- Email: [email protected]
Efficient biosynthesis of anticancer polysaccharide by a mutant Chaetomium globosum ALE20 via non-sterilized fermentation
International Journal of Biological Macromolecules 136 (2019) 1106–1111
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Efficient biosynthesis of anticancer polysaccharide by a mutant Chaetomium globosum ALE20 via non-sterilized fermentation Zichao Wang a, Xuyang Chen a, Siyu Liu a, Yingying Zhang a, Zhangtao Wu a, Wenwen Xu a, Qi Sun b,⁎, Libo Yang c, Huiru Zhang a,⁎ a b c
College of Biological Engineering, Henan University of Technology, Zhengzhou 450001, China College of Life Sciences, Chongqing Normal University, Chongqing 401331, China College of Landscape and Ecological Engineering, Hebei University of Engineering, Handan 056021, China
a r t i c l e
i n f o
Article history: Received 25 May 2019 Received in revised form 13 June 2019 Accepted 24 June 2019 Available online 25 June 2019 Keywords: Adaptive laboratory evolution Methanol-resistant strain Anticancer polysaccharide
a b s t r a c t The sterilization process, due to its immense energy consumption, high facilities investment, and loss of raw materials by caramelization, during industrial production has drawn much attention. In this study, a methanolresistant mutant strain, Chaetomium globosum ALE20, was obtained following 20 cycles of adaptive laboratory evolution process. The titer of anticancer polysaccharide (GCP-M) from C. globosum ALE20 reached 9.2 g/L with glycerol as sole carbon source using non-sterilized and fed-batch fermentation strategy. This titer represents a 200% increase compared with the 3.3 g/L attained with batch fermentation. The GCP-M monosaccharide was comprised of galactose, glucose, mannose and glucuronic acid, in a molar ratio of 3.83:66.37:3.26:1.95, respectively, and its weight-average molecular weight and polydispersity were 3.796 × 104 Da and 1.060, respectively. This work presents an ideal alternative and safer fermentation process without sterilization, and a useful approach for enhancing industrial production. © 2019 Published by Elsevier B.V.
1. Introduction Given the exhaustion of fossil fuel, growing demand for fossil oilbased products, and aggravation of global warming, alternative and renewable energy resources are being researched worldwide [1]. Biodiesel is regarded as one of the most promising alternatives to fossil fuel due to its reduced carbon dioxide emission and regeneration properties [2]. During the trans-esterification process for biodiesel production catalyzed by methanol, glycerol is produced inevitably and accounts for one-tenth of the total biodiesel yield [3]. However, the methanol, salts, fatty acids, soap, and other organic matter in crude glycerol make it difficult for use in conventional industries, such as foods, cosmetics, and pharmaceuticals. Adaptive laboratory evolution (ALE) has been considered an effective method for enhancing microbial strain tolerance to inhibiting environmental conditions and developing new biological functions in strains [4–6]. For instance, oxalic acid yield from corncob was enhanced by a methanol-resistant Aspergillus niger strain using semi-solid-state fermentation through the ALE process [7]. An Aspergillus niger MTCC 281 mutant with combined resistance to methanol and malic acid was successfully obtained via the ALE process [8]. Further, ALE combined with high salinity was performed to alleviate oxidative damage and ⁎ Corresponding authors. E-mail addresses: [email protected] (Q. Sun), [email protected] (H. Zhang).
https://doi.org/10.1016/j.ijbiomac.2019.06.186 0141-8130/© 2019 Published by Elsevier B.V.
improve lipid biosynthesis in the microalgae, Schizochytrium sp. ALE150 [9]. In this study, to improve the anti-cancer polysaccharide production capacity of Chaetomium globosum CGMCC 6882 from glycerol and crude glycerol [10], we first subjected C. globosum CGMCC 6882 to ALE by gradually increasing the methanol concentration to enrich methanol-resistant C. globosum strains, and the evolved C. globosum ALE20 was found to be more tolerant to methanol than the parent strain. Then, the fermenting properties of C. globosum ALE20 under sterilized and non-sterilized conditions, varying glycerol and methanol concentrations, and the fed-batch strategy were studied. Finally, high performance anion exchange chromatography, size exclusion chromatography, Fourier transform infrared spectroscopy (FT-IR), and nuclear magnetic resonance (NMR) were used to characterize the polysaccharide produced by C. globosum ALE20. 2. Materials and methods 2.1. Microorganisms and chemicals The parent strain, C. globosum CGMCC 6882, was isolated from the herb, G. pentaphyllum, and stored in China General Microbiological Culture Collection Center (China). The methanol-resistant strain C. globosum ALE20 was obtained through ALE from C. globosum CGMCC 6882 and collected in the Engineering Laboratory of Henan
Z. Wang et al. / International Journal of Biological Macromolecules 136 (2019) 1106–1111
Province for Industrial Culture Collection and Breeding Center. The chemicals (including glycerol) used in this work were analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (China). 2.2. Preparation and storage of spores C. globosum CGMCC 6882 spores were collected by scratching the PDA plates with a disposable spreader and washing with sterilized water. Then, a hemocytometer (Neubauer, Germany) was used to count the spores under a phase contrast microscope (Zeiss, Axio Imager 2, Jena, Germany). A stock of spore suspension (1 × 107 spores/mL) was maintained in 50% glycerol at −80 °C for long-term storage. 2.3. Adaptive laboratory evolution The evolution of methanol-resistant strain C. globosum ALE20 was performed as described by Iyyappan et al. [8] with some modifications. With the continuous ALE process, methanol concentration on the PDA plate increased stepwise from 0.5% (v/v) to 10% (v/v); about 1 × 105/mL spores of C. globosum CGMCC 6882 were inoculated on the PDA plate and incubated at 28 °C for 7 d. After 7 d, spores in the region showing better mycelial growth were harvested and inoculated onto a fresh medium containing higher methanol concentration. A cycle of spore transfers was implemented until 10% (v/v) methanol concentration was attained on the PDA plate.
1107
through a 0.2 μm filter and added into the fermenter. In non-sterilized fermentation, 400 mL methanol was added directly into the fermenter containing 3.5 L of non-sterilized culture medium. At 12 h intervals, 20 mL broth aliquots were collected for analysis during the fermentation process. All batch cultures were performed in three replications. 2.6. Batch fermentation with varying glycerol and methanol concentrations During the fermentation process involving different glycerol and methanol concentrations, the fermenter and fermentation medium were sterilized at 121 °C for 20 min. To vary the glycerol concentrations for fermentation, the composition of culture media remained the same as in Section 2.5, but glycerol concentrations were set to 10, 20, 30, 40, 50, 60, 70 or 80 g/L. Similarly, methanol concentrations were varied, such that, after the culture medium was sterilized and cooled to room temperature, 80, 160, 240, 320, 400 or 480 mL methanol was filtered through a 0.2 μm filter and added into the fermenter. 2.7. Fed-batch fermentation
The seed medium used for C. globosum ALE20 was potato dextrose medium, incubated at 28 °C and 120 rpm for 48 h. Then, a hemocytometer (Neubauer, Germany) was used to count the spores under a phase contrast microscope (Jena, Germany), and the spore suspension was diluted to a concentration of 1 × 107 spores/mL with distilled water and a 1% (v/v) inoculation volume.
For the sterilized fed-batch fermentation, the initial glycerol concentration was set to 20 g/L and the culture medium was sterilized at 121 °C for 20 min. After the fermentation medium was cooled to room temperature, 240 mL filtered methanol was added into the fermenter to increase the original methanol concentration to 6% (v/v). Then, glycerol and methanol concentrations increased to 40 g/L and 10% (v/v), respectively, by adding sterilized glycerol and filtered methanol at 2.5 d of fermentation process. For non-sterilized fed-batch fermentation, the initial glycerol and methanol concentrations were 20 g/L and 6% (v/v), respectively, and these were increased to 40 g/L and 10% (v/v), respectively, by adding non-sterilized glycerol and non-filtered methanol at 2.5 d of fermentation. Fermentation conditions were the same as in Section 2.5 and aliquots of the fermentation broth were collected for analysis at 12 h intervals.
