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Evaluation of Amycolatopsis mediterranei VA18 for production of rifamycin-B
Process Biochemistry 36 (2000) 305 – 309 www.elsevier.com/locate/procbio
Evaluation of Amycolatopsis mediterranei VA18 for production of rifamycin-B G. Venkateswarlu a, P.S. Murali Krishna a, Ashok Pandey b, L. Venkateshwar Rao a,* b
a Department of Microbiology, Osmania Uni6ersity, Hyderabad 500007, India Biotechnology Di6ision, Regional Research Laboratory, Council of Scientific and Industrial Research (CSIR), Tri6andrum 695019, India
Received 17 January 2000; received in revised form 24 May 2000; accepted 3 June 2000
Abstract Comparative studies were performed to develop a fermentation process for the production of rifamycin-B using Amycolatopsis mediterranei VA18. Wheat bran as substrate in solid state fermentation (SSF), and peanut meal and soybean with glucose were used as substrate for submerged fermentation (SmF). Various process parameters including moisture and pH of the substrate, inoculum size, temperature and period of incubation were optimised for rifamycin-B production. For SmF, the optimal conditions were 10% (v/v) inoculum, pH 7.2, incubation temperature 30°C with a fermentation period of 6 days. When SmF was carried out in fermenter with controlled aeration (1.5 vvm) and dissolved oxygen level (80%), rifamycin production increased to about 1.5-fold greater than the flasks. SSF was, in general, superior to SmF conducted in flasks or fermenter. Optimal conditions for SSF were 7.2 substrate pH, 30% (v/v) inoculum size and incubation at 32°C for 9 days. As the content of substrate moisture increased in SSF, rifamycin production also increased and highest yields were obtained when fermentation was carried out using 90% moisture, which led practically to semi-solid-to-slurry conditions of fermentation. With 70% substrate moisture (SSF conditions), rifamycin production was about 15 g/kg which was about 31 g/kg with 80% moisture. Maximum production (rifamycin-B, 39 g/kg substrate) was obtained with 90% substrate moisture, which was almost 16-fold higher than that obtained in the fermenter (SmF). Ethyl acetate appeared as the most suitable organic solvent for the extraction of the antibiotic from the fermented material. © 2000 Published by Elsevier Science Ltd. All rights reserved. Keywords: Solid state fermentation; Semi-solid fermentation; Submerged fermentation; Rifamycin-B; Wheat bran; Process parameters
1. Introduction In recent years, much attention has been directed towards the use of solid state fermentation (SSF) for the production of products of commercial interest including enzymes, antibiotics, alcohol, etc. [1 – 8]). SSF has also been found of potential significance for bacterial cultures as well, in addition to fungi and yeast [2,4,9,10]. Rifamycin-B is a broad-spectrum macrolide antibiotic used as a drug of choice against Mycobacterium tuberculosis and Mycobacterium leprae, the causative agents of tuberculosis and leprosy. It is pro-
* Corresponding author. Fax: +91-4-7019020. E-mail address: [email protected] (L. Venkateshwar Rao).
duced by Amycolatopsis mediterranei in submerged fermentation (SmF) [11,12]. For the large-scale production of rifamycin, the development of inexpensive culture substrates is essential. During this quest for cheaper substrates, agro-industrial residues, such as wheat bran, rice bran and other cereals appeared to be viable possibilities [13–15]. Application of agro-industrial residues in SSF has been found potentially useful for biotechnological processes [5,16,17]. Although, rifamycin-B is currently produced by SmF, there appears good scope for SSF based bioprocesses for its production, mainly due to the potential advantages SSF offers [2,3,18]. To the best of our knowledge, no reports are available on rifamycin-B production with A. mediterranei in SSF. Hence, an attempt was made to produce rifamycin-B by SSF using wheat bran as solid substrate.
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2. Materials and methods
2.1. Microorganisms A. mediterranei VA18, a mutant strain isolated in our laboratory, was used in present study [19,20]. It was maintained on a medium containing (g/l), yeast extract, 4.0; malt extract, 10.0; glucose, 4.0; agar, 15; pH 7.2. Agar slants were prepared by incubating at 28°C for 5 days and stored at 4°C.
