Growth Kinetics and Ganoderic Acid Production from Ganoderma lucidum GIRAN17: A Real-Time Monitoring Platform

Heydarian, Motaharehsadat; Hatamian-Zarmi, Ashrafalsadat; Amoabediny, Ghassem; Ebrahimi-Hosseinzadeh, Bahman; Alvandi, Hale; Doryab, Ali; Salehi, Amir

doi:10.30699/ijmm.15.1.67

year 15, Issue 1 (January - February 2021) Iran J Med Microbiol 2021, 15(1): 67-84 | Back to browse issues page

‎ 10.30699/ijmm.15.1.67

Mendeley

Zotero

RefWorks

Heydarian M, Hatamian-Zarmi A, Amoabediny G, Ebrahimi-Hosseinzadeh B, Alvandi H, Doryab A et al . Growth Kinetics and Ganoderic Acid Production from Ganoderma lucidum GIRAN17: A Real-Time Monitoring Platform. Iran J Med Microbiol 2021; 15 (1) :67-84
URL: http://ijmm.ir/article-1-1069-en.html

Growth Kinetics and Ganoderic Acid Production from Ganoderma lucidum GIRAN17: A Real-Time Monitoring Platform

Motaharehsadat Heydarian¹

, Ashrafalsadat Hatamian-Zarmi

², Ghassem Amoabediny³

, Bahman Ebrahimi-Hosseinzadeh¹

, Hale Alvandi¹

, Ali Doryab³

, Amir Salehi⁴

1- Department of Life Science Engineering, Faculty of New Sciences and Technologies, University of Tehran, Tehran, Iran
2- Department of Life Science Engineering, Faculty of New Sciences and Technologies, University of Tehran, Tehran, Iran , hatamian_a@ut.ac.ir
3- Research Center for New Technologies in Life Science Engineering, University of Tehran, Tehran, Iran
4- Department of Microbiology, School of Biology, College of Science, University of Tehran, Tehran, Iran

Keywords: Ganoderma lucidum, Monitoring, Growth kinetics, Oxygen transfer rate, Miniaturized bioreactor

Full-Text [PDF 2379 kb] (1325 Downloads) | Abstract (HTML) (2880 Views)

Full-Text: (1420 Views)

Introduction

Ganoderma lucidum is widely used in Asia to treat health problems and increase vitality and longevity. These mushrooms offer various health benefits, including immunomodulatory, antitumor, antibacterial, antiviral, anti-diabetic, and anti-oxidant properties [1, 2]. Ganoderic Acid (GA) is a type of triterpenoids produced by G. lucidum and is considered a secondary metabolite [3]. Some GAs can inhibit cholesterol synthesis and tumor growth, which can effectively cure cancer [4, 5]. However, due to the low efficacy of GA production from G. lucidum, these useful metabolite applications have been limited.
Therefore, investigations are growing to enhance the GA biosynthesis’ efficiency by evaluating diverse conditions, in desired parameters such as pH, oxygen pressure and shear stress [6-8]. Furthermore, several studies have been performed to find suitable culture media for optimizing the production of mycelial biomass and secondary metabolite. Although available studies suggests that the more complex media are superior in cell growth and production of secondary metabolites compared to other media, such as semi-synthetic [9], further research is needed to improve the efficacy of production. It has also been reported that the biomass and secondary metabolite yields are increased by improving the oxygen supply [10, 11]. On the other hand, before scale-up studies, the traditional cultivation process has required the screening of large numbers of samples, and consequently, a vast number of development cultivations should be performed. So far, many investigations have been done to enhance G. lucidum production; however, in all of these studies, the lack of an efficient monitoring system is apparent. In fact, we can find useful information through the cultivation and obtain the necessary data for process development by monitoring the procedure. Several small-scale platforms have been developed for high throughput screening and process development.
Nevertheless, one of the most efficient and useful platforms is miniaturized bioreactors. They are widely used as tools for drug screening and discovery, optimization of media, strain, and production [12]. Miniaturized shaken bioreactor systems are also applied to measure the Oxygen Transfer Rate (OTR) during fermentation. OTR is an essential factor in obtaining more data by culturing the strains [13].
In the present work, for the first time, online and offline monitoring using miniaturized bioreactors was used for G. lucidum production concurrently as well as GA biosynthesis. Many different media were used in earlier studies; however, we tried to evaluate their different components in this study.
Moreover we examined the effects of these media on the growth of G. lucidum, level of oxygen consumption, and GA production. In practice, G. lucidum and secondary metabolite production’s growth behavior using a miniaturized shaken bioreactor system suggested the best culture medium for maximal production of GA. These data are so beneficial for find a better idea to increase G. lucidum yields, and it would suggest solutions for challenges.