2.5. Batch fermentation under sterilized and non-sterilized conditions
2.8. Biomass, glycerol, and polysaccharide titer determination
The fermentation medium was composed of the following (g/L): glycerol 40, peptone 1.0, yeast extract 1.0, beef extract 1.0, MgSO4·7H2O 1.5, KH2PO4 1.0, K2HPO4 1.0, and FeSO4 7H2O 0.01. Fermentation temperature was 28 °C, cultivation time was 7 d, pH was maintained at 7.0 ± 0.2 with 2 mol/L NaOH and 2 mol/L HCl. All the fermentation experiments were conducted in a 7.0 L fermenter (BioFlo 115, New Brunswick, USA) with a 3.5 L working volume. During the fermentation process, agitation and aeration were set to 150 rpm and 1.0 vvm, respectively. For sterilized fermentation, after the culture medium was sterilized and cooled to room temperature, 400 mL methanol was filtered
Dry cell weight of C. globosum ALE20 was determined using the modified method of Bahaloo-Horeh et al. [11]. Fermentation broth was filtered through a Buchner funnel and the resulting biomass (mycelium and spores) was washed twice with deionized water, then transferred to a pre-weighed porcelain dish, dried in an oven for 24 h, cooled in a desiccator and weighed. The dry cell weight of C. globosum ALE20 was calculated by the weight difference method. Meanwhile, the filtered liquor was centrifuged at 12,000 ×g for 30 min to remove insoluble impurities, after which, 1 mL supernatant was filtered through a 0.22 μm membrane for glycerol concentration determination by high-
2.4. Seed culture medium
Fig. 1. Morphological images of C. globosum CGMCC 6882 (A) and C. globosum ALE20 (B) on PDA plates supplemented with 10% (v/v) methanol.
1108
Z. Wang et al. / International Journal of Biological Macromolecules 136 (2019) 1106–1111
Fig. 2. Sterilized (A) and non-sterilized (B) batch fermentation profiles of C. globosum ALE20 with 40 g/L glycerol and 10% (v/v) methanol. The symbols represent: glycerol (■), biomass (●), GCP-M titer (▲).
performance liquid chromatography, as we reported previously [12]. Simultaneously, 10 mL supernatant, to which four volumes of cold 95% alcohol was added, was collected and kept at 4 °C overnight to precipitate polysaccharide, then the supernatant was discarded and the precipitated polysaccharide was re-dissolved into 10 mL deionized water for polysaccharide yield determination using the phenol sulfuric acid method at 490 nm. 2.9. Polysaccharide characterization At the end of fermentation, culture broth was filtered and centrifuged at 12,000 ×g for 30 min to remove mycelium and cells. Then, the broth was concentrated to one-tenth of the original volume with rotary evaporation at 60 °C and 0.1 MPa vacuum. After that, the concentrated broth was de-proteinized by adding three volumes Sevag solution, and three times of cold 95% alcohol was added into the deproteinized supernatant and kept at 4 °C overnight to precipitate polysaccharide. The precipitated polysaccharide was re-dissolved in distilled water and de-pigmented with AB-8 macroporous resin, and then, the polysaccharide solution was filtered through a 0.22 μm filter and applied to a Sepharose CL-6B column (2.5 × 60 cm) for further purification, eluting with 0.1 mol/L NaCl solution at a flow rate of 0.6 mL/min. The fraction was collected and freeze dried for studying the molecular characteristics of polysaccharide. High performance anion exchange chromatography was used for analyzing the monosaccharide composition of polysaccharide. Briefly, polysaccharide was dissolved in 2 mol/L trifluoroacetic acid and hydrolyzed at 120 °C for 2 h, washed three times with methanol and evaporated to dryness for removing TFA. Then, the hydrolyzed material was transferred to a 25 mL volumetric flask, diluted to 25 mL by deionized water and subjected to Dionex ICS5000 system (Dionex, USA) equipped with CarboPac PA20 column (ID 3 mm × 150 mm). The mobile phase was deionized water (A), 0.25 mol/L NaOH (B), 1 mol/L NaAc (C) and eluted as follows (A%, B%, C%): 0 min: 99.2, 0.8, 0; 30 min: 99.2, 0.8, 0; 40 min: 79.2, 0.8, 20;40.1 min: 20, 80, 0; 60 min: 99.2, 0.8, 0. The flow rate was 0.45 mL/min and the injection volume was 25 μL, column temperature was 30 °C and detected by a pulsed ampere detector, Au electrode, Ag/AgCl reference electrode. Polysaccharide was dissolved in distilled water to a concentration of 2 mg/mL and analyzed by high performance size exclusion chromatography for its molecular weight. The system consisted of Waters 2695 HPLC system equipped with multiple detectors: refractive index detector (RI) and a UV detector for concentration determination, multiple angle laser light scattering detector (MALLS, DAWNHELEOS, Wyatt Technology, USA) for direct molecular weight determination and differential pressure viscometer (DP) for viscosity determination. The columns were TSK PWXL 6000 and 3000 gel filtration columns which were eluted with PB buffer (0.15 mol/L NaNO3 and 0.05 mol/L NaH2PO4, pH = 7) at the flow rate of 0.5 mL/min. The calibration of the laser photometer was
performed with ultrapure toluene. The normalization was conducted with a bovine serum albumin globular protein (Mw = 66.7 kDa, Rg = 2.9 nm). A value of 0.146 mL/g was used as refractive index increment (dn/dc) for molecular weight calculation. Astra software (Version 6.1.1) was utilized for data acquisition and analysis. The column temperature and RI detector temperature were maintained at 35 °C [10]. Nexus 470 FT-IR spectrophotometer (Nicolet, USA) was used to record the IR spectrum of polysaccharide with KBr between 400 and 4000 cm−1, and a Bruker Avance 400 MHz spectrometer (Bruker Inc., Germany) was used to record the 1H NMR spectra of polysaccharide at 30 °C. polysaccharide was dissolved in D2O in 5 mm OD NMR tube, the chemical shifts for 1H NMR spectra were recorded in parts per million from tetramethylsilane [13,14]. 2.10. Statistical analysis Each analytical result is expressed as mean ± SD after triplicate measurements. Data were analyzed by analysis of variance (ANOVA), using the Origin Pro 2017 software. 3. Results and discussion 3.1. Adaptive laboratory evolution Several short-chain alcohols, including methanol, ethanol, butanol, propanol, isopropanol, tert-butanol, branched alcohols and octanol, are utilized in the trans-esterification process as catalysts for biodiesel production [15]. However, methanol is particularly preferred because of its low cost, physical and chemical advantages, quick reaction with triglycerides and easy dissolution in NaOH [16]. Therefore, crude glycerol derived from the biodiesel industry is always contaminated with methanol, thus reducing the suitability of crude glycerol as a potential fermentation substrate in bioprocess applications [17,18]. As shown in Fig. 1B, after the ALE process, with stepwise increment in methanol concentrations from 0.5 to 10% (v/v) on PDA, an evolved methanolresistant strain, C. globosum ALE20, was obtained and could grow on Table 1 Effect of varying glycerol concentrations on C. globosum ALE20 growth, biomass, and GCPM titer. Glycerol (g/L)
Cell growth time (d)
Biomass (g/L)
GCP-M titer (g/L)
10 20 30 40 50 60 70 80
2.5 2.5 3.0 3.5 3.5 4 4.5 4.5
2.13 ± 0.05 2.42 ± 0.08 2.04 ± 0.06 1.81 ± 0.13 1.73 ± 0.04 1.20 ± 0.15 0.71 ± 0.09 0.23 ± 0.02
0.35 ± 0.03 1.38 ± 0.21 2.55 ± 0.07 3.38 ± 0.13 3.07 ± 0.06 1.78 ± 0.19 0.65 ± 0.03 0.1 ± 0.04
Z. Wang et al. / International Journal of Biological Macromolecules 136 (2019) 1106–1111 Table 2 Effects of varying methanol concentrations on C. globosum ALE20 growth, biomass, and GCP-M titer. Methanol (%, v/v)
Cell growth time (d)
Biomass (g/L)
GCP-M titer (g/L)
2 4 6 8 10 12 14
2.5 2.5 3 3.5 3.5 4 4.5
2.35 ± 0.12 2.28 ± 0.07 2.17 ± 0.05 2.03 ± 0.03 1.83 ± 0.08 0.72 ± 0.13 0.39 ± 0.04
3.05 ± 0.13 3.16 ± 0.08 3.41 ± 0.15 3.78 ± 0.09 3.41 ± 0.11 1.03 ± 0.07 0.46 ± 0.12
