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Repeated fed-batch process for improving lovastatin production
Process Biochemistry 36 (2000) 363 – 368 www.elsevier.com/locate/procbio
Repeated fed-batch process for improving lovastatin production M. Sitaram Kumar *, Swapan K. Jana, V. Senthil, V. Shashanka, S. Vijay Kumar, A.K. Sadhukhan Fermentation Laboratory, Biotechnology R&D, Dr Reddy’s Research Foundation, Bollaram Road, Miyapur, Hyderabad 500 050, India Received 19 April 2000; received in revised form 18 July 2000; accepted 27 July 2000
Abstract Submerged cultivation of a high yielding strain of Aspergillus terreus DRCC 122 for the production of lovastatin in the batch process had limited success with a maximum titre of 1270 mg l − 1 in 288 h and an overall volumetric productivity of 4.41 mg l − 1 h − 1 in a 1000 l bioreactor. A cost effective repeated fed-batch process with maltodextrin and corn steep liquor feed as carbon and nitrogen sources, respectively, showed a significant increase in lovastatin yield. The final titre was 2200 mg l − 1 in 288 h of fermentation, with overall volumetric productivity of 7.64 mg l − 1 h − 1, showing an increase of 73% over the batch process. The kinetic parameters were studied; maximum specific oxygen uptake rate (QO2) and volumetric mass transfer coefficient (KLa) were 0.24 m mole O2 g − 1 dry cell wt. h − 1 and 280 h − 1, respectively, in fed-batch process. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: Lovastatin; Fed-batch process; Oxygen transfer; Submerged cultivation; Volumetric productivity; Aspergillus terreus
1. Introduction Lovastatin (Mevinolin, Monacolin K and Mevacor®) is the first compound of its kind to become available for treatment of hypercholesterolemia [14]. This fungal secondary metabolite is produced by Aspergillus terreus [1], Monascus ruber [5], and Penicillium species [6]. The role of hypercholesterolemia as a risk factor for atherosclerosis, and ischemic heart disease was indicated by the clinical, epidemiologic and pathologic studies [14]. Lovastatin and related compounds inhibit cholesterol synthesis by inhibiting the rate limiting step in cholesterol biosynthesis, namely the conversion of hydroxymethyl glutaryl coenzyme A (HMG-CoA) into mevalonate, catalyzed by HMG-CoA reductase [3,2,5 – 7,13]. Lovastatin is a b-hydroxy lactone while the active form is the corresponding b-hydroxy acid. The lovastatin titre of the parent strain A. terreus ATCC 20541 was poor (150 mg l − 1). Continuous improvement in productivity of an industrial microorganism is essential for the commercial success of a fermentation process. In addition to im* Corresponding author. Tel.: +91-40-3045439; fax: + 91-403045438. E-mail address: [email protected] (M.S. Kumar).
provement of yield, maintenance of desired morphological characteristics and elimination of unwanted co-metabolites are the goals of an industrial strain improvement programme [15]. A classical strain improvement through random mutation and protoplast isolation [11] was attempted for short-term strain development. Productivity in the original media [1] with glucose as the main carbon source was not satisfactory. Media optimization was carried out following the Plackett–Burman system [9]. In our experiments, a combination of glucose, starch and maltodextrin were found as the best carbon sources for lovastatin production. In this paper we combine the above factors, i.e. strain improvement, seed stabilization, media optimization along with fed-batch addition of carbon and nitrogen sources in order to improve the overall titre of lovastatin.
2. Materials and methods
2.1. Organism, seed inoculum, media and culture conditions Strain improvement programme was initiated on A. terreus ATCC 20541 by random mutation. Spores of A.