Materials and Methods

Strains and Cultural Conditions
G. lucidum (Sp. GIRAN17) was obtained from bioscience faculty, Shahid Beheshti University, Tehran, Iran. It has been screened from Carpinus betulus L. (Corylaceae) from Mazandaran, Iran [14, 15]. The strain of G. lucidum was cultured on potato dextrose agar (PDA) plates according to prior study [16]. The optimum conditions for this fungus growth were temperature (25°C), aeration (130 rpm), and initial pH (6.5). Due to the effect of media pH on the stability of GA production and cell growth, the pH of media was kept constant during cultivation.
Studies show that high concentrations of primary glucose lead to increased biomass production and GA biosynthesis [17]. Adding vitamin B1 to the culture medium also increases productivity [18]. The aerated shaken flasks (online) and ventilation flasks (offline) included 20 mL broth culture [15, 17]. Four broth culture media were chosen to evaluate the growth kinetics of G. lucidum (Table 1), and inoculum size was 10% v/v for each broth flasks. In addition to online monitoring of the OTR during cultivation, we performed offline monitoring to measure biomass formation, residual sugar, and GA production using parallel flasks in given intervals during the fermentation time course. All the conditions of the online and offline flasks for each broth culture were kept constant.

The Miniaturized Shaken Bioreactor System
For online measurement of the respiration activity of G. lucidum in small scales bioreactors, aerated shaken flasks (AF) were used (Figure 1A). In this modified 250 ml Erlenmeyer flasks, all of the conditions except the culture medium were the same [19]. Each AF contained a different culture medium: AF₁ contained PDB (basal medium), AF₂ contained PDB and vitamin B₁, AF₃ contained YPG, and AF₄ contained complex media (Table 1). Briefly, on-line monitoring based on OTR was performed and the partial pressures were monitored using an oxygen sensor. The OTR was calculated by the following equation:
Equation 1
Where ΔpO₂is a difference of oxygen partial pressure (Pa), V_Gis the gas volume (L), V_L is the liquid volume (L), R is gas constant (L.Pa/K.mol), T is the temperature (K), and Δt is the time of the measuring phase (h).

Table 1. Operating conditions of aerated shaken flasks

Flow rate (L/h)	Flask volume (mL)	Gas volume (mL)	Working volume (mL)	Culture medium		Flask Number (AF and VF)
1.2	260	240	20	Potato dextrose broth (PDB)		1
1.2	260	240	20	PDB+ 0.05 g/L vitamin B₁		2
1.2	260	240	20	Yeast extract 2.5 g/L, peptone 5 g/L and glucose 35 g/L (YPG).		3
1.2	260	240	20	Complex media (glucose 35 g/L, peptone 5 g/L, yeast extract 2.5 g/L, KH₂PO₄·H₂O 1 g/L, MgSO₄·7H₂O 0.5 g/L, and vitamin B1 0.05 g/L)		4
AF₁, AF₂, AF₃ and AF₄: online monitoring					VF₁, VF₂, VF₃ and VF₄: offline monitoring