10% (v/v) methanol PDA plate, while the parent strain, C. globosum CGMCC 6882, could not (Fig. 1A). The methanol resistance ability of C. globosum ALE20 could well alleviate the inhibitory effect of methanol on its growth and improve its crude glycerol utilization capacity, thereby improving the suitability of crude glycerol as a potential fermentation substrate in bioprocess applications and relieving the burden on the biodiesel industry. 3.2. Batch fermentation under sterilized and non-sterilized conditions In general, except for utilization of extremophiles thriving under extreme conditions, including extreme pH, temperature and salinity, to reduce or prevent the growth of competing microorganisms, sterilization is a universal and preferred choice in industrial production [19]. However, the sterilization process requires immense energy and facility investments, and easily causes loss of raw materials due to caramelization [12]. However, high methanol concentration could inhibit the growth of microorganisms and achieve non-sterilized fermentation [8,19]. During non-sterilized fermentation, cell growth time and dry cell weight were 3.5 d and 1.8 g/L, respectively, and GCPM titer reached 3.3 g/L at the end of 7 d of fermentation (Fig. 2B). These results were similar to those obtained with sterilized fermentation (Fig. 2A), and indicated that 10% methanol (v/v) could adequately control the growth of contaminants in GCP-M production from glycerol under non-sterilized conditions. Meanwhile, there were no growth of original strain C. globosum CGMCC 6882 and synthesis of polysaccharide when non-sterilized fermentation was used. Chen et al. [19] reported that 5% methanol (w/v) was able to inhibit the growth of contaminants and achieve lipid production from Trichosporon oleaginosus under nonsterilized fermentation conditions, using crude glycerol as carbon source. 3.3. Effect of glycerol concentration on cell growth, biomass and GCP-M production The type and concentration of carbon source can affect cell growth and product synthesis by influencing the microbial energy metabolic pathway and the osmotic pressure of the fermentation system. In
1109
Table 3 Characteristics of the polysaccharide (GCP-M) produced by C. globosum ALE20. Items
Amount
Galactose (μmol/L) Glucose (μmol/L) Mannose (μmol/L) Glucuronic acid (μmol/L) Weight-average molecular weight (Mw, Da) Number-average molecular weight (Mn, Da) Polydispersity (Mw/Mn)
3.83 66.37 3.26 1.95 3.796 × 104 3.582 × 104 1.060
xanthan production, the inhibition of cell growth and xanthan titer were observed when glucose concentration was 50 g/L or higher; hence, glucose concentrations had to be controlled [12]. During lipid production by Pichia pastoris, lipid accumulation began when the carbon to nitrogen ratio was 20 and lipid yield increased with increasing carbon to nitrogen ratio, but lipid synthesis was inhibited when the carbon to nitrogen ratio was higher than 100 [20]. Cell growth time of C. globosum ALE20 was prolonged with increasing glycerol concentration, whereas the dry cell weight and GCP-M titer initially increased and later decreased (Table 1). The maximum dry cell weight reached 2.42 g/L at 20 g/L glycerol, but the highest GCP-M titer was 3.38 g/L at 40 g/L glycerol as the carbon source. These results suggest that glycerol concentration affects the cell growth of C. globosum ALE20, thus influencing the synthesis and production of GCP-M. In the work conducted by Yang et al. [21], glycerol was fed to reduce the inhibition of cell growth by osmotic pressure. 3.4. Effect of methanol concentration on cell growth, biomass and GCP-M production The tolerance of microorganisms to maximum methanol concentration is strain-dependent: 0.1%–1.0% (w/v) for bacteria, and 1.0%–4.0% (w/v) for yeast [19]. With the ability to grow over a wide range of pH values and high tolerance to toxic materials, fungi have a better potential to tolerate methanol than bacteria and yeast, and many researchers have obtained excellent results depicting the high methanol tolerance of fungi [7,8,19]. As shown in Table 2, with increased methanol concentration, cell growth duration and dry cell weight decreased, demonstrating the inhibitive effect of high methanol concentration on C. globosum ALE20 growth. When methanol concentration increased to 12% (v/v) or more, dry cell weight and GCP-M titer declined sharply to almost zero, indicating that 12% (v/v) methanol was above the tolerance limit of C. globosum ALE20. Other researchers reported a similar trend of effects of methanol concentration on cell growth and found that 4.6% (w/v) methanol was fatal to most microorganisms [22]. However, an increase in GCP-M titer was observed when methanol concentration increased from 2% (v/v) to 10% (v/v). This might be due to the improved permeability of the cell wall or membrane, and the properties of
Fig. 3. Sterilized (A) and non-sterilized (B) fed-batch fermentation profiles of C. globosum ALE20 with initial glycerol and methanol concentrations of 20 g/L and 6% (v/v), respectively. The symbols represent: glycerol (■), biomass (●), GCP-M titer (▲).
1110
Z. Wang et al. / International Journal of Biological Macromolecules 136 (2019) 1106–1111
Fig. 4. FT-IR (A) and 1H NMR (B) spectra of GCP-M produced from C. globosum ALE20.
microorganisms synthesizing exopolysaccharides in response to the adverse environment created by methanol [7,12]. At the same time, no other contaminated bacteria were detected during the fermentation process, suggesting the accuracy of this work, and which is in accordance with the tolerance of microorganisms to methanol [19]. 3.5. Fed-batch fermentation Previous researchers have revealed that fermentation strategy optimization could rapidly enhance the yield of target products [23]. In heterologous protein production by Pichia pastoris, glycerol is used as carbon source to produce a significant amount of biomass in the first batch growth phase, the second phase is designed to depress the alcohol oxidase promoter by glycerol-limited fed-batch growth, and the third stage is fed-batch growth on methanol to induce protein expression [18,23]. Therefore, based on the fermentation strategies applied by other researchers and the results obtained in Sections 3.3 and 3.4, a fed-batch fermentation strategy, which involved low glycerol and methanol concentrations during the cell growth stage, and high glycerol and methanol concentrations during polysaccharide synthesis, was used in this work for improving GCP-M titer. During sterilized fermentation, when initial glycerol and methanol concentrations were set to 20 g/L and 6% (v/v), respectively, cell growth time decreased from 3.5 d to 2.5 d, while dry cell weight increased from 1.75 g/L to 2.2 g/L (Fig. 3A). After glycerol and methanol concentrations increased to 40 g/L and 10% (v/v), respectively, at 2.5 d, GCP-M titer reached 9.2 g/L in the final fermentation broth, which was almost 3-fold that of batch fermentation. Moreover, similar results were obtained for both sterilized and non-sterilized fermentation (Fig. 3B). In a report by Chen et al. [19], lipid production reached 20.42 g/L with non-sterilized fed-batch fermentation. Jin et al. [24] demonstrated that the production of chondroitin and heparosan increased to 5.22 g/L and 5.82 g/L in 3-L fed-batch fermentation, respectively. 3.6. Characteristics of the polysaccharide produced from C. globosum ALE20 It is widely reported that the biological activities of a polysaccharide are correlated with its chemical structure, such as repeating units, monosaccharide types, glycosidic bond of main chain, chain length, branch degree of chain, flexibility and configuration of chains [25–27]. As shown in Table 3, the monosaccharide composition of GCP-M produced from glycerol by C. globosum ALE20 was galactose, glucose, mannose, and glucuronic acid in a molar ratio of 3.83:66.37:3.26:1.95, with a weight-average molecular weight (Mw) and polydispersity of 3.796 × 104 Da and 1.060, respectively. These were similar to those of the anticancer polysaccharide (GCP-1) produced from glycerol by C. globosum CGMCC 6882 [10]. Further, FT-IR and NMR results for GCP-M (Fig. 4) were consistent with those for GCP-1, further suggesting that GCP-M is similar to GCP-1.
