Using the mycelium-covered cereals as an efficient inoculation method for rapamycin fermentation in a 15-L fermenter using Streptomyces hygroscopicus
© Yen and Chiang. 2015
Received: 3 September 2015
Accepted: 4 November 2015
Published: 17 November 2015
Rapamycin is produced from Streptomyces hygroscopicus, and was initially identified as an antifungal antibiotic. More recently, rapamycin has been found to have various medical applications, including in relation to immunosuppression and anti-aging. Due to its complex structure, biological production is the major route for commercialized rapamycin production. The conventional fermentation process requires a large seed fermenter for the inoculation process (in general, the volume of the seed fermenter is equal to 5–10 % that of the production fermenter), which presents challenges with regard to scaling up production, due to the high investment costs of seed fermenters. This study explored different inoculation strategies for rapamycin production in a 15-L agitation fermenter.
The results indicated that solid-state fermentation (SSF) using barley as the substrate is a suitable method for the inoculation. The highest rapamycin concentration measured in the batch with SSF (barley) inoculated was about 520 mg/L, which was significantly higher than that of 400 mg/L obtained in the batch inoculated with 5 % liquid seed medium. Besides the higher rapamycin production, using SSF of barley as the inoculation method can greatly reduce both the labor and cost requirements.
The usage of mycelium-covered barley as the solid substrate for the inoculation of 15-L fermenter leading to a higher rapamycin production compared to that of conventional liquid seed medium. The solid-state inoculation method can avoid both the intensive labor requirement and costly seed fermenter needed with the latter approach. This inoculation method thus has the potential to be applied to the large-scale production of rapamycin.
Rapamycin was initially identified as having the medical function of an antifungal antibiotic, and was purified from an isolated Streptomyces hygroscopicus from Easter island soil in 1975 (Sehgal et al. 1975). Furthermore, rapamycin has been shown to have various medical applications, such as immunosuppression by inhibiting T cell activation and proliferation (Michaela Kuhnt et al. 1997). Of perhaps more interest is that rapamycin has been suggested to have a potent anti-aging function, by inhibiting mammalian targeting of rapamycin (mTOR). Due to its wide medical applications, the production of rapamycin through the biological process of S. hygroscopicus cultivation has attracted much attention in recent years.
Various fermentation strategies of S. hygroscopicus for the enhancement of rapamycin production have been explored, such as the optimum carbon source (Kojima et al. 1995) and nitrogen source screening (Lee et al. 1997). Besides the study of medium components, the use of a high dissolved oxygen (DO) environment is beneficial to rapamycin production. Nevertheless, the aim of high rapamycin production is to keep the intact pellet form of S. hygroscopicus, even under the high DO conditions. Therefore, the addition of pure oxygen to avoid the high shear force resulting from rapid agitation speed has been carried out, with 780 mg/L of rapamycin being obtained in a 5-L fermentor batch with DO controlled to over 30 % using pure oxygen (Yen and Hsiao 2013). In addition to DO, it has also been suggested that the pH value can alter the related metabolic pathway, thus changing the process of rapamycin production. More recently, a two-stage pH control strategy with no pH control in the first stage and with the pH controlled at 5.5 in the second stage was proposed to enhance rapamycin production (Yen et al. 2013).
Besides the effects of fermentation process parameters on rapamycin production, the morphologies of microorganisms also play a crucial role in the resulting metabolite products (Dobson et al. 2008; Ilić et al. 2008; Treskatis et al. 1997). It is well known that the cultivation conditions, including chemical and physical parameters, would change the morphology of actinomyces, and further affect their antibiotic productivity (Chen et al. 1999; Dobson et al. 2008; El-Sabbagh et al. 2006; Kanda et al. 2010). For example, a relationship between morphology and productivity of antibiotics by Streptomyces has been found, including with regard to the composition, structure, hydrophobicity or charge of the cell wall, as well as the presence of extracellular polymeric substances (Choi et al. 1998; Jonsbu et al. 2002; Pinto et al. 2004; Tamura et al. 1997). The morphology of Streptomyces may vary from a free filamentous suspension to pellets, depending on the degree of aggregation. The entanglement of prolonged filaments leads to the formation of pellets in submerged fermentations as a result of the aggregation of spores and/or hyphae. It was reported that such pellet-formed processes depend strongly on the species and the process conditions employed, and there may be a direct or indirect relationship with regard to the effects of morphology on the formation of metabolite products. Morphological control has been achieved by increasing the inoculum size, changing the initial pH value of the medium, and using additives, such as carboxymethylcellulose (CMC), chelators, Tween-80, or agar granules. For example, a study reported that adding CMC increased the viscosity of the medium and reduced pellet size, thus increasing rapamycin production in the cultivation of S. hygroscopicus (Yen and Li 2014). Similarly, smaller pellets can be beneficial to lovastatin production in the cultivation of Aspergillus terreus (Casas Lopez et al. 2005). The inoculation size can also affect the morphology by providing more growing points, due to the high initial cell density. A paper on the cultivation of Ganoderma lucidum found that a large inoculation density led to a small pellet size and high production of extracellular and intracellular polysaccharides (Fang et al. 2002). A review of the literature thus suggests that the production of rapamycin by S. hygroscopicus could be enhanced by the use of a more suitable inoculation method.
