3.1 Production of GAMG by C. globosum DX-THS3 using licorice straw as substrate
C. globosum DX-THS3 is an endophytic fungus, and it was isolated from Dongxiang wild rice in our previous work (Wang et al., 2015b). In this study, strain DX-THS3 can utilize licorice straw as a substrate for producing GAMG. Strain DX-THS3 was cultivated in licorice straw to produce GAMG by SSF (Fig. 1A). After 20 days of cultivation, C. globosum DX-THS3 covered almost all of the licorice straw (Fig. 1A, right). Solid-state medium was sampled, and the product was analyzed by TLC to detect the GAMG. As shown in Fig. 1B, only GL was detected in the licorice straw (b), but after 20 days of fermentation, considerable GAMG was detected using TLC (c). UPLC was performed for identification and further confirmation of these products. Our results confirmed that GAMG was the product in SSF (Fig. 1C). Thus, our results show that GL of licorice straw can be bio-transformed to produce GAMG using licorice straw as a substrate by C. globosum DX-THS3.
GAMG is an innovative functional sweetener with higher sweetness and stronger pharmacological activity than GL (Lin et al., 2009; Li et al., 2017; Mizutani et al., 1994). Biocatalysis of GAMG is a more environment-friendly and efficient than the chemical method (Brieskorn & Lang, 1978). To date, several microorganisms (mainly fungi) are screened for GL transformation to produce GAMG. However, a limited number of microorganisms have a GL-hydrolyzing ability. Especially, microorganisms that can selectively transform GL to generate GAMG are rarely found. The previously reported microorganisms with GL-hydrolyzing ability mainly include fungi, such as the filamentous fungus P. purpurogenum Li-3 (Zou et al., 2013) and Talaromyces pinophilus Li-93 (Xu et al., 2018), and screened from soil. However, the poor substrate specificity and low efficiency are the main disadvantages that limit the further application of these reported fungi. On the other hand, the production of GAMG by microorganic enzymes (GUS) involve tedious steps, including strain cultivation, fermentation, enzyme extraction, enzymatic reaction, and product separation, that seriously limit the application of microorganic enzymes in industries. Thus, screening microorganism from other sources and a novel strategy for high-efficient utilization of microorganic enzymes should be suggested. In this study, an endophytic fungi C. globosum DX-THS3 selectively and efficiently transformed GL to produce GAMG when using licorice straw as substrate in SSF. To our knowledge, this work is the first to report the GL production by SSF using endophytic fungi. This research also contributes to the application of endophytic fungi as potential industrial strains in food, biopharmaceutical, and biotechnological industries.
3.2 Optimization of SSF conditions for GAMG production by C. globosum DX-THS3 using licorice straw as substrate
We optimized the SSF conditions for GAMG production by C. globosum DX-THS3 using licorice straw as substrate in the present study. The particle size of the substrate is one of the key factors in SSF. Thus, we first investigated the effect of particle size of licorice straw on GAMG production by C. globosum DX-THS3. As shown in Fig. 2A, 4.56 mg/g (yield, Y) GAMG was produced by C. globosum DX-THS3 when using small-sized licorice straw (0–0.25 mm) as the substrate after 20 days of fermentation; 10.47 mg/g (Y) of GAMG was obtained using a medium particle size (0.26–0.85 mm) of licorice straw after 20 days of fermentation, and 10.35 mg/g (Y) GAMG was detected using licorice straw with a large particle size (0.86–3.0 mm). Our results show that the medium particle size of licorice straw was better than the other particle sizes for the production of GAMG by C. globosum DX-THS3. Furthermore, other fermentation conditions, including temperature, seed age, inoculum size, and solid–liquid ratio (substrate:water, m/v), were optimized. Our results show that 28 ℃ (Y = 10.47 mg/g, Fig. 2B), 96 h seed age (Y = 13.46 mg/g, Fig. 2C), 20% inoculum size (Y = 19.78 mg/g, v/w, Fig. 2D), and 1:3 solid–liquid ratio (Y = 11.75 mg/g, Fig. 2E) were the optimal conditions for the production of GAMG by SSF using C. globosum DX-THS3. These optimal SSF conditions (0.26–0.85 mm licorice straw, 28 °C, 96 h seed age, 20% inoculum size, and 1:3 solid–liquid ratio) were confirmed.
