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Scaling up for the industrial production of rifamycin B; optimization of the process conditions in bench-scale fermentor
Bulletin of Faculty of Pharmacy, Cairo University (2013) 51, 43–48
Cairo University
Bulletin of Faculty of Pharmacy, Cairo University www.elsevier.com/locate/bfopcu www.sciencedirect.com
ORIGINAL ARTICLE
Scaling up for the industrial production of rifamycin B; optimization of the process conditions in bench-scale fermentor Hewaida F. El-Sedawy a, Manal M.M. Hussein b, Tamer Essam Ossama M. El-Tayeb b, Fatma H.A. Mohammad c a b c
a,b,*
,
Microbial Biotechnology Center, Faculty of Pharmacy, Cairo University, Cairo, Egypt Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo, Egypt Microbial Chemical Engineering and Pilot Plant Department, National Research Center, Egypt
Received 28 January 2013; accepted 19 February 2013 Available online 28 March 2013
KEYWORDS Amycolatopsis mediterranei; Antibiotics; Fermentor; Rifamycin B; Scaling up
Abstract Optimization of fermentation process conditions using a gene amplified variant of Amycolatopsis mediterranei (NCH) was carried out. The use of aeration level 1.5 vvm increased the yield by 16.6% (from 13.81 to 16.1 g/l) upon controlling the temperature at 28 C. Adjustment of the aeration level at 1.5 vvm for 3 days then controlling the dissolved oxygen (DO) at 30% saturation further increased the yield to 17.8 g/l. The optimum pH was 6.5 for 3 days then 7 thereafter when a production yield of 16.1 g/l was recorded using an aeration rate of 1.5 vvm. Controlling the pH at constant value (6.5 or 7) all through the fermentation process decreased the yield by 5–21%. Controlling the temperature at 30 C for 3 days then 28 C thereafter slightly increased the yield by 5% upon using an aeration rate of 1 vvm while it decreased upon using an aeration rate of 1.5 vvm. Integration of the most optimum conditions increased the production yield by 22% from 13.81 to 17.8 g/l. ª 2013 Production and hosting by Elsevier B.V. on behalf of Faculty of Pharmacy, Cairo University.
* Corresponding author at: Microbial Biotechnology Center, Faculty of Pharmacy, Cairo University, Cairo, Egypt. Tel.: +20 01005661976. E-mail address: [email protected] (T. Essam). Peer review under responsibility of Faculty of Pharmacy, Cairo University.
Production and hosting by Elsevier
1. Introduction Rifamycins, mainly rifamycin B, belong to the ansamycin antibiotic family with pronounced anti-mycobacterial activity.1,2 They are used in the chemotherapeutic treatment of tuberculosis, leprosy and AIDS-related mycobacterial infections.3 These antibiotics regained importance due to their use in the treatment of AIDS associated tuberculosis.1,4 With the increasing demand of this antibiotic in terms of its utility spectrum in the current AIDS outbreak the world forced the scientific community to look for a novel antibiotic environment or increase
1110-0931 ª 2013 Production and hosting by Elsevier B.V. on behalf of Faculty of Pharmacy, Cairo University. http://dx.doi.org/10.1016/j.bfopcu.2013.02.002
44 the productivity with the existing microbial system by modifying the cultivation procedures. Usually, the productivity of the desired product is high in flask scale, and will be gradually reduced as the scale is enlarged because of the complexity of the fermentation processes.5 Accordingly, results obtained in the shake flasks should be taken only as preliminary indicators for the conditions necessary for successful industrial production through pre-designed scaling-up studies and must be verified in studies carried out in a bench scale fermentor. The method for scalingup a fermentation system is usually based on empirical criteria such as pH, temperature, constant power input per unit volume, a constant mass transfer coefficient, constant mixing time and a constant impeller tip velocity.5,6 Agitation power per unit volume is among the most frequently used parameters for scale-up. Where sensor technologies developed for dissolved oxygen (DO) concentration, the scale-up method to maintain DO at a constant level became popular.5,7 Culturing filamentous microorganisms such as fungi or actinomycetes in a large fermentor could subject cells to damage by the exerted shear stress,8 and consequently the product yield is reduced. Hence, a compromise shall be attempted to achieve a sufficient level of DO with minimum possible shear stress.9 Indeed, each organism has its optimum pH where the growth of a selected microbial strain and its cellular metabolism occur in optimum levels compared to other conditions.5 Rifamycin fermentation shows an interesting pH pattern where the initial neutral pH increases continuously to an alkaline pH region in the initial growth phase, followed by a decrease to acidic pH at the beginning of the production of rifamycin. The pH of the medium affected both cell growth and rifamycin B production. Several authors reported the effect of pH on the cell growth and rifamycin B production at different pH values.10 The influence of incubation temperature on antibiotic production was studied by several authors.11,12 These studies showed that lowering the temperature decreases the rate of glucose consumption. It has also often been observed that a lower rate of carbon metabolism in the initial growth phase results in higher antibiotic production.12,13 For instance, Mahalaxmi et al.,1 reported that maximum growth and antibiotic production were noticed at 28 C and variation of this temperature resulted in the reduction of both biomass and production. Hence, it could be concluded that the optimal temperature for rifamycin B production is strain specific.12,13 In this prospective, the present study aimed to improve and optimize the fermentation process conditions to achieve a better yield of rifamycin B. The significant conditions that govern the fermentation process, such as aeration, dissolved oxygen, pH as well as the temperature were studied. Final integration of the optimized conditions was attempted to investigate the maximum possible yield. 2. Materials and Methods 2.1. Bacterial strains The amplified variant NCH of Amycolatopsis mediterranei– RCP 1001, previously obtained by El-Tayeb et al.14 was maintained on Q/2 agar slants and stored at 4 C to be used within
H.F. El-Sedawy et al. 27 days. The strain was propagated on Bennett’s agar. For long-term storage, the surface growth on Q/2 agar slants was harvested in 10% skimmed milk and lyophilized. 2.2. Chemicals Chemicals used throughout this work were of laboratory reagent grade, unless otherwise indicated. Glucose monohydrate, sucrose, NH4NO3, NaNO2 were the products of ADWIC, Egypt. Sodium diethyl barbiturate (SDB) was the product of Grindstedvaerket A/S, Denmark. Potassium sodium tartarate tetrahydrate, 3,5_dinitrosalicylic acid, CaCO3, MgSO4.7H2O, KH2PO4, (NH4)2SO4, NaOH and HCl were the products of E. Merck, Darmstadt, Germany. Glacial acetic acid was the product of Aldrich Ltd, England. 2.3. Media Yeast extract, malt extract, beef extract, tryptone, soytone, skimmed milk and bacto agar were the products of Difco Laboratories, Detroit, U.S.A. Oat flakes and sunflower oil were obtained from commercial suppliers. Glucose syrup was obtained from the Egyptian Co. for Production of Starch and Glucose, Cairo. Media used for propagation, selection and maintenance as well as the vegetative medium V2 were those previously reported by El-Tayeb et al.15 The modified fermentation medium F2m3 was prepared and used according to El-Tayeb et al.13 2.4. Inoculum preparation and analytical regime The methods used for maintenance, propagation, selection and preparation of inoculums as well as the determination of the remaining reducing sugars concentration, biomass and assay of rifamycin B were previously reported by ElTayeb et al.13,15 2.5. Production of rifamycin B in the fermentor The production of rifamycin B was carried out in a 6.6 liter laboratory glass fermentor (Bioflo 3000 Benchtop fermentor, New Brunswick Scientific Co., NJ, USA) as previously described by El-Tayeb et al.13 The inoculum was prepared according to El-Tayeb et al.15 and was added (5% v/v) to 2.5 liters of F2m3 medium to which 5% glucose syrup was added at day 4. Unless otherwise indicated, agitation was kept at 500 rpm by using 2 six bladed Rushton impellers separated by 9.5 cm with the lower impeller 2.5 cm above the agitator tip. Aeration using a multiorifice ring sparger was controlled by a pressure regulator and monitored by a flowmeter. The% of dissolved oxygen (DO) was monitored and/or controlled through the fermentor control unit using the airflow and the agitation speed simultaneously. The impact of commonly known key parameters was studied through different sets of experiments (Table 1). Unless otherwise indicated, the temperature was adjusted at 28 C, the pH was automatically controlled at 6.5 for 3 days then 7 thereafter, an agitation rate at 500 rpm and aeration at 1 vvm. Samples of 25 ml were periodically withdrawn every 24 h for analysis of rifamycin B concentration, remaining reducing sugars concentration and dry cell weight.
