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Optimisation studies on neomycin production by a mutant strain of Streptomyces marinensis in solid state fermentation
Process Biochemistry 39 (2004) 529–534
Optimisation studies on neomycin production by a mutant strain of Streptomyces marinensis in solid state fermentation P. Ellaiah∗ , B. Srinivasulu, K. Adinarayana Pharmaceutical Biotechnology Division, Department of Pharmaceutical Sciences, Andhra University, Visakhapatnam 530 003, India Received 13 December 2001; received in revised form 18 January 2002; accepted 19 February 2002
Abstract Production of neomycin by a mutant strain of Streptomyces marinensis, under solid state fermentation was optimised. Various substrates were used to study their effect on neomycin production. Wheat rawa was found to be better for optimum production of neomycin. The maximum neomycin yield (10 755 g/g substrate) was achieved with optimised process parameters such as coarse size of wheat rawa with raspberry seed powder (10% w/w) as additional nutrient, initial moisture content of solid substrate 80%, initial pH of the medium 7.5, incubation temperature 30 ◦ C, inoculum size 0.5% w/w dry cell mass equivalent and 10 days of fermentation time. One hundred and forty percent of higher production was achieved after optimising the process parameters. © 2002 Published by Elsevier Ltd. Keywords: Solid state fermentation; Wheat rawa; Neomycin; Streptomyces marinensis
1. Introduction Solid state fermentation (SSF) has been known for centuries and used successfully for the production of oriental foods. More recently, it has gained much importance in the production of microbial enzymes due to several economic advantages over conventional submerged fermentation (SmF). Several reports on SSF have been published in the recent years on the production of fine chemicals [1–6], enzymes [7–9], antibiotics [10–19] and immunosuppresants [20–22]. Solid state processes are therefore of special economic interest for countries with an abundance of biomass and agro-industrial residues, as these can be used as cheap raw materials. SSF has generated much interest; because, it needs lower manufacturing costs by utilising unprocessed or moderately processed raw materials. SSF is generally a simpler process and requires less pre-processing energy than SmF. Further the initial capital costs are less for SSF. Other advantages are superior productivity, low waste water output and improved product recovery. Unfortunately, SSF is usually slower, be∗ Corresponding author. Tel.: +91-891-2701852; fax: +91-891-2755547. E-mail address: [email protected] (P. Ellaiah).
0032-9592/$ – see front matter © 2002 Published by Elsevier Ltd. doi:10.1016/S0032-9592(02)00059-6
cause of the diffusion barriers imposed by the solid nature of the fermented mass. However, the metabolic processes of the microorganisms are influenced to a great extent by the change of temperature, pH, substrate, moisture content, supply of air, inoculum concentration etc. These conditions vary widely from species to species for each of the organism. So it becomes very important to know the environmental conditions of the microorganisms for maximum production. In SSF, the selection of a suitable solid substrate for a fermentation process is a critical factor and thus involves the screening of a number of agro-industrial materials for microbial growth and product formation. Several studies have been conducted for the production of various antibiotics using SSF technique. Different substrates reported for various antibiotics production include wheat rawa for cephamycin [10,11], corn cobs for oxytetracycline [12,13], okara and wheat bran for iturin [14,15,18,19], soya bean residue okara for surfactin [16], sweet potato for tetracycline [19], barley for cephalosporin [19] and wheat bran for cyclosporin [20–22]. Neomycin is one of the important aminoglycoside antibiotics widely used in pharmaceutical preparations for local applications. It is effective against Gram-positive and Gram-negative bacteria, and mycobacteria. It also has wide applications in veterinary practice. It is used in the storage
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P. Ellaiah et al. / Process Biochemistry 39 (2004) 529–534
tanks of petroleum fuels where it prevents the formation of sludge and in rubber tree plantations where it increases the flow of latex by preventing bacterial infection of tapping wounds [23]. It is commercially produced by Streptomyces fradiae by SmF process where it requires high energy [24]. In the search for cheaper fermentation processes with a high yield of antibiotic, SSF was found to be more attractive. Hence, the authors decided to study the production of neomycin by a mutant strain of Streptomyces marinensis NUV 5, (an isolate of this laboratory) in a SSF process. In this paper, a number of factors, which influence the maximum production of neomycin, are reported.