4. Conclusions Compared with the high energy and facility investments required during sterilization, non-sterilized fermentation, using a methanolresistant strain, C. globosum ALE20, obtained through 20 cycles of ALE, represents an ideal alternative. C. globosum ALE20 could grow on 10% (v/v) methanol PDA plate and the anticancer polysaccharide titer increased from 3.3 g/L in batch fermentation to 9.2 g/L using a nonsterilized fed-batch strategy. Further genomic, transcriptomic, and metabonomic investigations of C. globosum ALE20 need to be carried out to decipher its methanol-resistance mechanism.
Declaration of Competing Interest The authors declare that there are no conflicts of interest. Acknowledgements This work is supported by the Natural Science Foundation of Youth Support Plan of Henan University of Technology (2017QNJH10), the High Level Research Fund for Qualified People of Henan University of Technology (2017BS011), the natural science foundation of Henan provincial education department (19A180015), the Science and Technology Research Program of Chongqing Municipal Education Commission (KJQN201800540) and the Natural Science Foundation of Hebei Province (C2018402265). References [1] R. Parate, R. Mane, M. Dharne, C. Rode, Mixed bacterial culture mediated direct conversion of bio-glycerol to diols, Bioresour. Technol. 250 (2018) 86–93. [2] A.E.F. Abomohra, H. Eladel, M. El-Esawi, S. Wang, Q. Wang, Z. He, Y. Feng, H. Shang, D. Hanelt, Effect of lipid-free microalgal biomass and waste glycerol on growth and lipid production of Scenedesmus obliquus: innovative waste recycling for extraordinary lipid production, Bioresour. Technol. 249 (2018) 992–999. [3] P.K. Dikshit, G.J. Kharmawlong, V.S. Moholkar, Investigations in sonication-induced intensification of crude glycerol fermentation to dihydroxyacetone by free and immobilized Gluconobacter oxydans, Bioresour. Technol. 256 (2018) 302–311. [4] H. Mundhada, J.M. Seoane, K. Schneider, A. Koza, H.B. Christensen, T. Klein, P.V. Phaneuf, M. Herrgard, A.M. Feist, A.T. Nielsen, Increased production of L-serine in Escherichia coli through adaptive laboratory evolution, Metab. Eng. 39 (2017) 141–150. [5] D. Li, L. Wang, Q. Zhao, W. Wei, Y. Sun, Improving high carbon dioxide tolerance and carbon dioxide fixation capability of Chlorella sp. by adaptive laboratory evolution, Bioresour. Technol. 185 (2015) 269–275. [6] L. Wang, C. Xue, L. Wang, Q. Zhao, W. Wei, Y. Sun, Strain improvement of Chlorella sp. for phenol biodegradation by adaptive laboratory evolution, Bioresour. Technol. 205 (2016) 264–268. [7] H.T.N. Mai, K.M. Lee, S.S. Choi, Enhanced oxalic acid production from corncob by a methanol-resistant strain of Aspergillus niger using semi solid-sate fermentation, Process Biochem. 51 (1) (2016) 9–15. [8] J. Iyyappan, B. Bharathiraja, G. Baskar, J. Jayamuthunagai, S. Barathkumar, R. Anna shiny, Malic acid production by chemically induced Aspergillus niger MTCC 281 mutant from crude glycerol, Bioresour. Technol. 251 (2018) 264–267.
Z. Wang et al. / International Journal of Biological Macromolecules 136 (2019) 1106–1111 [9] X.M. Sun, L.J. Ren, Z.Q. Bi, X.J. Ji, Q.Y. Zhao, H. Huang, Adaptive evolution of microalgae Schizochytrium sp. under high salinity stress to alleviate oxidative damage and improve lipid biosynthesis, Bioresour. Technol. 267 (2018) 438–444. [10] Z. Wang, P. Chen, N. Tao, H. Zhang, R. Li, X. Zhan, F. Wang, Y. Shen, Anticancer activity of polysaccharides produced from glycerol and crude glycerol by an endophytic fungus Chaetomium globosum CGMCC 6882 on human lung cancer A549 cells, Biomolecules 8 (4) (2018) 171. [11] N. Bahaloo-Horeh, S.M. Mousavi, Enhanced recovery of valuable metals from spent lithium-ion batteries through optimization of organic acids produced by Aspergillus niger, Waste Manag. 60 (2017) 666–679. [12] Z. Wang, J. Wu, L. Zhu, X. Zhan, Activation of glycerol metabolism in Xanthomonas campestris by adaptive evolution to produce a high-transparency and lowviscosity xanthan gum from glycerol, Bioresour. Technol. 211 (2016) 390–397. [13] Z. Wang, X. Zhao, X. Liu, W. Lu, S. Jia, T. Hong, R. Li, H. Zhang, L. Peng, X. Zhan, Antidiabetic activity evaluation of a polysaccharide extracted from Gynostemma pentaphyllum, Int. J. Biol. Macromol. 126 (2019) 209–214. [14] Z. Wang, R. Xue, J. Cui, J. Wang, W. Fan, H. Zhang, X. Zhan, Antibacterial activity of a polysaccharide produced from Chaetomium globosum CGMCC 6882, Int. J. Biol. Macromol. 125 (2019) 376–382. [15] N.N.A.N. Yusuf, S.K. Kamarudin, Z. Yaakub, Overview on the current trends in biodiesel production, Energ. Convers. Manage. 52 (7) (2011) 2741–2751. [16] I.A. Musa, The effects of alcohol to oil molar ratios and the type of alcohol on biodiesel production using transesterification process, Egypt. J. Petrol. 25 (1) (2016) 21–31. [17] D.J. Pyle, R.A. Garcia, Z. Wen, Producing docosahexaenoic acid (DHA)-rich algae from biodiesel-derived crude glycerol: effects of impurities on DHA production and algal biomass composition, J. Agri. Food Chem. 56 (11) (2008) 3933–3939. [18] J. Schenk, I.W. Marison, U. von Stockar, A simple method to monitor and control methanol feeding of Pichia pastoris fermentations using mid-IR spectroscopy, J. Biotechnol. 128 (2) (2007) 344–353.
1111
[19] J. Chen, X. Zhang, R.D. Tyagi, P. Drogui, Utilization of methanol in crude glycerol to assist lipid production in non-sterilized fermentation from Trichosporon oleaginosus, Bioresour. Technol. 253 (2018) 8–15. [20] J. Chen, X. Zhang, S. Yan, R.D. Tyagi, P. Drogui, Lipid production from fed-batch fermentation of crude glycerol directed by the kinetic study of batch fermentations, Fuel 209 (2017) 1–9. [21] L.B. Yang, X.B. Zhan, Z.Y. Zheng, J.R. Wu, M.J. Gao, C.C. Lin, A novel osmotic pressure control fed-batch fermentation strategy for improvement of erythritol production by Yarrowia lipolytica from glycerol, Bioresour. Technol. 151 (2014) 120–127. [22] D.R. Caldwell, Effects of methanol on the growth of gastrointestinal anaerobes, Can. J. Microbiol. 35 (2) (1989) 313–317. [23] Z. Wang, Y. Wang, D. Zhang, J. Li, Z. Hua, G. Du, J. Chen, Enhancement of cell viability and alkaline polygalacturonate lyase production by sorbitol co-feeding with methanol in Pichia pastoris fermentation, Bioresour. Technol. 101 (4) (2010) 1318–1323. [24] P. Jin, L. Zhang, P. Yuan, Z. Kang, G. Du, J. Chen, Efficient biosynthesis of polysaccharides chondroitin and heparosan by metabolically engineered Bacillus subtilis, Carbohydr. Polym. 140 (2016) 424–432. [25] Y. Yu, M. Shen, Q. Song, J. Xie, Biological activities and pharmaceutical applications of polysaccharide from natural resources: a review, Carbohydr. Polym. 183 (2018) 91–101. [26] Y. Ma, D. Mao, L. Geng, Z. Wang, C. Xu, Production, fractionation, characterization of extracellular polysaccharide from a newly isolated Trametes gibbosa and its hypoglycemic activity, Carbohydr. Polym. 96 (2) (2013) 460–465. [27] T. Chen, B. Li, Y. Li, C. Zhao, J. Shen, H. Zhang, Catalytic synthesis and antitumor activities of sulfated polysaccharide from Gynostemma pentaphyllum Makino, Carbohydr. Polym. 83 (2) (2011) 554–560.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Efficient biosynthesis of anticancer polysaccharide by a mutant Chaetomium globosum ALE20 via non-sterilized fermentation Zichao Wang a, Xuyang Chen a, Siyu Liu a, Yingying Zhang a, Zhangtao Wu a, Wenwen Xu a, Qi Sun b,⁎, Libo Yang c, Huiru Zhang a,⁎ a b c
College of Biological Engineering, Henan University of Technology, Zhengzhou 450001, China College of Life Sciences, Chongqing Normal University, Chongqing 401331, China College of Landscape and Ecological Engineering, Hebei University of Engineering, Handan 056021, China
a r t i c l e
i n f o
Article history: Received 25 May 2019 Received in revised form 13 June 2019 Accepted 24 June 2019 Available online 25 June 2019 Keywords: Adaptive laboratory evolution Methanol-resistant strain Anticancer polysaccharide
a b s t r a c t The sterilization process, due to its immense energy consumption, high facilities investment, and loss of raw materials by caramelization, during industrial production has drawn much attention. In this study, a methanolresistant mutant strain, Chaetomium globosum ALE20, was obtained following 20 cycles of adaptive laboratory evolution process. The titer of anticancer polysaccharide (GCP-M) from C. globosum ALE20 reached 9.2 g/L with glycerol as sole carbon source using non-sterilized and fed-batch fermentation strategy. This titer represents a 200% increase compared with the 3.3 g/L attained with batch fermentation. The GCP-M monosaccharide was comprised of galactose, glucose, mannose and glucuronic acid, in a molar ratio of 3.83:66.37:3.26:1.95, respectively, and its weight-average molecular weight and polydispersity were 3.796 × 104 Da and 1.060, respectively. This work presents an ideal alternative and safer fermentation process without sterilization, and a useful approach for enhancing industrial production. © 2019 Published by Elsevier B.V.
