Dissolved oxygen control strategy for improvement of TL1-1 production in submerged fermentation by Daldinia eschscholzii
© The Author(s) 2017
Received: 13 October 2016
Accepted: 26 December 2016
Published: 2 January 2017
2,3-Dihydro-5-hydroxy-2-methylchromen-4-one (TL1-1) is a phenolic compound with significant anti-fungal and anti-cancer activities produced by Daldinia eschscholzii (D. eschscholzii). However, studies have rarely been reported on the fermentation process of D. eschscholzii due to the urgent demand for its pharmaceutical researches and applications.
In this work, the optimal fermentation medium for improved TL1-1 yield was first obtained in a shake flask. As the fermentation process was scaling up, the marked effects of dissolved oxygen (DO) on cell growth and TL1-1 biosynthesis were observed and confirmed. Controlling a suitable DO level by the adjustment of agitation speed and aeration rate remarkably enhanced TL1-1 production in a lab-scale bioreactor. Moreover, the fermentation of D. eschscholzii was successfully applied in 500-L bioreactor, and TL1-1 production has achieved 873.63 mg/L, approximately 15.4-fold than its initial production (53.27 mg/L).
Dissolved oxygen control strategy for enhancing TL1-1 production was first proposed. Furthermore, control of the appropriate DO level has successfully performed for improving TL1-1 yield and scale-up of D. eschscholzii fermentation process.
KeywordsTL1-1 Dissolved oxygen control Submerged fermentation Daldinia eschscholzii
2,3-Dihydro-5-hydroxy-2-methyl-chrlchromen-4-one (TL1-1) is one kind of phenolic compound isolated from the filamentous fungus Daldinia eschscholzii IFB-TL01 (Zhang et al. 2011). According to previous studies, TL1-1 has revealed significant anti-fungal activity against plant pathogenic fungi, Microbotryum violaceum (Dai et al. 2006), and it suggests that TL1-1 should be developed as a potential candidate for agricultural antibiotic. It is also reported that it inhibits cell proliferation of various cancer cells (leukemia, melanoma and liver) with IC50 values of 8–55 μg/mL. Furthermore, the little cytotoxicity to normal cells indicates that TL1-1 could serve as a promising candidate for cancer treatment (Zilla et al. 2013; Pathania et al. 2015). However, low production would severely restrict its further pharmaceutical researches and applications. Moreover, little research has been reported on the submerged fermentation of D. eschscholzii. Therefore, it is urgent to develop an effective strategy to enhance the yield of TL1-1 from fermentation process.
In the fermentation processes, the medium composition and culture conditions play a crucial role in both cell growth and product formation (Guleria et al. 2016). The molasses used as carbon source significantly enhance Fumigaclavine C production (from 75.9 to 215.0 mg/L) by Aspergillus fumigatus CY018 in a lab-scale bioreactor (Zhu et al. 2015). Distinguished from shake flask, bioreactor requires for optimal conditions (agitation speed and aeration rate) to achieve the maximum productivity in industrial scale-up level (Zou et al. 2012). To satisfy cell growth and maintain desired product synthesis, it is requested to settle the problems of shear stress, mass transfer and mixing in submerged fermentation process (Lu et al. 2015; Formenti et al. 2014; Werner et al. 2014). In Cellulophaga lytica LBH-14 fermentation, cell growth and cellobiase formation are regulated by adjustment of agitation speed as well as aeration rate, and the maximum cellobiase activity (140.1 U/mL) is obtained in 100-L bioreactor, 1.52 times higher than that in shake flask (Gao et al. 2015).