250 mL Erlenmeyer shake flasks, called ventilation flasks (VF), were also cultivated simultaneously under the same condition as used in the aerated shaken flasks system [20]. There are several VFs that have different gas transfer resistance of the sterile closure as described and modeled [21]. Since the aeration value of online monitoring flasks or AF is 1.2 vvm, VF type (diameter, D=2.80 cm; height, H= 2.12 cm) was used for offline monitoring which has the same aeration value as AFs (Figure 1). Equations 2 and 3 relate the Oxygen Transfer Rate of the plug (OTR_plug) and the gas transfer coefficient (k_plug) in VF where V_Lis the filling volume, P_abs is the absolute pressure, P_O2 is oxygen partial pressure in headspace of the flask, P_O2,out is oxygen partial pressure in the surrounding environment and a, b and c are fitting parameters (dimensionless) of Eq.3.
OTR_plug = k_plug · (1/V_L · p_abs) · (pO_{2, out} − pO₂)
Equation 2
Equation 3
for VF type 1; a : 22.4×10⁶, b: 6.95×10³, c: 19.97 [21]
Therefore, to find more information through G. lucidum growth, four factors, including biomass formation, GA, residual sugar, and gene expression were evaluated on days 0, 2, 4, 6, 8, 9, 11, 13, and 14 by 35 pcs of VFs. Each run was performed at 130 rpm and the temperature was set at 25°C for fourteen days.

Figure 1. Miniaturized shaken bioreactor system. (A) Schematic drawing of gas transfer in an aerated flask (AF), (B) ventilation flask (VF) with sterile closures as offline technique which used in this research.

Determining dry biomass, residual sugar, specific growth rate, specific GA production rate, and oxygen uptake
As mentioned earlier, an offline monitoring flask or ventilation flask was used to measure four parameters namely dry biomass, GA, residual sugar, and gene expression. The selected ventilation flasks have the same aeration value (1.2 vvm) as the aerated flask used in online monitoring. Mycelium was isolated from liquid media by centrifugation and was dried by a freeze dryer (Alpha 1–2 LD Plus, Christ, Germany) to measure dry cell weight. Residual sugar concentration was also measured by the phenol–sulfuric acid method [18]. The average specific growth rate (µ) and the specific GA production rate (q_GA) were obtained from the following equations:
µ = Equation 4
q_GA= Equation 5
When X is cell concentration. Oxygen consumption during cultivation was also determined by the integration of the OTR profile as follows:
Oxygen uptake = Equation6

Assay of Ganoderic Acid

According to literature [18], GA was extracted after obtaining dried mycelium. Briefly, to extract GA, 100 mg of dried biomass is added to ethanol 50% (v/v) twice for one week, and then biomass was isolated by centrifugation (25 min, 12,000g). Additionally, the supernatants were dried at 50°C under vacuum conditions, and the residue was dissolved in a few milliliters of water besides extracting the aqueous solution with 2 mL of chloroform. The GA extraction from chloroform is performed with 5% NaHCO₃ (w/v) and the pH of the solution is adjusted by the addition of hydrochloric acid (below 3). GA residues were then extracted by chloroform and in the next step, the remaining chloroform was evaporated at 40°C. The GA content was dissolved in absolute ethanol and measured at 245 nm in a spectrophotometer (T80, PG Instruments Limited, London) as standard [22].

RNA Isolation and Quantitative real-time PCR

A 0.1g Aliquot of mycelium was separated from the culture medium and frozen by liquid nitrogen. Total RNA was extracted by RNX plus (Sinacolon, Iran). cDNA synthesis was performed by a synthesizing kit (Amplicon, Denmark). The random primer was used for cDNA synthesis. Then, transcript levels of hmgr and sqs were determined by quantitative real-time PCR using SYBR Green (Applied biosystem, USA). The 2 ^{- ΔΔ CT} method was employed to analyze relative gene expression [23]. 18srRNA primer was used as an internal control and to normalize the data for the gene expression. The sequences of the hmgr and sqs primers have been described in the literature. The primers sequences were as follows: hmgr forward: 5'-GTCATCCTCCTATGCCAAAC-3', hmgr reverse: 5'-GGGCGTAGTCGTAGTCCTTC-3', sqs forward: 5'-ACAGTTGTCAGCGAAGAGC-3', sqs reverse: 5'-CGTAGTGGCAGTAGAGGTTG-3', 18S rRNA forward: 5'-TATCGAGTTCTGACTGGGTTGT-3', 18S rRNA reverse: 5'-ATCCGTTGCTGAAAGTTGTAT-3' [24, 10].
The expression level 1 was selected for samples for the PDB culture and fold changes were done in comparison with it. An initial denaturation stage were at 95°C for 5 min, the amplification conditions were in a three-step procedure: 30s at 94°C (denaturation), 30 s at 56°C (annealing), and 30 s at 72°C (extension) followed by 40 cycles.