1. Introduction Given the exhaustion of fossil fuel, growing demand for fossil oilbased products, and aggravation of global warming, alternative and renewable energy resources are being researched worldwide [1]. Biodiesel is regarded as one of the most promising alternatives to fossil fuel due to its reduced carbon dioxide emission and regeneration properties [2]. During the trans-esterification process for biodiesel production catalyzed by methanol, glycerol is produced inevitably and accounts for one-tenth of the total biodiesel yield [3]. However, the methanol, salts, fatty acids, soap, and other organic matter in crude glycerol make it difficult for use in conventional industries, such as foods, cosmetics, and pharmaceuticals. Adaptive laboratory evolution (ALE) has been considered an effective method for enhancing microbial strain tolerance to inhibiting environmental conditions and developing new biological functions in strains [4–6]. For instance, oxalic acid yield from corncob was enhanced by a methanol-resistant Aspergillus niger strain using semi-solid-state fermentation through the ALE process [7]. An Aspergillus niger MTCC 281 mutant with combined resistance to methanol and malic acid was successfully obtained via the ALE process [8]. Further, ALE combined with high salinity was performed to alleviate oxidative damage and ⁎ Corresponding authors. E-mail addresses: [email protected] (Q. Sun), [email protected] (H. Zhang).
https://doi.org/10.1016/j.ijbiomac.2019.06.186 0141-8130/© 2019 Published by Elsevier B.V.
improve lipid biosynthesis in the microalgae, Schizochytrium sp. ALE150 [9]. In this study, to improve the anti-cancer polysaccharide production capacity of Chaetomium globosum CGMCC 6882 from glycerol and crude glycerol [10], we first subjected C. globosum CGMCC 6882 to ALE by gradually increasing the methanol concentration to enrich methanol-resistant C. globosum strains, and the evolved C. globosum ALE20 was found to be more tolerant to methanol than the parent strain. Then, the fermenting properties of C. globosum ALE20 under sterilized and non-sterilized conditions, varying glycerol and methanol concentrations, and the fed-batch strategy were studied. Finally, high performance anion exchange chromatography, size exclusion chromatography, Fourier transform infrared spectroscopy (FT-IR), and nuclear magnetic resonance (NMR) were used to characterize the polysaccharide produced by C. globosum ALE20. 2. Materials and methods 2.1. Microorganisms and chemicals The parent strain, C. globosum CGMCC 6882, was isolated from the herb, G. pentaphyllum, and stored in China General Microbiological Culture Collection Center (China). The methanol-resistant strain C. globosum ALE20 was obtained through ALE from C. globosum CGMCC 6882 and collected in the Engineering Laboratory of Henan
Z. Wang et al. / International Journal of Biological Macromolecules 136 (2019) 1106–1111
Province for Industrial Culture Collection and Breeding Center. The chemicals (including glycerol) used in this work were analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (China). 2.2. Preparation and storage of spores C. globosum CGMCC 6882 spores were collected by scratching the PDA plates with a disposable spreader and washing with sterilized water. Then, a hemocytometer (Neubauer, Germany) was used to count the spores under a phase contrast microscope (Zeiss, Axio Imager 2, Jena, Germany). A stock of spore suspension (1 × 107 spores/mL) was maintained in 50% glycerol at −80 °C for long-term storage. 2.3. Adaptive laboratory evolution The evolution of methanol-resistant strain C. globosum ALE20 was performed as described by Iyyappan et al. [8] with some modifications. With the continuous ALE process, methanol concentration on the PDA plate increased stepwise from 0.5% (v/v) to 10% (v/v); about 1 × 105/mL spores of C. globosum CGMCC 6882 were inoculated on the PDA plate and incubated at 28 °C for 7 d. After 7 d, spores in the region showing better mycelial growth were harvested and inoculated onto a fresh medium containing higher methanol concentration. A cycle of spore transfers was implemented until 10% (v/v) methanol concentration was attained on the PDA plate.
1107
through a 0.2 μm filter and added into the fermenter. In non-sterilized fermentation, 400 mL methanol was added directly into the fermenter containing 3.5 L of non-sterilized culture medium. At 12 h intervals, 20 mL broth aliquots were collected for analysis during the fermentation process. All batch cultures were performed in three replications. 2.6. Batch fermentation with varying glycerol and methanol concentrations During the fermentation process involving different glycerol and methanol concentrations, the fermenter and fermentation medium were sterilized at 121 °C for 20 min. To vary the glycerol concentrations for fermentation, the composition of culture media remained the same as in Section 2.5, but glycerol concentrations were set to 10, 20, 30, 40, 50, 60, 70 or 80 g/L. Similarly, methanol concentrations were varied, such that, after the culture medium was sterilized and cooled to room temperature, 80, 160, 240, 320, 400 or 480 mL methanol was filtered through a 0.2 μm filter and added into the fermenter. 2.7. Fed-batch fermentation
The seed medium used for C. globosum ALE20 was potato dextrose medium, incubated at 28 °C and 120 rpm for 48 h. Then, a hemocytometer (Neubauer, Germany) was used to count the spores under a phase contrast microscope (Jena, Germany), and the spore suspension was diluted to a concentration of 1 × 107 spores/mL with distilled water and a 1% (v/v) inoculation volume.
For the sterilized fed-batch fermentation, the initial glycerol concentration was set to 20 g/L and the culture medium was sterilized at 121 °C for 20 min. After the fermentation medium was cooled to room temperature, 240 mL filtered methanol was added into the fermenter to increase the original methanol concentration to 6% (v/v). Then, glycerol and methanol concentrations increased to 40 g/L and 10% (v/v), respectively, by adding sterilized glycerol and filtered methanol at 2.5 d of fermentation process. For non-sterilized fed-batch fermentation, the initial glycerol and methanol concentrations were 20 g/L and 6% (v/v), respectively, and these were increased to 40 g/L and 10% (v/v), respectively, by adding non-sterilized glycerol and non-filtered methanol at 2.5 d of fermentation. Fermentation conditions were the same as in Section 2.5 and aliquots of the fermentation broth were collected for analysis at 12 h intervals.
