Kinetic studies of penicillin production during batch and repeated batch in fluidized bed bioreactor with agar immobilized P.

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1 Indian Journal of Biotechnology Vol 3 July 2004, pp Kinetic studies of penicillin production during batch and repeated batch in fluidized bed bioreactor with agar immobilized P. chrysogenum cells A Swaroopa Rani 1, Annapurna Jetty 1 * and S V Ramakrishna 2 1 Biochemical and Environmental Engineering Centre, Indian Institute of Chemical Technology Hyderabad , India 2 Industrial Biotechnology, Module 9, Reliance Complex, Fosbery Road, Off Reay Road Station (E) Sewree, Mumbai , India Received 18 December 2001; revised 29 January 2003; accepted 2 September 2003 The fermentation kinetics of penicillin production by Penicillium chrysogenum was carried out at 27 C and ph 6.0. Batch and repeated batch fermentations using agar-immobilized cells in fluidized bed bioreactor were studied for their potential application in production of penicillin from lactose, a fermentable sugar. Kinetics of immobilized cell fermentation, showed the penicillin yield of ~ g/l, with highest penicillin concentration of ~57.09 mg/l and the high reactor productivity of ~23.8 mg/l h -1. Repeated batch fermentation experiments showed that the immobilized biocatalysts could be recycled effectively for 5 cycles. Penicillin yield was 4-5-fold greater by this method of immobilization, with high productivity as compared to free cells and other immobilization methods. Keywords: Penicillium chrysogenum, agar, penicillin, batch, repeated batch, fluidized bed reactor IPC Code: Int. Cl. 7 A 01 N 63/00; C 12 P 37/00 Introduction In industry, pharmaceutically important secondary matabolites such as antibiotics are produced by either the batch and fed batch submerged free cell fermentation of fungal cells 1. The morphology typically found in conventional suspended cultuers of these organisms often results in high viscosity, thereby leading to a decreaed mass transfer 2,3. Cell immobilization can provide a number of advantages like long-term operational stability significantly improving the productivity of secondary metabolites. In addition, it is also useful for product purification process. Earlier workers have reported immobilization of Penicillium chrysogenum in various hydrogels 4 and K.carrageenan 5. Production of penicillin by immobilized P. chrysogenum in urethane particles was studied in 160 l pilot plant fluidized bed 6. Calcium alginate beads in bubble column and conical bubble fermentor and urethane foam has also been reported 7-9. Fluidized-bed bioreactor has great potential in a wide range of bioprocesses due to their intrinsic advantages *Author for correspondence: Tel: ; Fax: annapurna@mail.iictnet.com and also the possibilities that they offer to the engineers to change their design in order to enhance its stable performance 10. The present paper describes the immobilization of P. chrysogenum using agar as the polymeric source, which resulted in 4-5-fold increase in penicillin. This demonstrates the improvement of the cultivation process by using fluidized bed bioreactor and the use of agar immobilized growing P. chrysogenum ATCC cells for the repeated production of penicillin. In the present study, a laboratory scale fluidized bed bioreactor (60 mm reactor diam and 600 mm height) with P. chrysogenum spores immobilized in agar has been operated for more than 960 hrs using agar beads 2 mm in diam under constant airflow. Materials and Methods Organism and Media P. chrysogenum ATCC was used throughout this study. Spore stocks were maintained on a sporulation medium containing (in g/l); malt extract, 20; glucose, 20; peptone, 1.0; ph maintained at with NaOH/HCl. The growth phase medium contained (in g/l); glycerol, 7.5; peptone, 5.0; molasses, 7.5; MgSO 4, 0.05; KH 2 PO 4, 0.06; NaCl, 4.0. The