2. Materials and methods 2.1. Culture A mutant strain of S. marinensis NUV 5, producer of neomycin was used in the present study. It was isolated from seawater from the Bay of Bengal, by the Department of Pharmaceutical Sciences, Andhra University, Visakhapatnam, India [25]. It was maintained on jowar starch agar slants at 4 ◦ C and subcultured at every 4 weeks. A test organism, Staphylococcus epedermidis NCIM 2493 was used for the microbiological assay of neomycin. 2.2. Inoculum preparation S. marinensis Nuv5 was grown on jowar starch agar slants at 30 ◦ C for 7 days for complete sporulation. Five millilitre of sterile water was added to the slant and the spores were scraped and transferred into 250 ml Erlenmeyer flask containing 50 ml of inoculum medium. The composition of the inoculum medium was; soluble starch 2.5%, corn steep liquor 1.0%, (NH)2 SO4 0.5%, NaCl 0.5%, CaCO3 0.5% and pH 7.5. The flasks were incubated at 30 ◦ C in shaker incubator (at 220 rpm) for 48 h. Cells were harvested and washed with sterile saline solution and resuspended in 25 ml sterile saline solution. This cell suspension was used as inoculum. 2.3. Solid state fermentation Experiments were conducted in 250 ml Erlenmeyer flask containing 10 g substrate moistened with distilled water to produce a 70% moisture level. The flasks were autoclaved for 30 min at 121 ◦ C. After cooling the flasks to room temperature, 2 ml inoculum (equivalent to 30 mg w/w dry cell mass) was added and the contents of the flasks were thoroughly mixed. Flasks were incubated at 30 ◦ C for 12 days. At the end of fermentation, neomycin was extracted with 100 ml of phosphate buffer (pH 8.0) and estimated by microbiological assay. All experiments were done in triplicate and average values are reported.
2.4. Effect of various solid substrates Various solid substrates such as wheat bran, wheat rawa, rice bran, rice rawa, rice husk, rice straw, maize bran, ragi bran, green gram bran, black gram bran, red gram bran, corn flour, jowar flour, sago and sugar cane bagasse, were procured from a local market and used to study their effects on neomycin production. The substrates were taken into different flasks and the fermentation was run as described earlier. 2.5. Effect of substrate particle size The effect of substrate particle size on neomycin production was studied by employing three categories of substrate particle sizes viz., coarse particles fraction is made out of particles larger than 0.84 mm, fine particles fraction are particles larger than 0.5 mm, and intermediate particle fraction between 0.5 and 1.0 mm. The fermentation was carried out under the same conditions as described earlier. 2.6. Effect of initial moisture content Various moisture levels were employed in the substrate medium to study their effect on neomycin production. Six flasks containing 10 g substrate were taken and distilled water was added to obtain various levels of moisture content (ranging from 50 to 100%). The fermentation was conducted as described earlier. 2.7. Effect of initial pH While optimising the initial pH of the substrate, the pH was varied from 6.0 to 9.0 with the 1 N KOH or 1 N phosphoric acid, to study their effect on neomycin production. The fermentation was carried out at 30 ◦ C for 12 days. 2.8. Effect of incubation temperature To study the effect of incubation temperature on the production of neomycin, the flasks were incubated at various temperatures (20, 25, 30, 35 and 40 ◦ C). The other parameters like moisture content, pH of the substrate were kept at their optimum level and the fermentation was run for 12 days. 2.9. Effect of inoculum The effects of the inoculum on neomycin production were studied by adding different concentrations of cell mass to the substrate and fermentation was carried out for 12 days. The moisture content, pH of the substrate and incubation temperature were kept at their optimum levels.
P. Ellaiah et al. / Process Biochemistry 39 (2004) 529–534
2.10. Effect of incubation period To study the effect of incubation period on neomycin production, flasks were incubated for varying periods (2, 4, 6, 8, 10, 12 and 14 days). Other parameters were kept at their optimum conditions. 2.11. Effect of additional nutrients Various additional nutrients like oil cakes (sesame, groundnut and coconut) and raspberry seed powder (source, Cleome viscosa Lin, Family: Capparidaceae; locally available abundantly) were employed at 10% level to study their effect on neomycin production. The fermentation was carried out for 10 days and other parameters were kept at their optimum level.
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Table 1 Effect of various substrates on neomycin production by S. marinensis under SSF Substrate
Neomycin yield (g/g substrate) ± S.D.
Wheat rawa Wheat bran Rice bran Rice rawa Rice husk Rice straw Maize bran Ragi bran Green gram bran Black gram bran Corn flour Jowar flour Red gram bran Sago Sugar cane bagasse
4471 2293 583 455 Nil Nil 1267 951 462 1341 675 1345 1439 1238 144
± ± ± ±
144 105 25 26
± ± ± ± ± ± ± ± ±
42 35 17 39 26 41 50 58 6
2.12. Analytical methods 3.2. Effect of substrate particle size At the end of the cultivation process, the solid mass was soaked with 100 ml sterile phosphate buffer (pH 8.0) by shaking for 30 min at 30 ◦ C and was kept for 6 h in the refrigerator. The resultant extract was centrifuged and clear supernatant was used for the estimation of neomycin content by microbiological assay using Staphylococcus epidermidis NCIM 2493 as test organism [26,27]. Standard neomycin sulphate (M/s Shanghai Pharmaceutical Industry Corporation, China) was used to construct the calibration curve. The moisture content of the wheat rawa was estimated by drying 10 g of wheat rawa to a constant weight at 105 ◦ C and the dry weight was recorded [28]. To fix the initial moisture content of the solid medium, wheat rawa was soaked with the desired quantity of additional water. After soaking, the sample was again dried as described earlier and moisture percentage was calculated as follows, Percent of moisture content (initial) of solid medium = (weight of the wheat rawa−dry wt.) × 100 per dry wt.