The fermentation conditions can markedly influence the products (not only the kinds but also the yields) when fermenting using microorganisms (Singh et al., 2017). Similar to liquid fermentation, temperature and seed age should be considered when using microorganisms (most of which belong to fungi) for SSF. However, special fermentation conditions, such as the particle size of substrate and water content, should be primarily considered. In general, oversized or extremely small particle size of substrate is unsuitable for SSF using fungi. Oversized particles can affect substrate release, especially that of lignocellulose, and significantly reduce the contact area of fungi with the substrate (Pandey, 2003). On the other hand, an extremely small particle size can influence fungal growth (Pandey, 2003). Water and substrate powder will mix to form a tight bulk or pellet, which not only significantly reduces oxygen transfer but also obstructs in-depth growth of fungal mycelium. In this study, the particle size of licorice straw was first optimized, and our results showed a suitable particle size of 0.26–0.85 mm (Fig. 2A) for the production of GAMG by SSF using C. globosum DX-THS3. Water content of substrate is another important factor for SSF by fungi. Low water content is a poor condition for fungal growth. However, a high water content can also inhibit fungal growth, that is, when oxygen transfer is obstructed by high water content of substrate. Thus, suitable water content is required in SSF, and a similar result was obtained in our study (Fig. 2E).
3.3 High activity of lignocellulosic enzymes during the degradation of licorice straw by C. globosum DX-THS3
C. globosum DX-THS3 was first cultured in pre-treated licorice straw under the above fermentation conditions to further investigate the feasibility of this novel strategy for GAMG production. C. globosum DX-THS3 mycelium was grown slowly in the early stage of fermentation (0–6 days). Then, the growth of C. globosum DX-THS3 gradually hastened in the middle stage of fermentation (7–12 days), and the mycelium covered the licorice straw after 20 days of fermentation. Then, deep fermentation was performed (Fig. 3A). The yields of GAMG by C. globosum DX-THS3 using licorice straw as substrate were analyzed during SSF to detect the production of GAMG by C. globosum DX-THS3. The production of GAMG was extremely low in the early and middle stages (0–18 days) of SFF (Fig. 3B). Until 18 days of fermentation, the yield of GAMG significantly increased. Our results demonstrate that the yield of GAMG reached 13.73 mg/g after 20 days, and the percent conversion of GL reached 90% (Y = 31.5 mg/g) after about 33 days of SSF (Fig. 3B). Furthermore, the total and reducing sugars were detected during SSF to investigate the utilization of carbon source by C. globosum DX-THS3. The total sugar of substrate continuously decreased during 0–30 days of SSF and stabilized afterward (Fig. 3C). Thus, the growth period of C. globosum DX-THS3 was mainly at 0–30 days of SSF. The reducing sugar increased rapidly at the early stage of SSF (0–7 days), and the highest concentration was detected at 7 days. Then, the reducing sugar was largely utilized by C. globosum DX-THS3 (Fig. 3C). The reducing sugar lowly increased again at the middle stage of SSF (20–22 days, Fig. 3C). The corresponding enzymatic activities, including those of CMCase, FPase, β-glucosidase, and xylanase, were analyzed during SSF to investigate the variation in reducing sugar during SSF by C. globosum DX-THS3. Lignocellulosic enzymatic activities were observed at the early stage of SSF, and the highest activities of CMCase (29.25 U/g), FPase (232.5 U/g), and xylanase (72.52 U/g) were detected at 10, 7, and 7 days of SSF, respectively (Table 1 and Fig. 3D). Meanwhile, our results showed rare β-glucosidase activity during SSF of C. globosum DX-THS3, whereas 6.42 U/g enzymatic activity was detected at 22 days of SSF.