2 SWAROOPA RANI et al: KINETIC STUDIES OF PENICILLIN PRODUCTION IN FBR 395 defined production phase medium for bioreactor contained (in g/l); lactose monohydrate, 10; KH 2 PO 4, 3.4; NH 4 Cl, 1.21; phenyl acetic acid, 0.3; K 2 SO 4, 3.95; MgSO 4.7H 2 O, 0.25; and ammonia, 30. Lactose was autoclaved separately. The salts were adjusted to ph 6.0 with NaOH/HCl prior to sterilization. Immobilization Procedure Aqueous spore suspension spores/ml of 10.5 ml was added to 2% agar solution swirled to obtain a homogeneous mixture; poured into paraffin oil in the bead form with the help of peristaltic pump. The suspension was cooled to below the setting temperature of agar. After the beads had solidified, preparation was allowed to settle overnight at 30ºC. Oil layer was decanted 11. Beads were filtered and first washed with phosphate buffer solution and then with water. All the materials used were sterilized in an autoclave. Ten per cent beads were used for inoculation of 1 l growth medium. Spore germination and mycelial pellet development were initiated by incubating the prepared biocatalyst in growth medium for 5 days at 27 C. Batch Fermentation Fluidized bed bioreactor was operated with above grown immobilized agar beads of P. chrysogenum. The total working volume of the bioreactor was made upto 500 ml and the fermentation was carried out in batch mode at 27 C. Repeated Batch Experiments A series of repeated batch fluidized-bed reactor experiments were performed to determine penicillin yield for the immobilized cells over an extended period. After every batch, the media was replaced with fresh full strength production medium. Analytical Procedures Dry Biomass Dry biomass was measured as the dry cell weight per litre of culture. Culture samples (5 ml) were centrifuged at 8,000 rpm for 10 min. The cell pellet was resuspended in distilled water and filtered on preweighed, dried Whatman No.1 filter paper discs. The cells were dried at 75ºC for 40 hrs, and the dry cell weight was determined. ph The ph of each culture sample was measured before any analysis was performed. Carbohydrate Estimation Total sugars present in fermented broth were measured by Anthrone method 12. Antibiotic Estimation Penicillin produced was estimated by biological assay 13 for fermented broth cultures using E. coli as test organism and thus reported in μg/ml in correlation with standard penicillin samples, which were determined with HPLC. Results Immobilization Spores of P. chrysogenum were immobilized in the laboratory scale fluidized bed bioreactor as described above. 500 ml broth containing immobilized P. chrysogenum spores in agar beads were transferred aseptically from growth medium and added to production medium. Maximum production of penicillin by the entrapped cells was obtained after cultivation for 5-6 days. Agar formed rigid gels when solution of these polygalactans was cooled to temperatures at about 45 C, the chemical resistance of these agar beads was extremely stable. The substrates of varying molecular sizes could diffuse freely through agar gel beads in well mixed solutions. Thus, the above natural polymer was used as the matrix for immobilization to entrap P. chrysogenum spores. A repeated batch culture of P. chrysogenum without cell recycle was carried out 5 times. At the end of each batch, decline in penicillin concentration was noticed due to the depletion in sugar concentration. Therefore, after each batch, fermented broth culture was replaced with fresh nutrient medium. In this way 5-repeated batch cultures of P. chrysogenum without cell recycle were carried out, which indicated the capability of using agar beads for penicillin production. Agar beads were stable for 5 repeated batches (40 days). Fermentor The operating volume of 500 ml laboratory fermentor (Fabricated) was used in this work. Fig. 1 represents the basic scheme of a fluidized bed bioreactor. Although various configurations are possible, the most extensively used is the gas-liquid cocurrent upflow reactor in which liquid usually comprises the continuous phase and is fed from the reactor bottom. Its flow is upwards in the reactor, promotes fluidization of the solid particles. The oxygen is provided by sparging air stream through the fermentor at the rate

3 396 INDIAN J BIOTECHNOL, JULY 2004 Fig. 1 Schematic diagram of fluidized-led bioreactor: 1, bioreactor vessel; 2, jacket for maintaining temperature of the vessel; 3, sintered glass frame; 4, calming section; 5, disengaging section; 6, circulation pump; 7, water bath at 27 C; 8, humidifier; 9, air flow meter; 10, medium feeding line; 11, sampling line; 12, inoculation part; 13, exhaust air line filter and condenser; & 14, immobilized biocatalyst. of 1VVM or 0.5 lpm. The gas stream is condensed by a reflex cooler; thus minimizing the evaporation loss during the course of experiment. Effect of Carbohydrate Utilization Fermentation capabilities of P. chrysogenum beads grown in growth media and fermented in production medium containing 10 g/l lactose as carbon and energy source was found to be suitable for penicillin production. After a lag phase of less than 24 hrs, biomass concentration rapidly increased to the maximum of 32 g/l in case of batch 1. As expected, the lactose concentration in Fig. 2 showed a drastic decline followed by slow consumption as the culture passed the log phase. About 40% of lactose was utilized during the exponential growth period. The yields of biomass and penicillin (based on lactose utilization) were experimentally determined to be Yp/s g/l and Yx/s 6.55 g/l, respectively. Figs 2 & 3 indicate that the biomass dry weight peak coincides with the point at which lactose becomes depleted. After 24 hrs onwards the growth of the organism remained steady, which was probably due to decline in sugar concentration. In repeated batches 2 and 3, biomass dry weight increased as compared to the batch 1, which followed the stationary phase behaviour as oxygen uptake rate continued long after sugar had been depleted from the medium. This implied the continued metabolic activity using an intracellular carbon source 14 and accounted for formation of stable biomass dry weight. In a fixed batch mode an optimal initial substrate concentration is added for maximum product yield. If there is too much substrate, a fast fermentation results, in which little product is formed and substrate is used primarily for biomass production. If there is too little substrate, not enough biomass is formed for product formation in the production phase. Effect of Dry Weight In this study, maximum biomass in the broth culture was 55 g/l in batch 3 as seen in Fig. 3, but most of this was formed at 24 hrs i.e 36 g/l. After a short lag phase, a period of rapid growth ensures, during which the cell number increases exponentially with time (exponential phase). In case of batch 1, maximum cell population in the broth achieved was 32 g/l Fig. 2 Utilization of sugar in fluidized-bed reactor Fig. 3 Mycelial dry weight during fermentation in fluidized-bed reactor

