Production of CPC using immobilized cells of Acremonium chrysogenum by entrapment technique in ALR.
Calcium, barium and strontium alginates were studied to develop a suitable gel matrix for the immobilization of A. chrysogenum (mold) cells for Cephalosporin C production. Calcium alginate gave best results in both gel stability and cell activity. Modified minimal production media for immobilized whole cell has been also evaluated. Cell growth rate of immobilized cells was about 39% of the growth rate of free cells. [beta] lactam antibiotic production rate of immobilized cells was also limited by mass transfer of oxygen and it was found to be 65% of free cells at oxygen saturation condition. Specific antibiotic production of immobilized cells was about 210% of free cells at saturated oxygen condition. The repeated fed batch fermentations were conducted (every 96 h) using the optimized alginate beads, employing the production media in 5 L Air Lift (Internal) Reactor. The increase in CPC production was observed up to the 5th cycle, and later gradual decrease in CPC production was observed.
Keywords: CPC, Immobilized cells, entrapment, and Oxygen concentration, repeated fed batch fermentation, ALR
Antibiotic production is one of the key areas in the field of applied microbiology. Cephalosporins are usually produced semi synthetically from Cephalosporin C (CPC), an important [beta]-lactam group of antibiotic, which is effective against gram positive and gram-negative bacteria and is exclusively produced by a mold Acremonium chrysogenum. Generally antibiotics are produced by fed batch fermentation using free cell cultures. To enhance the productivity and improve the economics, much attention has been paid on the improvement of the culture employed in the antibiotic production. Among the fermentation strategies adopted to improve the productivity, the whole cell immobilization technology appears to be more attractive for antibiotic fermentation. Free cell studies for the production of Cephalosporin C had some limitation such as pulpy growth of fungus causing an appreciable rise in the broth viscosity affecting the transfer of oxygen and other nutrients into the cells. High cell concentrations cannot be maintained because of wash out phenomenon at high dilution rates. The whole cell immobilization technique is a potentially important process for Cephalosporin C biosynthesis, where increase cell densities were maintained and broth-handling problems were reduced [1,2,3]. Cephalosporin C fermentation is a highly aerobic process. Whole cell immobilization technology is widely used with various microorganisms like bacteria and fungi for production of various metabolites. More than 2500 research papers on various aspects of whole cell immobilization have been published . Different method for the immobilization of whole cell microbial cells and their application to various production process have been developed including gel entrapment [5,6] the covalent binding of cells to inert materials  and adsorption onto inert surfaces .
The use of immobilized whole cells is promising and advantageous in several cases . Immobilized microbial cells offer several advantages over free-living cells for metabolite productions. It includes easier handling of cells, easier separation of products, higher dilution rates, and continuous operation. Cell immobilization, however, also creates problems. In the case of oxygen demanding cells, the primary reason for avoiding the use of immobilized cells is the mass transfer limitation of oxygen supply because of the solid material. An important first step in the use of immobilized cells is the quantitative determination of the effect of oxygen concentration on cell growth and product formation.
The need for a cheap and renewable strategy for immobilization for continuous CPC fermentation is still relevant. Besides the carrier material and process used for CPC fermentation, the bioreactor design will also markedly influence the final success of the proposed technology. The choice of an airlift reactor for our immobilized system is in agreement with the latest trends in continuous fermentation. As compared to packed-bed reactors, the systems with pneumatically forced circulation have the advantage of improved C[O.sub.2] removal, no channeling and clogging, better mass and heat transfer. The present article deals with fermentation of A. chrysogenum in Ba-alg, Ca-alg and Sr-alg in an airlift reactor for CPC production.
The present study demonstrates the optimization of A. chrysogenum whole cell immobilization to provide a suitable gel matrix for CPC production. The effect of alginate concentration, different cation and its concentration, curing time and reusability of the entrapped whole cells for the CPC production were studied. The subsequent production of [beta] lactam antibiotics by immobilized cells was quantified under various oxygen concentrations and compared to the antibiotic production of the free cells.