2.5. Batch fermentation under sterilized and non-sterilized conditions
2.8. Biomass, glycerol, and polysaccharide titer determination
The fermentation medium was composed of the following (g/L): glycerol 40, peptone 1.0, yeast extract 1.0, beef extract 1.0, MgSO4·7H2O 1.5, KH2PO4 1.0, K2HPO4 1.0, and FeSO4 7H2O 0.01. Fermentation temperature was 28 °C, cultivation time was 7 d, pH was maintained at 7.0 ± 0.2 with 2 mol/L NaOH and 2 mol/L HCl. All the fermentation experiments were conducted in a 7.0 L fermenter (BioFlo 115, New Brunswick, USA) with a 3.5 L working volume. During the fermentation process, agitation and aeration were set to 150 rpm and 1.0 vvm, respectively. For sterilized fermentation, after the culture medium was sterilized and cooled to room temperature, 400 mL methanol was filtered
Dry cell weight of C. globosum ALE20 was determined using the modified method of Bahaloo-Horeh et al. [11]. Fermentation broth was filtered through a Buchner funnel and the resulting biomass (mycelium and spores) was washed twice with deionized water, then transferred to a pre-weighed porcelain dish, dried in an oven for 24 h, cooled in a desiccator and weighed. The dry cell weight of C. globosum ALE20 was calculated by the weight difference method. Meanwhile, the filtered liquor was centrifuged at 12,000 ×g for 30 min to remove insoluble impurities, after which, 1 mL supernatant was filtered through a 0.22 μm membrane for glycerol concentration determination by high-
2.4. Seed culture medium
Fig. 1. Morphological images of C. globosum CGMCC 6882 (A) and C. globosum ALE20 (B) on PDA plates supplemented with 10% (v/v) methanol.
1108
Z. Wang et al. / International Journal of Biological Macromolecules 136 (2019) 1106–1111
Fig. 2. Sterilized (A) and non-sterilized (B) batch fermentation profiles of C. globosum ALE20 with 40 g/L glycerol and 10% (v/v) methanol. The symbols represent: glycerol (■), biomass (●), GCP-M titer (▲).
performance liquid chromatography, as we reported previously [12]. Simultaneously, 10 mL supernatant, to which four volumes of cold 95% alcohol was added, was collected and kept at 4 °C overnight to precipitate polysaccharide, then the supernatant was discarded and the precipitated polysaccharide was re-dissolved into 10 mL deionized water for polysaccharide yield determination using the phenol sulfuric acid method at 490 nm. 2.9. Polysaccharide characterization At the end of fermentation, culture broth was filtered and centrifuged at 12,000 ×g for 30 min to remove mycelium and cells. Then, the broth was concentrated to one-tenth of the original volume with rotary evaporation at 60 °C and 0.1 MPa vacuum. After that, the concentrated broth was de-proteinized by adding three volumes Sevag solution, and three times of cold 95% alcohol was added into the deproteinized supernatant and kept at 4 °C overnight to precipitate polysaccharide. The precipitated polysaccharide was re-dissolved in distilled water and de-pigmented with AB-8 macroporous resin, and then, the polysaccharide solution was filtered through a 0.22 μm filter and applied to a Sepharose CL-6B column (2.5 × 60 cm) for further purification, eluting with 0.1 mol/L NaCl solution at a flow rate of 0.6 mL/min. The fraction was collected and freeze dried for studying the molecular characteristics of polysaccharide. High performance anion exchange chromatography was used for analyzing the monosaccharide composition of polysaccharide. Briefly, polysaccharide was dissolved in 2 mol/L trifluoroacetic acid and hydrolyzed at 120 °C for 2 h, washed three times with methanol and evaporated to dryness for removing TFA. Then, the hydrolyzed material was transferred to a 25 mL volumetric flask, diluted to 25 mL by deionized water and subjected to Dionex ICS5000 system (Dionex, USA) equipped with CarboPac PA20 column (ID 3 mm × 150 mm). The mobile phase was deionized water (A), 0.25 mol/L NaOH (B), 1 mol/L NaAc (C) and eluted as follows (A%, B%, C%): 0 min: 99.2, 0.8, 0; 30 min: 99.2, 0.8, 0; 40 min: 79.2, 0.8, 20;40.1 min: 20, 80, 0; 60 min: 99.2, 0.8, 0. The flow rate was 0.45 mL/min and the injection volume was 25 μL, column temperature was 30 °C and detected by a pulsed ampere detector, Au electrode, Ag/AgCl reference electrode. Polysaccharide was dissolved in distilled water to a concentration of 2 mg/mL and analyzed by high performance size exclusion chromatography for its molecular weight. The system consisted of Waters 2695 HPLC system equipped with multiple detectors: refractive index detector (RI) and a UV detector for concentration determination, multiple angle laser light scattering detector (MALLS, DAWNHELEOS, Wyatt Technology, USA) for direct molecular weight determination and differential pressure viscometer (DP) for viscosity determination. The columns were TSK PWXL 6000 and 3000 gel filtration columns which were eluted with PB buffer (0.15 mol/L NaNO3 and 0.05 mol/L NaH2PO4, pH = 7) at the flow rate of 0.5 mL/min. The calibration of the laser photometer was
performed with ultrapure toluene. The normalization was conducted with a bovine serum albumin globular protein (Mw = 66.7 kDa, Rg = 2.9 nm). A value of 0.146 mL/g was used as refractive index increment (dn/dc) for molecular weight calculation. Astra software (Version 6.1.1) was utilized for data acquisition and analysis. The column temperature and RI detector temperature were maintained at 35 °C [10]. Nexus 470 FT-IR spectrophotometer (Nicolet, USA) was used to record the IR spectrum of polysaccharide with KBr between 400 and 4000 cm−1, and a Bruker Avance 400 MHz spectrometer (Bruker Inc., Germany) was used to record the 1H NMR spectra of polysaccharide at 30 °C. polysaccharide was dissolved in D2O in 5 mm OD NMR tube, the chemical shifts for 1H NMR spectra were recorded in parts per million from tetramethylsilane [13,14]. 2.10. Statistical analysis Each analytical result is expressed as mean ± SD after triplicate measurements. Data were analyzed by analysis of variance (ANOVA), using the Origin Pro 2017 software. 3. Results and discussion 3.1. Adaptive laboratory evolution Several short-chain alcohols, including methanol, ethanol, butanol, propanol, isopropanol, tert-butanol, branched alcohols and octanol, are utilized in the trans-esterification process as catalysts for biodiesel production [15]. However, methanol is particularly preferred because of its low cost, physical and chemical advantages, quick reaction with triglycerides and easy dissolution in NaOH [16]. Therefore, crude glycerol derived from the biodiesel industry is always contaminated with methanol, thus reducing the suitability of crude glycerol as a potential fermentation substrate in bioprocess applications [17,18]. As shown in Fig. 1B, after the ALE process, with stepwise increment in methanol concentrations from 0.5 to 10% (v/v) on PDA, an evolved methanolresistant strain, C. globosum ALE20, was obtained and could grow on Table 1 Effect of varying glycerol concentrations on C. globosum ALE20 growth, biomass, and GCPM titer. Glycerol (g/L)
Cell growth time (d)
Biomass (g/L)
GCP-M titer (g/L)
10 20 30 40 50 60 70 80
2.5 2.5 3.0 3.5 3.5 4 4.5 4.5
2.13 ± 0.05 2.42 ± 0.08 2.04 ± 0.06 1.81 ± 0.13 1.73 ± 0.04 1.20 ± 0.15 0.71 ± 0.09 0.23 ± 0.02
0.35 ± 0.03 1.38 ± 0.21 2.55 ± 0.07 3.38 ± 0.13 3.07 ± 0.06 1.78 ± 0.19 0.65 ± 0.03 0.1 ± 0.04
Z. Wang et al. / International Journal of Biological Macromolecules 136 (2019) 1106–1111 Table 2 Effects of varying methanol concentrations on C. globosum ALE20 growth, biomass, and GCP-M titer. Methanol (%, v/v)
Cell growth time (d)
Biomass (g/L)
GCP-M titer (g/L)
2 4 6 8 10 12 14
2.5 2.5 3 3.5 3.5 4 4.5
2.35 ± 0.12 2.28 ± 0.07 2.17 ± 0.05 2.03 ± 0.03 1.83 ± 0.08 0.72 ± 0.13 0.39 ± 0.04
3.05 ± 0.13 3.16 ± 0.08 3.41 ± 0.15 3.78 ± 0.09 3.41 ± 0.11 1.03 ± 0.07 0.46 ± 0.12