0032-9592/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 0 ) 0 0 2 2 2 - 3
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M.S. Kumar et al. / Process Biochemistry 36 (2000) 363–368
terreus ATCC 20541 were exposed to EMS followed by UV, NTG followed by UV at 254 nm for 10–20 min at a distance of 10 cms. After exposure the spores were diluted and pour plated. The plates were covered in black polythene bags and incubated at 28°C for 5 days. Colonies from plates having up to 90% kill were selected for further isolation. Primaries screening of the strains were carried out using a novel agar plug method [12]. Strains with improved yields were further subjected to a series of treatments as described above followed by monospore isolation of selected cultures. The high yielding strain was designated as DRCC 122 (Dr Reddy’s Culture Collection) and all subsequent experiments were carried out using A. terreus DRCC 122. Fig. 1 indicates the genealogy of the strain Primary seed inoculum was prepared in nutrient broth having the following composition (g l − 1), 5 peptone; 1.5 yeast extract; 1.5 beef extract; 5 NaCl; 10 ml trace element solution (composition of trace elements solution as per [1] and 1.25 ml polypropylene glycol 2000, 1.5 l of the above medium contained in a 5 l non-baffled aspirator bottle was inoculated with 3.5×109 spores. The aspirator bottles were incubated at 289 0.5°C for 22 – 24 h on a rotary shaker at 220 rpm. Growth was measured as percentage packed cell volume (PCV). The PCV was 10 – 15% (v/v) and pH was in the range 6.9 – 7.2. Secondary seed was developed for 24 – 26 h in 150 l SS bioreactor with on-line control of dissolved oxygen (DO2), pH and temperature. Ten percent (v/v) of primary seed developed in aspirator bottles were transferred to 60 l of the medium of the above
composition with the following process parameters. Fermentation was carried out with aeration 0.6–0.75 vvm, agitation 150–165 rpm, pH 6.5–7.2, DO2 ]40% of air saturation and temperature 2890.5°C. pH was automatically controlled by dosing either with 2 M NaOH or 2 M H2SO4. The pH of final seed was 7.1–7.2 and PCV 14–16% (v/v). Six percent (v/v) of secondary seed was aseptically transferred to 775 l of production media in a 1000 l SS bioreactor. The composition of the production media was as follows (g l − 1), 15 glucose; 32 maltodextrin; 20 starch; 25 peptonized milk; 2.5 yeast extract; 5 corn steep liquor (wet wt.); 1.5 ml polypropylene glycol as antifoaming agent. Stock solution of 50% maltodextrin and 37.5% corn steep liquor were used separately as the feeding substrate in the fed-batch process. An indigenously designed 1000 l (Dt = 780 mm) bioreactor was equipped with four baffles, three six-bladed Rushton turbines (di = 320 mm) with automatic control systems for temperature, DO2, back pressure and pH. The process parameters for production were as follows, temperature 2890.5°C; pH 5.8–6.3 maintained by adding 4 M NaOH or 2 M H2SO4, dissolved oxygen concentration (DO2)] 40% of air saturation was maintained by combination of aeration (Qp = 0.6–1 vvm) and agitation (Ni = 80–120 rpm, tip speed= 1.34–2.0 m s − 1) and head pressure of 0.3–0.5 bar. The course of fermentation was constantly monitored by off-line sampling at 12 h intervals. Each sample was subjected to tests for sterility, reducing sugars, total sugars, total fermentable nitrogen, growth (measured as percent PCV) and lovastatin titre.
3. Analytical methods
Fig. 1. Genealogy of A. terreus DRCC 122.
Growth/cell mass concentration was determined by centrifuging 10 ml of the sample in a graduated centrifuge tube at 2000 rpm for 10 min. The cell mass was washed twice with distilled water and centrifuged again and expressed as percent PCV. Reducing sugars were spectrophotometrically determined by dinitrosalicylic acid method [8]. Total sugar was determined by a phenol–sulphuric acid method [4]. Total nitrogen was determined by Kjeldahl method using a semi-automated Kjeldahl apparatus (M/s Buchi, Switzerland). Lovastatin was estimated by high performance liquid chromatography (HPLC). The broth sample was mixed with an equal volume of ethyl acetate and stirred on a rotary shaker at 220 rpm. After 2 h, the entire contents were filtered under vacuum and the residual mass was washed three to four times with ethyl acetate. The organic and aqueous phases were separated in a separating funnel. The volume of the organic phase was
M.S. Kumar et al. / Process Biochemistry 36 (2000) 363–368
365
Fig. 2. Kinetics of lovastatin production in batch process using A. terreus DRCC 122.
measured and recorded. The organic phase (20 ml) was dried in a Rotavapor (model, Buchi 461) under vacuum at 45°C. The dried residue was dissolved with a mobile phase having the following composition, acetonitrile (HPLC grade) 55%, methanol (HPLC grade) 12% and phosphate buffer (pH 4.0) 33%. HPLC was carried out in Perkin Elmer (series 200 pump, detector, Waters 486; integrater, Perkin Elmer 1022) using 250× 4.6 mm ID column packed with Hypersil BDF C18 of 5 mm particle size and thermostated at 30°C; flow rate of the mobile phase was 1 ml min − 1. An internal standard was incorporated to confirm the efficiency of the extraction process and the reproducibility and accuracy of the analytical procedure. A known quantity of the standard lovastatin was added to 100 ml of culture broth devoid of lovastatin. Extraction was done as per the above
mentioned procedure. HPLC estimation of the samples was carried out in triplicate to validate the reproducibility of assay method.