Optimization of rifamycin B production by NCH strain using F2m3 medium in batch mode in the fermentor.
Variable factors
Fermentation conditionsa Agitationb (rpm)
Aeration (vvm)
pH
Dissolved oxygen (DO)
Temperature (C)
Rifamycin B conc. (g/l)
(Control)
500
1
C
UC
28
13.8
Aerationc 1.5
500
Vf
C
UC
28
16.1
(+) 16.6
Vf
Vf
C C C C C
Vf
28 28 28 28 28
16.1 12.7 13.8 17.8 15.3
(+) 16.6 ( ) 8.0 0 (+) 29.0 (+) 10.8
pHd 6.5 7.0 6.5 for 3 days then 7.5 thereafter 6.5 for 3 days then 7 thereafter (control) 6.5 for 3 days then 7 thereafter
500 500 500 500 500
1 1 1 1 1.5
Vf
UC UC UC UC UC
28 28 28 28 28
13.1 10.8 13.5 13.8 16.1
( ) 5.0 ( ) 21.0 ( ) 2.0 . . .. . .. (+) 16.6
Temperaturee 28 C for 18 h then 24 thereafter 28 C for 18 h then 26 thereafter 26 C 28 C (control) 30 C 30 C for 3 days then 28 C thereafter 30 C for 3 days then 28 C thereafter
500 500 500 500 500 500 500
1 1 1 1 1 1 1.5
C C C C C C C
UC UC UC UC UC UC CDOg
Vf
7.5 13.2 12.7 13.8 11.5 14.5 15.5
( ) 45 ( ) 4.5 ( )8 . . .. . .. ( ) 16.5 (+) 5 (+) 12.5
Dissolved oxygen regimens (DO) 1 vvm for 3 days then controlled DO at 30% (control) 1 vvm for 2 days then controlled DO at 30% 1 vvm for 2 day then controlled DO at 60% 1.5 vvm for 3 days then controlled DO at 30%. DO control at 60% from zero time
F2m3; incubation temperature, 28 C; pH 6.5 for 3 days then 7 thereafter; inoculum size, 5% and the incubation period, 192 h unless otherwise indicated. (C) Control pH at 6.5 for 3 days then 7 thereafter. (UC) Uncontrolled. a The following conditions were kept constant in all investigations unless otherwise prescribed: the fermentation medium. b Agitation was kept at 500 rpm except in DO control regimes. c Without DO control. d All the tested pH regimes were carried out without DO control. e All the tested temperature regimes were carried out without DO control. f Variable. g The DO was controlled at 30% of saturation after 3 days.
% Change
Scaling up for the industrial production of rifamycin B; optimization of the process conditions in bench-scale fermentor
Table 1
45
46
H.F. El-Sedawy et al. (P) 1 vvm
(A)
(P) 1.5 vvm
(X) 1 vvm
(X) 1.5vvm
18
4
6 2 3
0
(P) Rifamycin B conc. (g/l)
9
(X)Dry cell weight (g%)
(P)Rifamycin Bconc. (g/l)
6
12
0 0
1
2
3
4
5
6
7
8
(P) 1vvm for 2 days then 30% DO
(P) 1 vvm for 2 days then 60% DO
(P) 60% DO
(P)1.5 vvm
(P) 1.5 vvm for 3 days then 30% DO
20 18 16 14 12 10 8 6 4 2 0
8
15
(P) 1 vvm (control)
0
9
1
2
3
(S) 1 vvm
(DO) 1.5 vvm 12
25
10
20
8
15
6
10
4
5
2
0
0
1
2
3
4
5
Time (days)
6
7
8
9
10
11
6
7
8
9
Figure 1 Effect of aeration rate 1.5 vvm on (A) rifamycin B concentration, dry cell weight and (B) remaining reducing sugars’ concentration and dissolved oxygen concentration using NCH strain in a 6.6 l fermentor under the following conditions: aeration, 1.5 vvm; agitation, 500 rpm; inoculum size, 5%; working volume, 2.5 l; medium used, F2m3; 5% glucose syrup was added at day 4; pH, 6.5 for 3 days then 7 thereafter; temperature, 28 C.
(X) 1 vvm for 2 days then 30% DO
(X) 1 vvm for 2 days then 60% DO
(X) 60% DO
(X) 1.5 vvm
(X) 1.5 vvm for 3 days then 30% DO
6 5 4 3 2 1 0
0
1
2
3
4 5 6 Time (days)
7
8
9
10
11
Figure 2 Effect of different DO control regimes on (A) rifamycin B production and (B) dry cell weight using an NCH strain in a 6.6 l fermentor under the following conditions: aeration, 1 or 1.5 vvm; agitation, 500 rpm; inoculum size, 5%; working volume, 2.5 l; medium used, F2m3; 5% glucose syrup was added at day 4; pH, 6.5 for 3 days then 7 thereafter; temperature, 28 C.