10% (v/v) methanol PDA plate, while the parent strain, C. globosum CGMCC 6882, could not (Fig. 1A). The methanol resistance ability of C. globosum ALE20 could well alleviate the inhibitory effect of methanol on its growth and improve its crude glycerol utilization capacity, thereby improving the suitability of crude glycerol as a potential fermentation substrate in bioprocess applications and relieving the burden on the biodiesel industry. 3.2. Batch fermentation under sterilized and non-sterilized conditions In general, except for utilization of extremophiles thriving under extreme conditions, including extreme pH, temperature and salinity, to reduce or prevent the growth of competing microorganisms, sterilization is a universal and preferred choice in industrial production [19]. However, the sterilization process requires immense energy and facility investments, and easily causes loss of raw materials due to caramelization [12]. However, high methanol concentration could inhibit the growth of microorganisms and achieve non-sterilized fermentation [8,19]. During non-sterilized fermentation, cell growth time and dry cell weight were 3.5 d and 1.8 g/L, respectively, and GCPM titer reached 3.3 g/L at the end of 7 d of fermentation (Fig. 2B). These results were similar to those obtained with sterilized fermentation (Fig. 2A), and indicated that 10% methanol (v/v) could adequately control the growth of contaminants in GCP-M production from glycerol under non-sterilized conditions. Meanwhile, there were no growth of original strain C. globosum CGMCC 6882 and synthesis of polysaccharide when non-sterilized fermentation was used. Chen et al. [19] reported that 5% methanol (w/v) was able to inhibit the growth of contaminants and achieve lipid production from Trichosporon oleaginosus under nonsterilized fermentation conditions, using crude glycerol as carbon source. 3.3. Effect of glycerol concentration on cell growth, biomass and GCP-M production The type and concentration of carbon source can affect cell growth and product synthesis by influencing the microbial energy metabolic pathway and the osmotic pressure of the fermentation system. In
1109
Table 3 Characteristics of the polysaccharide (GCP-M) produced by C. globosum ALE20. Items
Amount
Galactose (μmol/L) Glucose (μmol/L) Mannose (μmol/L) Glucuronic acid (μmol/L) Weight-average molecular weight (Mw, Da) Number-average molecular weight (Mn, Da) Polydispersity (Mw/Mn)
3.83 66.37 3.26 1.95 3.796 × 104 3.582 × 104 1.060
xanthan production, the inhibition of cell growth and xanthan titer were observed when glucose concentration was 50 g/L or higher; hence, glucose concentrations had to be controlled [12]. During lipid production by Pichia pastoris, lipid accumulation began when the carbon to nitrogen ratio was 20 and lipid yield increased with increasing carbon to nitrogen ratio, but lipid synthesis was inhibited when the carbon to nitrogen ratio was higher than 100 [20]. Cell growth time of C. globosum ALE20 was prolonged with increasing glycerol concentration, whereas the dry cell weight and GCP-M titer initially increased and later decreased (Table 1). The maximum dry cell weight reached 2.42 g/L at 20 g/L glycerol, but the highest GCP-M titer was 3.38 g/L at 40 g/L glycerol as the carbon source. These results suggest that glycerol concentration affects the cell growth of C. globosum ALE20, thus influencing the synthesis and production of GCP-M. In the work conducted by Yang et al. [21], glycerol was fed to reduce the inhibition of cell growth by osmotic pressure. 3.4. Effect of methanol concentration on cell growth, biomass and GCP-M production The tolerance of microorganisms to maximum methanol concentration is strain-dependent: 0.1%–1.0% (w/v) for bacteria, and 1.0%–4.0% (w/v) for yeast [19]. With the ability to grow over a wide range of pH values and high tolerance to toxic materials, fungi have a better potential to tolerate methanol than bacteria and yeast, and many researchers have obtained excellent results depicting the high methanol tolerance of fungi [7,8,19]. As shown in Table 2, with increased methanol concentration, cell growth duration and dry cell weight decreased, demonstrating the inhibitive effect of high methanol concentration on C. globosum ALE20 growth. When methanol concentration increased to 12% (v/v) or more, dry cell weight and GCP-M titer declined sharply to almost zero, indicating that 12% (v/v) methanol was above the tolerance limit of C. globosum ALE20. Other researchers reported a similar trend of effects of methanol concentration on cell growth and found that 4.6% (w/v) methanol was fatal to most microorganisms [22]. However, an increase in GCP-M titer was observed when methanol concentration increased from 2% (v/v) to 10% (v/v). This might be due to the improved permeability of the cell wall or membrane, and the properties of
Fig. 3. Sterilized (A) and non-sterilized (B) fed-batch fermentation profiles of C. globosum ALE20 with initial glycerol and methanol concentrations of 20 g/L and 6% (v/v), respectively. The symbols represent: glycerol (■), biomass (●), GCP-M titer (▲).
1110
Z. Wang et al. / International Journal of Biological Macromolecules 136 (2019) 1106–1111
Fig. 4. FT-IR (A) and 1H NMR (B) spectra of GCP-M produced from C. globosum ALE20.
microorganisms synthesizing exopolysaccharides in response to the adverse environment created by methanol [7,12]. At the same time, no other contaminated bacteria were detected during the fermentation process, suggesting the accuracy of this work, and which is in accordance with the tolerance of microorganisms to methanol [19]. 3.5. Fed-batch fermentation Previous researchers have revealed that fermentation strategy optimization could rapidly enhance the yield of target products [23]. In heterologous protein production by Pichia pastoris, glycerol is used as carbon source to produce a significant amount of biomass in the first batch growth phase, the second phase is designed to depress the alcohol oxidase promoter by glycerol-limited fed-batch growth, and the third stage is fed-batch growth on methanol to induce protein expression [18,23]. Therefore, based on the fermentation strategies applied by other researchers and the results obtained in Sections 3.3 and 3.4, a fed-batch fermentation strategy, which involved low glycerol and methanol concentrations during the cell growth stage, and high glycerol and methanol concentrations during polysaccharide synthesis, was used in this work for improving GCP-M titer. During sterilized fermentation, when initial glycerol and methanol concentrations were set to 20 g/L and 6% (v/v), respectively, cell growth time decreased from 3.5 d to 2.5 d, while dry cell weight increased from 1.75 g/L to 2.2 g/L (Fig. 3A). After glycerol and methanol concentrations increased to 40 g/L and 10% (v/v), respectively, at 2.5 d, GCP-M titer reached 9.2 g/L in the final fermentation broth, which was almost 3-fold that of batch fermentation. Moreover, similar results were obtained for both sterilized and non-sterilized fermentation (Fig. 3B). In a report by Chen et al. [19], lipid production reached 20.42 g/L with non-sterilized fed-batch fermentation. Jin et al. [24] demonstrated that the production of chondroitin and heparosan increased to 5.22 g/L and 5.82 g/L in 3-L fed-batch fermentation, respectively. 3.6. Characteristics of the polysaccharide produced from C. globosum ALE20 It is widely reported that the biological activities of a polysaccharide are correlated with its chemical structure, such as repeating units, monosaccharide types, glycosidic bond of main chain, chain length, branch degree of chain, flexibility and configuration of chains [25–27]. As shown in Table 3, the monosaccharide composition of GCP-M produced from glycerol by C. globosum ALE20 was galactose, glucose, mannose, and glucuronic acid in a molar ratio of 3.83:66.37:3.26:1.95, with a weight-average molecular weight (Mw) and polydispersity of 3.796 × 104 Da and 1.060, respectively. These were similar to those of the anticancer polysaccharide (GCP-1) produced from glycerol by C. globosum CGMCC 6882 [10]. Further, FT-IR and NMR results for GCP-M (Fig. 4) were consistent with those for GCP-1, further suggesting that GCP-M is similar to GCP-1.