2.2. Inoculum preparation A. mediterranei VA18 was grown in 50 ml medium, held in 250 ml Erlenmeyer flasks, containing (g/l), meat extract, 5.0; peptone, 5.0; yeast extract, 5.0; enzymatic hydrolysate of casein, 2.9; glucose, 20.0; sodium chloride, 1.5; sodium citrate, 0.0259; and pH, 7.2. Flasks were incubated on a rotary shaker incubator at 30°C for 5 days at 200 rpm. After incubation, the culture was transferred to a sterile tube and centrifuged for 5 min at 2000× g. The supernatant obtained was discarded and the culture pellet suspended in 0.1-M phosphate buffer. This process was repeated and the total number of cells adjusted to 4.2× 109 cfu/ml. These cells were used as inoculum for SSF and SmF.
matter basis) 14.9 protein, 6.1 fat, 30.6 starch, 7.5 free sugars, 20.2 pentosans, 7.5 cellulose, 3.1 lignin, 5.6 uronic acids and 4.5 ash [20]. Ten g of wheat bran were taken in 250 ml conical flasks and were supplemented with peanut de-oiled cake (1 g), CaCO3, 600 mg; (NH4)2SO4, 700 mg; KH2PO4, [S1] mm 300 mg; MgSO 7H2O, 200 mg; sodium barbiturate, 200 mg; and moistened with sterile distilled water containing trace elements solution (as in SmF). The contents of the flasks were mixed and autoclaved at 121°C for 20 min. After cooling, the flasks were inoculated with 30% (v/w) inoculum and incubated at 30°C for 12 days.
2.4.2. Effect of moisture of the substrate The effect of moisture level of the substrate on rifamycin-B production was studied by adjusting the moisture level of the medium between 40 and 110%. Sterile distilled water and mineral solution were used as moistening agents to adjust the moisture content of the substrate. 2.4.3. Effect of inoculum size The 5-day-old inoculum ranging 5–60% (v/w, initial dry weight of wheat bran) was inoculated into the sterile medium containing 10 g of wheat bran with supplements, pH 7.2 and incubated at 30°C. Substrate moisture was 90%.
2.3. Submerged fermentation The medium used for SmF contained (g/l), peanut meal, 20.0; soybean meal, 20.0; glucose, 25.0; (NH4)2SO4, 7.0; CaCO3, 6.0; KH2PO4, 3.0; MgSO4 · 7H2O, 2.0; and sodium barbiturate, 2.0. Fifty ml of this medium was placed in 250-ml Erlenmeyer flasks and autoclaved at 121°C for 15 min. After adjusting the pH to 7.2, 1000 ml of the sterilised trace elements solution containing (g/l) CuSO4 · 5H2O, 3.3; FeSO4 · 7H2O, 10.0; ZnSO4 · 7H2O, 50.0; MnSO4 · 2H2O, 4.0; CoCl2, 2.0; (NH4)2 · MoO4, 1.0 were added. The flasks were inoculated using 10% inoculum size. Fermentation was carried out on a rotary incubator at 30°C for 12 days (200 rpm). SmF was also performed in a 2-l lab fermenter (New Brunswick, Bioflo 2000) using the same medium as used for flasks. The aeration rate was 1.5 vvm, maintaining the dissolved oxygen at 80% by regulation and 300 rpm agitation. The pH of the medium was maintained at 7.2 by the addition of 1 N NaOH/HCl. Samples were withdrawn at regular intervals for the analysis of rifamycin.
2.4. Solid state fermentation 2.4.1. Preparation of solid substrate Wheat bran was used as solid substrate for the production of rifamycin-B. It contained (percent of dry
2.4.4. Effect of initial pH and temperature The effect of pH of the substrate and temperature of incubation on the production of rifamycin-B by SSF was studied by growing the culture at different initial pH values (6.0–9.0) and temperatures (24–40°C). The substrate moisture was 90%. 2.5. Extraction of rifamycin-B In order to optimise the extraction of rifamycin from the fermented bran, different solvents such as methanol, ethyl acetate, isopropyl alcohol and butyl acetate were used in the ratio of 1:3 (bran:solvent, w/v). Extraction was done overnight on a rotary shaker at 200 rpm at 32°C. Contents were then filtered through Whatman No. 1 filter paper and the solvent collected was evaporated under vacuum to achieve the concentrated product.
2.6. Analytical methods The pH of the samples was determined as described previously [19,20]. To determine the moisture content of the substrate, samples were dried in an air-oven for 12 h at 60°C. Rifamycin-B in the extract was estimated by reverse phase of high performance liquid chromatography (Watery’s double pumping HPLC system) using C18 ODS column with ammonium formate and
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Fig. 1. Growth and fermentation profile of A. mediterranei in SmF.
methanol as solvent system [21]. Rifamycin production was quantified as described elsewhere [22].