3. Results
(P) 6.5 (P) 6.5 for 3 days then 7 (X) 6.5 (X) 6.5 for 3 days then 7
(P) Rifamycin B conc. (g/l)
Application of 1.5 vvm aeration rate increased the yield of rifamycin B to 16.1 g/l (16.6%) compared to 13.8 g/l (Fig. 1A). This yield was accompanied by a higher biomass and higher substrate consumption (Fig. 1). Moreover, the DO reached zero at day 7 upon the use of 1.5 vvm compared to zero value at day 5 in case of the use of an aeration rate of 1 vvm. (Fig. 1B). Different regimes for controlling DO were tested (Table 1 and Fig. 2). The best regime was the use of 1.5 vvm for 3 days, then controlling DO at 30% of saturation to the end of the process, where the yield increased by 10.5% (from 16.1 to 17.8 g/l). This was followed by the application of 1.5 vvm then by controlling DO at 60% saturation from zero time (Fig. 2A). The other regimes resulted in comparable production as control (Table 1 and Fig. 2A). The recorded dry cell weight was higher than the control in all the tested regimes (Fig. 2B), while the remaining reducing sugars concentration showed almost the same pattern as the control (data not shown). In general, all 4 tested regimes for controlling pH at an aeration rate of 1 vvm had either no or negative impact of
(X) 1 vvm (control)
7
(X) Dry cell weight (g%)
30
(DO)dissolved oxygen conc.(%)*10
(S)Remaining reducing sugars conc.(g/l)
(B)
(S) 1.5 vvm
DO (1vvm)
0
5
Time (days)
Time (days)
(B)
4
(P) 7 (P) 6.5 for 3 days then 7.5 (X) 7 (X) 6.5 for 3 days then 7.5
16
8
14
7
12
6
10
5
8
4
6
3
4
2
2
1
0
(X) Dry cell weight (g%)
(A)
0 0
1
2
3
4 5 Time (days)
6
7
8
9
Figure 3 Effect of different pH control regimes on rifamycin B production and dry cell weight using an NCH strain in a 6.6 l fermentor under the following conditions: aeration, 1 vvm; agitation, 500 rpm; inoculum size, 5%; working volume, 2.5 l; medium used, F2m3; 5% glucose syrup was added at day 4; temperature, 28 C.
Scaling up for the industrial production of rifamycin B; optimization of the process conditions in bench-scale fermentor
28°C (Control)
26°C
30°C
28°C then 24°C
28°C then 26°C
30°C then 28°C
16 14 12 10 8 6 4 2 0 0
1
2
3
4
5
6
7
8
9
Time (days)
Dry cell weight (g%)
(B)
28°C (Control)
26°C
30°C
28°C then 24°C
28°C then 26°C
30°C then 28°C
7 6 5 4 3 2 1 0 0
1
2
3
4
5
6
7
8
9
Time (days)
Remaining reducing sugars conc.(g/l)
(C)
28°C (Control)
26°C
30°C
28°C then 24°C
28°C then 26°C
30°C then 28°C
35 30 25 20 15 10 5 0 0
1
2
3
4
5
6
7
8
9
Time (days)
Figure 4 Effect of different temperature control regimes on (A) rifamycin B concentration, (B), dry cell weight and (C) remaining reducing sugars concentration using an NCH strain in 6.6 l fermentor under the following conditions: aeration, 1 vvm; agitation, 500 rpm; inoculum size, 5%; working volume, 2.5 l; medium used, F2m3; 5% glucose syrup was added at day 4; pH, 6.5 for 3 days then 7 thereafter.
4. Discussion Optimization of antibiotic production necessitates the studying of different physical and physiological factors affecting its production; taking into concern that the optimal fermentation conditions are usually strain specific.12,13 The use of an aeration rate of 1.5 vvm increased the production yield by almost 17%. A characteristic of A. mediterranei fermentation is the very high oxygen requirement in a definite period of the growth–production cycle.16,17 The time at which this occurs varies with the strain and the medium composition, but it always coincides with the phase in which glucose is rapidly metabolized. If this oxygen demand is not met by an adequate oxygen supply through aeration, very low final yields of rifamycin B are obtained.16 Although previously, El-Tayeb et al.13 studied the effect of different aeration levels on rifamycin B production and the results showed that the maximum rifamycin B production was obtained upon using 1 vvm, in the present study the highest yield was recorded at a 1.5 vvm aeration rate. Controlling the dissolved oxygen (DO) at different regimes resulted in an increase in the production yield with the highest yield (10.5%) obtained by the use of 1.5 vvm for 3 days then DO control at 30% of saturation thereafter. These results were in accordance with those reported by El-Tayeb et al.13 where the yield increased by 8% upon using 1 vvm for 3 days then controlling DO at 30% thereafter. In this respect, Jin et al.5 reported that rifamycin B fermentation was dependent on a high amount of dissolved oxygen (DO), and that the DO must be
(P) 28°C
(P) 30°C for 3 days then 28°C
(S) 28°C
(S) 30°C for 3 days then 28°C
20
25
16
20
12
15
8
10
4
5
0
(S) Remaining reducing sugars conc.(g/l)
Rifamycin B conc. (g/l)
(A)
The optimum conditions obtained from previous sets of experiments suggested that a road forward for the estabilishment of an optimized set of conditions. Integration of all conditions which lead to the highest yield (aeration rate of 1.5 vvm for 3 days, and controlling the DO at 30% of saturation to the end of the process, controlling the pH at 6.5 for 3 days then at 7 thereafter, controlling the temperature at 30 C for 3 days then 28 C thereafter) was tested. This combination decreased the yield at day 8 from 16.1 to 15.5 g/l compared to the control (aeration, 1.5 vvm; agitation, 500 rpm; temperature, 28 C and pH control at 6.5 then 7 thereafter) with the reducing sugars reaching zero at day 6 (Fig. 5).
(P) Rifamycin B conc. (g/l)
rifamycin B production compared to the control (Table 1). The lowest production yield was recorded when the pH was adjusted at 7 (Fig. 3). Other attempted regimens resulted in a production yield of almost the same order of magnitude and dry cell weight especially at the end of the production (Fig. 3). The remaining reducing sugars showed comparable pattern as control (data not shown). The highest yield and dry cell weight were recorded upon adjusting the temperature at 30 C for 3 days then 28 C thereafter at 1 vvm aeration rate (Fig. 4). The lowest yield and dry cell weight were recorded when the temperature was adjusted at 28 C for 18 h. then 24 C thereafter (Fig. 4). The other regimes decreased the yield and the recorded the dry cell weight (Fig. 4B). The remaining reducing sugars’ concentration was comparable to the control except for using 30 C for 3 days then 28 C thereafter (Fig. 4C).