Accumulative studies demonstrate that dissolved oxygen has become a significant factor in the scale-up of industrial aerobic fermentation process (Pollard et al. 2007). Due to inadequate oxygen supply capacity, various technologies are employed to improve DO level (Häusler et al. 2016; Garcia-Ochoa and Gomez 2009). For example, in Ralstonia eutropha fermentation, off-gas recycle pressure swing adsorption is applied to provide high-purity oxygen continuously, and higher titer of poly(3-hydroxybutyrate) is acquired in industrial scale (Chang et al. 2010). However, the excessive oxygen concentration caused by increase of agitation speed and aeration rate could have negative impacts on bioprocess because of oxygen toxicity or shear stress for sensitive cells (Nagy 2002). That means the demand for oxygen in microorganisms varies among different fermentation processes. To ensure the appropriate oxygen supplement, DO is commonly controlled at a suitable level (Cao et al. 2013). Take submerged fermentation of Saccharopolyspora spinosa, for example, DO is kept at 40–50% during 0–96 h for cell growth and at 25–35% during 96–240 h for spinosad formation (Bai et al. 2015). In consequence, the application of DO control strategy could effectively enhance desired product yield.
In this paper, the optimal medium for higher yield of TL1-1 was first obtained. Subsequently, the significant effects of the oxygen concentration on cell growth and TL1-1 biosynthesis were discovered. In the scale-up process, DO level was further confirmed to have played a critical role in desired product formation. The strategy of controlling the appropriate DO level was conducted through the adjustment of agitation speed and aeration rate, which enhanced TL1-1 production and was scaled up in 500-L bioreactor successfully. The information obtained would contribute to the industrial application of submerged fermentation of filamentous fungus.
The fungus, D. eschscholzii IFB-TL01 (CCTCCM 207198) from the Tenodera aridifolia gut, was friendly provided by Nanjing University (Zhang et al. 2008). This strain was preserved on potato dextrose agar (PDA) and sub-cultured every month. The strain was incubated on PDA at 28 °C for 7 days. Subsequently, two agar tablets (size of 1.0 cm × 1.0 cm) were cut and inoculated in a 1-L Erlenmeyer flask containing 400 mL seed medium (potato dextrose broth) (PDB) (Pan et al. 2015).
Optimization of fermentation media in shake flask
Seven fermentation media (Medium 1–Medium 7) (data not shown) were screened for TL1-1 accumulation (Zhang et al. 2008; Atlas 2010; Cai et al. 2009). Subsequently, the carbon sources with same molar amount of C (glucose, sucrose, mannitol and soluble starch) were chose to investigate the influence of biomass and TL1-1 production in M5.
The details of culture conditions including inoculation ages, inoculums volume, and initial pH of fermentation medium were optimized in our previous work (data not shown and detailed data in Additional file 1: Figure S1). For the shake flask fermentation, the seed was cultured at 180 rpm, 28 °C for 72 h. Subsequently, 7 mL seed broth was inoculated into a 250-mL shake flask with 50 mL fermentation medium of which initial pH was 9.0 ± 0.2 (before sterilization). The above fermentations for TL1-1 production were performed at 180 rpm, 28 °C for 144 h in a shake flask.
Investigation of fermentation conditions in shake flask
In the test of shaking speed, fermentation was performed for 144 h at 28 °C and 80, 180 and 280 rpm, respectively. To investigate effects of shear stress, 0, 2, 6 and 10 glass beads (diameter of 2 mm) were added into fermentation medium before sterilization. In the study on various oxygen concentrations, the seed broth prepared was transferred into a 250-mL shake flask with 30, 50, 70, 90 and 150 mL medium, respectively. Additionally, 200% silicone oil, 1% n-dodecane and 3% n-dodecane were added into 50 mL fermentation medium before sterilization, respectively. All data obtained in this work were the mean of triplicate experiments, and the error bars indicated the corresponding standard deviation.
Optimization of agitation speed in 5-L bioreactor
Optimization of agitation speed was performed in 5-L stirred bioreactor equipped with two layers of six flat-blade disk turbine impeller based on the optimal fermentation medium. 400 mL seed broth, prepared under the same conditions as previously described, was inoculated into the 5-L bioreactor containing 2.9 L medium. The fungus was cultivated at 28 °C for 120 h with aeration rate of 1.2 air volume/culture volume/min (VVM) during the fermentation. The agitation speed was set as 180, 230 and 280 rpm, respectively. Antifoam (mixture of organic polyether dispersions) was added with 0.08% (v/v). Samples were taken at an interval of 12 h.