Table 1
Analysis of lignocellulose-degrading enzymatic activities and production of GAMG under optimal fermentation conditions.
| Enzymes | Max enzymatic activity (U/g) | Days (d) |
Non-optimization | CMCase | 29.25 ± 1.05 | 10 |
FPase | 232.5 ± 30 | 7 |
β-glucosidase | 6.42 ± 0.5175 | 22 |
Xylanase | 72.52 ± 4.00 | 7 |
GUSase | 264 ± 12 | 20 |
Optimization | CMCase | 33.67 ± 2.48 | 5 |
FPase | 245.8 ± 13.4 | 3 |
β-glucosidase | 5.78 ± 0.69 | 20 |
Xylanase | 83.44 ± 3.76 | 3 |
GUSase | 271.42 ± 6.54 | 10 |
Biotransformation has more potential than chemical approaches because of its high yield, high selectivity, and environmental compatibility. Compared with liquid fermentation, SSF has many advantages, including low cost and simple processing, for the production of certain compounds. Meanwhile, excellent lignocellulose-degrading activities for utilization of complex substrates (usually crop straw) by microorganisms are needed. Thus, fungi that harbor rich genes coding lignocellulosic enzymes and excellent lignocellulose-degrading activities are usually used in SSF for the production of certain compounds in most previous reports; such fungi include Ceratocystis paradoxa TT1 (Nutongkaew et al., 2019) and Trichoderma koningiopsis TM3 (Nutongkaew et al., 2019) for the degradation of oil palm trunk to produce reducing sugar. Trichoderma asperellum UC1 (Ezeilo et al., 2019) utilizes raw oil palm frond leaves to produce cellulase and xylanase. Similarly, high lignocellulose-degrading enzymatic activities were found during SSF (Table 1 and Fig. 3D) using C. globosum DX-THS3 (Table 2). The 234.6 U/g FPase activity was detected during SSF, and this value is substantially higher than the other reported FPase activities in SSF using fungi. T. asperellum UC1 (Ezeilo et al., 2019) showed a relative high FPase (26.02 U/g) activity when using oil palm frond leaves as substrate for SSF, and most FPase activity was detected at 0.09–5 U/g (Table 2). High FPase activity indicates that complex cellulose can be degraded to generate easily hydrolyzed cello-oligomers, which are then further utilized by fungi. Furthermore, compared with the results of other studies, lignocellulose-degrading enzymes, including CMCase, xylanase, and β-glucosidase, from C. globosum DX-THS3 during SSF exhibited relative better enzymatic activities (Tables 1 and 2). Thus, high FPase activity and relative excellent CMCase, xylanase, and β-glucosidase activities were detected during SSF by C. globosum DX-THS3, demonstrating their wide application in SSF. Our results also strongly consider C. globosum DX-THS3 as a potential producer for producing lignocellulose-degrading enzymes.
Table 2
Comparison of CMCase, FPase, β-glucosidase, xylanase, and GUS activities by C. globosum DX-THS3 and other fungi under SSF.