4 SWAROOPA RANI et al: KINETIC STUDIES OF PENICILLIN PRODUCTION IN FBR 397 at the exponential phase, which was followed by stationary growth. When Figs 3 & 4 were compared, it could be seen that in all five batches the penicillin produced was higher at the exponential phase Among the five batches as seen in Fig. 3, batch 3 gave high dry biomass concentration because of the maximum utilization of nutrients for the growth but which was not contributing to the penicillin production. An optimum amount of biomass formation resulted in higher yield of penicillin. Using agar as the polymer beads showed good design to minimize the length of the lag phase, as could be seen that maximum product was formed at 24 hrs of fermentation itself when compared to the reports available in literature. Agar immobilized beads also maximized the rate and length of the exponential phase. This was achieved by slowing the onset of the transition to stationary growth. In batch 1, specific growth rate at 24 hrs was hr -1 whereas in repeated batches i.e. batch 2 it was hr -1 ; batch hr -1, batch hr -1 and batch hr -1, respectively. In repeated batch when fresh medium was replaced, the lag phase was minimized, which depended on the age and size of inoculum, thereby an exponential rate might resume immediately. Not much decline in biomass and no lag phase upon transfer into fresh medium rich in metabolic intermediates, indicated that the agar beads could be used repeatedly for nearly 40 days. The stability of beads, depends on the culture medium used to grow the inoculum should be as close as possible to the full scale fermentation composition 15,16. Effect of ph The cells consumed components, which influenced the acidity of the environment and the interplay of cellular composition with acid base equilibrium determined medium ph, which in turn influenced cellular activities and transport processes 15,16. Fig. 5 indicates that during the course of time, broth ph may not change with time, and it remained almost stable in all the 5 batches. Our initial laboratory studies were carried with reported production medium containing 6.8 g/l of KH 2 PO Experimental results presented a decrease in medium ph, which had inhibited penicillin production. Amount of phosphate in the medium was further reduced to 3.8 g/l in the present investigation for the higher stability of penicillin production as it also reduced the risk for the disintegration of beads. In this experiment KH 2 PO 4 level was maintained at 3.4 g/l, since phosphate was required for growth 18 and Fig. 4 Penicillin production in fluidized-bed reactor Fig. 5 ph profile during fermentation in fluidized-bed reactor this optimum phosphate level presumably existed. Discussion Current processes for the production of penicillin is primarily dependent upon the use of very slow growing free cells and is not designed for the repeated use of the cells. This study clearly indicated that immobilized cells have potential for the batch and repeated batch production of penicillin. The problems posed by the deleterious effects of some immobilization processes 19 on the antibiotic producing activity of cells and the prolonged operations of a long secondary metabolic pathway were inherently difficult, which could be circumvented by using agar as a natural polymer for immobilization. The 4-5-fold enhanced penicillin production by immobilized cells as compared with that of free cells suggests that free suspended mycelia have higher growth rates. If the growth is diminished this mycelial layer does not seem to limit diffusion. This could be observed by the stability of the specific penicillin production. In immobilized beads, the mycelial cells confined to biosupport allow a switch from extended mycelial growth to particulate growth, thereby improving the mass transfer capacity as a result of a lower viscosity in the immobilized-cell broth 20. The authors selected