Adjustment of aeration rate in 50-L bioreactor
Adjustment of aeration rate was operated in a 50-L stirred bioreactor equipped with three turbine impellers (Zhang et al. 2014). Based on the amplified standard of similar blade and linear velocity, agitation speed was set at 130 rpm in 50-L bioreactor. The aeration rate was set as 0.6, 0.9 and 1.2 VVM, respectively. Samples were taken at an interval of 24 h.
Scale-up of the fermentation process
4 L preliminary seed broth (prepared under the same conditions as previously described) was transferred into a 50-L bioreactor containing 40 L PDB; secondary seed was cultivated at 28 °C for 24 h (agitation speed of 150 rpm and aeration rate of 1.0 VVM). Then, the secondary seed broth was inoculated into a 500-L stirred bioreactor equipped with three turbine impellers at inoculums ratio of 14% (v/v). The fungus was cultivated at 28 °C for 192 h with agitation speed of 70 rpm and aeration rate of 0.9 VVM during the fermentation process. Samples were taken at an interval of 24 h.
Measurement of fungus growth, residual sugar and TL1-1 production
The whole fermentation broth was pretreated as report (Zhu et al. 2014). The qualitative and quantitative measurements of TL1-1 were analyzed by HPLC (Agilent 1290 Infinity, DAD-G4212B, USA) with a ZORBAX Eclipse SB-C18 column (4.6 × 250 mm, 5 µm). Each injected sample (20 µL) was eluted with a mobile phase made up of methanol/water (60:40) for 25 min. The flow rate was set as 1.0 mL/min, the operating temperature 25 °C, and the detection wavelength 272 nm, respectively. The standard calibration curve equation was Y = 42.95X − 341.77 with high linearity in the range of 62.5–750.0 mg/L (linear relative coefficients up to 0.9999), where Y is the peak area and X is the concentration of standard TL1-1 (mg/L) (data not shown and detailed data in Additional file 1: Figure S2).
Results and discussion
Effects of fermentation medium on production of TL1-1
Effects of fermentation conditions on cell growth and TL1-1 production in shake flask
According to previous reports, high shear stress environment may be stimulated by the addition of glass beads in shake flask (Dobson et al. 2008). To study the effects of shear force on TL1-1 synthesis, the test of glass beads addition was performed. The data showed that total titer of TL1-1 (750.60 mg/L) was the highest without addition of glass beads, and it declined with the addition amounts of glass beads being increased. TL1-1 concentration was decreased by only 27.2% when 10 glass beads were added (Fig. 2b). This indicated that shear stress had a negative effect on TL1-1 biosynthesis to a certain extent and another critical factor may be to explore.
Previous studies showed oxygen concentration was declined with the increase of medium volume in shake flask due to change of oxygen transfer area (Dou et al. 2013). To study the effects of oxygen concentration on TL1-1 biosynthesis, the test of loading volume was operated. It was observed that the increase of fermentation medium volume led to biomass reduction (Fig. 2c). And TL1-1 production was greatly improved in the range of filling volume from 30 to 90 mL, while sharply decreased from 90 to 150 mL (Fig. 2c). To further confirm the effects of oxygen concentration on TL1-1 synthesis, n-dodecane and silicone oil were added into fermentation medium before sterilization. Previous studies illustrated that n-dodecane as oxygen vector could enhance volumetric mass transfer coefficients to improve oxygen concentration, and a significant amount of silicone oil could isolate oxygen to reduce oxygen concentration during fermentation process (Da Silva et al. 2006; Xu et al. 2015). As shown in Fig. 2d, compared to control (763.49 mg/L and 5.06 g/L), TL1-1 production was significantly declined to 469.35 mg/L and biomass was increased to 6.77 g/L when adding 3% n-dodecane. Additionally, double volume of silicone oil was added into medium, both TL1-1 production and biomass were reduced by 97.2 and 38.4%, respectively. These results evidently indicated that higher oxygen concentration was beneficial for cell growth but failed to get higher TL1-1 yield, while extremely low one inhibited both. Oxygen concentration stress played a critical role on TL1-1 biosynthesis. Increasing studies showed many secondary metabolite syntheses were sensitive to oxygen concentration, such as cephamycin C, tylosin and erythromycin (Pollard et al. 2002). Hence, different shaking speeds mainly caused the change of oxygen concentration, as well as shear stress as a secondary factor, which resulted in variation of TL1-1 production.