4. Results and discussion
4.1. Batch fermentation Carbon and nitrogen sources were central to fermentation of secondary metabolites, not only should they take care of growth and production but cost factors play a major role in their selection. Various C and N sources were examined for their effectiveness in increasing lovastatin titre (data not shown). Use of readily consumable C and N sources like glucose and peptone
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M.S. Kumar et al. / Process Biochemistry 36 (2000) 363–368
led to uncontrolled filamentous type growth of the culture within 24–48 h of fermentation, increase in viscosity, drastic drop of DO2, very poor mass transfer and low lovastatin titres. This promoted a search for alternative, slowly metabolizable, cheap C and N sources. Maltodextrin and Corn Steep liquor were found to be the best, not only at arresting uncontrolled growth but established the required pelletal morphology. Based on these experiments in pilot scale, free elongated mycelial growth resulted in poor lovastatin yield. Ideally small, compact pellets with short peripheral mycelia was a prerequisite for production of high titres of lovastatin. Increasing the concentration of the maltodextrin and corn steep liquor at the initial batch medium also gave adverse results, leading to the possibility of fed-batch methodology. To our knowledge there have been no reports of using maltodextrin and corn steep liquor in lovas-
tatin fermentation. Fig. 2 shows the kinetics of a batch fermentation in a 1000 l bioreactor. The maximum titre of lovastatin was 1270 mg l − 1 at 288 h and the corresponding overall volumetric productivity was 4.41 mg l − 1 h − 1. It was also observed that the rate of lovastatin biosynthesis in 12 h varied at different time intervals. Although there was a sudden drop in the utilization of both sugar and total nitrogen up to 96 h, the rate of consumption of these nutrients slowed down thereafter and remained constant throughout the fermentation cycle. The PCV reached a peak at 53% and thereafter remained constant at 50% throughout the batch. The corresponding DO2 dropped to approximately 45% of air saturation and then gradually increased and was maintained at 60– 70% of air saturation throughout the process. Initial pH of the cultivation process was 6.1, which dropped to 5.7 and maintained between 5.7 and 6.5.
Fig. 3. Kinetics of lovastatin production in fed-batch process using A. terreus DRCC 122.
M.S. Kumar et al. / Process Biochemistry 36 (2000) 363–368
367
Fig. 4. Comparison of volumetric productivity (mg l − 1 h − 1) of batch and fed-batch processes (A) and kinetic parameters of specific oxygen uptake rate and (m mole O2 g − 1 dry cell wt. h − 1) and volumetric mass transfer coefficient (h − 1) for fed-batch process (B).
4.2. Fed-batch fermentation In the fed-batch fermentation (Fig. 3), the maltodextrin feed was commenced from 72 h (indicates feed addition in Fig. 3) when the total sugar concentration of the broth dropped to 18 g l − 1. Maltodextrin was added at 5 h intervals in order to increase the total sugar concentration by approximately 0.377 g l − 1 h − 1. From 72 h onwards the rate of total sugar consumption was 0.445 g l − 1 h − 1. The feed strategy was designed such that the total sugar in the broth gradually reduced, so that at 12 h before the termination of fermentation it was kept at a minimal concentration. Similarly, addition of corn steep liquor (pH adjusted to 6 before sterilization) was commenced at 72 h and continued till 144 h at intervals of 24 h, to raise the total nitrogen of broth by approximately 90 mg l − 1. The feeding strategy was changed afterwards with half of the initial dose, at 168, 180, 192 and 216 h, respectively. The above feeding profile was found optimum, because maintaining a higher concentra-
tion of total sugar (18 g l − 1) and corn steep liquor (90 mg l − 1) from 72 h onwards to the end of fermentation did not improve lovastatin production (data not shown). Other operating parameters, e.g. pH, DO2, temperature etc. were same in both batch and fed-batch processes. The PCV increased to 45% (v/v) and then remained constant at 42% (v/v) until the end of the process. DO2 decreased to 25% of air saturation, gradually increased and was maintained between 60 and 70% of air saturation throughout the process. The initial pH was 6.2 and was then subsequently maintained between 6.0 and 6.7 until the end. The final titre of lovastatin in the fed-batch was 2000 mg l − 1. Additional maltodextrin and corn steep liquor supplemented in the fed-batch where 7.74 and 1.74%, respectively. The fed-batch fermentation was continued until 288 h; no significant increase in lovastatin titre was observed thereafter. An increased productivity in a reduced time B37.5% was achieved as compared with Novak et al. where the fermentation time was 396 h [10].