Materials & Methods
Acremonium chrysogenum (a gift from J.K. Pharmaceuticals Limited, Cuddalore, Pondichery, India) was used throughout the studies. It was maintained on Potato-Dextrose-Agar after incubation for 5-8 days at 28[degrees] C. The surface growth from a 7-day old slant was suspended in 5.0 ml sterilized distilled water and was used for inoculation of the seed media. The reactivation (seed) medium was used for inoculm preparation to inoculate production media for production of cephalosporin C by free as well as immobilized cells .
After three days of growth in free cell defined media, cells were harvested by centrifugation at 3,000 rpm for 10 min and washed with phosphate buffer. The concentrated cells were mixed with sodium alginate. Using a peristaltic pump, the cell alginate mixtures were dropped through a needle (gauge # 18) into various hardening solution containing Ca[Cl.sub.2], Ba[Cl.sub.2] and Sr[Cl.sub.2]. The cells were immobilized in calcium alginate by the traditional external gelation method .
Acremonium chrysogenum Cells in seed culture were transferred several times in fresh medium until the cell dry weight of seed culture became 2.5 + 0.2 g/l. 10% V/V seed medium were used as inoculum. Free cells were cultured in production media containing Sucrose 3% (W/V), (N[H.sub.4])2S[O.sub.4] 0.75%, [K.sub.2]HP[O.sub.4] 1.53%, K[H.sub.2]P[O.sub.4] 1.56%, NaS[O.sub.4] and DL-Methionine 3% at pH 6.5. Fermentation with immobilized cells was cultured in modified defined media (described below). In repeated batch process with all modified optimum conditions, after attaining the maximum production of CPC (96 h), the medium was replaced with fresh production medium. The process was repeated for several batches until the beads started disintegrating. The CPC titre and cell leakage of each cycle were determined. The temperature and pH were maintained at 28[degrees] C and 6.5 respectively.
The CPC content was quantitatively determined by Hydroxyl amine assay method  and confirmed by HPLC method using a [C.sub.18] column and mobile phase containing aceticacid:methanol:acetonitrile:water (2:4:7.5:86.5). Purified CPC (sigma) used as a standard.
For the determination of bead disintegration time, 20 beads were placed in a 100 mL conical flask containing 20 mL of 0.1 M phosphate buffer (pH 7) and agitated on a rotary shaker at 140 rpm. The criterion to evaluate the strength of the bead was to determine the time required for the complete disintegration of beads.
The free cells and cell leaked from the matrix were collected by centrifugation at 3000 rpm for 10 min and dried up to constant weight i.e. at 105[degrees]C for 3 h. The amount of the entrapped cells in alginate was determined as follows: the specified number of alginate beads were taken and washed with distilled water. They were dissolved in 2% (w/v) sodium hexametaphosphate, and then the cell mass was collected by centrifugation and dried .
One milliliter of the 100 fold diluted sample was analyzed using the Lowry method for total Protein in the sample. Bovine serum albumin (Sigma) was used for a standard. Dissolved oxygen was measured using an electrode with a replaceable membrane.
Air Lift Reactor
5L Air lift (Internal) Reactor used for free as well as immobilized whole cell fermentation. Filtered and humidified sterile air was introduced at a rate to maintain dissolved oxygen concentration around 40% or above with appropriate airflow. Silicone oil was added to control foaming whenever necessary.
Results & Discussion
Effect of media composition on fermentation with alginate beads
Alginate gels are known to be easily dissolved in phosphate buffered media. Because chemically defined media prepared for free cells of A. chrysogenum usually contained high concentrations of phosphate as well as other monovalent cations , such as ammonium and sodium, the effects of these monovalent cations on Ca-, Ba- and Sr-alginate gel matrices were investigated in order to produce a modified defined medium for immobilized cells. Measuring bead diameters swollen in each monovalent cation solution quantitatively compared the gel stabilities. As listed in Table 1, all beads seemed to be stable in K[H.sub.2]P[O.sub.4] and N[H.sub.4]Cl solutions, but unstable in [K.sub.2]HP[O.sub.4], (N[H.sub.4])2S[O.sub.4] and [Na.sub.2]S[O.sub.4] solutions, which may be possibly attributed to solvation effect of potassium and ammonium. Thus free cell defined media were modified for the cultivation of immobilized cells as follows: [(N[H.sub.4]).sub.2]S[O.sub.4] was replaced with N[H.sub.4]Cl; on the other hand [Na.sub.2]S[O.sub.4] and [K.sub.2]HP[O.sub.4] were omitted.