3. Results and discussion 3.1. Selection of substrate In the present study, various substrates, viz; wheat bran, wheat rawa, rice bran, rice rawa, rice husk, rice straw, maize bran, ragi bran, green gram bran, black gram bran, red gram bran, corn flour, jowar flour, sago and sugar cane bagasse were used for the neomycin production by S. marinensis. The results were shown in the Table 1. The data show that the highest neomycin production (4471 g/g substrate) was achieved with wheat rawa. Negligible yields were observed with rice husk and rice straw. Poor yields were observed with other supports. Hence, wheat rawa was selected and used for subsequent studies. Prasad and Sridhar reported similar data while studying cephamycin C production [10,11].
Among the several factors in SSF processes, which are important for microbial growth and activity, the particular substrate particle size is the most critical [17,29,30]. Generally, smaller substrate particles will provide a larger surface area for microbial attack and thus it should be considered as a desirable factor. However, too small substrate particles may result in substrate agglomeration in most cases, which may interfere with aeration and may thus result in poor growth. At the same time, larger particles provide better aeration efficiency (due to increased inter-particle space) but provide limited surface for microbial attack. Thus it may be necessary to provide a compromised particle size [31]. In the present study, the effect of substrate (wheat rawa) particle size on neomycin production, with three different sizes (fine, intermediate and coarse) were tried. The results indicate that the coarse size of the substrate was the best substrate for neomycin production (4478 g/g) than the intermediate and fine size substrates, which yielding 4043 and 3427 g/g substrate, respectively. The coarse size of wheat rawa was found to be optimal size of the substrate for higher neomycin production. In the subsequent experiments, therefore coarse wheat rawa was used for the production of neomycin. 3.3. Effect of initial moisture content The effect of initial moisture content of the substrate on neomycin production was presented in Fig. 1. The highest antibiotic production (5227 g/g) was achieved at 80% initial moisture content. A decrease in neomycin production was observed when the moisture level is higher or lower than the optimum. Substrate moisture is a critical factor in SSF and its importance for the production of secondary metabolites has been well established [19]. It is reported that higher substrate moisture in SSF resulted in suboptimal product
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the optimum level at pH 7.5 (5780 g/g). A further increase in pH resulted in decrease in neomycin production. As the metabolic activities of the microorganisms are very much sensitive to the pH change, neomycin production by S. marinensis is found to be affected if pH level of the substrate is higher or lower compared with optimum value. 3.5. Effect of incubation temperature
Fig. 1. Effect of initial moisture content on neomycin production by S. marinensis NUV 5 under SSF.
formation due to reduced mass transfer process such as diffusion of solutes and gas to cell during fermentation. The decrease in moisture level results reduced solubility, minimises heat exchange, oxygen transfer and low availability of nutrients to the culture. This effects microbial activity and results in decreased productivity [32,33]. 3.4. Influence of initial pH The effect of initial pH of the substrate on neomycin production was shown in Fig. 2. When the initial pH was 6.0, there was less antibiotic production. As the pH was increased, neomycin production was increased and reached to
Fig. 2. Effect of initial pH on neomycin production by S. marinensis NUV 5 under SSF.
Different incubation temperatures, viz., 20, 25, 30, 35, 40 ◦ C were used to grow the culture for neomycin production. The fermentation was carried out for 12 days. The results were presented in Fig. 3. Maximum antibiotic production (5760 g/g) was attained at 30 ◦ C. A decrease in the yield of neomycin was observed when the incubation temperature was higher or lower than the optimum incubation temperature. Higher temperature were found to have adverse effects on the metabolic activities of the microorganism and it is also reported that the metabolic activities of the microorganisms become slower at lower temperature. Hence, incubation temperature and its control in SSF process is crucial as the heat evolved during SSF processes is accumulated due to poor heat dissipation in solid media. This results in reduced microbial activity, thereby decreasing the yield of product formation [34,35]. 3.6. Effect of inoculum To evaluate the effect of inoculum level on neomycin production, varying cell concentrations (equivalent to 0.1, 0.2, 0.4, 0.5, 0.6% w/w dry cell mass of 48 h culture) were added to different flasks. Fermentations were carried out for 12 days and results are shown in Fig. 4. Results indicate that optimum neomycin production (6880 mg/g) was
Fig. 3. Effect of temperature on neomycin production by S. marinensis NUV 5 under SSF.
P. Ellaiah et al. / Process Biochemistry 39 (2004) 529–534
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Table 2 Effect of various supplements on the neomycin production by S. marinensis under SSF Additional nutrient supports
Weight (g)
Neomycin yield (g/g substrate) ± S.D.