Effects of DO on cell growth and TL1-1 production by optimization of agitation speed in 5-L bioreactor
Effects of DO on cell growth and TL1-1 production by adjustment of aeration rate in 50-L bioreactor
Based on our previous results, to control the relatively low DO level, the strategy of reducing the aeration rate was applied to 50-L bioreactor for further improvement of TL1-1 production. The aeration rate was adjusted from 1.2 to 0.6 VVM at the same agitation speed (130 rpm) in 50-L bioreactor. When aeration rate was declined from 1.2 to 0.9 VVM, the value of DO fell to less than 10% during 48–168 h in Fig. 5a and TL1-1 concentration reached 745.58 mg/L at 168 h in Fig. 5b, correspondingly. TL1-1 production was equivalent to shake flake level. Similarly, in Streptomyces griseorubens fermentation, DO was kept at a constant level by adjusting agitation speed and aeration rate, which accelerated cellulase and xylanase formation (Zhang et al. 2016). In the next study, aeration rate was decreased from 0.9 to 0.6 VVM, and DO was maintained constant afterward at an extremely low level during 24–192 h (Fig. 5a). It was observed that cell growth got restricted under oxygen-limited conditions in Fig. 5c and TL1-1 yield was decreased to 371.72 mg/L in Fig. 5b. The cell physiologic state has been recognized to have impacts on secondary metabolisms of microorganisms. For example, the cell growth of Porphyridium purpureum was significantly inhibited and the production of total fatty acids as well as arachidonic acid was declined with limited amount of oxygen (Su et al. 2016). The level of oxygen available played an important role in control of the competition between the biomass formation and product synthesis. These operating conditions (agitation speed 130 rpm and aeration rate 0.9 VVM) in 50-L bioreactor seemed to be favorable for TL1-1 formation and the hypothesis that DO control strategy promoted target product biosynthesis was further confirmed.
Scale-up TL1-1 production in 500-L bioreactor
In this study, the fermentation medium and conditions for high yield of TL1-1 were first attained. In the scale-up process, the correlation between oxygen concentration and TL1-1 synthesis was discovered and discussed. The present study suggested higher production should be obtained under the appropriate DO level. The DO control strategy by adjusting agitation speed and aeration rate was proposed and significantly enhanced TL1-1 yield in bioreactor. Furthermore, the fermentation process of D. eschscholzii was successfully scaled up to 500-L bioreactor and TL1-1 production has achieved 873.63 mg/L, approximately 15.4-fold than its initial one (53.27 mg/L). This work demonstrated that DO control strategy was an economic and effective approach for enhancement of TL1-1 productivity and would be helpful in the common large-scale submerged fermentation process of filamentous fungus.
potato dextrose agar
potato dextrose broth
air volume/culture volume/min
- D. eschscholzii :
Daldinia eschscholzii IFB-TL01 (CCTCCM 207198)
Medium 1–Medium 7
dry cell weight
XCW was in charge of the design of the study, performing the experimental work and drafting the manuscript. LT participated in the experiments in 500-L bioreactor. YHL directed the study as the tutor. All authors read and approved the final manuscript.
This work is financially supported by the National High Technology Research and Development Program of China (2013AA092901), the Fundamental Research Funds for the Central Universities, as well as the National Special Fund for State Key Laboratory of Bioreactor Engineering (2060204).
The authors declare that they have no competing interests.
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