3. Results and discussions Rifamycins are primarily produced by the Gram-positive bacterium A. mediterranei, which belongs to the order Actinomycetales. The growth and fermentation profile of rifamycin production in SmF is shown in Fig. 1. Data recorded in Table 1 present the comparative profile of rifamycin-B production in SmF in flasks and fermenter, and in SSF using wheat bran by A. mediterranei VA18. As is evident, antibiotic production in flasks was minimal (1.8 g/l), but this was about 1.5-fold higher when SmF was carried out in the fermenter under improved controlled conditions. In the former case, the maximum yield was obtained in 7 days while in the latter; it took only 6 days to attain the maximum yields Table 1. In both cases the same medium was used and the pH of the medium, temperature of incubation, size and age of inoculum and the period of fermentation were similar. There were, however, differences in aeration rate and pattern. In the fermenter, there was continuous inflow of air at a rate of 1.5 vvm and dissolved oxygen was maintained at 80% saturation. The increase in the production of antibiotic could be
307
Fig. 2. Effect of initial moisture content of substrate on rifamycin-B production.
attributed to this factor. However, higher antibiotic production occurred in SSF using wheat bran as the substrate. With an increase in moisture of the substrate in SSF, an increase in the yield of antibiotic was noticed. However, a gradual increase in substrate moisture simultaneously changed the conditions of SSF to semi-solid and finally to those comparable with SmF when 90% substrate moisture resulted in the highest antibiotic production. At 70% moisture, which could be considered as the substrate in SSF condition, antibiotic production was about 26 g/kg substrate. A substrate with higher than 70% moisture and up to 90% could be termed as semi-solid conditions and resulted in a further increase in the rifamycin production. The yield was highest with 90% substrate moisture, which was almost 16-fold higher than SmF conducted in the fermenter Fig. 2. Interestingly, in SSF and semi-solid fermentation, the time required to attain the highest production was 9 days which was higher than SmF. In SSF, the moisture level of the substrate has been considered as an important factor for the growth and activity of the microbial culture. In these studies, higher rifamycin production was observed with an increase in the substrate moisture and the maximum was at 90% substrate moisture Fig. 1in 9 days. Further increases in the moisture content of the substrate retarded antibiotic
Table 1 Production profile of rifamycin-B with different fermentation systems using A. mediterranei VA18a Fermentation type SmF (flask) SmF (fermenter 2-l) SSF (90% moistureb) 80% moisturec 70% moistured a
Time (days) 7 6 9 9 9
*, g/l Medium; **, g/kg substrate. Semi-solid to slurry conditions. c Semi-solid conditions. d True SSF. b
Aeration (vvm) 0 1.5 0 0 0
Agitation 200 300 0 0 0
pH 7.2 7.2 7.2 7.2 7.2
Temperature (°C) 30 30 32 32 32
Yield 1.8* 2.45* 39** 32.8** 26**
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production. It has been reported that higher moisture levels cause particle agglomeration, which interferes with heat and mass transfer, in turn resulting in decreased microbial activity [2,3]. However, if there is insufficient water (lower moisture levels), it causes decreased solubility and lower availability of nutrients to the microbial culture which, in turn, affects harmfully the microbial activity and causes a decrease in product yields. The influence of inoculum size on rifamycin-B production by A. mediterranei in SSF is shown in Fig. 3. Inoculum size was based on v/w ratio of the substrate and took into account the final moisture as 90%, inFig. 5. Effect of incubation temperature on rifamycin-B production.
Fig. 3. Effect of inoculum size on rifamycin-B production.
Fig. 4. (a) Effect of substrate pH on rifamycin-B production, (b) change of pH during the course of fermentation.
cluding that contributed by the inoculum. At 10% inoculum size, the culture produced antibiotic about 12 g/kg substrate, which increased to 35 g/kg with 30% inoculum size. A higher inoculum size resulted in decreased production of the antibiotic. The time required to achieve the highest production in this case was also 9 days. The effect of pH of the medium and temperature of incubation on the production of rifamycin-B by A. mediterranei in SSF is shown in Fig. 4a and b Fig. 5. The effect of pH was studied with a range 6.0–9.0. Evidently, media with pH near to neutrality were most suitable for antibiotic production and maximum production occurred at pH 7.2 (36 g/kg substrate in 9 days). Higher or lower media pH resulted in lowering of rifamycin-B. At pH 6.0, the strain could not produce even one-third of the quantity of rifamycin produced under the most suitable pH conditions. As is evident from Fig. 4a, there was not much change in the pH of the substrate during the course of fermentation, which, however, showed a fluctuating trend, but did not vary much at the end of fermentation. The optimum temperature for rifamycin production was 32°C, which resulted in antibiotic substrate of 39 g/kg. This was higher by 2°C than SmF. The temperature of incubation and its control in SSF processes is crucial as the heat evolved during SSF process is accumulated in the medium due to poor heat dissipation. This results in reduced microbial activity, thereby decreasing the yield of product formation [23,24]. The yield of rifamycin-B extraction with different solvents viz. methanol, ethyl acetate, isopropyl alcohol and butyl acetate is shown in Fig. 6. Optimum extraction of rifamycins was obtained by using ethyl acetate (39 g/kg) followed by methanol (36 g/kg). The order of their effectiveness was ethyl acetate\ methanol\butyl acetate\ isopropyl alcohol. The crude extract obtained by extraction with butyl acetate and isopropyl alcohol was darker than other extracts and on concentration contained higher solid contents.
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Fig. 6. Extraction of rifamycin-B with different solvents.