Control Sesame oil cake Ground nut oil cake Coconut oil cake Raspberry seed powder
Nil 1 1 1 1
7457 ± 216 8834 ± 185 8709 ± 264 7100 ± 155 10 755 ± 256
cell mass of inoculum level) was studied for a period of 14 days (Fig. 5). Detectable neomycin production occurred on day 4 and maximum neomycin (7457 g/g) production was achieved on day 10, after which there was decrease in neomycin production. Fig. 4. Effect of inoculum on neomycin production by S. marinensis NUV 5 under SSF.
observed at 0.5% w/w dry cell mass of inoculum. At lower and higher inoculum levels, poor neomycin production was observed. It is important to provide an optimum inoculum level in fermentation processes. A low inoculum density may give insufficient biomass causing reduced product formation, whereas a higher inoculum may produce too much biomass and deplete the substrate of nutrients necessary for product formation [24]. 3.7. Fermentation cycle The production of neomycin by S. marinensis with optimised parameters (such as 80% substrate moisture level, pH 7.5, incubation temperature 30 ◦ C, with 0.5% w/w dry
3.8. Effect of additional nutrients To study the effect of various additional nutrients on neomycin production, various oil cakes and raspberry seed powder at 10% level were tried. The results are shown in Table 2. The maximum neomycin yield (10 755 g/g substrate) was obtained with raspberry seed powder followed by sesame and groundnut oil cakes whereas decreased yield was observed with coconut oil cake.
4. Conclusions SmF is usually employed for commercial production of neomycin. In this SSF process, wheat rawa was found to be most suitable substrate for neomycin production by S. marinensis. The maximum antibiotic production (10 755 g/g substrate) was achieved by employing wheat rawa with raspberry seed powder as supplementary nutrient and with optimised process parameters such as 80% of initial moisture content, pH 7.5, incubation temperature 30 ◦ C, inoculum 0.5% w/w dry cell mass and incubation period of 10 days. With all optimum parameters as high as 140%, an increase in neomycin production was achieved over control. Earlier studies with submerged fermentation (SmF) employing a synthetic medium showed 5813 g/ml neomycin yield [36]. The comparative production of neomycin by SSF and SmF indicated better production with SSF technique. The accumulation of neomycin by SSF was 1.85 times higher than the SmF. This study has shown the feasibility of SSF for the first time for neomycin production employing the S. marinensis culture.
Acknowledgements
Fig. 5. Kinetics of neomycin production by S. marinensis NUV 5 under SSF.
The authors would like to acknowledge the Council for Scientific and Industrial Research, Government of India, New Delhi for providing a fellowship to B. Srinivasulu.
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References [1] Vandenberghe LPS, Soccol CR, Pandey A, Lebeault JM. Citric acid production by Aspergillus niger in solid state fermentation. Biores Technol 2000;74:175–8. [2] Roukas T. Citric acid production from pod by solid fermentation. Enzyme Microb Biotechnol 1999;24:54–9. [3] Leangon S, Maddox IS, Brooks JD. Influence of glycolytic rate on production of citric acid and oxalic acid by Aspergillus niger in solid state fermentation. World J Microbiol Biotechnol 1999;15:493–5. [4] Kar B, Benerjee R, Bhattacharya BC.Biotransformation of tannic acid to gallic acid by solid state fermentation. In: Pandey A, editor. Advances in biotechnology. New Delhi: Educational Publishers, 1998:67–74. [5] Nagampoothiri KM, Pandey A. Solid state fermentation for lglutamic acid production using Brevibacterium sp. Biotechnol Lett 1996;6(2):199–204. [6] Soccol CR, Marrin B, Raimbault M, Lebeault JM. Potential of solid state fermentation for production of l-(+) lactic acid by Rhizopus oryzae. Appl Microbiol Biotechnol 1994;41:286–90. [7] Adreas KG, Annette I, Pinto LRC, Denise MGF. Lipase production by Pencillium restrictum in solid state fermentation using babassu oil cake as substrate. Process Biochem 1999;35(1–2):85–90. [8] Selvakumar P, Pandey A. Solid state fermentation for the synthesis of inulinase from Straphylococcus sp. and Kluyveromyces marxianus. Process Biochem 1999;34(8):851–5. [9] Suresh PV, Chandrasekaran M. Impact of process parameters on chitinase production by an alkalophilic marine Beauveria bassiana in solid state fermentation. Process Biochem 1999;34(3):257–67. [10] Kota KP, Sridhar P. Solid state cultivation of Streptomyces clavuligerus for cephamycin C production. Process Biochem 1999;34:325–8. [11] Kota KP, Sridhar P. Solid state cultivation of Streptomyces clavuligerus for production cephamycin C. J Sci Ind Res 1998;58:587–90. [12] Yang SS, Swei WJ. Oxytetracycline production by Streptomyces rimosus in solid state fermentation of corn-cob. World J Microbiol Biotechnol 1996;12:43–6. [13] Yang SS. Antibiotics production of cellulosic wastes with solid state fermentation by Streptomyces. Renew Energy 1996;9:876–979. [14] Ohno A, Ano T, Shoda M. Use of soybean curd residue, okara, for the solid state substrate in the production of lipopeptide antibiotic, iturin A by Bacillus subtilis NB 22. Process Biochem 1996;31:801–6. [15] Ohno A, Ano T, Shoda M. Effect of temperature on production of lipopeptide antibiotics iturin A and surfactin by a dual producer, Bacillus subtilis RB 14, in solid state fermentation. J Ferment Bioeng 1995;80:517–9. [16] Ohno A, Ano T, Shoda M. Production of lipopeptide antibiotic, surfactin, by recombinant Bacillus in solid state fermentation. Biotechnol Bioeng 1995;47:209–14. [17] Ohno A, Ano T, Shoda M. Production of antifungal peptide antibiotic, iturin by Bacillus subtilis NB 22 in solid state fermentation. J Ferment Bioeng 1993;75:23–7.