Moreover, a suitable inoculation method not only can enhance rapamycin production, but also simplify the commercial fermentation process. In a scaled-up fermentation process a high inoculation density means a long inoculation time is required, which increases costs and reduces the economic feasibility of such an approach. Therefore, this study aimed to examine the effects of inoculation strategy on rapamycin production, including the inoculation ratio, amount of spores and type of seeding. The aim is to find a suitable inoculation strategy for rapamycin production in a scaled-up fermentation process that is both efficient and convenient.
Strain and medium
The strain used in the experiment, Streptomyces hygroscopicus, was purchased from the Bioresource Collection and Research Center, Taiwan, under the catalog number BCRC 16270 (the same as ATCC 29253). A spore suspension was used for the inoculation of S. hygroscopicus cultivation in this study. The spore suspension aliquot was prepared according to the following procedure; agar plates with the sporulation medium (4 g/L dextrose, 20 g/L agar, 4 g/L yeast extract and 10 g/L malt extract) were plated out with the prepared spore solution and incubated at 28 °C for 20–25 days. After the black spores were seen to spread on the surface of agar plates, 1 mL of sterilized water was added to the plates to harvest the spore suspension, and preserved in frozen stock (stored in 25 % glycerin at −20 °C). The count of the prepared spore suspension was about 108 spores/mL, as measured in the colony-forming unit.
Three inoculation methods were performed in this study, including liquid seed medium inoculation, spore suspension inoculation and mycelium inoculation via solid-state fermentation. Liquid seed medium inoculation was performed using conventional liquid seed medium preparation procedure. A seed culture was initiated by adding l mL of the thawed cell suspension to a 250-mL Erlenmeyer flask containing 100 mL of seed medium (4 g/L dextrose, 4 g/L yeast extract, 10 g/L malt extract) at the initial pH 7.3 for 48 h (Yen and Hsiao 2013). A defined inoculation ratio of 2, 5 and 10 % (v/v) of seed medium was then inoculated into a 15-L agitation fermentor. The spore suspension was prepared in the petri-dishes containing the solid seed medium, as described above. After 20–25 days of cultivation, the black spores were observed on the surface of agar plate. Sterile water was used to collect the spores from the surface for the preparation of spore suspension. After the spore suspension was collected, it was inoculated into the 15-L fermentor directly, without the seed medium stage. Two concentrations of spore suspension were examined in this study, 1 × 1010/mL (denoted as the low SS batch) and 2 × 1010/mL (denoted as the high SS batch). The batch with mycelium inoculation of cereals cultured by solid-state fermentation (SSF) was performed as following: a seed culture was initiated by adding l mL of the thawed cell suspension to a 250-mL Erlenmeyer flask containing 100 mL of seed medium (4 g/L dextrose, 4 g/L yeast extract, 10 g/L malt extract) (Yen and Hsiao 2013) at the initial pH 7.3 for 48 h. A 1 mL aliquot of the seed culture was then transferred into a solid medium containing 10 g of cereal or bean and 10 mL of water, incubated at 28 °C for 10 days. Eight crops that often be investigated as substrates for solid fermentation, namely barley, buckwheat, brown rice, polished rice, red beans, soybean and green beans (mung bean) were examined in this study (Yang et al. 2013). After 10 days of cultivation, the white mycelium covered the cereals, and the whole 10 g of each crops along with the mycelium was liquefied with some sterile water using a homogenizer. The homogenized mycelium solution (with the cereals or beans) was then inoculated into the 15-L fermentor.
The fermentation in a 15-L agitation fermentor
The steps used to prepare the seed cultivation and medium components were the same as described in the previous section, except for the addition of 0.05 % antifoaming agent in the fermentation medium, which prevented foaming under severe agitation.
