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Bioresource Technology 99 (2008) 2087–2091
Eﬀect of liquid culture requirements on antifungal antibiotic production by Streptomyces rimosus MY02 Jicheng Yu, Qiu Liu *, Qiao Liu, Xiangdong Liu, Qiang Sun, Jianfang Yan, Xiaohui Qi, Shengdi Fan Department of Bioengineering, Dalian Nationalities University, Dalian 116600, PR China Received 12 February 2007; received in revised form 14 March 2007; accepted 17 March 2007 Available online 1 May 2007
Abstract Streptomyces rimosus MY02 was isolated from a soil sample which was collected from the northeast of China. The eﬀect of medium components (i.e. carbon and nitrogen sources) and other culture requirements (i.e. initial pH and temperature) on production of antifungal antibiotics by S. rimosus MY02 was investigated in our work. The best conditions for the strain MY02 in 250-ml Erlenmeyer ﬂask, for example, initial pH, temperature, medium capacity, agitation rate, seed age, inoculum size and growth period, were 6.0, 28 C, 50 ml, 180 rpm, 4 days, 10% (v/v) and 120 h, respectively. Components and dosage of the medium, which eﬀect antibiotic production, were determined by uniform design combined with regression analysis; meanwhile, a regression model was established. The components and dosage of the best medium were starch, 53.313 g; defatted peanut powder, 9.376 g; (NH4)2SO4, 6.244 g; and NaCl, 5.836 g; in 1 l of distilled water. Residual values obtained between the observed values by experiments and predicted values by the model are very low, and this result showed that the experimental results were well in consistence with the calculation results via the model. The antifungal antibiotic production by S. rimosus MY02 was improved by optimization of the components and culture requirements. The diameter of inhibition zone of the culture supernatant from S. rimosus MY02 against Fusarium oxysporium f sp. cucumarinum was 33.19 mm. 2007 Published by Elsevier Ltd. Keywords: Liquid culture requirements; Antifungal antibiotic production; Design method; Regression model; Best media components
1. Introduction Agricultural antibiotic, which is produced by diﬀerent species of actinomyces, is a kind of biological product from natural resource. They have been attracting growing interest with the development of environmentally friendly and safe integrated crop management. Marten et al. (2001) reported that RhizovitR from Streptomyces rimosus is used in the control of a wide range of fungi such as Pythium spp., Phytophthora spp., Rhizoctonia solani, Alternaria brassicola, and Botrytis sp. Liu et al. (2004a) also reported that S. rimosus showed a high antagonism activity against Fusarium solani, F. oxysporium f sp. *
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(Q. Liu). 0960-8524/$ - see front matter 2007 Published by Elsevier Ltd. doi:10.1016/j.biortech.2007.03.023
cucumarinum, Verticillium dahliae, R. solani, Fulvia fulva, Botrytis cinearia, A. alternat, Sclerotinia sclerotiorum and Bipolaris maydis. The antifungal antibiotic, which is produced by S. rimosus, was puriﬁed by silica gel column chromatography. Its ultraviolet (UV) spectrum was consistent with that of polyene macrolide, which had the same absorption peaks at 291, 305, and 318 nm. Antifungal activity can be kept for 20 months at room temperature (12–30 C, pH 5.4) (Liu et al., 2004b). So S. rimosus will be employed as a target to search for new biocontrol agents or drugs to satisfy public demands, and much interests will be generated. Many investigators have attempted to obtain optimal liquid culture requirements for antibiotic production by various methods. The uniform design is an eﬃcient fractional design which was proposed by Professor Fang Kai-Tai (Fang, 1994). The uniform design has been
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successfully used in various ﬁelds such as chemistry and chemical engineering, pharmaceutics, quality engineering, survey design, computer science and natural science (Fang, 2002). The uniform design not only involves relatively less experiments, but it also allows the study of the relationships between the culture requirements and their eﬀect on metabolites (Huang et al., 2003). Many investigators have obtained optimal liquid culture requirements for microbe growth by uniform design (Tian and Fang, 1999; Xu and Yun, 2003). At present, agricultural antibiotics have been gaining increasing attention. Several workers have reported that in vitro studies have