Enzymes | Strains | Enzymatic activity (U/g) | Substrate | Reference |
CMCase | T. viridae PAJ 01 | 64.56 | Sugarcane bagasse/wheat bran | N.P. Marques et al. |
| Chaetomium sp. TCF 01 | 12.13 | Sugarcane bagasse/wheat bran | N.P. Marques et al. |
| A. fumigatus | 16.90 | Wheat straw | A. Shenef et al. |
| Botryosphaeria sp. | 8.13 | Empty fruit bunch | E. K. Bahrin et al. |
| Fomitopsis sp. RCK2010 | 71.70 | Wheat bran | D. Deswal et al. |
| Hypocreanigricans TT2 | 6.10 | Oil palm trunk | Nutongkaew T. et al. |
| T. koningiopsis TM3 | 7.13 | Oil palm trunk | Nutongkaew T. et al. |
| T. asperellum RCK2011 | 10.25 | Wheat bran | Raghuwanshi et al. |
| T. asperellum UC1 | 136.12 | Oil palm frond leaves | Ezeilo, U. R. et al. |
| C. globosum DX-THS3 | 29.25 | Licorice straw | This work |
FPase | Chaetomium sp. TCF 01 | 0.09 | Sugarcane bagasse/wheat bran | N.P. Marques et al. |
| A. fumigates | 0.98 | Wheat straw | Sherief, A. A. et al. |
| A. tubingensis NKBP-55 | 3.8 | Copra meal | Prajapati et al. |
| Botryosphaeria sp. | 3.30 | Empty fruit bunch | Bahrin E.K. et al. |
| C. paradoxa TT1 | 1.64 | Oil palm trunk | Nutongkaew T. et al. |
| Fomitopsis sp. RCK2010 | 3.50 | Wheat bran | Deswal, D. et al. |
| T. auraticus | 4.40 | Wheat straw | Kalogeris E. et al. |
| T. asperellum MR 1 | 0.72 | Pressed oil palm petiole fiber | Ikubar M.R.M. et al. |
| T. asperellumUC1 | 26.02 | Oil palm frond leaves | Ezeilo, U. R. et al. |
| C. globosum DX-THS3 | 234.6 | Licorice straw | This work |
β-glucosidase | Chaetomium sp. TCF 01 | 3.81 | Sugarcane bagasse/wheat bran | N.P. Marques et al. |
| A. tubingensis NKBP-55 | 71.0 | Copra meal | Prajapati et al. |
| I. obliquus | 2.58 | Wheat bran | Xu X. et al. |
| T. asperellum MR 1 | 0.43 | Pressed oil palm petiole fiber | Ikubar M.R.M. et al. |
| T. asperellum UC1 | 130.09 | Oil palm frond leaves | Ezeilo, U. R. et al. |
| C. globosum DX-THS3 | 6.42 | Licorice straw | This work |
Xylanase | T. viridae PAJ 01 | 351.74 | Sugarcane bagasse/wheat bran | N.P. Marques et al. |
| Chaetomium sp. TCF 01 | 39.75 | Sugarcane bagasse/wheat bran | N.P. Marques et al. |
| A.niger USM Al 1 | 35.00 | Palm kernel cake | Kheng P.P. et al. |
| A. fumigatus | 56.40 | Wheat straw | Sherief A. et al. |
| A. tubingensis TSIP9 | 59.30 | Empty fruit bunch | Kitcha S. et al. |
| A. tubingensis NKBP-55 | 167 | Copra meal | Prajapati et al. |
| T. koningiopsis TM3 | 56.46 | Oil palm trunk | Nutongkaew T. et al. |
| T. asperellum MR 1 | 5.69 | Pressed oil palm petiole fiber | Ikubar M.R.M. et al. |
| T. asperellum UC1 | 255.01 | Oil palm frond leaves | Ezeilo, U. R. et al. |
| C. globosum DX-THS3 | 72.52 | Licorice straw | This work |
GUSase | A. terreus Li-20 | 1.86a | -- | Xu Y. et al. |
| Streptococcus LJ-22 | 0.77a | -- | Park H. Y. et al. |
| P. purpurogenum. Li-3 | 5.90 ×104a | -- | Zou S. et al. |
| C. globosum DX-THS3 | 264.17b | Licorice straw | This work |
a: GUS proteins were purified and enriched, and enzymatic activities were detected; |
b: GUS activity of solid-state medium was detected. |
3.4 Fructose can significantly improve GAMG production by C. globosum DX-THS3
Nitrogen and carbon sources play key roles for production of specific products by fermentation using microorganism. Additional nitrogen and carbon sources were added to the licorice straw to further increase the productivity of GAMG by using SSF. First, the nitrogen source was optimized for GAMG production by SSF using C. globosum DX-THS3. NH4NO3, peptone, yeast powder, and yeast extract were used as nitrogen sources for the production of GAMG by C. globosum DX-THS3 (S Fig. 1A). Our results also show that the yield of GAMG was 1.44- (20.21 mg/g) and 1.19-fold (16.79 mg/g) higher than those of the control (14.02 mg/g) when using NH4NO3 and yeast extract as nitrogen sources after 20 days of fermentation, respectively. The GAMG yields were lower than that of the