4. Conclusions The results obtained with different types of fermentation and substrates used in this study indicated the superiority of SSF over SmF and that of wheat bran as substrate. The highest production with wheat bran as substrate was 39 g antibiotic per kg substrate, which, however, was with 90% substrate moisture, leading practically to SmF conditions. This was followed by SmF in the fermenter (2.45 g/l). The lowest yield (1.8 g/l) was obtained in flasks (SmF). Although, the duration of fermentation with wheat bran was longer (9 days) than in SmF (6 days), there was almost 16-fold increase in the product titres.
Acknowledgements We are grateful to the University Grants Commission, New Delhi and the Department of Biotechnology, New Delhi for the financial support during this investigation.
References [1] Mitchell DA, Lonsane BK. Definition, characteristics and potential. In: Doelle HW, Mitchell DA, Rolz C, editors. Solid Substrate Cultivation. London, UK: Elsevier, 1993:1–16. [2] Pandey A. Recent developments in solid state fermentation. Process Biochem 1992;27:109–16. [3] Pandey A. Solid state fermentation: an overview. In: Pandey A, editor. Solid State Fermentation. New Delhi: Wiley Eastern, 1994:3 – 10. [4] Pandey A, Selvakumar P, Nigam P, Soccol CR. Solid state fermentation for production of industrial enzymes. Curr Sci 1999;77:149 – 62.
.
309
[5] Pandey A, Soccol CR. Bioconversion of biomass — a case study of lignocellulosics bioconversions in solid state fermentation. Brazilian Arch Biol Technol 1998;42:377 – 90. [6] Pandey A, Azmi W, Banerjee UC. Types of fermentation and factors affecting it. In: Joshi VK, Pandey A, editors. Biotechnology: Food Fermentation. New Delhi: Educational Publishers, 1999:383 – 426. [7] Kiran Sree N, Sridhar M, Rao LV, Pandey A. Ethanol production in solid substrate fermentation using thermotolerant yeast. Process Biochem 1999;34:115 – 9. [8] Pandey A, Soccol CR, Mitchell D. New developments in solid state fermentation. I-bioprocesses and products. Process Biochem 2000, in press. [9] Nampoothiri KM, Pandey A. Solid state fermentation for production of glutamic acid using Bre6ibacterium sp. Biotechnol Lett 1996;18:199 – 204. [10] Carrizales V, Jaffe W. Solid state fermentation: an appropriate biotechnology for developing countries. Intern Sci 1986;11:9–15. [11] Hartmann G, Honikel KO, Knusel F, Nuesh J. The specific inhibition of the DNA directed RNA synthesis by rifamycin. Biochem Biothysic Acta 1967;145:843 – 4. [12] Kunin CM. Clinical infectious diseases, antimicrobial activity of rifabutin. 1986. [13] Pandey A, Soccol CR, Nigam P, Soccol VT, Mohan R. Biotechnological potential of agro-industrial residues: I sugarcane bagasse. Biores Technol 2000;74:69 – 80. [14] Pandey A, Soccol CR, Nigam P, Soccol VT, Vandenberghe LPS. Biotechnological potential of agro-industrial residues: II cassava bagasse. Biores Technol 2000;74:81 – 7. [15] Pandey A, Soccol CR. Economic utilization of crop residues — a futuristic approach. J Sci Ind Res 2000;59:12 – 22. [16] Fan L, Pandey A, Soccol CR. Solid state culturing: an efficient technique to use toxic agro-industrial residues. J Basic Microbiol 2000, in press. [17] Pandey A, Soccol CR. Potential applications of cellulosic residues for the production of bulk chemicals and value added products. In: Soni PL, Kumar V, editors. Trends in Carbohydrate Chemistry, vol. 5. Dehradun, India: Surya International Publications, 1998:83 – 8. [18] Hesseltine CW. Solid state fermentation. Process Biochem 1977;12:24 – 9. [19] Venkateswarlu G, Krishna PSM, Venkateshwar Rao L. Production of rifamycin using Amycolatopsis mediterranei. Bioprocess Eng 1999;20:27 – 30. [20] Venkateswarlu G, Krishna PSM, Sharma G, Venkateshwar Rao L. Improvement of rifamycin B production using Amycolatopsis mediterranei. Bioprocess Eng 1999, in press. [21] Vohra RM, Dube S. Identification and quantification of rifamycins by reversed phase high performance liquid chromatography. J Chromatogr 1986;477:463 – 6. [22] Passqualucci CR, Vigaveni A, Radaelli P, Galleo CG. Improved differential spectrophotometric determination of rifamycins. J Pharm Sci 1970;59:685 – 7. [23] Raimbault M, Alazard D. Culture method to study fungal growth in solid fermentation. Eur J Appl Microbiol Biotechnol 1980;9:199 – 209. [24] Pandey A, Radhakrishnan S. The production of glucomylase in solid state fermentation. Process Biochem 1993;28:305 –8.