Statistical analysis
Experimental results were analyzed statistically using Student’s t-test (S). All runs were carried out in triplicate. A probability (p) value of less than 0.05 (P<0.05) was taken as the level of significance

Results

Online monitoring

Miniaturized shaken bioreactor system provides a rich source of data by studying the OTR profile in each fermentation period, such as physiological responses of aerobic microorganisms to specific culture conditions, inhibition of the product, diauxic growth, and other biological phenomena. The OTR evolutions of AF₁, AF₂, AF₃, and AF₄during the cultivation of G. lucidum are shown in Figure 2. At first, in the first 100 hr of cultivation, no fundamental changes were detected. The OTR for AF₁ and AF₂ achieved their maximal values of 2 and 3.2 mmol/Lh at 150 hr and 160 hr, respectively. Although by culturing under AF₁ and AF₂ the growth behavior of G. lucidum was the same, the maximal OTR of AF₂ was more than that of AF₁, which can be attributed to vitamin B₁. By analyzing the AF₃OTR profile, we found that two maximal values of 3.5 and 4.9 mmol/Lh occurred at 140 hr and 210 hr, respectively. Furthermore, with fermentation in complex media (AF₄), maximal values of 3.8 and 10 mmol/Lh occurred at 155 hr and 220 hr, respectively.
The OTR evaluation results also indicated that with culturing in AF₁ and AF₂, the OTR curve sloped downward after achieving their maximum value.

Figure 2. Evolution of oxygen transfer rate, expressed in mol/L^/h using aerated shake flask (AF) and GA production expressed in mg/mL using ventilation flask (VF) during culturing of G. lucidum with different media culture; AF₁ and VF₁: PDB; AF₂and VF₂: PDB + vitamin B₁; AF₃ and VF₃: YPG; AF₄and VF₄: complex medium (n=3).

Offline Monitoring
Cultivation of G. lucidum using offline monitoring was also carried out to examine the biomass formation, GA biosynthesis, residual sugar, and gene expression during certain times. The combination of an offline and online monitoring can be beneficial for kinetic analysis of biomass formation and GA production.
Figure 3 shows the biomass production of G. lucidum during cultivation. After 48 hr, the biomass formation increased under VF₁-VF₄, and the maximum cell concentration was achieved after 192 hr. Then, the biomass almost remained constant until the end of the period despite the corresponding depletion of sugar concentration. During cultivation, a lag phase of two-day was considered for all the flasks. It is also observed that the culturing of G. lucidum under VF₄ yielded the maximum biomass (18.5 g DW/L) compared to other flasks. The maximum biomass accumulation under VF₃, VF₂, and VF₁ were 15.25, 12.25, and 11.25 g DW/L, respectively. Evaluation of the total cell concentration during fermentation demonstrated that biomass production in a more complex broth culture (VF₄) was 1.6-times higher than that in the basal medium (VF₁). The sugar consumption profiles in all flasks have the same permanent declining trend. Further analysis shows that on the thirteenth day, residual sugar was almost depleted for all cases. Moreover, more complex media (flasks 3-4) that contain richer carbon sources experienced higher cell growth than less complex ones (flasks 1-2).
GA production analysis demonstrated that GA was produced after 144 h in all flasks and then by the rest of culturing this substance was accumulated continuously (Figure 2). Maximum GA production of VF₄was obtained equal to 270 mg/L 4 times more than the VF₁.
Further analysis demonstrated that by culturing G. lucidum under different media (flasks 1-4), gene expression of hmgr and sqs had been experienced a sharp increase after 96 hr (Figure 4), which coincides with the starting of GA biosynthesis (Figure 2). For more complex media (flasks 3-4), another increase is observed after 192 hr. Also, gene expression of hmgr and sqs at the end of cultivation were 1.45- and 2-times higher than their initial value at beginning of culturing, respectively. The final gene expression analysis under different culture media demonstrated this parameter was increased 1.25 and 1.81 times in VF₃, and 1.2 and 1.6 times in VF₂ for hmgr and sqs, respectively. In all cases, the gene expression of sqs was higher than that of hmgr.