documented satisfactory results in the use of antibiotics against phytopathogens. For example, Streptomyces sp. strain 5406 has been used in China for the past 35 years to protect cotton crops against soilborne pathogens (Valois et al., 1996). Blasticidin-S from S. griseochromogenes and Kasugamycine from S. kasugaensis are used in the control of rice blast pathogen, Piricularia oryzae (Agrios, 1988). Antifungal antibiotics from S. rimosus MY02 also play a key role in controlling plant pathogen. To the best of our knowledge, the eﬀect of nutritional requirements and environmental conditions on the production of antifungal antibiotics by S. rimosus has not been demonstrated so far. Therefore, the optimization of the medium and fermentation requirements via uniform design for S. rimosus MY02 will rapidly achieve higher product yield, increase economic beneﬁt and provide opportunities of practical use. 2. Methods 2.1. Micro-organism The strain MY02 was isolated from the soil samples from the northeast of China, which was identiﬁed as S. rimosus MY02 based on morphological, physiological characteristics and analysis of the 16S rDNA sequence. The culture was maintained on potato dextrose agar slant (PDA, potato 200 g, glucose 20 g and agar 18 g in 1 l of distilled water) at 4 C and transferred once every 3 months or kept in glycerol suspension (40%, v/v) at 20 C for 1 year. F. oxysporium f sp. cucumarinum was obtained from the Laboratory of Plant Pathology of Shenyang Agricultural University, China. 2.2. Inoculum preparation and liquid culture The strain MY02 was grown on PDA plate for 5 days at 28 C. Spores were harvested and resuspended in water. Spore suspension was inoculated in 250-ml Erlenmeyer ﬂasks containing 50 ml of the basal fermentation medium and incubated on a rotatory shaker at 180 rpm and 28 C for 120 h. The basal medium composed of starch (33 g), defatted peanut powder (17 g), (NH4)2SO4 (2.3 g) and NaCl (2 g) in 1 l of distilled water, and the pH was adjusted to 7.0. The fermentation medium based on basal medium
was used to screen out better carbon source, nitrogen source and other factors by shaking ﬂask culture experiments. 2.3. Culture requirements In order to ﬁnd the optimal liquid culture requirements for antifungal antibiotic production by the strain MY02, the following factors were investigated, i.e. carbon resource, nitrogen resource, temperature, initial pH, growth period, medium capacity, seed age and inoculum size (the spore suspension of the strain MY02, 106 spores/ml). Then components and dosage of the medium were determined by uniform design combined with regression analysis. 2.4. Preparation of crude extract To prepare crude extract, the culture was harvested when it reached the maximum activity, and heated at 100 C for 5 min, the cooled culture was centrifuged at 6000 rpm for 10 min. The supernatant was stored at 4 C. 2.5. Bioassay of antifungal antibiotic production by S. rimosus MY02 Antifungal antibiotic production by the strain MY02 was determined by cup-plate diﬀusion method. PDA medium (60 ml) was heated till it was completely melted and then gradually cooled to 50 C, rapidly mixed with 10 ml of the suspension of F. oxysporium f sp. cucumarinum (106 spores/ml) before being poured onto the plates (200 mm · 150 mm) and was solidiﬁed at room temperature. The above supernatant (0.1 ml) was added into each oxford-cup on the plate. Then the plates were incubated at 25 C for 72 h and antifungal antibiotic production was determined by measuring the diameter of inhibition zones (DIZ, mm). The corresponding liquid medium instead of the supernatant was used as control. All experiments were carried out in triplicate to minimize errors. 2.6. Uniform design method and statistical analysis According to the above results, starch, defatted peanut powder, (NH4)2SO4 and NaCl were designated as Xl, X2, X3 and X4, respectively. The four components (factors) were further optimized by uniform design method U17(174) (Fang and Ma, 2001). The concentration range of each factor was designated as follows: starch (0.05– 0.8 g/l), defatted peanut powder (0–0.225 g/l), (NH4)2SO4 (0–0.075 g/l) and NaCl (0–0.075 g/l). Each factor was equally divided into 16 levels, and is listed in Table 1. According to uniform design, only 16 experiments were required to be done. The software (STATISTICA 6.0) was used to analyzed all data, and a best regression model was established. The best components and dosage of the media were obtained by the regression model.