47
0 0
1
2
3
4 5 Time (days)
6
7
8
9
Figure 5 Effect of integration of optimum conditions in batch mode on rifamycin B production (P) and remaining reducing sugars concentration (S) by NCH strain using F2m3 medium in a 6.6 l laboratory fermentor.
48 kept above 25% of the saturation to meet the oxygen requirement for mycelia growth and rifamycin B production. A. mediterranei has a characteristic pH behavior pattern. Thus controlling the pH during the fermentation may be a good way to improve rifamycin B production. Thus, El-Tayeb et al.13 reported a characteristic pH behavior pattern of A. mediterranei and recommended a pH strategy of 6.5 at the trophophase (day 1–3) and 7.0 at the idiophase (day 4–8). In the present study all the tested regimes did not increase the yield of rifamycin B. In addition the results showed reduction in the recorded biomass. These results were in agreement with El-Tayeb et al.,13 who reported that the optimum pH for rifamycin B production was the use of pH 6.5 at the trophophase and pH 7.0 at the idiophase. The optimal temperature reported for rifamycin B fermentation was 28 C.10 Data obtained by Krishna et al.10 suggested that the fermentation carried out at 26–30 C gave the maximum yield of rifamycin and any deviation from the optimum temperature resulted in growth retardation and decreased the yield of antibiotic. The results of the present study showed a slight increase in production upon using 30 C for 3 days then 28 C thereafter. Although Sensi and Thiemann18 have reported that shifting the temperature from 28 C to 24 C after 18 h of fermentation increased the yield of rifamycin B production, in the present study, a marked decrease was observed in productivity, biomass and glucose consumption associated with the use of 28 C for 18 h then 24 C thereafter. However, this could be due to the difference in the strain used. Although the use of 30 C throughout the fermentation time decreased productivity and biomass, glucose consumption was much faster than all the tested temperature regimes. Indeed, lowering the temperature decreases the rate of glucose consumption. The lower the rate of carbon metabolism in the initial growth phase the higher the antibiotic production.12,16 Integration of the most optimum conditions previously mentioned above (1.5 vvm for 3 days then DO control at 30% thereafter, pH control at 6.5 for 3 days then 7 thereafter, temperature control at 30 C for 3 days then 28 C thereafter) decreased the yield from 17.8 to 15.5 g/l. Interestingly, the main difference started to obviously appear at days 7 and 8, where the remaining reducing sugars’ concentration reached zero from day 6. This may be explained by the observation of Jin et al.5 during rifamycin B fermentation that the reducing sugar was still consumed in the stationary phase although the mycelium growth almost stopped in this phase, which meant that the consumed sugar was primarily used for rifamycin B biosynthesis and mycelium maintenance. 5. Conclusion Application of 1.5 vvm aeration rate increased the production yield. None of the tested pH regimens had a positive impact on the production. The overall optimization of rifamycin B production by NCH strain in batch mode in a 6.6 l laboratory fermentor increased the yield by almost 30% from 13.81 to 17.8 g/l. 6. Conflict of interest None.
H.F. El-Sedawy et al. References 1. Mahalaxmi Y, Sathish T, Subba Rao Ch, Prakasham RS. Corn husk as a novel substrate for the production of rifamycin B by isolated Amycolatopsis sp. RSP 3 under SSF. J Process Biochem 2010;45:47–53. 2. Oppolzer W, Prelog V. Ueber die konstitution und die konfiguration des rifamycin B, O, S and SV. Helv Chim Acta 1973;56:2214–87, Cited in: Biotech of Ind Antibiot Vandamme, E. ed. Basel: Marcel Decker, Inc. New York, p. 291. 3. Floss HG, Yu T. Rifamycin-mode of action, resistance, and biosynthesis. Chem Rev 2005;105(2):621. 4. Sepkowitz KA, Rafalli J, Riley L, Kien TE, Armstrong D. Tuberculosis in the AIDA era. Clin Microbiol Rev 1995;8:180–99. 5. Jin ZH, Lin JP, Xu ZN, Cen PL. Scale-up of rifamycin B fermentation with Amycolatopsis mediterranei. J Zhejiang Univ Sci 2004;5(12):1590–6. 6. Shuler FL, Kargi F. Biopro eng basic concepts. Englewood Cliffs (NJ): Prentice-Hall; 1992. 7. Oosterhuis NMG, Kossen NWF. Dissolved oxygen concentration profiles in a production scale bioreactor. J Biotechnol Bioeng 1983;26:546–50. 8. Ziegler H, Dunn IJ, Bourine J. Oxygen transfer and mycelial growth in a tubular loop fermentor. J Biotechnol Bioeng 1980;22:1613–30. 9. Dobson LF, O’Cleirigh CC, O’Shea DG. The influence of morphology on geldanamycin production in submerged fermentations of Streptomyces hygroscopicus var. geldanus. J Appl Microbiol Biotechnol 2008;79:859–66. 10. Mahalaxmi Y, Subba Rao Ch, Suvarnalaxmi G, Satish T, Sudhakar P, Prakasham RS. Rifamycin B production pattern in Nocardia RSP-3 strain and influence of barbital on antibiotic production. Curr Trends Biotechnol Pharm 2008;2(1):173–81. 11. Krishna PSM, Venkateswarlu G, Venkateswar RL. Studies on fermentative production of rifamycin using Amycolatopsis mediterranei. World J Microbiol Biotechnol 1998;14(5):689–91. 12. Venkateswarlu G, Murali PSK, Sharma G, Venkateswar RL. Improvement of rifamycin B production using mutant strains of Amycolatopsis mediterranei. J Biopro Eng 2000;23:315–8. 13. El-Tayeb OM, Salama AA, Hussein MMM, El-Sedawy HF. Optimization of industrial production of rifamycin B by Amycolatopsis mediterranei IV. Production in the fermentor. Afr J Biotechnol 2004;3(9):432–40. 14. El-Tayeb OM, Salama AA, Hussein MMM, El-Sedawy HF. Optimization of industrial production of rifamycin B by Amycolatopsis mediterranei II. The role of gene amplification and physiological factors in productivity. Afr J Biotechnol 2004;3(5):273–80. 15. El-Tayeb OM, Salama AA, Hussein MMM, El-Sedawy HF. Optimization of industrial production of rifamycin B by Amycolatopsis mediterranei I. The role of colony morphology and nitrogen sources in productivity. Afr J Biotechnol 2004;3(5):266–72. 16. Lancini G, Cavalleri B. Rifamycins. In: Strohl WR, editor. Biotech Ind Antibiot. 2nd ed. New York: Marcel Decker; 1997. p. 521–49. 17. Lee KJ, Rho YT. Quantitative analysis of mycelium morphological characteristics and rifamycin B production using Nocardia mediterranei. J Biotechnol 1994;36:239–45. 18. Sensi P, Thiemann JE. Production of rifamycins. J Prog Ind Microbiol 1967;6:21–59.