[18] Ohno A, Ano T, Shoda M. Production of antifungal antibiotic, iturin in solid state fermentation by Bacillus subtilis NB 22 using wheat bran as substrate. Biotechnol Lett 1992;14:817–22. [19] Balakrishna K, Pandey A. Production of biologically active secondary metabolites in solid state fermentation. J Sci Ind Res 1996;55:365– 72. [20] Murthy MVR, Mohan EVS, Sadhukhan AK. Cyclosporin A production by Tolypocladium inflatum using solid state fermentation. Process Biochem 1999;34:269–80. [21] Sekhar C, Balaraman K. Optimization studies on the production of cyclosporin A by solid state fermentation. Bioprocess Eng 1998;18:293–6. [22] Sekhar C, Rajasekar VW, Balaraman K. Production of cyclosporin A by solid state fermentation. Bioprocess Eng 1997;17:257–9. [23] Lechevalier HA. The 25 years of neomycin. CRC critical reviews in microbiology, vol. 3 (issue number 4). FL, USA: CRC Press; 1975:359–97 (issue number 4). [24] Mudgetti RE. In: Demain Arnolds L, Solmen Nadine A, editors. Manual of industrial biotechnology. Washington, DC: American Society for Microbiology, 1986:66–83. [25] Sambamurthy K, Ellaiah P. A new Streptomycete producing neomycin (B&C) complex—S. marinensis (part I). Hind Antibiot Bull 1974;17:24–7. [26] Grove DC, Randall WA. Assay methods of antibiotics: a laboratory manual. New York: Medical Encyclopedia, 1955:91. [27] Pharmacopoeia of India, Government of India, Ministry for Health and Welfare, vol. 2. New Delhi: The Controller of Publications, 1985:A88. [28] Tunga R, Banerjee R, Battacharyya BC. Optimizing some factors affecting protease production under solid state fermentation. Bioprocess Eng 1998;19:187–90. [29] Zadrazil F, Punia AK. Studies on the effect of particle size on solid state fermentation of sugar cane bagasse into animal feed using white rot fungi. Biores Technol 1995;54:85–7. [30] Pandey A. Effect of particle size of substrate on enzyme production in solid state fermentation. Biores Technol 1991;37:169–72. [31] Pandey A, Soccol CR, Mitchell D. New developments in solid state fermentation: 1-bioprocess and products. Process Biochem 2000;35:1153–69. [32] Carrizales V, Rodriguez H, Sardina . Determination of specific growth rate of molds as semi solid cultures. Biotechnol Bioeng 1981;23: 321–33. [33] Hesseltine CW. SSF: an overview. Int Biodeterioration 1979;23: 79–89. [34] Rainbauet M, Alazrd D. Culture method to study fungal growth in solid state fermentation. Eur J Appl Microbiol Biotechnol 1980;9:199–209. [35] Pandey A. Recent developments in solid state fermentation. Process Biochem 1992;27:109–17. [36] Ellaiah P, Srinivasulu B. Studies on the production of neomycin in a fluidized bed reactor with immobilized cells of S. marinensis, paper presented at the 51st Indian Pharmaceutical Congress, Indore, India, 18–20th, December, 1999.
Optimisation studies on neomycin production by a mutant strain of Streptomyces marinensis in solid state fermentation P. Ellaiah∗ , B. Srinivasulu, K. Adinarayana Pharmaceutical Biotechnology Division, Department of Pharmaceutical Sciences, Andhra University, Visakhapatnam 530 003, India Received 13 December 2001; received in revised form 18 January 2002; accepted 19 February 2002
Abstract Production of neomycin by a mutant strain of Streptomyces marinensis, under solid state fermentation was optimised. Various substrates were used to study their effect on neomycin production. Wheat rawa was found to be better for optimum production of neomycin. The maximum neomycin yield (10 755 g/g substrate) was achieved with optimised process parameters such as coarse size of wheat rawa with raspberry seed powder (10% w/w) as additional nutrient, initial moisture content of solid substrate 80%, initial pH of the medium 7.5, incubation temperature 30 ◦ C, inoculum size 0.5% w/w dry cell mass equivalent and 10 days of fermentation time. One hundred and forty percent of higher production was achieved after optimising the process parameters. © 2002 Published by Elsevier Ltd. Keywords: Solid state fermentation; Wheat rawa; Neomycin; Streptomyces marinensis
1. Introduction Solid state fermentation (SSF) has been known for centuries and used successfully for the production of oriental foods. More recently, it has gained much importance in the production of microbial enzymes due to several economic advantages over conventional submerged fermentation (SmF). Several reports on SSF have been published in the recent years on the production of fine chemicals [1–6], enzymes [7–9], antibiotics [10–19] and immunosuppresants [20–22]. Solid state processes are therefore of special economic interest for countries with an abundance of biomass and agro-industrial residues, as these can be used as cheap raw materials. SSF has generated much interest; because, it needs lower manufacturing costs by utilising unprocessed or moderately processed raw materials. SSF is generally a simpler process and requires less pre-processing energy than SmF. Further the initial capital costs are less for SSF. Other advantages are superior productivity, low waste water output and improved product recovery. Unfortunately, SSF is usually slower, be∗ Corresponding author. Tel.: +91-891-2701852; fax: +91-891-2755547. E-mail address: [email protected] (P. Ellaiah).