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Overall volumetric productivity was 7.64 mg l − 1 h in fed-batch as compared with 4.41 mg l − 1 h − 1 in batch fermentation (Fig. 4A). The volumetric produc-tivity (mg l − 1 h − 1) in fed-batch sharply increased and reached a peak at 132 h thereafter the productivity was constant up to 216 h followed by a decline. In the batch process, it followed the same pattern, however the volumetric productivity was less when compared with fed-batch, this may be due to the insufficient availability of both carbon and nitrogen sources. The profile of the kinetic parameters, specific oxygen uptake rate (QO2) and volumetric mass transfer coefficient (KLa) are shown in Fig. 4B. At low biomass concentrations the volumetric O2 uptake rate was low even though the cells may be respiring at their maximum specific O2 uptake rate and then QO2 (m mole O2 g − 1 dry cell wt. h − 1) gradually decreased with time and reached to minimum at 72 h. Thereafter, it maintains a constancy of specific oxygen uptake rate — suggesting that the metabolic activity of the cells remained constant, as cell mas was more or less constant. The maximum KLa was 280 (h − 1) at 24 h of fermentation.
References
−1
5. Conclusion The present study shows an appreciable increase of lovastatin by intermittent feeding of maltodextrin and corn steep liquor as carbon and nitrogen sources, respectively. A significant increase (73%) in production of lovastatin was achieved in 288 h in repeated fedbatch process as compared with batch process. By fine tuning of feeding strategy, there may be a scope for further enhancement of the lovastatin titre.
Acknowledgements We are thankful to Hemant Sarnaik and P.V. Prashanti for media preparation and D. Srinivasa Rao for analytical support. We are also thankful to Dr A. Venkateswarlu, President, DRF for the encouragement.
.
[1] Alberts AW, Chen J, Kuron G, Hunt V, Huff J, Hoffman C, Rothrock J, Lopez M, Joshua H, Harris E, Patchett A, Monaghan R, Currie S, Stapley E, Albers-Schonberg G, Hensens O, Hirshchfield J, Hoogsteen K, Liesch J, Springer J. Mevinolin: a highly potent competitive inhibitor of hydroxymethyl glutaryl-coenzyme A reductase and a cholesterol-lowering agent. Proc Natl Acad Sci USA 1980;77:3957 – 61. [2] Brown MS, Faust JR, Goldstein JL, Kaneko I, Endo A. Induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in human firboblasts incubated with copactin (ML-236 B), a competitive inhibitor of the reductase. J Biol Chem 1978;253:1121–8. [3] Buckland B, Gbewonyo K, Hallada T, Kaplan L, Masurekar P. Production of lovastatin, an inhibitor of cholesterol accumulation in humans. In: Demain AL, Somkuti GA, Hunter-Cevera, JC, Rossmoore HW, editors. Novel Microbial Products for Medicine and Agriculture. Amsterdam, 1989. pp. 161 – 69. [4] Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal Chem 1956;28:350 – 6. [5] Endo A, Kuroda M, Tanazawa K. Competitive inhibition of 3-hydroxy-3-methyl glutaryl coenzyme A reductase by ML-236 A and ML-236 B fungal metabolites having hypocholesterolemic activity. FEBS Lett 1976;72:323 – 6. [6] Endo A, Kuroda M, Tsujita Y. ML-236 A, ML-236 B and ML-236 C, new inhibitors of cholesterogenesis produced by Penicillium citrinum. J Antibiot 1976;29:1346 – 8. [7] Endo A. Monacolin K, a new hypocholesterolemic agent produced by a Monascus spp. J Antibiot 1979;32:852 – 4. [8] Miller GL. Use of DNS reagent for determination of reducing sugar. Anal Chem 1959;31:426 – 8. [9] Monaghan RL, Koupal LR. Use of the Plackett and Burman technique in a discovery program for new natural products. In: Demain AL, Somkuti GA, Hunter-Cevera JC, Rossmoore HW, editors. Novel Microbial products for