Cairo University
Bulletin of Faculty of Pharmacy, Cairo University www.elsevier.com/locate/bfopcu www.sciencedirect.com
ORIGINAL ARTICLE
Scaling up for the industrial production of rifamycin B; optimization of the process conditions in bench-scale fermentor Hewaida F. El-Sedawy a, Manal M.M. Hussein b, Tamer Essam Ossama M. El-Tayeb b, Fatma H.A. Mohammad c a b c
a,b,*
,
Microbial Biotechnology Center, Faculty of Pharmacy, Cairo University, Cairo, Egypt Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo, Egypt Microbial Chemical Engineering and Pilot Plant Department, National Research Center, Egypt
Received 28 January 2013; accepted 19 February 2013 Available online 28 March 2013
KEYWORDS Amycolatopsis mediterranei; Antibiotics; Fermentor; Rifamycin B; Scaling up
Abstract Optimization of fermentation process conditions using a gene amplified variant of Amycolatopsis mediterranei (NCH) was carried out. The use of aeration level 1.5 vvm increased the yield by 16.6% (from 13.81 to 16.1 g/l) upon controlling the temperature at 28 C. Adjustment of the aeration level at 1.5 vvm for 3 days then controlling the dissolved oxygen (DO) at 30% saturation further increased the yield to 17.8 g/l. The optimum pH was 6.5 for 3 days then 7 thereafter when a production yield of 16.1 g/l was recorded using an aeration rate of 1.5 vvm. Controlling the pH at constant value (6.5 or 7) all through the fermentation process decreased the yield by 5–21%. Controlling the temperature at 30 C for 3 days then 28 C thereafter slightly increased the yield by 5% upon using an aeration rate of 1 vvm while it decreased upon using an aeration rate of 1.5 vvm. Integration of the most optimum conditions increased the production yield by 22% from 13.81 to 17.8 g/l. ª 2013 Production and hosting by Elsevier B.V. on behalf of Faculty of Pharmacy, Cairo University.
* Corresponding author at: Microbial Biotechnology Center, Faculty of Pharmacy, Cairo University, Cairo, Egypt. Tel.: +20 01005661976. E-mail address: [email protected] (T. Essam). Peer review under responsibility of Faculty of Pharmacy, Cairo University.
Production and hosting by Elsevier
1. Introduction Rifamycins, mainly rifamycin B, belong to the ansamycin antibiotic family with pronounced anti-mycobacterial activity.1,2 They are used in the chemotherapeutic treatment of tuberculosis, leprosy and AIDS-related mycobacterial infections.3 These antibiotics regained importance due to their use in the treatment of AIDS associated tuberculosis.1,4 With the increasing demand of this antibiotic in terms of its utility spectrum in the current AIDS outbreak the world forced the scientific community to look for a novel antibiotic environment or increase
1110-0931 ª 2013 Production and hosting by Elsevier B.V. on behalf of Faculty of Pharmacy, Cairo University. http://dx.doi.org/10.1016/j.bfopcu.2013.02.002
44 the productivity with the existing microbial system by modifying the cultivation procedures. Usually, the productivity of the desired product is high in flask scale, and will be gradually reduced as the scale is enlarged because of the complexity of the fermentation processes.5 Accordingly, results obtained in the shake flasks should be taken only as preliminary indicators for the conditions necessary for successful industrial production through pre-designed scaling-up studies and must be verified in studies carried out in a bench scale fermentor. The method for scalingup a fermentation system is usually based on empirical criteria such as pH, temperature, constant power input per unit volume, a constant mass transfer coefficient, constant mixing time and a constant impeller tip velocity.5,6 Agitation power per unit volume is among the most frequently used parameters for scale-up. Where sensor technologies developed for dissolved oxygen (DO) concentration, the scale-up method to maintain DO at a constant level became popular.5,7 Culturing filamentous microorganisms such as fungi or actinomycetes in a large fermentor could subject cells to damage by the exerted shear stress,8 and consequently the product yield is reduced. Hence, a compromise shall be attempted to achieve a sufficient level of DO with minimum possible shear stress.9 Indeed, each organism has its optimum pH where the growth of a selected microbial strain and its cellular metabolism occur in optimum levels compared to other conditions.5 Rifamycin fermentation shows an interesting pH pattern where the initial neutral pH increases continuously to an alkaline pH region in the initial growth phase, followed by a decrease to acidic pH at the beginning of the production of rifamycin. The pH of the medium affected both cell growth and rifamycin B production. Several authors reported the effect of pH on the cell growth and rifamycin B production at different pH values.10 The influence of incubation temperature on antibiotic production was studied by several authors.11,12 These studies showed that lowering the temperature decreases the rate of glucose consumption. It has also often been observed that a lower rate of carbon metabolism in the initial growth phase results in higher antibiotic production.12,13 For instance, Mahalaxmi et al.,1 reported that maximum growth and antibiotic production were noticed at 28 C and variation of this temperature resulted in the reduction of both biomass and production. Hence, it could be concluded that the optimal temperature for rifamycin B production is strain specific.12,13 In this prospective, the present study aimed to improve and optimize the fermentation process conditions to achieve a better yield of rifamycin B. The significant conditions that govern the fermentation process, such as aeration, dissolved oxygen, pH as well as the temperature were studied. Final integration of the optimized conditions was attempted to investigate the maximum possible yield. 2. Materials and Methods 2.1. Bacterial strains The amplified variant NCH of Amycolatopsis mediterranei– RCP 1001, previously obtained by El-Tayeb et al.14 was maintained on Q/2 agar slants and stored at 4 C to be used within
H.F. El-Sedawy et al. 27 days. The strain was propagated on Bennett’s agar. For long-term storage, the surface growth on Q/2 agar slants was harvested in 10% skimmed milk and lyophilized. 2.2. Chemicals Chemicals used throughout this work were of laboratory reagent grade, unless otherwise indicated. Glucose monohydrate, sucrose, NH4NO3, NaNO2 were the products of ADWIC, Egypt. Sodium diethyl barbiturate (SDB) was the product of Grindstedvaerket A/S, Denmark. Potassium sodium tartarate tetrahydrate, 3,5_dinitrosalicylic acid, CaCO3, MgSO4.7H2O, KH2PO4, (NH4)2SO4, NaOH and HCl were the products of E. Merck, Darmstadt, Germany. Glacial acetic acid was the product of Aldrich Ltd, England. 2.3. Media Yeast extract, malt extract, beef extract, tryptone, soytone, skimmed milk and bacto agar were the products of Difco Laboratories, Detroit, U.S.A. Oat flakes and sunflower oil were obtained from commercial suppliers. Glucose syrup was obtained from the Egyptian Co. for Production of Starch and Glucose, Cairo. Media used for propagation, selection and maintenance as well as the vegetative medium V2 were those previously reported by El-Tayeb et al.15 The modified fermentation medium F2m3 was prepared and used according to El-Tayeb et al.13 2.4. Inoculum preparation and analytical regime The methods used for maintenance, propagation, selection and preparation of inoculums as well as the determination of the remaining reducing sugars concentration, biomass and assay of rifamycin B were previously reported by ElTayeb et al.13,15 2.5. Production of rifamycin B in the fermentor The production of rifamycin B was carried out in a 6.6 liter laboratory glass fermentor (Bioflo 3000 Benchtop fermentor, New Brunswick Scientific Co., NJ, USA) as previously described by El-Tayeb et al.13 The inoculum was prepared according to El-Tayeb et al.15 and was added (5% v/v) to 2.5 liters of F2m3 medium to which 5% glucose syrup was added at day 4. Unless otherwise indicated, agitation was kept at 500 rpm by using 2 six bladed Rushton impellers separated by 9.5 cm with the lower impeller 2.5 cm above the agitator tip. Aeration using a multiorifice ring sparger was controlled by a pressure regulator and monitored by a flowmeter. The% of dissolved oxygen (DO) was monitored and/or controlled through the fermentor control unit using the airflow and the agitation speed simultaneously. The impact of commonly known key parameters was studied through different sets of experiments (Table 1). Unless otherwise indicated, the temperature was adjusted at 28 C, the pH was automatically controlled at 6.5 for 3 days then 7 thereafter, an agitation rate at 500 rpm and aeration at 1 vvm. Samples of 25 ml were periodically withdrawn every 24 h for analysis of rifamycin B concentration, remaining reducing sugars concentration and dry cell weight.