Evaluation of Amycolatopsis mediterranei VA18 for production of rifamycin-B G. Venkateswarlu a, P.S. Murali Krishna a, Ashok Pandey b, L. Venkateshwar Rao a,* b
a Department of Microbiology, Osmania Uni6ersity, Hyderabad 500007, India Biotechnology Di6ision, Regional Research Laboratory, Council of Scientific and Industrial Research (CSIR), Tri6andrum 695019, India
Received 17 January 2000; received in revised form 24 May 2000; accepted 3 June 2000
Abstract Comparative studies were performed to develop a fermentation process for the production of rifamycin-B using Amycolatopsis mediterranei VA18. Wheat bran as substrate in solid state fermentation (SSF), and peanut meal and soybean with glucose were used as substrate for submerged fermentation (SmF). Various process parameters including moisture and pH of the substrate, inoculum size, temperature and period of incubation were optimised for rifamycin-B production. For SmF, the optimal conditions were 10% (v/v) inoculum, pH 7.2, incubation temperature 30°C with a fermentation period of 6 days. When SmF was carried out in fermenter with controlled aeration (1.5 vvm) and dissolved oxygen level (80%), rifamycin production increased to about 1.5-fold greater than the flasks. SSF was, in general, superior to SmF conducted in flasks or fermenter. Optimal conditions for SSF were 7.2 substrate pH, 30% (v/v) inoculum size and incubation at 32°C for 9 days. As the content of substrate moisture increased in SSF, rifamycin production also increased and highest yields were obtained when fermentation was carried out using 90% moisture, which led practically to semi-solid-to-slurry conditions of fermentation. With 70% substrate moisture (SSF conditions), rifamycin production was about 15 g/kg which was about 31 g/kg with 80% moisture. Maximum production (rifamycin-B, 39 g/kg substrate) was obtained with 90% substrate moisture, which was almost 16-fold higher than that obtained in the fermenter (SmF). Ethyl acetate appeared as the most suitable organic solvent for the extraction of the antibiotic from the fermented material. © 2000 Published by Elsevier Science Ltd. All rights reserved. Keywords: Solid state fermentation; Semi-solid fermentation; Submerged fermentation; Rifamycin-B; Wheat bran; Process parameters
1. Introduction In recent years, much attention has been directed towards the use of solid state fermentation (SSF) for the production of products of commercial interest including enzymes, antibiotics, alcohol, etc. [1 – 8]). SSF has also been found of potential significance for bacterial cultures as well, in addition to fungi and yeast [2,4,9,10]. Rifamycin-B is a broad-spectrum macrolide antibiotic used as a drug of choice against Mycobacterium tuberculosis and Mycobacterium leprae, the causative agents of tuberculosis and leprosy. It is pro-
* Corresponding author. Fax: +91-4-7019020. E-mail address: [email protected] (L. Venkateshwar Rao).
duced by Amycolatopsis mediterranei in submerged fermentation (SmF) [11,12]. For the large-scale production of rifamycin, the development of inexpensive culture substrates is essential. During this quest for cheaper substrates, agro-industrial residues, such as wheat bran, rice bran and other cereals appeared to be viable possibilities [13–15]. Application of agro-industrial residues in SSF has been found potentially useful for biotechnological processes [5,16,17]. Although, rifamycin-B is currently produced by SmF, there appears good scope for SSF based bioprocesses for its production, mainly due to the potential advantages SSF offers [2,3,18]. To the best of our knowledge, no reports are available on rifamycin-B production with A. mediterranei in SSF. Hence, an attempt was made to produce rifamycin-B by SSF using wheat bran as solid substrate.
0032-9592/00/$ - see front matter © 2000 Published by Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 0 ) 0 0 2 0 4 - 1
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2. Materials and methods
2.1. Microorganisms A. mediterranei VA18, a mutant strain isolated in our laboratory, was used in present study [19,20]. It was maintained on a medium containing (g/l), yeast extract, 4.0; malt extract, 10.0; glucose, 4.0; agar, 15; pH 7.2. Agar slants were prepared by incubating at 28°C for 5 days and stored at 4°C.
2.2. Inoculum preparation A. mediterranei VA18 was grown in 50 ml medium, held in 250 ml Erlenmeyer flasks, containing (g/l), meat extract, 5.0; peptone, 5.0; yeast extract, 5.0; enzymatic hydrolysate of casein, 2.9; glucose, 20.0; sodium chloride, 1.5; sodium citrate, 0.0259; and pH, 7.2. Flasks were incubated on a rotary shaker incubator at 30°C for 5 days at 200 rpm. After incubation, the culture was transferred to a sterile tube and centrifuged for 5 min at 2000× g. The supernatant obtained was discarded and the culture pellet suspended in 0.1-M phosphate buffer. This process was repeated and the total number of cells adjusted to 4.2× 109 cfu/ml. These cells were used as inoculum for SSF and SmF.