Medicine and Agriculture. Amsterdam, 1989. pp. 161 – 69. [10] Novak N, Gerdin S, Berovic M. Increased lovastatin formation by Aspergillus terreus using repeated fed-batch process. Biotechnol Lett 1997;19:947 – 8. [11] Peberdy JF, Gibson RK. Regeneration of Aspergillus nidulans protoplasts. J Gen Microbiol 1971;69:325 – 30. [12] Kumar SM, Kumar PM, Sarnaik HM, Sadhukhan AK. A rapid technique for screening of lovastatin producing strains of Aspergillus terreus by agar plug and Neurospora crassa bioassay. J Microbiol Methods 2000;40:99 – 104. [13] Tanazawa K, Endo A. Kinetic analysis of the reaction catalyzed by rat-liver 3-hydroxy-3-methylglutaryl coenzyme A reductase using two specific inhibitor. Eur J Biochem 1979;98:195–201. [14] Tobert JA. New development in lipid lowering therapy: the role of inhibitors of hydroxymethyl glutaryl co-enzyme A reductase. Circulation 1987;76:534 – 8. [15] Vinci VA, Hoerner TD, Coffman AD, Schimmel TG, Dabora RL, Kirpekar AC, Ruby CL, Stieber RW. Mutants of a lovastatin hyper producing Aspergillus terreus deficient in the production of sulochrin. J Ind Microbiol 1991;8:113 – 20.
Repeated fed-batch process for improving lovastatin production M. Sitaram Kumar *, Swapan K. Jana, V. Senthil, V. Shashanka, S. Vijay Kumar, A.K. Sadhukhan Fermentation Laboratory, Biotechnology R&D, Dr Reddy’s Research Foundation, Bollaram Road, Miyapur, Hyderabad 500 050, India Received 19 April 2000; received in revised form 18 July 2000; accepted 27 July 2000
Abstract Submerged cultivation of a high yielding strain of Aspergillus terreus DRCC 122 for the production of lovastatin in the batch process had limited success with a maximum titre of 1270 mg l − 1 in 288 h and an overall volumetric productivity of 4.41 mg l − 1 h − 1 in a 1000 l bioreactor. A cost effective repeated fed-batch process with maltodextrin and corn steep liquor feed as carbon and nitrogen sources, respectively, showed a significant increase in lovastatin yield. The final titre was 2200 mg l − 1 in 288 h of fermentation, with overall volumetric productivity of 7.64 mg l − 1 h − 1, showing an increase of 73% over the batch process. The kinetic parameters were studied; maximum specific oxygen uptake rate (QO2) and volumetric mass transfer coefficient (KLa) were 0.24 m mole O2 g − 1 dry cell wt. h − 1 and 280 h − 1, respectively, in fed-batch process. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: Lovastatin; Fed-batch process; Oxygen transfer; Submerged cultivation; Volumetric productivity; Aspergillus terreus
1. Introduction Lovastatin (Mevinolin, Monacolin K and Mevacor®) is the first compound of its kind to become available for treatment of hypercholesterolemia [14]. This fungal secondary metabolite is produced by Aspergillus terreus [1], Monascus ruber [5], and Penicillium species [6]. The role of hypercholesterolemia as a risk factor for atherosclerosis, and ischemic heart disease was indicated by the clinical, epidemiologic and pathologic studies [14]. Lovastatin and related compounds inhibit cholesterol synthesis by inhibiting the rate limiting step in cholesterol biosynthesis, namely the conversion of hydroxymethyl glutaryl coenzyme A (HMG-CoA) into mevalonate, catalyzed by HMG-CoA reductase [3,2,5 – 7,13]. Lovastatin is a b-hydroxy lactone while the active form is the corresponding b-hydroxy acid. The lovastatin titre of the parent strain A. terreus ATCC 20541 was poor (150 mg l − 1). Continuous improvement in productivity of an industrial microorganism is essential for the commercial success of a fermentation process. In addition to im* Corresponding author. Tel.: +91-40-3045439; fax: + 91-403045438. E-mail address: [email protected] (M.S. Kumar).