Optimization of rifamycin B production by NCH strain using F2m3 medium in batch mode in the fermentor.
Variable factors
Fermentation conditionsa Agitationb (rpm)
Aeration (vvm)
pH
Dissolved oxygen (DO)
Temperature (C)
Rifamycin B conc. (g/l)
(Control)
500
1
C
UC
28
13.8
Aerationc 1.5
500
Vf
C
UC
28
16.1
(+) 16.6
Vf
Vf
C C C C C
Vf
28 28 28 28 28
16.1 12.7 13.8 17.8 15.3
(+) 16.6 ( ) 8.0 0 (+) 29.0 (+) 10.8
pHd 6.5 7.0 6.5 for 3 days then 7.5 thereafter 6.5 for 3 days then 7 thereafter (control) 6.5 for 3 days then 7 thereafter
500 500 500 500 500
1 1 1 1 1.5
Vf
UC UC UC UC UC
28 28 28 28 28
13.1 10.8 13.5 13.8 16.1
( ) 5.0 ( ) 21.0 ( ) 2.0 . . .. . .. (+) 16.6
Temperaturee 28 C for 18 h then 24 thereafter 28 C for 18 h then 26 thereafter 26 C 28 C (control) 30 C 30 C for 3 days then 28 C thereafter 30 C for 3 days then 28 C thereafter
500 500 500 500 500 500 500
1 1 1 1 1 1 1.5
C C C C C C C
UC UC UC UC UC UC CDOg
Vf
7.5 13.2 12.7 13.8 11.5 14.5 15.5
( ) 45 ( ) 4.5 ( )8 . . .. . .. ( ) 16.5 (+) 5 (+) 12.5
Dissolved oxygen regimens (DO) 1 vvm for 3 days then controlled DO at 30% (control) 1 vvm for 2 days then controlled DO at 30% 1 vvm for 2 day then controlled DO at 60% 1.5 vvm for 3 days then controlled DO at 30%. DO control at 60% from zero time
F2m3; incubation temperature, 28 C; pH 6.5 for 3 days then 7 thereafter; inoculum size, 5% and the incubation period, 192 h unless otherwise indicated. (C) Control pH at 6.5 for 3 days then 7 thereafter. (UC) Uncontrolled. a The following conditions were kept constant in all investigations unless otherwise prescribed: the fermentation medium. b Agitation was kept at 500 rpm except in DO control regimes. c Without DO control. d All the tested pH regimes were carried out without DO control. e All the tested temperature regimes were carried out without DO control. f Variable. g The DO was controlled at 30% of saturation after 3 days.
% Change
Scaling up for the industrial production of rifamycin B; optimization of the process conditions in bench-scale fermentor
Table 1
45
46
H.F. El-Sedawy et al. (P) 1 vvm
(A)
(P) 1.5 vvm
(X) 1 vvm
(X) 1.5vvm
18
4
6 2 3
0
(P) Rifamycin B conc. (g/l)
9
(X)Dry cell weight (g%)
(P)Rifamycin Bconc. (g/l)
6
12
0 0
1
2
3
4
5
6
7
8
(P) 1vvm for 2 days then 30% DO
(P) 1 vvm for 2 days then 60% DO
(P) 60% DO
(P)1.5 vvm
(P) 1.5 vvm for 3 days then 30% DO
20 18 16 14 12 10 8 6 4 2 0
8
15
(P) 1 vvm (control)
0
9
1
2
3
(S) 1 vvm
(DO) 1.5 vvm 12
25
10
20
8
15
6
10
4
5
2
0
0
1
2
3
4
5
Time (days)
6
7
8
9
10
11
6
7
8
9
Figure 1 Effect of aeration rate 1.5 vvm on (A) rifamycin B concentration, dry cell weight and (B) remaining reducing sugars’ concentration and dissolved oxygen concentration using NCH strain in a 6.6 l fermentor under the following conditions: aeration, 1.5 vvm; agitation, 500 rpm; inoculum size, 5%; working volume, 2.5 l; medium used, F2m3; 5% glucose syrup was added at day 4; pH, 6.5 for 3 days then 7 thereafter; temperature, 28 C.
(X) 1 vvm for 2 days then 30% DO
(X) 1 vvm for 2 days then 60% DO
(X) 60% DO
(X) 1.5 vvm
(X) 1.5 vvm for 3 days then 30% DO
6 5 4 3 2 1 0
0
1
2
3
4 5 6 Time (days)
7
8
9
10
11
Figure 2 Effect of different DO control regimes on (A) rifamycin B production and (B) dry cell weight using an NCH strain in a 6.6 l fermentor under the following conditions: aeration, 1 or 1.5 vvm; agitation, 500 rpm; inoculum size, 5%; working volume, 2.5 l; medium used, F2m3; 5% glucose syrup was added at day 4; pH, 6.5 for 3 days then 7 thereafter; temperature, 28 C.