0032-9592/$ – see front matter © 2002 Published by Elsevier Ltd. doi:10.1016/S0032-9592(02)00059-6
cause of the diffusion barriers imposed by the solid nature of the fermented mass. However, the metabolic processes of the microorganisms are influenced to a great extent by the change of temperature, pH, substrate, moisture content, supply of air, inoculum concentration etc. These conditions vary widely from species to species for each of the organism. So it becomes very important to know the environmental conditions of the microorganisms for maximum production. In SSF, the selection of a suitable solid substrate for a fermentation process is a critical factor and thus involves the screening of a number of agro-industrial materials for microbial growth and product formation. Several studies have been conducted for the production of various antibiotics using SSF technique. Different substrates reported for various antibiotics production include wheat rawa for cephamycin [10,11], corn cobs for oxytetracycline [12,13], okara and wheat bran for iturin [14,15,18,19], soya bean residue okara for surfactin [16], sweet potato for tetracycline [19], barley for cephalosporin [19] and wheat bran for cyclosporin [20–22]. Neomycin is one of the important aminoglycoside antibiotics widely used in pharmaceutical preparations for local applications. It is effective against Gram-positive and Gram-negative bacteria, and mycobacteria. It also has wide applications in veterinary practice. It is used in the storage
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P. Ellaiah et al. / Process Biochemistry 39 (2004) 529–534
tanks of petroleum fuels where it prevents the formation of sludge and in rubber tree plantations where it increases the flow of latex by preventing bacterial infection of tapping wounds [23]. It is commercially produced by Streptomyces fradiae by SmF process where it requires high energy [24]. In the search for cheaper fermentation processes with a high yield of antibiotic, SSF was found to be more attractive. Hence, the authors decided to study the production of neomycin by a mutant strain of Streptomyces marinensis NUV 5, (an isolate of this laboratory) in a SSF process. In this paper, a number of factors, which influence the maximum production of neomycin, are reported.
2. Materials and methods 2.1. Culture A mutant strain of S. marinensis NUV 5, producer of neomycin was used in the present study. It was isolated from seawater from the Bay of Bengal, by the Department of Pharmaceutical Sciences, Andhra University, Visakhapatnam, India [25]. It was maintained on jowar starch agar slants at 4 ◦ C and subcultured at every 4 weeks. A test organism, Staphylococcus epedermidis NCIM 2493 was used for the microbiological assay of neomycin. 2.2. Inoculum preparation S. marinensis Nuv5 was grown on jowar starch agar slants at 30 ◦ C for 7 days for complete sporulation. Five millilitre of sterile water was added to the slant and the spores were scraped and transferred into 250 ml Erlenmeyer flask containing 50 ml of inoculum medium. The composition of the inoculum medium was; soluble starch 2.5%, corn steep liquor 1.0%, (NH)2 SO4 0.5%, NaCl 0.5%, CaCO3 0.5% and pH 7.5. The flasks were incubated at 30 ◦ C in shaker incubator (at 220 rpm) for 48 h. Cells were harvested and washed with sterile saline solution and resuspended in 25 ml sterile saline solution. This cell suspension was used as inoculum. 2.3. Solid state fermentation Experiments were conducted in 250 ml Erlenmeyer flask containing 10 g substrate moistened with distilled water to produce a 70% moisture level. The flasks were autoclaved for 30 min at 121 ◦ C. After cooling the flasks to room temperature, 2 ml inoculum (equivalent to 30 mg w/w dry cell mass) was added and the contents of the flasks were thoroughly mixed. Flasks were incubated at 30 ◦ C for 12 days. At the end of fermentation, neomycin was extracted with 100 ml of phosphate buffer (pH 8.0) and estimated by microbiological assay. All experiments were done in triplicate and average values are reported.
2.4. Effect of various solid substrates Various solid substrates such as wheat bran, wheat rawa, rice bran, rice rawa, rice husk, rice straw, maize bran, ragi bran, green gram bran, black gram bran, red gram bran, corn flour, jowar flour, sago and sugar cane bagasse, were procured from a local market and used to study their effects on neomycin production. The substrates were taken into different flasks and the fermentation was run as described earlier. 2.5. Effect of substrate particle size The effect of substrate particle size on neomycin production was studied by employing three categories of substrate particle sizes viz., coarse particles fraction is made out of particles larger than 0.84 mm, fine particles fraction are particles larger than 0.5 mm, and intermediate particle fraction between 0.5 and 1.0 mm. The fermentation was carried out under the same conditions as described earlier. 2.6. Effect of initial moisture content Various moisture levels were employed in the substrate medium to study their effect on neomycin production. Six flasks containing 10 g substrate were taken and distilled water was added to obtain various levels of moisture content (ranging from 50 to 100%). The fermentation was conducted as described earlier. 2.7. Effect of initial pH While optimising the initial pH of the substrate, the pH was varied from 6.0 to 9.0 with the 1 N KOH or 1 N phosphoric acid, to study their effect on neomycin production. The fermentation was carried out at 30 ◦ C for 12 days. 2.8. Effect of incubation temperature To study the effect of incubation temperature on the production of neomycin, the flasks were incubated at various temperatures (20, 25, 30, 35 and 40 ◦ C). The other parameters like moisture content, pH of the substrate were kept at their optimum level and the fermentation was run for 12 days. 2.9. Effect of inoculum The effects of the inoculum on neomycin production were studied by adding different concentrations of cell mass to the substrate and fermentation was carried out for 12 days. The moisture content, pH of the substrate and incubation temperature were kept at their optimum levels.