matter basis) 14.9 protein, 6.1 fat, 30.6 starch, 7.5 free sugars, 20.2 pentosans, 7.5 cellulose, 3.1 lignin, 5.6 uronic acids and 4.5 ash [20]. Ten g of wheat bran were taken in 250 ml conical flasks and were supplemented with peanut de-oiled cake (1 g), CaCO3, 600 mg; (NH4)2SO4, 700 mg; KH2PO4, [S1] mm 300 mg; MgSO 7H2O, 200 mg; sodium barbiturate, 200 mg; and moistened with sterile distilled water containing trace elements solution (as in SmF). The contents of the flasks were mixed and autoclaved at 121°C for 20 min. After cooling, the flasks were inoculated with 30% (v/w) inoculum and incubated at 30°C for 12 days.
2.4.2. Effect of moisture of the substrate The effect of moisture level of the substrate on rifamycin-B production was studied by adjusting the moisture level of the medium between 40 and 110%. Sterile distilled water and mineral solution were used as moistening agents to adjust the moisture content of the substrate. 2.4.3. Effect of inoculum size The 5-day-old inoculum ranging 5–60% (v/w, initial dry weight of wheat bran) was inoculated into the sterile medium containing 10 g of wheat bran with supplements, pH 7.2 and incubated at 30°C. Substrate moisture was 90%.
2.3. Submerged fermentation The medium used for SmF contained (g/l), peanut meal, 20.0; soybean meal, 20.0; glucose, 25.0; (NH4)2SO4, 7.0; CaCO3, 6.0; KH2PO4, 3.0; MgSO4 · 7H2O, 2.0; and sodium barbiturate, 2.0. Fifty ml of this medium was placed in 250-ml Erlenmeyer flasks and autoclaved at 121°C for 15 min. After adjusting the pH to 7.2, 1000 ml of the sterilised trace elements solution containing (g/l) CuSO4 · 5H2O, 3.3; FeSO4 · 7H2O, 10.0; ZnSO4 · 7H2O, 50.0; MnSO4 · 2H2O, 4.0; CoCl2, 2.0; (NH4)2 · MoO4, 1.0 were added. The flasks were inoculated using 10% inoculum size. Fermentation was carried out on a rotary incubator at 30°C for 12 days (200 rpm). SmF was also performed in a 2-l lab fermenter (New Brunswick, Bioflo 2000) using the same medium as used for flasks. The aeration rate was 1.5 vvm, maintaining the dissolved oxygen at 80% by regulation and 300 rpm agitation. The pH of the medium was maintained at 7.2 by the addition of 1 N NaOH/HCl. Samples were withdrawn at regular intervals for the analysis of rifamycin.
2.4. Solid state fermentation 2.4.1. Preparation of solid substrate Wheat bran was used as solid substrate for the production of rifamycin-B. It contained (percent of dry
2.4.4. Effect of initial pH and temperature The effect of pH of the substrate and temperature of incubation on the production of rifamycin-B by SSF was studied by growing the culture at different initial pH values (6.0–9.0) and temperatures (24–40°C). The substrate moisture was 90%. 2.5. Extraction of rifamycin-B In order to optimise the extraction of rifamycin from the fermented bran, different solvents such as methanol, ethyl acetate, isopropyl alcohol and butyl acetate were used in the ratio of 1:3 (bran:solvent, w/v). Extraction was done overnight on a rotary shaker at 200 rpm at 32°C. Contents were then filtered through Whatman No. 1 filter paper and the solvent collected was evaporated under vacuum to achieve the concentrated product.
2.6. Analytical methods The pH of the samples was determined as described previously [19,20]. To determine the moisture content of the substrate, samples were dried in an air-oven for 12 h at 60°C. Rifamycin-B in the extract was estimated by reverse phase of high performance liquid chromatography (Watery’s double pumping HPLC system) using C18 ODS column with ammonium formate and
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Fig. 1. Growth and fermentation profile of A. mediterranei in SmF.
methanol as solvent system [21]. Rifamycin production was quantified as described elsewhere [22].
3. Results and discussions Rifamycins are primarily produced by the Gram-positive bacterium A. mediterranei, which belongs to the order Actinomycetales. The growth and fermentation profile of rifamycin production in SmF is shown in Fig. 1. Data recorded in Table 1 present the comparative profile of rifamycin-B production in SmF in flasks and fermenter, and in SSF using wheat bran by A. mediterranei VA18. As is evident, antibiotic production in flasks was minimal (1.8 g/l), but this was about 1.5-fold higher when SmF was carried out in the fermenter under improved controlled conditions. In the former case, the maximum yield was obtained in 7 days while in the latter; it took only 6 days to attain the maximum yields Table 1. In both cases the same medium was used and the pH of the medium, temperature of incubation, size and age of inoculum and the period of fermentation were similar. There were, however, differences in aeration rate and pattern. In the fermenter, there was continuous inflow of air at a rate of 1.5 vvm and dissolved oxygen was maintained at 80% saturation. The increase in the production of antibiotic could be
307
Fig. 2. Effect of initial moisture content of substrate on rifamycin-B production.