3. Results
(P) 6.5 (P) 6.5 for 3 days then 7 (X) 6.5 (X) 6.5 for 3 days then 7
(P) Rifamycin B conc. (g/l)
Application of 1.5 vvm aeration rate increased the yield of rifamycin B to 16.1 g/l (16.6%) compared to 13.8 g/l (Fig. 1A). This yield was accompanied by a higher biomass and higher substrate consumption (Fig. 1). Moreover, the DO reached zero at day 7 upon the use of 1.5 vvm compared to zero value at day 5 in case of the use of an aeration rate of 1 vvm. (Fig. 1B). Different regimes for controlling DO were tested (Table 1 and Fig. 2). The best regime was the use of 1.5 vvm for 3 days, then controlling DO at 30% of saturation to the end of the process, where the yield increased by 10.5% (from 16.1 to 17.8 g/l). This was followed by the application of 1.5 vvm then by controlling DO at 60% saturation from zero time (Fig. 2A). The other regimes resulted in comparable production as control (Table 1 and Fig. 2A). The recorded dry cell weight was higher than the control in all the tested regimes (Fig. 2B), while the remaining reducing sugars concentration showed almost the same pattern as the control (data not shown). In general, all 4 tested regimes for controlling pH at an aeration rate of 1 vvm had either no or negative impact of
(X) 1 vvm (control)
7
(X) Dry cell weight (g%)
30
(DO)dissolved oxygen conc.(%)*10
(S)Remaining reducing sugars conc.(g/l)
(B)
(S) 1.5 vvm
DO (1vvm)
0
5
Time (days)
Time (days)
(B)
4
(P) 7 (P) 6.5 for 3 days then 7.5 (X) 7 (X) 6.5 for 3 days then 7.5
16
8
14
7
12
6
10
5
8
4
6
3
4
2
2
1
0
(X) Dry cell weight (g%)
(A)
0 0
1
2
3
4 5 Time (days)
6
7
8
9
Figure 3 Effect of different pH control regimes on rifamycin B production and dry cell weight using an NCH strain in a 6.6 l fermentor under the following conditions: aeration, 1 vvm; agitation, 500 rpm; inoculum size, 5%; working volume, 2.5 l; medium used, F2m3; 5% glucose syrup was added at day 4; temperature, 28 C.
Scaling up for the industrial production of rifamycin B; optimization of the process conditions in bench-scale fermentor
28°C (Control)
26°C
30°C
28°C then 24°C
28°C then 26°C
30°C then 28°C
16 14 12 10 8 6 4 2 0 0
1
2
3
4
5
6
7
8
9
Time (days)
Dry cell weight (g%)
(B)
28°C (Control)
26°C
30°C
28°C then 24°C
28°C then 26°C
30°C then 28°C
7 6 5 4 3 2 1 0 0
1
2
3
4
5
6
7
8
9
Time (days)
Remaining reducing sugars conc.(g/l)
(C)
28°C (Control)
26°C
30°C
28°C then 24°C
28°C then 26°C
30°C then 28°C
35 30 25 20 15 10 5 0 0
1
2
3
4
5
6
7
8
9
Time (days)
Figure 4 Effect of different temperature control regimes on (A) rifamycin B concentration, (B), dry cell weight and (C) remaining reducing sugars concentration using an NCH strain in 6.6 l fermentor under the following conditions: aeration, 1 vvm; agitation, 500 rpm; inoculum size, 5%; working volume, 2.5 l; medium used, F2m3; 5% glucose syrup was added at day 4; pH, 6.5 for 3 days then 7 thereafter.
4. Discussion Optimization of antibiotic production necessitates the studying of different physical and physiological factors affecting its production; taking into concern that the optimal fermentation conditions are usually strain specific.12,13 The use of an aeration rate of 1.5 vvm increased the production yield by almost 17%. A characteristic of A. mediterranei fermentation is the very high oxygen requirement in a definite period of the growth–production cycle.16,17 The time at which this occurs varies with the strain and the medium composition, but it always coincides with the phase in which glucose is rapidly metabolized. If this oxygen demand is not met by an adequate oxygen supply through aeration, very low final yields of rifamycin B are obtained.16 Although previously, El-Tayeb et al.13 studied the effect of different aeration levels on rifamycin B production and the results showed that the maximum rifamycin B production was obtained upon using 1 vvm, in the present study the highest yield was recorded at a 1.5 vvm aeration rate. Controlling the dissolved oxygen (DO) at different regimes resulted in an increase in the production yield with the highest yield (10.5%) obtained by the use of 1.5 vvm for 3 days then DO control at 30% of saturation thereafter. These results were in accordance with those reported by El-Tayeb et al.13 where the yield increased by 8% upon using 1 vvm for 3 days then controlling DO at 30% thereafter. In this respect, Jin et al.5 reported that rifamycin B fermentation was dependent on a high amount of dissolved oxygen (DO), and that the DO must be
(P) 28°C
(P) 30°C for 3 days then 28°C
(S) 28°C
(S) 30°C for 3 days then 28°C
20
25
16
20
12
15
8
10
4
5
0
(S) Remaining reducing sugars conc.(g/l)
Rifamycin B conc. (g/l)
(A)
The optimum conditions obtained from previous sets of experiments suggested that a road forward for the estabilishment of an optimized set of conditions. Integration of all conditions which lead to the highest yield (aeration rate of 1.5 vvm for 3 days, and controlling the DO at 30% of saturation to the end of the process, controlling the pH at 6.5 for 3 days then at 7 thereafter, controlling the temperature at 30 C for 3 days then 28 C thereafter) was tested. This combination decreased the yield at day 8 from 16.1 to 15.5 g/l compared to the control (aeration, 1.5 vvm; agitation, 500 rpm; temperature, 28 C and pH control at 6.5 then 7 thereafter) with the reducing sugars reaching zero at day 6 (Fig. 5).
(P) Rifamycin B conc. (g/l)
rifamycin B production compared to the control (Table 1). The lowest production yield was recorded when the pH was adjusted at 7 (Fig. 3). Other attempted regimens resulted in a production yield of almost the same order of magnitude and dry cell weight especially at the end of the production (Fig. 3). The remaining reducing sugars showed comparable pattern as control (data not shown). The highest yield and dry cell weight were recorded upon adjusting the temperature at 30 C for 3 days then 28 C thereafter at 1 vvm aeration rate (Fig. 4). The lowest yield and dry cell weight were recorded when the temperature was adjusted at 28 C for 18 h. then 24 C thereafter (Fig. 4). The other regimes decreased the yield and the recorded the dry cell weight (Fig. 4B). The remaining reducing sugars’ concentration was comparable to the control except for using 30 C for 3 days then 28 C thereafter (Fig. 4C).
47
0 0
1
2
3
4 5 Time (days)
6
7
8
9
Figure 5 Effect of integration of optimum conditions in batch mode on rifamycin B production (P) and remaining reducing sugars concentration (S) by NCH strain using F2m3 medium in a 6.6 l laboratory fermentor.