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Table 1 Factors, design levels and results of uniform design U17(174)a Test no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Factors and concentration (g/100 ml) Starch X1
Defatted peanut powder X2
0.50b 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00
0.9 1.95 0.45 1.50 0 1.05 2.10 0.60 1.65 0.15 1.20 2.25 0.75 1.80 0.30 1.35
0.65 0.5 0.35 0.20 0.05 0.75 0.60 0.45 0.30 0.15 0 0.70 0.55 0.40 0.25 0.10
0.05 0.15 0.25 0.35 0.45 0.55 0.65 0.75 0 0.10 0.20 0.30 0.40 0.50 0.60 0.70
Observed value (y) or DIZ (mm)
29.51c 18.17 25.92 29.93 10.11 27.41 17.32 29.84 32.23 21.12 24.85 13.11 29.2 19.01 19.22 21.33
29.54d 19.71 24.46 28.45 11.48 27.27 17.25 30.05 32.02 21.19 24.99 11.74 30.68 20.47 17.68 21.30
0.03e 1.54 1.46 1.48 1.37 0.14 0.07 0.21 0.21 0.07 0.14 1.37 1.48 1.46 1.54 0.03
DIZ means diameter of inhibition zones. a Fermentation was carried out for 120 h at 28 C with initial pH 6.0. b The arrangement of columns X1–X4 were decided by uniform design for U17(174). c The observed diameter of inhibition zones via uniform design, represents one experimental replicate, and every experimental group was replicated thrice. d The predicted diameter of inhibition zones by the model. e Residual values obtained between the observed values and the predicted values.
2.7. Investigation of the calculated results via the regression model To investigate the error of calculated results via the regression model on components and dosage of the best medium, the strain MY02 was inoculated in 250-ml Erlenmeyer ﬂasks containing 50 ml of the best medium and incubated on a rotatory shaker in the following conditions: (i) the fermentation medium included the following: starch (53.313 g), defatted peanut powder (9.376 g), (NH4)2SO4 (6.244 g), and NaCl (5.836 g) in 1 l of distilled water; (ii) other conditions such as initial pH, temperature, medium capacity, agitation rate, seed age, inoculum size and growth period were 6.0, 28 C, 50 ml, 180 rpm, 4 days, 10% (v/v) and 120 h, respectively. Antifungal antibiotic production by the strain MY02 in the best media was compared with that in the basal medium.
3. Results and discussion
defatted soya powder, corn powder, yeast extract, peptone, (NH4)2SO4 and ammonia were provided at a concentration of 1.7% in the basal medium, respectively. Amongst the six kinds of carbon sources, maltose and starch were propitious to antifungal antibiotic production by the strain MY02. The medium including maltose gave the maximum antifungal activity (DIZ = 21.53 mm), and the medium including starch gave higher antifungal activity (DIZ = 20.96 mm). To consider the economic eﬃciency, the cost of maltose is higher than that of starch, so starch was determined as a factor in uniform design. Of all examined nitrogen sources, defatted peanut powder, defatted soya powder, and corn powder were favorable for antifungal antibiotic production by the strain MY02, whereas the maximum antifungal antibiotic production (DIZ = 21.31 mm) was obtained in the medium containing defatted peanut powder. In comparison with inorganic nitrogen sources, organic nitrogen sources gave relatively higher antifungal antibiotic production. So defatted peanut powder was selected as organic nitrogen resource of the medium.
3.1. Eﬀect of carbon and nitrogen source In order to screen a suitable carbon and nitrogen source, the strain MY02 was incubated in the basal medium containing various carbon and nitrogen sources. Each carbon source was added into the basal medium at a concentration of 3.3% (w/v) instead of starch, and the carbon source was as follows: maltose, sucrose, dextrin, glucose, and lactose. Various nitrogen sources such as defatted peanut powder,
3.2. Eﬀect of initial pH The strain MY02 was cultivated in the above improved medium with diﬀerent initial pH values (3.5–8.5). The results showed that the best pH for antifungal antibiotic production was 6.0, and the corresponding maximum diameter of inhibition zones was 22.37 mm. As we know,
