 to 2600 mg/L in three consecutive cycles. This result indicates that, even when such a small size was not efficient in degrading phenol at higher initial concentrations (Figure 4b), higher phenol concentration could be tolerated and degraded by cells when the initial concentration is increased gradually. One more important result that can be observed in Figure 5 is the instantaneous reduction in phenol concentration in the

Figure 6. Phenol degradation kinetics with repeated use of immobilized cells in two runs for an initial concentration of 1700 mg/L, compared to phenol concentration with a blank system (beads without cells).

three runs. This instantaneous reduction could be attributed to the adsorption process on the powdered activated carbon content. To determine if this obtained observation is due to adsorption and biodegradation processes, a similar reuse experiment was conducted with two sets of 4-mm beads of the matrix containing clay and PAC, where one of the sets was without biomass (blank). Figure 6 shows the kinetics of phenol removal versus the time for beads with biomass and for beads without cells in two successive runs. As this figure shows, the instantaneous reduction of phenol concentration due to the adsorption process that was obtained at the first run with both beads (with and without biomass) was observed only with the beads with biomass at the second run. However, we did not observe any reduction of phenol with the beads that do not contain biomass, which indicates a saturated sorbed site of the PAC content of these beads. The observed instantaneous reduction of phenol concentration in the second run (see Figure 6) indicates that this reduction is due to adsorption processes that were followed by a biodegradation process, causing a cleaning-up of the sorbed site of any organic matter (either phenol or metabolites of phenol). A similar observation was reported in the previous experiment in the three successive runs (see Figure 5), showing an instantaneous adsorption step in each run. This interpretation was confirmed by a TOC analysis of the aqueous phase at the end of each experiment, resulting in a TOC of 3-4 mg/L. This is additional proof for the full mineralization of phenol when using either immobilized or free cells of the studied isolated microorganisms.

6824

Ind. Eng. Chem. Res., Vol. 46, No. 21, 2007

Conclusion The results show that free cells tolerate initial phenol concentrations up to 1300 mg/L, whereas immobilized cells in a modified immobilizer tolerate and completely mineralize phenol at initial concentrations of 2000 mg/L and higher when using the beads of alginate, clay, and powdered activated carbon (PAC). Using the same beads for more fresh runs, the results showed that a higher initial concentration (∼2600 mg/L) was utilized during the same time frame of the experiments. The results of reuse and total organic carbon (TOC) analysis indicate full mineralization of the phenol. Acknowledgment The authors are grateful to the Israeli Ministry of Science, Sport and Culture for the financial support. They also thank Prof. Avi Shaviv for his valuable advice and Mrs. Ahlam Saliba for her help in the experimental work. Literature Cited (1) Dabrowski, A.; Podkoscielny, P.; Hubicki, M.; Barczak, M. Adsorption of phenolic compounds by activated carbonsa critical review. Chemosphere 2005, 58, 1049. (2) Aksu, Z. Application of biosorption for removal of organic pollutants: a review. Process Biochem. 2005, 40, 997. (3) Sabbah, I.; Yazbak, A.; Haj, J.; Saliba, A.; Basheer, S. Biomass selection for optimal anaerobic treatment of olive mill wastewater. EnViron. Technol. 2005, 26, 47. (4) Atlow, S. C.; Bonadonna-Aparo, L.; Klibanov, A. M. Dephenolization of industrial wastewater catalyzed by polyphenol oxidase. Biotechnol. Bioeng. 1984. 26, 599. (5) Collins, L. D.; Daugulis, A. J. Characterization and optimization of a two-phase partitioning bioreactor for the biodegradation of phenol. Appl. Microbiol. Biotechnol. 1997, 48, 18. (6) Prpich, G. P.; Daugulis, A. J. Enhanced biodegradation of phenol by microbial consortium in a solid-liquid two-phase partitioning bioreactor. Biodegradation 2005, 16, 329.

(7) Bandhyopadadhyay, K.; Das, D.; Bhattacharyya, P.; Maiti, B. R. Reaction engineering studies on biodegradation of phenol by Pseudomonas Putida MTCC 1194 immobilized on Ca-alginate. Biochem. Eng. J. 2001, 8, 179. (8) Trevors, J. T.; Lee, H.; Wolters, A. C.; Van Elsas, J. D. Survival of alginate-encapsulated Pseudomonas fluorescens cells in soil. Appl. EnViron. Microbiol. 1993, 30, 637. (9) Cassidy, M. B.; Lee, H.; Trevors, J. T. Environmental applications of immobilized microbial cells: a review. J. Ind. Microbial Biotechnol. 1996, 16, 79. (10) Aksu, Z.; Bulbul, G. Determination of the effective diffusion coefficient of phenol in Ca-alginate immobilized P. Putida beads. Enzyme Microbiol. Technol. 1999, 25, 344. (11) Tanaka, H.; Matsumura, M.; Veliky, I. A. Diffusion characteristics of substrate in Ca-alginate gel beads. Biotechnol. Bioeng. 1984, 26, 53. (12) Chen, D.; Lewandowski, Z.; Roe, F.; Surapaneni, P. Diffusivity of Cu+2 in Ca-alginate beads. Biotechnol. Bioeng. 1993, 41, 755. (13) Dursun, A. Y.; Tepe, O. Internal mass transfer effect on biodegradation of phenol by Ca-alginate immobilized Ralstonia eutropha. J. Hazard. Mater. 2005, 11 (126), 1-3. (14) Chung, T. P.; Tseng, H. Y.; Juang, R. S. Mass transfer effect and intermediate detection for phenol degradation in immobilized Pseudomonas putida systems. Process Biochem. 2003, 38, 1497-1507. (15) Sheeja, R. Y.; Murugesan, T. Studies on biodegradation of phenol using response surface methodology. J. Chem. Technol. Biotechnol. 2002, 77, 1219-1230. (16) Mofidi, N.; Aghai-Moghadam, M.; Sarbolouki, M. N. Mass preparation and characterization of alginate microspheres. Process Biochem. 2000, 35 (9), 885. (17) Haldane, J. B. S. Enzymes; MIT Press: Cambridge, MA, 1965; p 84. (18) Fan, L. S.; Leyva-Ramos, R.; Wisecarver, K. D.; Zahner, B. J. Diffusion of phenol through a biofilm grown on activated carbon particles in a draft-tube three-phase fluidized-bed bioreactor. Biotechnol. Bioeng. 1990, 35, 279-286.

ReceiVed for reView January 10, 2007 ReVised manuscript receiVed May 1, 2007 Accepted May 2, 2007 IE070057V