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Journal of Sol-Gel Science and Technology 13, 283–287 (1998) c 1998 Kluwer Academic Publishers. Manufactured in The Netherlands. °
Encapsulation of Microbial Cells into Silica Gel ´ Sˇ BRANYIK ´ ´ TOMA AND GABRIELA KUNCOVA Institute of Chemical Process Fundamentals, Academy of Sciences of the Czech Republic, Rozvojov´a 135, 16502 Praha 6-Suchdol, Czech Republic
[email protected]
´ ˇ ´ JAN PACA AND KATERINA DEMNEROVA Institute of Chemical Technology Prague, Technick´a 5, 166 28 Praha 6, Czech Republic
Abstract. This work deals with changes in microbial phenol degradation and cell proliferation caused by immobilization into silica gel. Mixed microbial culture and the yeast Candida tropicalis were immobilized in silica layers and pieces prepared by mixing of prepolymerized tetraethoxysilane with cell suspension. The phenol degradation rate of cells entrapped in silica gel was compared with those immobilized into an organic polymer-polyurethane. The phenol degradation efficiency decreased in the following order: free cell suspension > cells entrapped into polyurethane foam > cells entrapped into prepolymerized TEOS. Inside the silica there was no growth observed by optical microscope. The immobilization of bacterium Pseudomonas species 2 into silica gel, cells which cometabolize PCBs with biphenyl, did not result in substantial change of intermediate concentration. Keywords:
1.
sol-gel method, cell encapsulation, phenol biodegradation, PCB biodegradation
Introduction
Decontamination of waste waters containing phenols and polychlorinated biphenyls (PCBs) is of a great practical importance. Some microorganisms are able to utilize phenol as a sole carbon and energy source [1]. PCBs are co-metabolized with biphenyl [2]. Biodegradation is often complicated by unexpected difficulties and immobilized cells offer solutions via increasing and retention of bioreactors catalytic activity, protection of cells against wash out and substrate inhibitory effects, separation ease and storage stability. Among the common immobilization materials used in biodegradations [3] silica gels belong to those with high chemical and biological resistance. Microbial cells, plant cells and pancreatic islets [4–6] have been successfully incorporated into silica gel derived materials. The aim of this work was to investigate the influence of immobilization into silica gel by sol-gel processing on phenol and PCB degrading cells. The phenol degrading cells were an undefined mixed culture and
pure microbial culture of yeast Candida tropicalis. The PCB degrading cells were the bacteria Pseudomonas species 2. The applicability of biocatalysts formed by encapsulation of cells into silica gel and into polyurethane foam commonly used in environmental technologies was studied.
2. 2.1.
Experimental Part Microorganisms and Medium
A mixed culture adapted to phenol was used. The microbes were cultured under aerobic conditions (rotary shaker, 120 rpm) at 30◦ C for 72 h in a mineral medium. 175 mg/l of phenol was added at the beginning of the cultivation 350 mg/l after 24 h and 525 mg/l after 48 h. Yeasts Candida tropicalis were cultured under aerobic conditions (rotary shaker, 120 rpm) at 30◦ C for 120 h in a mineral medium. 250 mg/l of phenol was added daily. Bacterium Pseudomonas species 2
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was cultured under aerobic conditions (rotary shaker, 120 rpm) at 30◦ C for 120 h in a mineral medium. Substrate (biphenyl) was added at the beginning of the cultivation at a concentration of 20 mg/l. The mineral medium contained in 1 liter: 4.3 g of K2 HPO4 ; 3.4 g of KH2 PO4 ; 2.0 g of (NH4 )2 SO4 ; 0.34 g of MgCl2 · 6H2 O and 1 ml of trace element solution. 2.2.
Chemicals and Analytical Methods
The following chemicals were used: colloidal SiO2 (250 g/l), 4-aminoantipyrene (Aldrich), tetraethoxysilane (TEOS) (Fluka Chemie AG), polyurethane (Hypol 2002; Hampshire Chemical Limited, Middlesbrough, UK), Biphenyl (Lachema, Brno) and polychlorinated biphenyls (PCBs) that were mixtures of approximately 40 congeners containing 2–6 chlorine molecules (Chemko, Strazske, Czech Republic). Phenol content was determined by the photometric method [7]. Yellow intermediates (chlorinated derivates of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid) [2] of the PCB biodegradation were followed by UV-Vis spectrophotometer Hewlett Packard 8452A at 600 nm. The influence of light losses by cells and medium was eliminated by subtraction of the absorbance measured at 400 nm (A600 − A400 ). The concentration of cells in suspension (optical density, OD) was determined by measuring the absorbance at 400 nm (Pseudomonas species 2) and 500 nm (mixed culture, Candida tropicalis). The growth of individual cells or colonies was observed by optical microscopy (Leitz Wetzlar). 2.3.