attributed to this factor. However, higher antibiotic production occurred in SSF using wheat bran as the substrate. With an increase in moisture of the substrate in SSF, an increase in the yield of antibiotic was noticed. However, a gradual increase in substrate moisture simultaneously changed the conditions of SSF to semi-solid and finally to those comparable with SmF when 90% substrate moisture resulted in the highest antibiotic production. At 70% moisture, which could be considered as the substrate in SSF condition, antibiotic production was about 26 g/kg substrate. A substrate with higher than 70% moisture and up to 90% could be termed as semi-solid conditions and resulted in a further increase in the rifamycin production. The yield was highest with 90% substrate moisture, which was almost 16-fold higher than SmF conducted in the fermenter Fig. 2. Interestingly, in SSF and semi-solid fermentation, the time required to attain the highest production was 9 days which was higher than SmF. In SSF, the moisture level of the substrate has been considered as an important factor for the growth and activity of the microbial culture. In these studies, higher rifamycin production was observed with an increase in the substrate moisture and the maximum was at 90% substrate moisture Fig. 1in 9 days. Further increases in the moisture content of the substrate retarded antibiotic
Table 1 Production profile of rifamycin-B with different fermentation systems using A. mediterranei VA18a Fermentation type SmF (flask) SmF (fermenter 2-l) SSF (90% moistureb) 80% moisturec 70% moistured a
Time (days) 7 6 9 9 9
*, g/l Medium; **, g/kg substrate. Semi-solid to slurry conditions. c Semi-solid conditions. d True SSF. b
Aeration (vvm) 0 1.5 0 0 0
Agitation 200 300 0 0 0
pH 7.2 7.2 7.2 7.2 7.2
Temperature (°C) 30 30 32 32 32
Yield 1.8* 2.45* 39** 32.8** 26**
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production. It has been reported that higher moisture levels cause particle agglomeration, which interferes with heat and mass transfer, in turn resulting in decreased microbial activity [2,3]. However, if there is insufficient water (lower moisture levels), it causes decreased solubility and lower availability of nutrients to the microbial culture which, in turn, affects harmfully the microbial activity and causes a decrease in product yields. The influence of inoculum size on rifamycin-B production by A. mediterranei in SSF is shown in Fig. 3. Inoculum size was based on v/w ratio of the substrate and took into account the final moisture as 90%, inFig. 5. Effect of incubation temperature on rifamycin-B production.
Fig. 3. Effect of inoculum size on rifamycin-B production.
Fig. 4. (a) Effect of substrate pH on rifamycin-B production, (b) change of pH during the course of fermentation.
cluding that contributed by the inoculum. At 10% inoculum size, the culture produced antibiotic about 12 g/kg substrate, which increased to 35 g/kg with 30% inoculum size. A higher inoculum size resulted in decreased production of the antibiotic. The time required to achieve the highest production in this case was also 9 days. The effect of pH of the medium and temperature of incubation on the production of rifamycin-B by A. mediterranei in SSF is shown in Fig. 4a and b Fig. 5. The effect of pH was studied with a range 6.0–9.0. Evidently, media with pH near to neutrality were most suitable for antibiotic production and maximum production occurred at pH 7.2 (36 g/kg substrate in 9 days). Higher or lower media pH resulted in lowering of rifamycin-B. At pH 6.0, the strain could not produce even one-third of the quantity of rifamycin produced under the most suitable pH conditions. As is evident from Fig. 4a, there was not much change in the pH of the substrate during the course of fermentation, which, however, showed a fluctuating trend, but did not vary much at the end of fermentation. The optimum temperature for rifamycin production was 32°C, which resulted in antibiotic substrate of 39 g/kg. This was higher by 2°C than SmF. The temperature of incubation and its control in SSF processes is crucial as the heat evolved during SSF process is accumulated in the medium due to poor heat dissipation. This results in reduced microbial activity, thereby decreasing the yield of product formation [23,24]. The yield of rifamycin-B extraction with different solvents viz. methanol, ethyl acetate, isopropyl alcohol and butyl acetate is shown in Fig. 6. Optimum extraction of rifamycins was obtained by using ethyl acetate (39 g/kg) followed by methanol (36 g/kg). The order of their effectiveness was ethyl acetate\ methanol\butyl acetate\ isopropyl alcohol. The crude extract obtained by extraction with butyl acetate and isopropyl alcohol was darker than other extracts and on concentration contained higher solid contents.
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Fig. 6. Extraction of rifamycin-B with different solvents.