Parameters Investigated for Optimization of Alginate Matrix for Maximum CPC Production
Different cationic solutions such as Ba[Cl.sub.2] and Sr[Cl.sub.2] in addition to Ca[Cl.sub.2] were used to prepare the alginate beads for the production of the antibiotic. Among all these after 96 h, which was found to be the optimum time for maximum antibiotic production, calcium alginate beads was observed to produce highest CPC (Fig 1). The strength of the beads was determined by stirring an equal number of beads prepared from different cationic solutions in 0.1M-phosphate buffer. The calcium alginate beads disintegrated within two hours, whereas the barium alginate and strontium alginate beads did not disintegrate until 12 hours. Activities of A. chrysogenum cells entrapment in Ca- Ba- and Sr alginate beads were determined by measuring the amount of total proteins dissolved in culture. The total proteins production rates of Ca-alginate was higher than Ba- and Sr alginate, (Fig. 2) lower protein content in Barium and Strontium alginate beads indicates that [Ba.sup.2+] and [Sr.sup.2+] were inhibitory towards the growth of A. chrysogenum cells. It could be inferred that the gel stability was higher with Ba[Cl.sub.2] and Sr[Cl.sub.2] when compared with Ca[Cl.sub.2]. However, Ca[Cl.sub.2] gave better porous beads, and total protein production rates of Ba- alginate and Sr-alginate were lower than that of Ca-alginate and higher production of CPC was also observed in calcium alginate beads when compared with other cations. The effect of several parameters, such as the effect of alginate concentration, the effect of cation, and its concentration on cephalosporin C production and bead stability were studied. From this study, it may be concluded that the antibiotic titre was reduced with increasing alginate concentration, which may be due to reduced porosity of the beads which limits the nutrient supply and oxygen diffusion.
[FIGURE 1 OMITTED]
The alginate beads were prepared with various concentrations of cation (0.01M, 0.125M, 0.25M, 0.375M, and 0.5M) and used for the production of cephalosporin C. After fermentation, the cephalosporin C titer and disintegration time of beads were determined. The beads prepared with 0.25M cation were found to be relatively more stable and showed better cephalosporin C production. The data indicated that the cation has a significant effect on gelling behavior of alginate (Fig. 1). In order to evaluate the stability of calcium alginate beads, various concentration of alginate (1%, 2%, 3% 4% & 5% w/v) were used for the immobilization of Acremonium chrysogenum cells. The results indicated that the calcium alginate concentration for the immobilization of cells play a prominent role in the production of cephalosporin C. Alginate at 2% w/v was found to be the optimum concentration for formulation of spherical and stable beads with better antibiotic production (Fig. 3). When 0.125M solutions were used, the gelling was relatively slow and it required more time for curing. A central core was observed in-side the bead. At higher concentrations of cation (0.375M) the gelling was instantaneous and a case- hardening effect (gelling outside, leaving slurry as it was inside) was noticed. As the concentration of cation increased, it seems that the microporous structure of the bead was altered. The pore size and number of pores might have decreased. The beads prepared with 0.01M and 0.125M cation solution were irregular in shape, whereas the beads prepared with 0.25M and higher concentrations of cation were spherical in shape. The beads prepared with 0.01M were found to be better antibiotic producers when compared with the beads prepared with other higher concentrations. However, the beads prepared with 0.01M and 0.125M cation had disintegrated at the end of the first cycle itself. The beads prepared with 0.25M cation were found to be relatively more stable and showed better cephalosporin C production.
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
CPC Production with A. chrysogenum in ALR
The CPC and cell mass production profile of free and immobilized cells of A. chrysogenum were investigated in an ALR. The fermentation was conducted for 168 h. The cell mass and CPC titer was determined during the fermentation cycle are evaluated and the results are shown in Fig. 4 for free cells and leaked cell as well as CPC concentrations for immobilized cells are shown in Fig. 5.