5 398 INDIAN J BIOTECHNOL, JULY 2004 agar as the matrix because changes in ph due to the interaction with the immobilization matrix affects the metabolic behaviour of immobilized cells with respect to free cells. Hence, in this study it could be seen that agar plays no negative role for the change in ph (Fig. 5). However, behaviour of the immobilized cells and how it is affected by operational conditions is very important as any optimized fluidized bed bioreactor operation will require the fundamental knowledge about the biocatalyst. The present study concentrates on the bioreactor and biocatalytic particles, in order to enhance the stability and operation of the reactor. Andrews 21 addressed very clearly the importance of selecting the right particle in order to correctly design fluidized bed bioreactor. The size and density of the biocatalyst particles are the only free variables for a given kinetic behaviour, liquid flow rate, inlet substrate concentration and desired conversion. Once they are fixed, the superficial liquid velocity to keep the bed fluidized is also fixed. Antibiotics are secondary metabolites and, therefore, can be produced at low growth rates, that in turn be achieved by limitation of a key compound for cell growth, for example, phosphate. Therefore, keeping this in view the production media was modified by limiting the phosphate and adding ammonia solution as described in materials and methods. Fluidized-bed bioreactors present a number of advantages that make them an attractive alternative in processes involving biocatalysts. Selection of fluidized bed bioreactors provide a much lower attrition of the solid particle, and almost any kind of immobilized biocatalysts preparations can be used without physical disruption. Fluidized-bed bioreactor can be operated with smaller particle size which minimizes the internal diffusional resistances and the level of mixing enhances external mass and heat transfer from liquid to solid phase. This favours the enhancement of bioreactor performance, in terms of overall productivity, biocatalytic stability, or product separation. Kinetic profiles of sugar uptake, dry weight, protein and penicillin production varied with fermentation time and stable operation for more than 40 days was achieved in the present study. Penicillin productivity based on the total reactor volume approached g/l h -1, and sugar conversion exceeded 80%. With continued research, even higher production rates would be possible as conditions are optimized and scale-up to larger systems would allow the establishment of technical feasibility. Acknowledgement The authors thank the Director IICT for his cooperation. ASR thanks CSIR for the financial support. References 1 Vandamme E J, Peptide antibiotic production through immobilized biocatalyst technology, Enzyme Microb Technol, 5 (1983) Moo-Young M, Halard B, Allen D G, Burrel R & Kawase Y, Oxygen transfer to mycelial fermentation broths in airlilft fermentor, Biotechnol Bioeng, 30 (1987) Olsvik E S & Kristiansen B, Influence of oxygen tension, biomass concentration, and specific growth rate on the rheological properties of a filamentous fermentation broth, Biotechnol Bioeng, 40 (1992) Gaucher G M & Behie L A, Cell immobilization in the production of patulin and penicillin by P. urticae and P. chrysogenum, in Immobilized enzymes and cells. A volume of Methods in Enzymology (1986). 5 Wood J A, Razniewska D N T, Gaucher G M & Behie L A, Continuons production of penicillin G by Penicillium chrysogenum cells immobilized on celite biocatalyst support particles, Can J Chem Eng, 64 (1986) Fan L S, Gas-liquid-solid fluidization engineering (Butterworths, Stoneham) El-Sayed A H M & Rehm H J, Semi-continuous penicillin production by two Penicillium chrysogenum strains immobilized in calcium alginate beads, Appl Microbiol Biotechnol, 26 (1987a) El-Sayed A H M & Rehm H J, Continuous penicillin production by Penicillium chrysogenum immobilized in calcium alginate beads, Appl Microbiol Biotechnol, 26 (1987b) Kobayashi T, Tachi K, Nagamune T & Endo I, Production of penicillin in a fluidized bed bioreactor using urethane foams as carriers, JPN J Chem Eng, 23 (1990) Godia & Carles Sola, Fluidized-bed bioreactors, Rev Biotechnol Prog, 11 (1995) Nilsson K & Mosbach K, Preparation of immobilized animal cells, FEBS Lett, 118 (1980) Loewus F A, Improvement in Anthone method for determination of carbohydrates, Anal Chem, 24 (1952) Bandhopadhyay B, Humphery A E & Tayukhi M, Dynamic measurement of the volumetric oxygen transfer coefficient in fermentation systems, Biotechnol Bioeng, 9 (1967) Drapean D, Blanch H W & Wilke C R, Growth kinetics of Dioscorea deltoidea and Catharanthus roseus in batch culture, Biotechnol Bioeng, 28 (1986) Baily J E. & Ollis D F (Eds), Biochemical engineering fundamentals, 2 nd edn, Chemical Engg Series (McGraw-Hill International Editions, New York) 1986, Baily J E & Ollis D F (Eds), Biochemical engineering fundamentals, Chemical Engg Series (McGraw-Hill International Editions, New York) 1996, Flanagan W P, Klei H E, Sundstrom D W & Lawton C W, Optimization of a pelicular biocatalyst for penicillin G production by Penicillium chrysogenum, Biotechnol Bioeng, 36 (1990) Oh D K, Hyn L K, Kim J H & Park Y H, Production of penicillin in a fluidized bed bioreactor: Control of cell growth and penicillin production by phosphate limitation, Biotechnol Bioeng, 32 (1988) Deo Y M & Gaucher G M, Semi-continuous and continuous

6 SWAROOPA RANI et al: KINETIC STUDIES OF PENICILLIN PRODUCTION IN FBR 399 production of penicillin G by P. chrysogenum cells immobilized in K. carrageenan beads, Biotechnol Bioeng, 26 (1984) Gbewonyo K & Wang D I C, Enhancing gas-liquid mass transfer rates in non-newtonian fermentations by confining mycelial growth to microbeads in a bubble column, Biotechnol Bioeng, 25 (1983) Andrews G F & Przezdziedeki J, Design of fluidized bed fermenters, Biotechnol Bioeng, 28 (1986)

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