control when using peptone and yeast powder as nitrogen sources after 20 days of fermentation, with values reaching 13.17 and 13.37 mg/g, respectively. We further detected the GUS activity after adding NH4NO3, peptone, yeast powder, and yeast extract as nitrogen sources to the medium. Our results also showed the higher GUS activity when using NH4NO3 and yeast extract as nitrogen source than the control, whereas those obtained with peptone and yeast powder were lower after 20 days of fermentation (S Fig. 1B). Thus, NH4NO3 as additional nitrogen source can increase the yield of GAMG by C. globosum DX-THS3 in SSF. Meanwhile, the carbon source for GAMG production was investigated (S Fig. 2). The addition of fructose and glucose can produce 18.38 and 17.12 mg/g of GAMG after 20 days of fermentation (S Fig. 2A and 2B), respectively, which denoted increases of 33.9% and 24.7% than those obtained without a carbon source (13.73 mg/g). The addition of sucrose can generate 13.98 mg/g GAMG but showed no significant effect on the production of GAMG using C. globosum DX-THS3 (S Fig. 2C). Our results showed the significant inhibition of GAMG production when adding lactose to the medium for SSF using C. globosum DX-THS3 after 20 days of fermentation. A total of 9.23 mg/g GAMG was produced, which was a 34% reduction in GAMG compared with that obtained without a carbon source (S Fig. 2D). Furthermore, GUS activities with the addition of carbon sources were further detected after 20 days of SSF. Our results showed similarity to those of GAMG production with carbon source (S Fig. 2E). GUS activity with the addition of fructose was 338 U/g, which was significantly higher than that without carbon source (167 U/g). GUS activity with the addition of glucose was 1.5-fold (250 U/g, S Fig. 2F) higher than that without a carbon source (control, 167 U/g). Compared with the control, the addition of sucrose showed no significant effect on GUS activity (155 U/g, S Fig. 2G), but the addition of lactose significantly inhibited GUS activity (132 U/g, S Fig. 2H). Thus, NH4NO3 and fructose can significantly promote GAMG production of C. globosum DX-THS3 by SSF using licorice straw as a medium.
The yield of GAMG during the SSF period with or without carbon and nitrogen sources was detected to further investigate the production of GAMG using C. globosum DX-THS3. First, we detected the yield of GAMG with or without the addition of carbon source (Fig. 4A). The variation trends of GAMG yields were similar. All the test yields of GAMG slowly increased at the initial stage of SSF (0–15 days), whereas those at the middle stage rapidly increased (15–30 days). Then, the yields of GAMG stabilized at about 32 mg/g at the late stage of SSF (after 30 days). Although all the test yields of GAMG exhibited no significant difference at the later stage of SSF (S Fig. 3), the productivities of GAMG by SSF with different carbon sources or without a carbon source presented significant differences. Compared with the control (33 days), the addition of fructose was the fastest for GAMG production, and 25 days of SSF was used to reach 90% conversion (Y = 31.52 mg/g), 28 days for the addition of glucose, and 30 days for the addition of sucrose. The addition of lactose to the medium can inhibit GAMG production by C. globosum DX-THS3 in SSF, requiring 38 days to reach 90% conversion. Meanwhile, the addition of nitrogen source also showed significant effect on the production of GAMG by SSF using C. globosum DX-THS3. As shown in Fig. 4B, compared with that without nitrogen source, the addition of NH4NO3 to the medium achieved improved production of GAMG by C. globosum DX-THS3, with a reduction of 9 days in the production period to transform 90% of GL compared with that without a nitrogen source. Yeast extract showed no effect on the production of GAMG, whereas yeast powder and peptone can inhibit GAMG