48 kept above 25% of the saturation to meet the oxygen requirement for mycelia growth and rifamycin B production. A. mediterranei has a characteristic pH behavior pattern. Thus controlling the pH during the fermentation may be a good way to improve rifamycin B production. Thus, El-Tayeb et al.13 reported a characteristic pH behavior pattern of A. mediterranei and recommended a pH strategy of 6.5 at the trophophase (day 1–3) and 7.0 at the idiophase (day 4–8). In the present study all the tested regimes did not increase the yield of rifamycin B. In addition the results showed reduction in the recorded biomass. These results were in agreement with El-Tayeb et al.,13 who reported that the optimum pH for rifamycin B production was the use of pH 6.5 at the trophophase and pH 7.0 at the idiophase. The optimal temperature reported for rifamycin B fermentation was 28 C.10 Data obtained by Krishna et al.10 suggested that the fermentation carried out at 26–30 C gave the maximum yield of rifamycin and any deviation from the optimum temperature resulted in growth retardation and decreased the yield of antibiotic. The results of the present study showed a slight increase in production upon using 30 C for 3 days then 28 C thereafter. Although Sensi and Thiemann18 have reported that shifting the temperature from 28 C to 24 C after 18 h of fermentation increased the yield of rifamycin B production, in the present study, a marked decrease was observed in productivity, biomass and glucose consumption associated with the use of 28 C for 18 h then 24 C thereafter. However, this could be due to the difference in the strain used. Although the use of 30 C throughout the fermentation time decreased productivity and biomass, glucose consumption was much faster than all the tested temperature regimes. Indeed, lowering the temperature decreases the rate of glucose consumption. The lower the rate of carbon metabolism in the initial growth phase the higher the antibiotic production.12,16 Integration of the most optimum conditions previously mentioned above (1.5 vvm for 3 days then DO control at 30% thereafter, pH control at 6.5 for 3 days then 7 thereafter, temperature control at 30 C for 3 days then 28 C thereafter) decreased the yield from 17.8 to 15.5 g/l. Interestingly, the main difference started to obviously appear at days 7 and 8, where the remaining reducing sugars’ concentration reached zero from day 6. This may be explained by the observation of Jin et al.5 during rifamycin B fermentation that the reducing sugar was still consumed in the stationary phase although the mycelium growth almost stopped in this phase, which meant that the consumed sugar was primarily used for rifamycin B biosynthesis and mycelium maintenance. 5. Conclusion Application of 1.5 vvm aeration rate increased the production yield. None of the tested pH regimens had a positive impact on the production. The overall optimization of rifamycin B production by NCH strain in batch mode in a 6.6 l laboratory fermentor increased the yield by almost 30% from 13.81 to 17.8 g/l. 6. Conflict of interest None.
H.F. El-Sedawy et al. References 1. Mahalaxmi Y, Sathish T, Subba Rao Ch, Prakasham RS. Corn husk as a novel substrate for the production of rifamycin B by isolated Amycolatopsis sp. RSP 3 under SSF. J Process Biochem 2010;45:47–53. 2. Oppolzer W, Prelog V. Ueber die konstitution und die konfiguration des rifamycin B, O, S and SV. Helv Chim Acta 1973;56:2214–87, Cited in: Biotech of Ind Antibiot Vandamme, E. ed. Basel: Marcel Decker, Inc. New York, p. 291. 3. Floss HG, Yu T. Rifamycin-mode of action, resistance, and biosynthesis. Chem Rev 2005;105(2):621. 4. Sepkowitz KA, Rafalli J, Riley L, Kien TE, Armstrong D. Tuberculosis in the AIDA era. Clin Microbiol Rev 1995;8:180–99. 5. Jin ZH, Lin JP, Xu ZN, Cen PL. Scale-up of rifamycin B fermentation with Amycolatopsis mediterranei. J Zhejiang Univ Sci 2004;5(12):1590–6. 6. Shuler FL, Kargi F. Biopro eng basic concepts. Englewood Cliffs (NJ): Prentice-Hall; 1992. 7. Oosterhuis NMG, Kossen NWF. Dissolved oxygen concentration profiles in a production scale bioreactor. J Biotechnol Bioeng 1983;26:546–50. 8. Ziegler H, Dunn IJ, Bourine J. Oxygen transfer and mycelial growth in a tubular loop fermentor. J Biotechnol Bioeng 1980;22:1613–30. 9. Dobson LF, O’Cleirigh CC, O’Shea DG. The influence of morphology on geldanamycin production in submerged fermentations of Streptomyces hygroscopicus var. geldanus. J Appl Microbiol Biotechnol 2008;79:859–66. 10. Mahalaxmi Y, Subba Rao Ch, Suvarnalaxmi G, Satish T, Sudhakar P, Prakasham RS. Rifamycin B production pattern in Nocardia RSP-3 strain and influence of barbital on antibiotic production. Curr Trends Biotechnol Pharm 2008;2(1):173–81. 11. Krishna PSM, Venkateswarlu G, Venkateswar RL. Studies on fermentative production of rifamycin using Amycolatopsis mediterranei. World J Microbiol Biotechnol 1998;14(5):689–91. 12. Venkateswarlu G, Murali PSK, Sharma G, Venkateswar RL. Improvement of rifamycin B production using mutant strains of Amycolatopsis mediterranei. J Biopro Eng 2000;23:315–8. 13. El-Tayeb OM, Salama AA, Hussein MMM, El-Sedawy HF. Optimization of industrial production of rifamycin B by Amycolatopsis mediterranei IV. Production in the fermentor. Afr J Biotechnol 2004;3(9):432–40. 14. El-Tayeb OM, Salama AA, Hussein MMM, El-Sedawy HF. Optimization of industrial production of rifamycin B by Amycolatopsis mediterranei II. The role of gene amplification and physiological factors in productivity. Afr J Biotechnol 2004;3(5):273–80. 15. El-Tayeb OM, Salama AA, Hussein MMM, El-Sedawy HF. Optimization of industrial production of rifamycin B by Amycolatopsis mediterranei I. The role of colony morphology and nitrogen sources in productivity. Afr J Biotechnol 2004;3(5):266–72. 16. Lancini G, Cavalleri B. Rifamycins. In: Strohl WR, editor. Biotech Ind Antibiot. 2nd ed. New York: Marcel Decker; 1997. p. 521–49. 17. Lee KJ, Rho YT. Quantitative analysis of mycelium morphological characteristics and rifamycin B production using Nocardia mediterranei. J Biotechnol 1994;36:239–45. 18. Sensi P, Thiemann JE. Production of rifamycins. J Prog Ind Microbiol 1967;6:21–59.