[FIGURE 4 OMITTED]
[FIGURE 5 OMITTED]
Cellular growth rate of free cell in ALR: Cell growth rate depend upon oxygen concentration up to a critical point, above which it was independent of oxygen level. The relationship between oxygen level and growth of A. chrysogenum is shown in Fig. 6.
[FIGURE 6 OMITTED]
[FIGURE 7 OMITTED]
Cellular growth of Immobilized Cells in ALR: Growth rate of immobilized cell was found to be lower than that of free cells. This is attributed to the mass transfer limitation of oxygen and limited space to grow. The growth rate of free cells and immobilized cells were 0.045[h.sup.-1] and 0.019[h.sup.-1] respectively at saturation condition of oxygen (0.27mM [O.sub.2]). The cell growth rate of immobilized cells was about 36.4% of that of free cell when oxygen was saturated.
Correlation between CPC production and Oxygen level: Oxygen is required both in Trophophase and Idiophase of A. chrysogrnum . Fig 7 shows specific CPC production rate as a function of oxygen concentration. The production profiles are parallel following a greater slope for the free cell system under condition of lower oxygenation. These results demonstrate oxygen limitation phenomena associated with the immobilized cell system for low oxygen levels.
The experimental effectiveness factor, which is defined as the ratio of CPC production rate of immobilized cells to that of free cells, was 0.6. This means that when A. chrysogenum cells were immobilized in calcium alginate, CPC production rate was not limited as much as cellular growth rate. Specific CPC production of immobilized cells, which was obtained by dividing CPC production rate by cell growth rate, was about 210% of that of free cells at saturated oxygen level. This implies a reduced level of volume handling, which is attractive industrially.
[FIGURE 8 OMITTED]
The results showed that CPC production started by 24 h and reached a maximum titre (1860 mg/l) by 96 h with immobilized cells, while the maximum titre (3100 mg/l) was obtained at 144 h with free cells. From the data, it was observed that CPC production started at 24 h fermentation and reached a maximum titre by 96 h by immobilized cells; upon further incubation, no improvement in CPC production was noticed. The leakage of cell mass increased with fermentation time.
Repeated Batch Fermentation in ALR
The repeated batch fermentations were conducted (every 96 h) using the optimized alginate beads, employing the modified production medium. The fermentation was continued for 36 days until the beads disintegrated. The data showed that the increase in antibiotic production was observed up to the 5th cycle, and later a gradual decrease in antibiotic production was noticed (Fig. 8). Gradual cell leakage was observed with each other.
From the result, it has been concluded that the immobilized cells of A. chrysogenum in calcium alginate are more efficient for the production of CPC with repeated batch fermentation in ALR. Cell growth rate was reduced when cells are immobilized in calcium alginate matrix. However, specific [beta] lactam antibiotic production of immobilized cells was about 210% of that of free cells when oxygen was saturated. This results indicates that immobilization of whole cells of A. chrysogenum is desirable for [beta] lactam antibiotic production since the cell growth rate was severely limited but not the antibiotic production rate.
This research was supported by a grant from the University Grants Commission, India.
 Cruz, AJG. Pan, T. Roberto, C. Giordano. Maria Lucia, Araujo , GC. Hokka, Carlos O. Cephalosporin C production by immobilized Cephalosporium acremonium cells in a repeated batch tower bioreactor. Biotechnology & Bioengineering. 2004, 85, pp. 96-102.
 Nigam, VK.Kundu, S.Ghosh, P. Single-Step Conversion of Cephalosporin-C to 7-Aminocephalosporanic Acid by Free and Immobilized Cells of Pseudomonas diminuta. Applied Biochemistry and Biotechnology. 2005,126 (1), pp. 13-22.
 Kundu, S. Mahapatra ,AC. Nigam, VK. Kundu, K. Continuous Production of Cephalosporin-C by Immobilized Microbial Cells Using Symbiotic Mode in a Packed Bed Bioreactor. Artificial Cells, Blood Substitutes, and Biotechnology (formerly known as Artificial Cells, Blood Substitutes, and Immobilization Biotechnology). 2003, 31 (3), pp. 313-327.