4. Conclusions The results obtained with different types of fermentation and substrates used in this study indicated the superiority of SSF over SmF and that of wheat bran as substrate. The highest production with wheat bran as substrate was 39 g antibiotic per kg substrate, which, however, was with 90% substrate moisture, leading practically to SmF conditions. This was followed by SmF in the fermenter (2.45 g/l). The lowest yield (1.8 g/l) was obtained in flasks (SmF). Although, the duration of fermentation with wheat bran was longer (9 days) than in SmF (6 days), there was almost 16-fold increase in the product titres.
Acknowledgements We are grateful to the University Grants Commission, New Delhi and the Department of Biotechnology, New Delhi for the financial support during this investigation.
References [1] Mitchell DA, Lonsane BK. Definition, characteristics and potential. In: Doelle HW, Mitchell DA, Rolz C, editors. Solid Substrate Cultivation. London, UK: Elsevier, 1993:1–16. [2] Pandey A. Recent developments in solid state fermentation. Process Biochem 1992;27:109–16. [3] Pandey A. Solid state fermentation: an overview. In: Pandey A, editor. Solid State Fermentation. New Delhi: Wiley Eastern, 1994:3 – 10. [4] Pandey A, Selvakumar P, Nigam P, Soccol CR. Solid state fermentation for production of industrial enzymes. Curr Sci 1999;77:149 – 62.
.
309
[5] Pandey A, Soccol CR. Bioconversion of biomass — a case study of lignocellulosics bioconversions in solid state fermentation. Brazilian Arch Biol Technol 1998;42:377 – 90. [6] Pandey A, Azmi W, Banerjee UC. Types of fermentation and factors affecting it. In: Joshi VK, Pandey A, editors. Biotechnology: Food Fermentation. New Delhi: Educational Publishers, 1999:383 – 426. [7] Kiran Sree N, Sridhar M, Rao LV, Pandey A. Ethanol production in solid substrate fermentation using thermotolerant yeast. Process Biochem 1999;34:115 – 9. [8] Pandey A, Soccol CR, Mitchell D. New developments in solid state fermentation. I-bioprocesses and products. Process Biochem 2000, in press. [9] Nampoothiri KM, Pandey A. Solid state fermentation for production of glutamic acid using Bre6ibacterium sp. Biotechnol Lett 1996;18:199 – 204. [10] Carrizales V, Jaffe W. Solid state fermentation: an appropriate biotechnology for developing countries. Intern Sci 1986;11:9–15. [11] Hartmann G, Honikel KO, Knusel F, Nuesh J. The specific inhibition of the DNA directed RNA synthesis by rifamycin. Biochem Biothysic Acta 1967;145:843 – 4. [12] Kunin CM. Clinical infectious diseases, antimicrobial activity of rifabutin. 1986. [13] Pandey A, Soccol CR, Nigam P, Soccol VT, Mohan R. Biotechnological potential of agro-industrial residues: I sugarcane bagasse. Biores Technol 2000;74:69 – 80. [14] Pandey A, Soccol CR, Nigam P, Soccol VT, Vandenberghe LPS. Biotechnological potential of agro-industrial residues: II cassava bagasse. Biores Technol 2000;74:81 – 7. [15] Pandey A, Soccol CR. Economic utilization of crop residues — a futuristic approach. J Sci Ind Res 2000;59:12 – 22. [16] Fan L, Pandey A, Soccol CR. Solid state culturing: an efficient technique to use toxic agro-industrial residues. J Basic Microbiol 2000, in press. [17] Pandey A, Soccol CR. Potential applications of cellulosic residues for the production of bulk chemicals and value added products. In: Soni PL, Kumar V, editors. Trends in Carbohydrate Chemistry, vol. 5. Dehradun, India: Surya International Publications, 1998:83 – 8. [18] Hesseltine CW. Solid state fermentation. Process Biochem 1977;12:24 – 9. [19] Venkateswarlu G, Krishna PSM, Venkateshwar Rao L. Production of rifamycin using Amycolatopsis mediterranei. Bioprocess Eng 1999;20:27 – 30. [20] Venkateswarlu G, Krishna PSM, Sharma G, Venkateshwar Rao L. Improvement of rifamycin B production using Amycolatopsis mediterranei. Bioprocess Eng 1999, in press. [21] Vohra RM, Dube S. Identification and quantification of rifamycins by reversed phase high performance liquid chromatography. J Chromatogr 1986;477:463 – 6. [22] Passqualucci CR, Vigaveni A, Radaelli P, Galleo CG. Improved differential spectrophotometric determination of rifamycins. J Pharm Sci 1970;59:685 – 7. [23] Raimbault M, Alazard D. Culture method to study fungal growth in solid fermentation. Eur J Appl Microbiol Biotechnol 1980;9:199 – 209. [24] Pandey A, Radhakrishnan S. The production of glucomylase in solid state fermentation. Process Biochem 1993;28:305 –8.