4. Conclusions Compared with the high energy and facility investments required during sterilization, non-sterilized fermentation, using a methanolresistant strain, C. globosum ALE20, obtained through 20 cycles of ALE, represents an ideal alternative. C. globosum ALE20 could grow on 10% (v/v) methanol PDA plate and the anticancer polysaccharide titer increased from 3.3 g/L in batch fermentation to 9.2 g/L using a nonsterilized fed-batch strategy. Further genomic, transcriptomic, and metabonomic investigations of C. globosum ALE20 need to be carried out to decipher its methanol-resistance mechanism.
Declaration of Competing Interest The authors declare that there are no conflicts of interest. Acknowledgements This work is supported by the Natural Science Foundation of Youth Support Plan of Henan University of Technology (2017QNJH10), the High Level Research Fund for Qualified People of Henan University of Technology (2017BS011), the natural science foundation of Henan provincial education department (19A180015), the Science and Technology Research Program of Chongqing Municipal Education Commission (KJQN201800540) and the Natural Science Foundation of Hebei Province (C2018402265). References [1] R. Parate, R. Mane, M. Dharne, C. Rode, Mixed bacterial culture mediated direct conversion of bio-glycerol to diols, Bioresour. Technol. 250 (2018) 86–93. [2] A.E.F. Abomohra, H. Eladel, M. El-Esawi, S. Wang, Q. Wang, Z. He, Y. Feng, H. Shang, D. Hanelt, Effect of lipid-free microalgal biomass and waste glycerol on growth and lipid production of Scenedesmus obliquus: innovative waste recycling for extraordinary lipid production, Bioresour. Technol. 249 (2018) 992–999. [3] P.K. Dikshit, G.J. Kharmawlong, V.S. Moholkar, Investigations in sonication-induced intensification of crude glycerol fermentation to dihydroxyacetone by free and immobilized Gluconobacter oxydans, Bioresour. Technol. 256 (2018) 302–311. [4] H. Mundhada, J.M. Seoane, K. Schneider, A. Koza, H.B. Christensen, T. Klein, P.V. Phaneuf, M. Herrgard, A.M. Feist, A.T. Nielsen, Increased production of L-serine in Escherichia coli through adaptive laboratory evolution, Metab. Eng. 39 (2017) 141–150. [5] D. Li, L. Wang, Q. Zhao, W. Wei, Y. Sun, Improving high carbon dioxide tolerance and carbon dioxide fixation capability of Chlorella sp. by adaptive laboratory evolution, Bioresour. Technol. 185 (2015) 269–275. [6] L. Wang, C. Xue, L. Wang, Q. Zhao, W. Wei, Y. Sun, Strain improvement of Chlorella sp. for phenol biodegradation by adaptive laboratory evolution, Bioresour. Technol. 205 (2016) 264–268. [7] H.T.N. Mai, K.M. Lee, S.S. Choi, Enhanced oxalic acid production from corncob by a methanol-resistant strain of Aspergillus niger using semi solid-sate fermentation, Process Biochem. 51 (1) (2016) 9–15. [8] J. Iyyappan, B. Bharathiraja, G. Baskar, J. Jayamuthunagai, S. Barathkumar, R. Anna shiny, Malic acid production by chemically induced Aspergillus niger MTCC 281 mutant from crude glycerol, Bioresour. Technol. 251 (2018) 264–267.
Z. Wang et al. / International Journal of Biological Macromolecules 136 (2019) 1106–1111 [9] X.M. Sun, L.J. Ren, Z.Q. Bi, X.J. Ji, Q.Y. Zhao, H. Huang, Adaptive evolution of microalgae Schizochytrium sp. under high salinity stress to alleviate oxidative damage and improve lipid biosynthesis, Bioresour. Technol. 267 (2018) 438–444. [10] Z. Wang, P. Chen, N. Tao, H. Zhang, R. Li, X. Zhan, F. Wang, Y. Shen, Anticancer activity of polysaccharides produced from glycerol and crude glycerol by an endophytic fungus Chaetomium globosum CGMCC 6882 on human lung cancer A549 cells, Biomolecules 8 (4) (2018) 171. [11] N. Bahaloo-Horeh, S.M. Mousavi, Enhanced recovery of valuable metals from spent lithium-ion batteries through optimization of organic acids produced by Aspergillus niger, Waste Manag. 60 (2017) 666–679. [12] Z. Wang, J. Wu, L. Zhu, X. Zhan, Activation of glycerol metabolism in Xanthomonas campestris by adaptive evolution to produce a high-transparency and lowviscosity xanthan gum from glycerol, Bioresour. Technol. 211 (2016) 390–397. [13] Z. Wang, X. Zhao, X. Liu, W. Lu, S. Jia, T. Hong, R. Li, H. Zhang, L. Peng, X. Zhan, Antidiabetic activity evaluation of a polysaccharide extracted from Gynostemma pentaphyllum, Int. J. Biol. Macromol. 126 (2019) 209–214. [14] Z. Wang, R. Xue, J. Cui, J. Wang, W. Fan, H. Zhang, X. Zhan, Antibacterial activity of a polysaccharide produced from Chaetomium globosum CGMCC 6882, Int. J. Biol. Macromol. 125 (2019) 376–382. [15] N.N.A.N. Yusuf, S.K. Kamarudin, Z. Yaakub, Overview on the current trends in biodiesel production, Energ. Convers. Manage. 52 (7) (2011) 2741–2751. [16] I.A. Musa, The effects of alcohol to oil molar ratios and the type of alcohol on biodiesel production using transesterification process, Egypt. J. Petrol. 25 (1) (2016) 21–31. [17] D.J. Pyle, R.A. Garcia, Z. Wen, Producing docosahexaenoic acid (DHA)-rich algae from biodiesel-derived crude glycerol: effects of impurities on DHA production and algal biomass composition, J. Agri. Food Chem. 56 (11) (2008) 3933–3939. [18] J. Schenk, I.W. Marison, U. von Stockar, A simple method to monitor and control methanol feeding of Pichia pastoris fermentations using mid-IR spectroscopy, J. Biotechnol. 128 (2) (2007) 344–353.
1111
[19] J. Chen, X. Zhang, R.D. Tyagi, P. Drogui, Utilization of methanol in crude glycerol to assist lipid production in non-sterilized fermentation from Trichosporon oleaginosus, Bioresour. Technol. 253 (2018) 8–15. [20] J. Chen, X. Zhang, S. Yan, R.D. Tyagi, P. Drogui, Lipid production from fed-batch fermentation of crude glycerol directed by the kinetic study of batch fermentations, Fuel 209 (2017) 1–9. [21] L.B. Yang, X.B. Zhan, Z.Y. Zheng, J.R. Wu, M.J. Gao, C.C. Lin, A novel osmotic pressure control fed-batch fermentation strategy for improvement of erythritol production by Yarrowia lipolytica from glycerol, Bioresour. Technol. 151 (2014) 120–127. [22] D.R. Caldwell, Effects of methanol on the growth of gastrointestinal anaerobes, Can. J. Microbiol. 35 (2) (1989) 313–317. [23] Z. Wang, Y. Wang, D. Zhang, J. Li, Z. Hua, G. Du, J. Chen, Enhancement of cell viability and alkaline polygalacturonate lyase production by sorbitol co-feeding with methanol in Pichia pastoris fermentation, Bioresour. Technol. 101 (4) (2010) 1318–1323. [24] P. Jin, L. Zhang, P. Yuan, Z. Kang, G. Du, J. Chen, Efficient biosynthesis of polysaccharides chondroitin and heparosan by metabolically engineered Bacillus subtilis, Carbohydr. Polym. 140 (2016) 424–432. [25] Y. Yu, M. Shen, Q. Song, J. Xie, Biological activities and pharmaceutical applications of polysaccharide from natural resources: a review, Carbohydr. Polym. 183 (2018) 91–101. [26] Y. Ma, D. Mao, L. Geng, Z. Wang, C. Xu, Production, fractionation, characterization of extracellular polysaccharide from a newly isolated Trametes gibbosa and its hypoglycemic activity, Carbohydr. Polym. 96 (2) (2013) 460–465. [27] T. Chen, B. Li, Y. Li, C. Zhao, J. Shen, H. Zhang, Catalytic synthesis and antitumor activities of sulfated polysaccharide from Gynostemma pentaphyllum Makino, Carbohydr. Polym. 83 (2) (2011) 554–560.