provement of yield, maintenance of desired morphological characteristics and elimination of unwanted co-metabolites are the goals of an industrial strain improvement programme [15]. A classical strain improvement through random mutation and protoplast isolation [11] was attempted for short-term strain development. Productivity in the original media [1] with glucose as the main carbon source was not satisfactory. Media optimization was carried out following the Plackett–Burman system [9]. In our experiments, a combination of glucose, starch and maltodextrin were found as the best carbon sources for lovastatin production. In this paper we combine the above factors, i.e. strain improvement, seed stabilization, media optimization along with fed-batch addition of carbon and nitrogen sources in order to improve the overall titre of lovastatin.
2. Materials and methods
2.1. Organism, seed inoculum, media and culture conditions Strain improvement programme was initiated on A. terreus ATCC 20541 by random mutation. Spores of A.
0032-9592/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 0 ) 0 0 2 2 2 - 3
364
M.S. Kumar et al. / Process Biochemistry 36 (2000) 363–368
terreus ATCC 20541 were exposed to EMS followed by UV, NTG followed by UV at 254 nm for 10–20 min at a distance of 10 cms. After exposure the spores were diluted and pour plated. The plates were covered in black polythene bags and incubated at 28°C for 5 days. Colonies from plates having up to 90% kill were selected for further isolation. Primaries screening of the strains were carried out using a novel agar plug method [12]. Strains with improved yields were further subjected to a series of treatments as described above followed by monospore isolation of selected cultures. The high yielding strain was designated as DRCC 122 (Dr Reddy’s Culture Collection) and all subsequent experiments were carried out using A. terreus DRCC 122. Fig. 1 indicates the genealogy of the strain Primary seed inoculum was prepared in nutrient broth having the following composition (g l − 1), 5 peptone; 1.5 yeast extract; 1.5 beef extract; 5 NaCl; 10 ml trace element solution (composition of trace elements solution as per [1] and 1.25 ml polypropylene glycol 2000, 1.5 l of the above medium contained in a 5 l non-baffled aspirator bottle was inoculated with 3.5×109 spores. The aspirator bottles were incubated at 289 0.5°C for 22 – 24 h on a rotary shaker at 220 rpm. Growth was measured as percentage packed cell volume (PCV). The PCV was 10 – 15% (v/v) and pH was in the range 6.9 – 7.2. Secondary seed was developed for 24 – 26 h in 150 l SS bioreactor with on-line control of dissolved oxygen (DO2), pH and temperature. Ten percent (v/v) of primary seed developed in aspirator bottles were transferred to 60 l of the medium of the above
composition with the following process parameters. Fermentation was carried out with aeration 0.6–0.75 vvm, agitation 150–165 rpm, pH 6.5–7.2, DO2 ]40% of air saturation and temperature 2890.5°C. pH was automatically controlled by dosing either with 2 M NaOH or 2 M H2SO4. The pH of final seed was 7.1–7.2 and PCV 14–16% (v/v). Six percent (v/v) of secondary seed was aseptically transferred to 775 l of production media in a 1000 l SS bioreactor. The composition of the production media was as follows (g l − 1), 15 glucose; 32 maltodextrin; 20 starch; 25 peptonized milk; 2.5 yeast extract; 5 corn steep liquor (wet wt.); 1.5 ml polypropylene glycol as antifoaming agent. Stock solution of 50% maltodextrin and 37.5% corn steep liquor were used separately as the feeding substrate in the fed-batch process. An indigenously designed 1000 l (Dt = 780 mm) bioreactor was equipped with four baffles, three six-bladed Rushton turbines (di = 320 mm) with automatic control systems for temperature, DO2, back pressure and pH. The process parameters for production were as follows, temperature 2890.5°C; pH 5.8–6.3 maintained by adding 4 M NaOH or 2 M H2SO4, dissolved oxygen concentration (DO2)] 40% of air saturation was maintained by combination of aeration (Qp = 0.6–1 vvm) and agitation (Ni = 80–120 rpm, tip speed= 1.34–2.0 m s − 1) and head pressure of 0.3–0.5 bar. The course of fermentation was constantly monitored by off-line sampling at 12 h intervals. Each sample was subjected to tests for sterility, reducing sugars, total sugars, total fermentable nitrogen, growth (measured as percent PCV) and lovastatin titre.