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suspension (1 ml, ca. 25 g/l dry cell weight of the mixed culture or Candida tropicalis, ca. 15 gl/l dry cell weight of Pseudomonas species 2) and 0.1 ml of KOH solution was rapidly added and mixed. Gelation occurred within a few seconds. The gels were then dried at room temperature for 20 min and subsequently cut into pieces, approximately 3 × 3 × 4 mm in size, which were dried at 4◦ C for 120 min. (b) Immobilization of cells into polyurethane (PU): 1.5 g of polyurethane was mixed with 1 ml of cell suspension (ca. 25 g/l dry cell weight) and allowed to polymerize in a silicone tube at 25◦ C. The resulting foam was cut to obtain cylinders with an average volume of 0.66 cm3 in the swollen state. 2.4.
Batch Experiments
Immobilized cells were placed into 250 ml Erlenmeyer flasks with 100 ml of mineral medium and the experiments were carried out under aerobic conditions on a rotary shaker (120 rpm) at 30◦ C. After phenol depletion in the medium and before starting a new cycle, the carriers with cells were rinsed several times with sterile distilled water and then transferred into a sterile medium together with an appropriate amount of phenol. In the first batch the newly prepared polyurethane with entrapped cells floated until it soaked up the medium. Batch experiments with free cells and with PCB degradation were carried out under the same conditions. Pseudomonas species 2 were entrapped into prepolymerized TEOS and placed into mineral medium with 0.1 g biphenyl and 4 µl PCB.
Immobilization
Biomass was harvested by centrifuging the cell suspension for 10 min at 10,000 rpm, after which the recovered microorganisms were resuspended in appropriate amounts of distilled water. The microorganisms were then immobilized by one of the following methods: (a) Immobilization of cells into TEOS: TEOS (3.5 ml) was vigorously mixed with 2.8 ml of slightly acid distilled water (pH 2.8) until the turbid mixture became a clear solution. The solution was then kept at 4◦ C for approximately 72 h until a viscous (ca. 1000 cP) solution, containing 25–27 wt.% Si, was formed. 1.5 g of the viscous solution was then transferred into a polyethylene test tube where the cell
3.
Results and Discussion
The influence of the encapsulation on phenol consumption and cell outgrowth is shown in Fig. 1. The mixed culture was immobilized either into prepolymerized TEOS (TEMC) or into polyurethane (PUMC). The most significant difference between the biocatalysts was in the duration of the phenol degradation lag phase. Twelve hours were observed for PUMC and less then one hour for TEMC. This means that cells suffered greater stress during the PU formation probably caused by toxic isocyanate groups present in the PU prepolymer [8]. The immediate phenol consumption in TEMC implies that the alcohol concentration in prepolymerized TEOS was below critical and
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Figure 1. Biodegradation of phenol after immobilization by TEMC ( • ), PUMC ( ¥ ) and yeast Candida tropicalis entrapped into prepolymerized TEOS ( 1 ). The outgrowths of cells are indicated with appropriate dotted lines.
therefore did not cause serious cell damage. Nevertheless, the average phenol degradation rate (APDR) for PUMC, after the lag phase, is higher (6.25 mg/l · h) than in TEOS (5.2 mg/l · h). Immobilization of Candida tropicalis into prepolymerized TEOS caused an 8-hour lag phase followed by a phenol degradation rate of 4.36 mg/l · h. The highest outgrowth was observed for TEMC probably as a result of partial gel dissolution during the first batch operation (Fig. 1). In subsequent batch operations the values of the outgrowth are similar for TEMC and PUMC. Candida tropicalis in TEOS showed no outgrowth due to the disadvantageous medium composition reducing its growth to a
Figure 2.
minimum and making the cultivation sensitive towards contamination. The APDR of PUMC increased during the subsequent batch operations while for TEMC the maximum APDR was reached after three batch operations (Fig. 2). It is thought that the rigidity of the silica gel did not allow a significant growth of the biomass inside the silica gel. By contrast the flexibility of the polyurethane allowed cell growth in the polymer itself, and the macroscopic pores on the surface enabled the cells to colonize the surface and thus increase the biomass content. The effect of initial phenol concentration on APDR is shown in Fig. 3. Two concentrations of cells in
The growth of APDR at 150 mg/l initial phenol concentration during subsequent batch cycles.