P. Ellaiah et al. / Process Biochemistry 39 (2004) 529–534
2.10. Effect of incubation period To study the effect of incubation period on neomycin production, flasks were incubated for varying periods (2, 4, 6, 8, 10, 12 and 14 days). Other parameters were kept at their optimum conditions. 2.11. Effect of additional nutrients Various additional nutrients like oil cakes (sesame, groundnut and coconut) and raspberry seed powder (source, Cleome viscosa Lin, Family: Capparidaceae; locally available abundantly) were employed at 10% level to study their effect on neomycin production. The fermentation was carried out for 10 days and other parameters were kept at their optimum level.
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Table 1 Effect of various substrates on neomycin production by S. marinensis under SSF Substrate
Neomycin yield (g/g substrate) ± S.D.
Wheat rawa Wheat bran Rice bran Rice rawa Rice husk Rice straw Maize bran Ragi bran Green gram bran Black gram bran Corn flour Jowar flour Red gram bran Sago Sugar cane bagasse
4471 2293 583 455 Nil Nil 1267 951 462 1341 675 1345 1439 1238 144
± ± ± ±
144 105 25 26
± ± ± ± ± ± ± ± ±
42 35 17 39 26 41 50 58 6
2.12. Analytical methods 3.2. Effect of substrate particle size At the end of the cultivation process, the solid mass was soaked with 100 ml sterile phosphate buffer (pH 8.0) by shaking for 30 min at 30 ◦ C and was kept for 6 h in the refrigerator. The resultant extract was centrifuged and clear supernatant was used for the estimation of neomycin content by microbiological assay using Staphylococcus epidermidis NCIM 2493 as test organism [26,27]. Standard neomycin sulphate (M/s Shanghai Pharmaceutical Industry Corporation, China) was used to construct the calibration curve. The moisture content of the wheat rawa was estimated by drying 10 g of wheat rawa to a constant weight at 105 ◦ C and the dry weight was recorded [28]. To fix the initial moisture content of the solid medium, wheat rawa was soaked with the desired quantity of additional water. After soaking, the sample was again dried as described earlier and moisture percentage was calculated as follows, Percent of moisture content (initial) of solid medium = (weight of the wheat rawa−dry wt.) × 100 per dry wt.
3. Results and discussion 3.1. Selection of substrate In the present study, various substrates, viz; wheat bran, wheat rawa, rice bran, rice rawa, rice husk, rice straw, maize bran, ragi bran, green gram bran, black gram bran, red gram bran, corn flour, jowar flour, sago and sugar cane bagasse were used for the neomycin production by S. marinensis. The results were shown in the Table 1. The data show that the highest neomycin production (4471 g/g substrate) was achieved with wheat rawa. Negligible yields were observed with rice husk and rice straw. Poor yields were observed with other supports. Hence, wheat rawa was selected and used for subsequent studies. Prasad and Sridhar reported similar data while studying cephamycin C production [10,11].
Among the several factors in SSF processes, which are important for microbial growth and activity, the particular substrate particle size is the most critical [17,29,30]. Generally, smaller substrate particles will provide a larger surface area for microbial attack and thus it should be considered as a desirable factor. However, too small substrate particles may result in substrate agglomeration in most cases, which may interfere with aeration and may thus result in poor growth. At the same time, larger particles provide better aeration efficiency (due to increased inter-particle space) but provide limited surface for microbial attack. Thus it may be necessary to provide a compromised particle size [31]. In the present study, the effect of substrate (wheat rawa) particle size on neomycin production, with three different sizes (fine, intermediate and coarse) were tried. The results indicate that the coarse size of the substrate was the best substrate for neomycin production (4478 g/g) than the intermediate and fine size substrates, which yielding 4043 and 3427 g/g substrate, respectively. The coarse size of wheat rawa was found to be optimal size of the substrate for higher neomycin production. In the subsequent experiments, therefore coarse wheat rawa was used for the production of neomycin. 3.3. Effect of initial moisture content The effect of initial moisture content of the substrate on neomycin production was presented in Fig. 1. The highest antibiotic production (5227 g/g) was achieved at 80% initial moisture content. A decrease in neomycin production was observed when the moisture level is higher or lower than the optimum. Substrate moisture is a critical factor in SSF and its importance for the production of secondary metabolites has been well established [19]. It is reported that higher substrate moisture in SSF resulted in suboptimal product
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the optimum level at pH 7.5 (5780 g/g). A further increase in pH resulted in decrease in neomycin production. As the metabolic activities of the microorganisms are very much sensitive to the pH change, neomycin production by S. marinensis is found to be affected if pH level of the substrate is higher or lower compared with optimum value. 3.5. Effect of incubation temperature
Fig. 1. Effect of initial moisture content on neomycin production by S. marinensis NUV 5 under SSF.