3. Analytical methods
Fig. 1. Genealogy of A. terreus DRCC 122.
Growth/cell mass concentration was determined by centrifuging 10 ml of the sample in a graduated centrifuge tube at 2000 rpm for 10 min. The cell mass was washed twice with distilled water and centrifuged again and expressed as percent PCV. Reducing sugars were spectrophotometrically determined by dinitrosalicylic acid method [8]. Total sugar was determined by a phenol–sulphuric acid method [4]. Total nitrogen was determined by Kjeldahl method using a semi-automated Kjeldahl apparatus (M/s Buchi, Switzerland). Lovastatin was estimated by high performance liquid chromatography (HPLC). The broth sample was mixed with an equal volume of ethyl acetate and stirred on a rotary shaker at 220 rpm. After 2 h, the entire contents were filtered under vacuum and the residual mass was washed three to four times with ethyl acetate. The organic and aqueous phases were separated in a separating funnel. The volume of the organic phase was
M.S. Kumar et al. / Process Biochemistry 36 (2000) 363–368
365
Fig. 2. Kinetics of lovastatin production in batch process using A. terreus DRCC 122.
measured and recorded. The organic phase (20 ml) was dried in a Rotavapor (model, Buchi 461) under vacuum at 45°C. The dried residue was dissolved with a mobile phase having the following composition, acetonitrile (HPLC grade) 55%, methanol (HPLC grade) 12% and phosphate buffer (pH 4.0) 33%. HPLC was carried out in Perkin Elmer (series 200 pump, detector, Waters 486; integrater, Perkin Elmer 1022) using 250× 4.6 mm ID column packed with Hypersil BDF C18 of 5 mm particle size and thermostated at 30°C; flow rate of the mobile phase was 1 ml min − 1. An internal standard was incorporated to confirm the efficiency of the extraction process and the reproducibility and accuracy of the analytical procedure. A known quantity of the standard lovastatin was added to 100 ml of culture broth devoid of lovastatin. Extraction was done as per the above
mentioned procedure. HPLC estimation of the samples was carried out in triplicate to validate the reproducibility of assay method.
4. Results and discussion
4.1. Batch fermentation Carbon and nitrogen sources were central to fermentation of secondary metabolites, not only should they take care of growth and production but cost factors play a major role in their selection. Various C and N sources were examined for their effectiveness in increasing lovastatin titre (data not shown). Use of readily consumable C and N sources like glucose and peptone
366
M.S. Kumar et al. / Process Biochemistry 36 (2000) 363–368
led to uncontrolled filamentous type growth of the culture within 24–48 h of fermentation, increase in viscosity, drastic drop of DO2, very poor mass transfer and low lovastatin titres. This promoted a search for alternative, slowly metabolizable, cheap C and N sources. Maltodextrin and Corn Steep liquor were found to be the best, not only at arresting uncontrolled growth but established the required pelletal morphology. Based on these experiments in pilot scale, free elongated mycelial growth resulted in poor lovastatin yield. Ideally small, compact pellets with short peripheral mycelia was a prerequisite for production of high titres of lovastatin. Increasing the concentration of the maltodextrin and corn steep liquor at the initial batch medium also gave adverse results, leading to the possibility of fed-batch methodology. To our knowledge there have been no reports of using maltodextrin and corn steep liquor in lovas-
tatin fermentation. Fig. 2 shows the kinetics of a batch fermentation in a 1000 l bioreactor. The maximum titre of lovastatin was 1270 mg l − 1 at 288 h and the corresponding overall volumetric productivity was 4.41 mg l − 1 h − 1. It was also observed that the rate of lovastatin biosynthesis in 12 h varied at different time intervals. Although there was a sudden drop in the utilization of both sugar and total nitrogen up to 96 h, the rate of consumption of these nutrients slowed down thereafter and remained constant throughout the fermentation cycle. The PCV reached a peak at 53% and thereafter remained constant at 50% throughout the batch. The corresponding DO2 dropped to approximately 45% of air saturation and then gradually increased and was maintained at 60– 70% of air saturation throughout the process. Initial pH of the cultivation process was 6.1, which dropped to 5.7 and maintained between 5.7 and 6.5.