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the range of pH for the proper growth of Streptomyces is between 7 and 7.5. But in our works, the best pH for antifungal antibiotic production was 6.0, maybe because the strain MY02 adapts to the acid soil in the northeast of China. 3.3. Eﬀect of cultural temperature and growth period In order to investigate the eﬀect of incubating temperature on antifungal antibiotic production, the strain MY02 was cultivated at 20–35 C, the best temperature was 28 C, and the maximum diameter of inhibition zone was 23.11 mm. In fact, antifungal antibiotic production was slightly ﬂuctuated at 25–28 C, the result showed that the eﬀect of temperature between 25 C and 28 C on antifungal antibiotic production was inapparent. The best growth period of the strain MY02 incubated in the improved medium at 28 C was 120 h, and the maximum diameter of inhibition zones was 25.50 mm. But after 120 h, the diameter of the inhibition zone dropped slowly. 3.4. Eﬀect of medium capacity and agitation rate The eﬀect of medium capacity and agitation rate on antifungal antibiotic production was investigated. The strain MY02 was cultivated at 28 C for 120 h in 250-ml Erlenmeyer ﬂask containing 30–150 ml medium with pH 6.0. Antifungal antibiotic production had no remarkable change in 30–100 ml, and declined sharply over 100 ml. So the best medium capacity was determined as 50– 100 ml in 250-ml Erlenmeyer ﬂask. In contrast, antifungal antibiotic production increased slightly corresponding to agitation rate. But antifungal antibiotic production has no signiﬁcant diﬀerences at 150–200 rpm. In our experiments, the medium capacity and agitation rate were 50 ml and 180 rpm, respectively. 3.5. Eﬀect of seed age and inoculum size To determine the best seed age and inoculum size, the strain MY02 was pre-cultivated for 4, 6, 8, and 10 days, respectively. Then the spore suspension (106 spores/ml) with diﬀerent inoculum sizes (2.5%, 5%, 10%, and 15% v/ v) was inoculated in the improved medium at 28 C for 120 h. When using the spore suspension of 4–6 days seed age, the antifungal antibiotic production was higher (DIZ = 22.39–26.76 mm). And the inoculum size with 10– 15% (v/v) were ﬁt for the antifungal antibiotic production by the strain MY02. In order to reduce the probability of contamination, the best inoculum size was determined, 10% (v/v). 3.6. Uniform design method Based on the above results, the dosage of starch, defatted peanut powder, (NH4)2SO4, and NaCl was further optimized via uniform design method U17(174). The culture
conditions such as temperature, initial pH, medium capacity, agitation rate, seed age, inoculum size, and growth period were ﬁxed to be 28 C, 6.0, 50 ml, 180 rpm, 4 days, 10% and 120 h, respectively. Factors, test number, method of uniform design, experimental conditions for each experimental group and experimental results are listed in Table 1. And the experimental results were analyzed by the software STATISTICA 6.0. When X1, X2, X3, X4, X1X2, X1X3, X1X4, X2X3, X2X4, X3X4, X 21 , X 22 , X 23 , X 24 are the independent variables and Y is a dependent variable, a regression model was established as follows: Y ¼ 11:182 þ 3:826X 1 þ 27:979X 2 þ 20:869X 3 30:859X 4 0:599X 1 X 2 þ 4:022X 1 X 3 þ 1:796X 1 X 4 12:013X 2 X 3 þ 5:502X 2 X 4 26:588X 3 X 4 0:64X 21 10:929X 22 12:435X 23 þ 28:038X 24 Components and dosage of the best medium were starch, 53.313 g; defatted peanut powder, 9.376 g; (NH4)2SO4, 6.244 g; and NaCl, 5.836 g in 1 l of distilled water via the regression model. Predicted value via the model (X1 = 5.3313, X2 = 0.9376, X3 = 0.6244 X4 = 0.5836) is 32.009, and R value is 0.98661. Residual values between the observed values via experiments and predicted values by the model are very low (Table 1), and these results showed that the experimental results were well in consistence with the calculation results via the model. 3.7. Investigation of the regression model The error between the observed values by experiments and predicted values via the model was investigated. The antifungal antibiotic production in the best medium, which was obtained from the regression model, was higher than that in the basal medium (20.96 mm). The diameter of inhibition zone of the culture supernatant from S. rimosus MY02 against F. oxysporium f sp. cucumarinum was 33.19 mm. The antifungal antibiotic production by S. rimosus MY02 was improved by optimization of the components and culture requirements. Production of antifungal antibiotic has been known to be eﬀected by media components and cultural conditions, such as carbon resource, nitrogen resource, aeration, agitation, pH and temperature. Deviation from optimal initial pH and temperature for antifungal antibiotic production severely aﬀects the yield of the antifungal antibiotic. Agitation rate and medium capacity aﬀect aeration and mixing of the nutrients in the fermentation medium, so adequate agitation was found to increase the yield of antifungal antibiotic (Sanchez and Brana, 1996). In our works, most cultural conditions were found to aﬀect antifungal antibiotic production by S. rimosus MY02, but agitation rate had a little eﬀect on antifungal antibiotic production between 150 and 200 rpm. Nutritional requirements of Streptomyces play an important role during metabolite synthesis process.