A seed medium was prepared and inoculated into a 15-L fermentor containing a 10-L working volume (4 g/L of yeast extract, 10 g/L of malt extract and 25 g/L of glucose). The fermentor was maintained at 28 °C and an aeration rate of 1 vvm was adopted. The DO level was controlled at about 30 % by adjusting the agitation rate automatically. However, the maximum agitation rate was limited to be no higher than 200 rpm to avoid damaging the pellets.
The glucose concentration was estimated using a glucose analysis instrument assay (YSI 2300 STAT Glucose Analyzer). Dry biomass was estimated using the moisture analyzer (IR-35, DENVER) assay; 5 mL of whole broth was centrifuged at 7000 rpm for 5 min to harvest the precipitated biomass. Following this, 1 mL of purified water was added into the tube to complete the aqueous mixing of the biomass. The whole aqueous biomass was transferred onto the IR dryer, and water was evaporated by infrared radiation to estimate the proportion of dry biomass. The measurement of rapamycin titer was performed by the HPLC method. A 1-mL aliquot of fermentation broth was centrifuged at 7000 rpm for 5 min. The supernatant was carefully transferred by pipette into a test tube, and the precipitate pellet was extracted again by shaking it with 1 mL of methanol for 20 min at room temperature. The extractant was subjected to HPLC analysis (Hitachi), using a Vercopak C18 column under the following conditions: mobile phase:methanol/water/acetic acid, 80/20/0.1; flow rate: 1.2 mL/min; detector: UV 254 nm (this method was suggested by LC Laboratories) (Yen et al. 2013). The standard rapamycin was purchased from LC Laboratories, USA (catalog number 53123-88-9, purity >99 %).
Results and discussion
Effects of inoculation ratio of liquid seed in a 15-L agitation fermentor
Using spore suspension for the inoculation
The inoculation using mycelia-covered cereals
The comparison of rapamycin production parameters between batches with various inoculation criteria into a 15-L fermenter: liquid seed medium at the ratios of 2, 5 and 10 % (v/v), spore suspension at low and high spore concentrations and mycelium-covered barley inoculated
Max. biomass (g/L)
Max. rapamycin (mg/L)
Rapamycin content (mg/g cells)
Rapamycin productivity (mg/L h)
Liquid seed mediuma
26 ± 3
41 ± 3
56 ± 5
33 ± 2
28 ± 3
36 ± 3
A comparison of rapamycin production by using SSF inoculation with other conventional liquid seed fermentations
Max. rapamycin (mg/L)
Rapamycin productivity (mg/L h)
(Yen and Hsiao 2013)
(Yen and Li 2014)
(Chen et al. 1999)
(Chen et al. 2008)
(Zhu et al. 2010)
Batch + SSF
This study explored different inoculation strategies for rapamycin production in a 15-L agitation fermenter, to reduce the scale-up production costs resulting from the conventional expensive inoculation method. The results indicated that solid-state fermentation (SSF) using barley as the substrate is a suitable method for inoculation. This led to higher rapamycin production compared to that seen with 5 % liquid seed medium, and avoided both the intensive labor requirement and costly seed fermenter needed with the latter approach. This inoculation method thus has the potential to be applied to the large-scale production of rapamycin.
MH carried out the most experiments and analysis. HW conceived of the study, participated in the experimental design and also be responsible for the preparation of this manuscript. Both authors read and approved the final manuscript.
The authors gratefully acknowledge the financial support this study received from Taiwan’s Ministry of Science and Technology (MOST) under Grant Number 103-2623-E-029-001-ET and 102-2221-E-029-017-MY2.
The authors declare that they have no competing interests.