production. Thus, NH4NO3 and fructose were further optimized. First, different concentrations of NH4NO3 and fructose (3, 5, 7, 9, and 11 mg/g) were added to the SSF medium. Then, GAMG production was detected after 20 days of SSF. Our results show that 7 mg/g NH4NO3 and 5 mg/g fructose were the optimum conditions for GAMG production by C. globosum DX-THS3 using SSF (Fig. 4C and 4D, respectively). Based on the above results, GAMG was produced faster under these optimal conditions than the control: 0.26–0.85 mm particle size, 28 ℃, 96 h seed age, 20% inoculum size, 1:3 solid–liquid ratio, 7 mg/g NH4NO3, and 5 mg/g fructose (Fig. 4E). Under these optimal conditions, the percent conversion of GL reached 90% within 15 days, whereas the control needed 35 days, that is, an additional 20 days needed for 90% conversion (Table 1 and Fig. 4E). The productivity of optimization (P = 2.1 mg•g− 1•day− 1) increased by 133.33% compared with that in non-optimized conditions (P = 0.9 mg•g− 1•day− 1).
Microorganic fermentation can be classified into three stages: early, middle, and late stages. In general, low-cost and easily available substrates, such as crop straw, are usually used as medium for SSF using fungi. However, complex and adequate enzymes are needed for the degradation of polysaccharides in these substrates (Prajapati et al., 2018; Deswal et al., 2011; Pandey, 2003; Nutongkaew et al., 2019; Ezeilo et al., 2019). Compared with liquid fermentation, more time is needed for fungal cell growth to secrete a complex enzyme system in SSF. Thus, fast accumulation of biomass is the key for shortening the early stage period and increasing the productivity of SSF. To reduce this period, we first optimized the fermentation conditions, including the particle size, temperature, seed age, inoculum size. and water content. However, the early stage of GAMG production was long (about 25 days) (Fig. 3B). Thus, several nitrogen sources that are easily utilized by microorganisms were considered. Glucose, fructose, and several monosaccharides (popular carbon sources) can be directly entered into glycolysis and TCA cycle for fungal growth (Fig. 5A). Therefore, we considered adding these carbon sources to the medium to increase productivity when using microorganisms for SSF. Our results demonstrate that the early stage period of SSF was reduced by about 10 days when adding fructose and NH4NO3 to the medium, thus significantly increasing productivity (Table 1 and Fig. 4E). In this study, lignocellulose and GL of licorice straw were used as carbon sources for C. globosum DX-THS3 growth, and high lignocellulose-degrading enzymatic activities were detected during 5–15 days of fermentation (Fig. 3D). High GUS activities were observed at 20–25 days (Fig. 3C), and the concentration of reducing sugar abruptly increased at 20 days of fermentation. These findings demonstrate that lignocellulose was first utilized, followed by GL, by C. globosum DX-THS3. Thus, certain carbon sources were added to the medium to reduce the early stage period and increase the GAMG production. In summary, at the initial period of SSF, several lignocellulose-degrading enzymes were secreted because of the rare biomass of C. globosum DX-THS3. C. globosum DX-THS3 slowly grew. Thus, a long period was needed for the accumulation of C. globosum DX-THS3 to secrete sufficient lignocellulose-degrading enzymes. Subsequently, GUS was rapidly secreted by C. globosum DX-THS3 for the utilization of GL as a carbon source to generate GAMG and glucuronic acid (Gur). If several popular carbon sources, such as fructose, are added to medium, C. globosum DX-THS3 will grow fast, which can promote the secretion of lignocellulose-degrading enzymes and fast utilization of lignocellulose, significantly reducing the time for production of GAMG (Fig. 5B). Thus, our study provides a novel, fast, and low-cost method for the production of GAMG.