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Amongst various nutritional requirements, carbon and nitrogen resource are the most important impact factors. In order to obtain the best liquid culture requirements for antifungal antibiotic production by the strain MY02, the uniform design method was used in our work. And a regression model was established. In comparison with full factors experimental design and orthogonal design, uniform design requires only one experiment for every factor at one level, and it can reduce experimental times. So it has been successfully applied in the optimization of culture medium for metabolites and increases the yield of the metabolites in fermentation. 4. Conclusion In our study, in the case of single carbon source, maltose and starch were eﬃcient carbon for antifungal antibiotic production by the strain MY02. And organic nitrogen such as defatted peanut powder, defatted soya powder, and corn powder sources could give relatively higher antifungal antibiotic production than inorganic nitrogen sources. According to the primary experimental results, the dosage of starch, defatted peanut powder, (NH4)2SO4 and NaCl was further optimized via uniform design method U17(174). The culture conditions such as temperature, initial pH, medium capacity, agitation rate, seed age, inoculum size, and growth period were 28 C, 6.0, 50 ml, 180 rpm, 4 days, 10% and 120 h, respectively. Components and dosage of the medium were determined via uniform design combined with regression analysis. The best media ingredients were starch (53.313 g), defatted peanut powder (9.376 g), (NH4)2SO4 (6.244 g), NaCl (5.836 g) in 1 l of distilled water. The experimental results were well in consistence with the calculation results via the model. The antifungal antibiotic production by S. rimosus MY02 was improved by optimization of the components and culture requirements. In conclusion, the ﬁndings of the present study showed that naturally occurring actinomycetes have a great potential, which help in the production of metabolites against plant pathogen, and optimization of liquid culture requirements can enhance yield of metabolites.
Acknowledgements This work was supported by National Natural Science Foundation of China (30671398), Natural Science Foundation of Liaoning Province (20022085, 20062189), Technology Research Fund, Educational Oﬃce of Liaoning Province (2004F079) and Youth Science Fund of Dalian City (2005J22JH040). References Agrios, G.N., 1988. Plant Pathology. Academic Press, San Diego. Fang, K.T., 1994. Uniform Design and Uniform Design Tables. Science Publishing Company, Beijing (in Chinese). Fang, K.T., 2002. Theory, methods and applications of the uniform designs. Int. J. Reliab. Qual. Safety Eng. 9, 305–316. Fang, K.T., Ma, C.X., 2001. The Orthogonal Layout and Uniform Design Experiment Methods. Science Publishing Company, Beijing (in Chinese). Huang, S.W., Yu, L.Q., Tang, Q.Y., Watson, A.K., 2003. Using uniform design to screen media ingredients for conidia production of barny. Acta Phytopath. Sinica 33, 493–497 (in Chinese). Liu, Q., Wu, Y.H., Yu, J.C., 2004a. Screening for antagonistic actionmyces isolates from greenhouse soil in northeast china. Soil 36, 573– 575 (in Chinese). Liu, Q., Wu, Y.H., Yu, J.C., 2004b. Puriﬁcation of active components SN06 in fermentation of Streptomyces rimosus MY02. Acta Phytopath. Sinica 31, 353–358 (in Chinese). Marten, P., Bruckner, S., Minkwitz, A., Luth, P., Bergm G., 2001. RhizovitR: Impact and formulation of a new bacterial product. In: Koch, E., Leinonen P. (Eds.), Formulation of Microbial Inoculants: Proceedings of a meeting held in Braunschweig, Germany. COST Action 830/Microbial inoculants for agriculture and environment, Germany, pp. 78–82. Sanchez, L., Brana, A.F., 1996. Cell density inﬂuences antibiotic biosynthesis in Streptomyces clavuligerus. Microbiology+ 142, 1209–1220. Tian, G.L., Fang, K.T., 1999. Uniform design for mixture-amount experiments and for mixture experiments under order restrictions. Sci. China Ser. A. 42, 456–470. Valois, D., Fayad, K., Barasubiye, T., Garon, M., Dery, C., Brzezinski, R., Beaulieu, C., 1996. Glucanolytic actinomycetes antagonistic to Phytophthora fragariae var. rubi, the causal agent of raspberry root rot. Appl. Environ. Microb. 62, 1630–1635. Xu, C.P., Yun, J.W., 2003. Optimization of submerged-culture conditions for mycelial growth and exo-biopolymer production by Auricularia polytricha (wood ears fungus) using the methods of uniform design and regression analysis. Biotechnol. Appl. Bioc. 38, 193–199.