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- Casas Lopez JL, Sanchez Perez JA, Fernandez Sevilla JM, Rodriguez Porcel EM, Chisti Y (2005) Pellet morphology, culture rheology and lovastatin production in cultures of Aspergillus terreus. J Biotechnol 116:61–77View ArticleGoogle Scholar
- Chen Y et al (1999) New process control strategy used in a rapamycin fermentation. Process Biochem 34:383–389View ArticleGoogle Scholar
- Chen Y, Krol J, Huang W, Cino JP, Vyas R, Mirro R et al (2008) DCO2 on-line measurement used in rapamycin fed-batch fermentation process. Process Biochem 43:351–355View ArticleGoogle Scholar
- Choi DB, Park EY, Okabe M (1998) Improvement of tylosin production from Streptomyces fradiae culture by decreasing the apparent viscosity in an air-lift bioreactor. J Ferment Bioeng 86:413–417View ArticleGoogle Scholar
- Dobson LF, O’Cleirigh CC, O’Shea DG (2008) The influence of morphology on geldanamycin production in submerged fermentations of Streptomyces hygroscopicus var. geldanus. Appl Microbiol Biotechnol 79:859–866View ArticleGoogle Scholar
- El-Sabbagh N, McNeil B, Harvey LM (2006) Dissolved carbon dioxide effects on growth, nutrient consumption, penicillin synthesis and morphology in batch cultures of Penicillium chrysogenum. Enzym Microbial Technol 39:185–190View ArticleGoogle Scholar
- Fang QH, Tang YJ, Zhong JJ (2002) Significance of inoculation density control in production of polysaccharide and ganoderic acid by submerged culture of Ganoderma lucidum. Process Biochem 37:1375–1379View ArticleGoogle Scholar
- Ilić SB, Konstantinović SS, Veljković VB, Savić DS, Lazić, Gojgić-Cvijović G (2008) Impact of carboxymethylcellulose on morphology and antibiotic production by Streptomyces hygroscopicus. Curr Microbiol 57:8–11View ArticleGoogle Scholar
- Jonsbu E, McIntyre M, Nielsen J (2002) The influence of carbon sources and morphology on nystatin production by Streptomyces noursei. J Biotechnol 95:133–144View ArticleGoogle Scholar
- Kanda M, Yamamoto E, Hayashi A, Yabutani T, Yamashita M, Honda H (2010) Scale-up fermentation of echinocandin type antibiotic FR901379. J Biosci Bioeng 109:138–144View ArticleGoogle Scholar
- Kojima I, Cheng YR, Mohan V, Demain AL (1995) Carbon source nutrition of rapamycin biosynthesis in Streptomyces hygroscopicus. J Ind Microbiol Biotechnol 14:436–439Google Scholar
- Kuhnt Michaela, Bitsch F, Ponelle M, Fehr T, Sanglier J-J (1997) Microbial conversion of rapamycin. Enzym Microbial Technol 21:405–412View ArticleGoogle Scholar
- Lee M, Kojima I, Demain A (1997) Effect of nitrogen source on biosynthesis of rapamycin by Streptomyces hygroscopicus. J Ind Microbiol Biotechnol 19:83–86View ArticleGoogle Scholar
- Pinto LS, Vieira LM, Pons MN, Fonseca MM, Menezes JC (2004) Morphology and viability analysis of Streptomyces clavuligerus in industrial cultivation systems. Bioprocess Biosyst Eng 26:177–184View ArticleGoogle Scholar
- Sehgal SN, Baker H, Vezina C (1975) Rapamycin (AY-22,989), A new antifungal antibiotic II. Fermentation, isolation and characterization. J Antibio 28:727–732View ArticleGoogle Scholar
- Tamura S, Park Y, Toriyama M, Okabe M (1997) Change of mycelial morphology in tylosin production by batch culture of Streptomyces fradiae under various shear conditions. J Ferment Bioeng 83:523–528View ArticleGoogle Scholar
- Treskatis SK, Orgeldinger V, Wolf H, Gilles ED (1997) Morphological characterization of filamentous microorganisms in submerged cultures by on-line digital image analysis and pattern recognition. Biotechnol Bioeng 53:191–201View ArticleGoogle Scholar
- Yang FC, Yang YH, Lu HC (2013) Enhanced antioxidant and antitumor activities of Antrodia cinnamomea cultured with cereal substrates in solid state fermentation. Biochem Eng J 78:108–113View ArticleGoogle Scholar
- Yen HW, Hsiao HP (2013) Effects of dissolved oxygen level on rapamycin production by pellet-form of Streptomyces hygroscopicus. J Biosci Bioeng 116:366–370View ArticleGoogle Scholar
- Yen HW, Li YL (2014) The effects of viscosity and aeration rate on rapamycin production in an airlift bioreactor by using Streptomyces hygroscopicus. J Taiwan Inst Chem Eng 45:1149–1153View ArticleGoogle Scholar
- Yen HW, Hsiao HP, Chen LJ (2013) The enhancement of rapamycin production using Streptomyces hygroscopicus through a simple pH-shifted control. J Taiwan Inst Chem Eng 44:743–747View ArticleGoogle Scholar
- Zhu X, Zhang W, Chen X, Wu H, Duan Y, Xu Z (2010) Generation of High Rapamycin Producing Strain via Rational Metabolic Pathway-Based Mutagenesis and Further Titer Improvement With Fed-Batch Bioprocess Optimization. Biotechnol Bioeng 107:506–515View ArticleGoogle Scholar