formation due to reduced mass transfer process such as diffusion of solutes and gas to cell during fermentation. The decrease in moisture level results reduced solubility, minimises heat exchange, oxygen transfer and low availability of nutrients to the culture. This effects microbial activity and results in decreased productivity [32,33]. 3.4. Influence of initial pH The effect of initial pH of the substrate on neomycin production was shown in Fig. 2. When the initial pH was 6.0, there was less antibiotic production. As the pH was increased, neomycin production was increased and reached to
Fig. 2. Effect of initial pH on neomycin production by S. marinensis NUV 5 under SSF.
Different incubation temperatures, viz., 20, 25, 30, 35, 40 ◦ C were used to grow the culture for neomycin production. The fermentation was carried out for 12 days. The results were presented in Fig. 3. Maximum antibiotic production (5760 g/g) was attained at 30 ◦ C. A decrease in the yield of neomycin was observed when the incubation temperature was higher or lower than the optimum incubation temperature. Higher temperature were found to have adverse effects on the metabolic activities of the microorganism and it is also reported that the metabolic activities of the microorganisms become slower at lower temperature. Hence, incubation temperature and its control in SSF process is crucial as the heat evolved during SSF processes is accumulated due to poor heat dissipation in solid media. This results in reduced microbial activity, thereby decreasing the yield of product formation [34,35]. 3.6. Effect of inoculum To evaluate the effect of inoculum level on neomycin production, varying cell concentrations (equivalent to 0.1, 0.2, 0.4, 0.5, 0.6% w/w dry cell mass of 48 h culture) were added to different flasks. Fermentations were carried out for 12 days and results are shown in Fig. 4. Results indicate that optimum neomycin production (6880 mg/g) was
Fig. 3. Effect of temperature on neomycin production by S. marinensis NUV 5 under SSF.
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Table 2 Effect of various supplements on the neomycin production by S. marinensis under SSF Additional nutrient supports
Weight (g)
Neomycin yield (g/g substrate) ± S.D.
Control Sesame oil cake Ground nut oil cake Coconut oil cake Raspberry seed powder
Nil 1 1 1 1
7457 ± 216 8834 ± 185 8709 ± 264 7100 ± 155 10 755 ± 256
cell mass of inoculum level) was studied for a period of 14 days (Fig. 5). Detectable neomycin production occurred on day 4 and maximum neomycin (7457 g/g) production was achieved on day 10, after which there was decrease in neomycin production. Fig. 4. Effect of inoculum on neomycin production by S. marinensis NUV 5 under SSF.
observed at 0.5% w/w dry cell mass of inoculum. At lower and higher inoculum levels, poor neomycin production was observed. It is important to provide an optimum inoculum level in fermentation processes. A low inoculum density may give insufficient biomass causing reduced product formation, whereas a higher inoculum may produce too much biomass and deplete the substrate of nutrients necessary for product formation [24]. 3.7. Fermentation cycle The production of neomycin by S. marinensis with optimised parameters (such as 80% substrate moisture level, pH 7.5, incubation temperature 30 ◦ C, with 0.5% w/w dry
3.8. Effect of additional nutrients To study the effect of various additional nutrients on neomycin production, various oil cakes and raspberry seed powder at 10% level were tried. The results are shown in Table 2. The maximum neomycin yield (10 755 g/g substrate) was obtained with raspberry seed powder followed by sesame and groundnut oil cakes whereas decreased yield was observed with coconut oil cake.
4. Conclusions SmF is usually employed for commercial production of neomycin. In this SSF process, wheat rawa was found to be most suitable substrate for neomycin production by S. marinensis. The maximum antibiotic production (10 755 g/g substrate) was achieved by employing wheat rawa with raspberry seed powder as supplementary nutrient and with optimised process parameters such as 80% of initial moisture content, pH 7.5, incubation temperature 30 ◦ C, inoculum 0.5% w/w dry cell mass and incubation period of 10 days. With all optimum parameters as high as 140%, an increase in neomycin production was achieved over control. Earlier studies with submerged fermentation (SmF) employing a synthetic medium showed 5813 g/ml neomycin yield [36]. The comparative production of neomycin by SSF and SmF indicated better production with SSF technique. The accumulation of neomycin by SSF was 1.85 times higher than the SmF. This study has shown the feasibility of SSF for the first time for neomycin production employing the S. marinensis culture.
Acknowledgements
Fig. 5. Kinetics of neomycin production by S. marinensis NUV 5 under SSF.
The authors would like to acknowledge the Council for Scientific and Industrial Research, Government of India, New Delhi for providing a fellowship to B. Srinivasulu.
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