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•
free cell suspension (OD500 = 0.5), ¥ free cell suspension (OD500 =
suspension (OD500 = 0.5, OD500 = 0.04) were compared with TEMC. The higher cell concentration represents the same level of cells that were entrapped into TEMC. The free cells exhibited a much higher APDR in the whole concentration range compared to TEMC. Ignoring cell damage caused by immobilization, the lower oxygen and substrate supply to the cells inside the particles, compared to cells near surface, could decrease the effectiveness of the phenol oxidation. This limitation could be the result of the diffusion resistance of the silica gel, or could be caused by complete oxygen and substrate consumption by cells near the surface. Moreover, inside the rigid silica gel the growth of
cells coupled with phenol consumption is considerably restricted and this could also lead to decreased degradation rate. The lower cell concentration is an average concentration of cells that outgrew from the TEMC during the degradation of approximately 150 mg/l phenol. The lower APDR of this cell suspension proved that other cells than just those that had escaped from the TEMC into the medium, consumed phenol. The evolution of the yellow intermediate products of PCB degradation and the cell outgrowth is depicted in Fig. 4. Equal initial amounts of bacteria Pseudomonas species 2 were compared in immobilized form and in cell suspension. The time of the greatest
Figure 3. The effect of initial phenol concentration on APDR. 0.04), N TEMC.
Figure 4. The evolution of yellow intermediates ( A600 − A400 ) during the degradation of PCBs by Pseudomonas species 2 ( ¥ free cell suspension, N cells immobilized into prepolymerized TEOS). The growth of cells (OD400 ) is indicated with appropriate dotted lines.
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increase in the intermediate concentration differed. However, there was no evidence of changes in the rate of biodegradation due to immobilization. This experiment, which was repeated several times using the initial silica gel with immobilized bacteria, also proved that co-substrates such as PCB were effectively metabolized. Observation of living cells in silica layers, made of colloidal SiO2 [9], showed that neither single cells nor groups of cells grew in the direction parallel to the silica layer. Under experimental conditions, there was no formation of colonies observed in silica gel pieces, unlike what happens in organic polymers [10]. It could be an effect of the material tensile strength that prevented cell reproduction [11]. The cell growth into the medium was probably a consequence of the cell leakage from the surface and/or the outgrowth in the direction vertical to the silica layer. However, local cell accumulations were observed in silica gel during PCB degradation. Air bubbles in the sol, which appeared after stirring, were captured by the rapid gelation. These open spaces were often filled with growing bacteria, giving dark spots inside the transparent silica gel. 4.
Conclusions
The immobilization of living microbial cells into prepolymerized TEOS and polyurethane foams proved that the sol-gel process influenced the mixed culture by providing a less stressful environment than the cross-linking of the polyurethane prepolymer. The rigidity and the small pore size of the silica gel did not allow the cell growth inside the silica material. Cell proliferation was possible only on the surface of the carrier. The biodegradation capacity decreased in the order free cells > encapsulation in polyurethane foam > encapsulation in prepolymerized TEOS. The total volume of the swollen polyurethane foam with cells is
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at least six times larger than that of the silica gel with cells. This means that use of silica gel would enable an increase in biocatalyst concentration in a bioreactor. High densities of living cells, and the chemical and biological stability of the silica material also makes these biocatalysts attractive for in situ bioremediation of contaminated soil and water. Acknowledgments Financial support by the Czech Grant Agency (Grant 104/96/0459 and 104/97/1212) is gratefully acknowledged. References 1. M. Krug, H. Ziegler, and G. Straube, Journal of Basic Microbiol. 2, 103 (1985). 2. K. Furukawa, N. Tomizuka, and A. Kamibayashi, Applied and Environmental Microbiology 38, 301 (1979). 3. M.B. Cassidy, H. Lee, and J.T. Trevors, Journal of Industrial Microbiology 16, 79 (1996). 4. E.J.A. Pope, Journal of Sol-Gel Science and Technology 4, 225 (1995). 5. R. Campostrini, G. Carturan, R. Caniato, A. Piovan, R. Filippini, G. Innocenti, and E.M. Cappelletti, Journal of Sol-Gel Science and Technology 7, 87 (1996). 6. E.J.A. Pope, K. Braun, and C.M. Peterson, Journal of Sol-Gel Science and Technology 8, 635 (1997). 7. R.W. Martin, Anal. Chem. 21, 1419 (1949). 8. J. Klein and F. Wagner, Appl. Biochem. Bioeng. 4, 11 (1983). 9. T. Br´anyik, G. Kuncov´a, J. P´aca, K. Jurek, and F. Kaˇst´anek, in Immobilized Cells: Basics and Applications, edited by R.H. Wijffels, R.M. Buitelaar, C. Bucke, and J. Tramper (Elsevier, Amsterdam, 1996), p. 757. 10. L.E. Huskens, J. Tramper, and R.H. Wijffels, in Immobilized Cells: Basics and Applications, edited by R.H. Wijffels, R.M. Buitelaar, C. Bucke, and J. Tramper (Elsevier, Amsterdam, 1996), p. 336. 11. L. Inama, S. Dir´e, G. Carturan, and A. Cavazza, Journal of Biotechnology 30, 197 (1993).