 Ramakrishna, SV. Prakasham, RS. Microbial fermentation with immobilized cells, Curr Sci, 1999, 77, pp. 87-100.
 Guzide, C., Savasu, H., Calik, P., Ozolamar, TH. Growth and carrageenan immobilization of Pseudomonas dacunha cells for L-alanine production. Enzy Microbial Cells. Ann. Rev. Microbial, 1982, 36, pp. 145-172.
 Elibol, M., Ozer, D. Lipase production by immobilized Rhyzopus arrhizus, Process Biochem, 2000, 36, 219-223. Gottschal, JC., Morris, JG. Continuous production of acetone and butanol by Clostridium acetobutylicum growing in turbidostat culture, Biotechnol Lett, 1982, 4, pp. 477-482.
 Beg, QK, Bhusan, B., Kapoor, M, Hoondal, GS. Enhanced production of thermostable xylanase from Streptomyces sp. QG-11-3 and its application in biotechnology of eucalyptus kraft pulp, Enzy. Microbiol Technol, 2000, 27, pp. 459-466.
 Chibata, I., Tosa, T. Use of immobilized cells. Ann. Rev. Biophys Bioengg, 1981, 10, pp. 197-216.
 Kundu, S., Mahapatra, AC., Srivastava, P., and Kundu, K. Studies on Cephalosporin C production using immobilized cells of C. acremonium in packed bed reactor. Process Biochem, 1992, 27, pp. 347-350.
 Johnsen A & Flink JM, Influence of alginate properties and gel reinforcement on fermentation characteristics of immobilized yeast cells, Enz Micrb Technol, 1986, 8, pp. 737-748.
 Boxer, GE & Everet,P. Cephalosporin C estimation by hydroxylamine assay method. Analytical Chemistry. 1949, 21, pp. 670-678.
 Mahmoud W, Rehm HJ. Chlortetracycline production with immobilized Streptomyces aureofaciens. I. Batch culture. Appl Microbiol Biotechnol.1987, 26, pp. 333-337.
 Demain, AL and Wolfe, S. In: Developments in Industrial Microbiology, G. Pierce, ed., New York, Elsevier Science Press, 1985, 27, pp. 175-182.
Punita Mishra, Pradeep Srivastava * and Subir Kundu School of Biochemical Engineering Institute of Technology, Banaras, Hindu University, Varanasi, 221005, U.P., India E-mail: firstname.lastname@example.org
Table 1: Diameter of alginate bead swollen in monovalent cation solutions for evaluation of Modified Production media Bead Diameter (mm) in Bead K[H.sub.2]P[O.sub.4] [K.sub.2]HP[O.sub.4] N[H.sub.4]Cl Ba-alg 2.59 [+ or -] 0.27 D 2.81 [+ or -] 0.1 Ca-alg 3.15 [+ or -] 0.15 D 2.80 [+ or -] .06 Sr-alg 3.07 [+ or -] 0.15 D 3.14 [+ or -] .03 Bead Diameter (mm) in Bead [Na.sub.2]S[O.sub.4] (N[H.sub.4])2SO4 N[H.sub.4]Cl Ba-alg D D 2.81 [+ or -] 0.1 Ca-alg 4.01 [+ or -] .09 3.76 [+ or -] .15 2.80 [+ or -] .06 Sr-alg 4.90 [+ or -] .07 5.46 [+ or -] .23 3.14 [+ or -] .03 * Abbreviations: D, completely dissolved beads.
|Printer friendly Cite/link Email Feedback|
|Title Annotation:||cephalosporin C|
|Author:||Mishra, Punita; Srivastava, Pradeep; Kundu, Subir|
|Publication:||International Journal of Biotechnology & Biochemistry|
|Date:||Jan 1, 2007|
|Previous Article:||Molecular identification of a virus causing banana chlorosis disease from Marathwada region.|
|Next Article:||Isolation and cloning of cDNA for both IGF-1 R and FSHR genes from mature bovine oocytes of using a few numbers of cells.|
|Cell immobilization technology facilitates meat fermentation.|
|Immobilize cells to facilitate meat fermentation.|
|Immobilized yeast shortens beer production time.|
|Carrier-bound immobilized enzymes; principles, applications and design.|