Fig. 3. Kinetics of lovastatin production in fed-batch process using A. terreus DRCC 122.
M.S. Kumar et al. / Process Biochemistry 36 (2000) 363–368
367
Fig. 4. Comparison of volumetric productivity (mg l − 1 h − 1) of batch and fed-batch processes (A) and kinetic parameters of specific oxygen uptake rate and (m mole O2 g − 1 dry cell wt. h − 1) and volumetric mass transfer coefficient (h − 1) for fed-batch process (B).
4.2. Fed-batch fermentation In the fed-batch fermentation (Fig. 3), the maltodextrin feed was commenced from 72 h (indicates feed addition in Fig. 3) when the total sugar concentration of the broth dropped to 18 g l − 1. Maltodextrin was added at 5 h intervals in order to increase the total sugar concentration by approximately 0.377 g l − 1 h − 1. From 72 h onwards the rate of total sugar consumption was 0.445 g l − 1 h − 1. The feed strategy was designed such that the total sugar in the broth gradually reduced, so that at 12 h before the termination of fermentation it was kept at a minimal concentration. Similarly, addition of corn steep liquor (pH adjusted to 6 before sterilization) was commenced at 72 h and continued till 144 h at intervals of 24 h, to raise the total nitrogen of broth by approximately 90 mg l − 1. The feeding strategy was changed afterwards with half of the initial dose, at 168, 180, 192 and 216 h, respectively. The above feeding profile was found optimum, because maintaining a higher concentra-
tion of total sugar (18 g l − 1) and corn steep liquor (90 mg l − 1) from 72 h onwards to the end of fermentation did not improve lovastatin production (data not shown). Other operating parameters, e.g. pH, DO2, temperature etc. were same in both batch and fed-batch processes. The PCV increased to 45% (v/v) and then remained constant at 42% (v/v) until the end of the process. DO2 decreased to 25% of air saturation, gradually increased and was maintained between 60 and 70% of air saturation throughout the process. The initial pH was 6.2 and was then subsequently maintained between 6.0 and 6.7 until the end. The final titre of lovastatin in the fed-batch was 2000 mg l − 1. Additional maltodextrin and corn steep liquor supplemented in the fed-batch where 7.74 and 1.74%, respectively. The fed-batch fermentation was continued until 288 h; no significant increase in lovastatin titre was observed thereafter. An increased productivity in a reduced time B37.5% was achieved as compared with Novak et al. where the fermentation time was 396 h [10].
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Overall volumetric productivity was 7.64 mg l − 1 h in fed-batch as compared with 4.41 mg l − 1 h − 1 in batch fermentation (Fig. 4A). The volumetric produc-tivity (mg l − 1 h − 1) in fed-batch sharply increased and reached a peak at 132 h thereafter the productivity was constant up to 216 h followed by a decline. In the batch process, it followed the same pattern, however the volumetric productivity was less when compared with fed-batch, this may be due to the insufficient availability of both carbon and nitrogen sources. The profile of the kinetic parameters, specific oxygen uptake rate (QO2) and volumetric mass transfer coefficient (KLa) are shown in Fig. 4B. At low biomass concentrations the volumetric O2 uptake rate was low even though the cells may be respiring at their maximum specific O2 uptake rate and then QO2 (m mole O2 g − 1 dry cell wt. h − 1) gradually decreased with time and reached to minimum at 72 h. Thereafter, it maintains a constancy of specific oxygen uptake rate — suggesting that the metabolic activity of the cells remained constant, as cell mas was more or less constant. The maximum KLa was 280 (h − 1) at 24 h of fermentation.
References
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5. Conclusion The present study shows an appreciable increase of lovastatin by intermittent feeding of maltodextrin and corn steep liquor as carbon and nitrogen sources, respectively. A significant increase (73%) in production of lovastatin was achieved in 288 h in repeated fedbatch process as compared with batch process. By fine tuning of feeding strategy, there may be a scope for further enhancement of the lovastatin titre.
Acknowledgements We are thankful to Hemant Sarnaik and P.V. Prashanti for media preparation and D. Srinivasa Rao for analytical support. We are also thankful to Dr A. Venkateswarlu, President, DRF for the encouragement.
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