Figure 3. Monitoring of residual sugar and biomass formation expressed in g/L and g DW/L, respectively using ventilation flasks (VF) during culturing of G. lucidum with different media culture (n=3).

Figure 4. Comparison analysis of gene expression of hmgr and sqs in flasks 1-4 (*indicates P < 0.05 compared to PDB culture).

Discussion

Kinetic Analysis of Biomass Formation and GA Production
The kinetic profiles of the mycelial growth and the GA accumulation of G. lucidum in the miniaturized shaken bioreactor systems in addition to residual sugar and gene expression were monitored and shown in four different media simultaneously.
According to the OTR profile of AFs through fermentation, the oxygen consumption for AF₄, AF₃, AF₂, and AF₁ were calculated to equal with 1.47, 0.76, 0.40, and 0.17 mol/L, respectively. It is concluded that the oxygen consumption of AF₄including more complex media is 8.6-fold higher than that of AF₁ as a basal broth culture. Residual sugar analysis suggested that reducing in OTR values can be attributed to the depletion of carbon sources from media cultivation.
Measuring the average specific growth rate (μ) indicated that the μ for VF₄, VF₃, VF₂, and VF₁, was 0.237, 0.211, 0.190, and 0.175 d^-1, respectively. Culturing under VF₄ was approximately 1.4-times higher in growth compared to VF₁.
With culturing by a more complex carbon medium (flask 4), the specific cell growth rate (μ) achieved two maximal values of 0.276 d^-1 and 0.247 d^-1 at 6 d and 8 d respectively. Moreover, culturing by using more than one carbon source (flask 3) the specific cell growth rate (μ) of VF₄obtained two maximal values equal to 0.252 d^-1 and 0.190 d^-1 at the same time. However, with fermentation under VF₂ and VF₁ (basal medium), the specific cell growth rate (μ) achieved maximal value of 0.190 d^-1 and 0.175 d^-1at 8 d. At maximum GA production, the growth curve can be divided into two phases: growth and production. Each phase has a specific growth rate (μ).
Also, by cultivation under VF₄, the maximal specific GA production rate (q_GA) was obtained 1.84×10^-3 (g GA. g/DW.h), which was almost 2.8-times higher than VF₁ 0.65×10^-3 (g GA. g/DW.h). Consequently, compared to VF₂and VF₁, the growth rate of VF₄ and VF₃sharply increased, and the main GA was produced between 144-216 h.
Transcript levels of hmgr and sqs indicated that the maximum gene expression is accessible when GA starts to produce and after that, they have a steady trend, until the end of fermentation. Furthermore, more gene expression levels of flasks 3-4 can be attributed to enhancing in GA production.
Table 2 presents the growth behavior of G. lucidum in flask 4 during fermentation. The critical time courses are highlighted. It seems that the interval between hours 134 and 312 when the changes of biomass and GA production coincided with the OTR evolution, was the crucial time for G. ludicum. Therefore, this interval is suggested as the critical time for G. lucidum growth.

Table 2. Growth behavior of G. lucidum under AF₄ and VF₄

Time range (h) Monitoring		0-96	96-134	134-168		168-216	216-312		312-336
AF (online)	OTR (mmol/Lh)	0	increase up to 3.5	constant 3.5		increase up to 10 OTR _max: 10	decrease to 2		constant 2
		→	↑	→		↑	↓		→
VF (offline)	biomass (g DW/L)	increase up to 11.3 µ₁ :0.276 (d^-1)		increase up to µ₂ :0.247 (d^-1)			almost constant
		↑		↑			→
	GA (mg/L)	almost no production		increase up to 270						constant
		---		↑						→
	Residual sugar (g/L)	almost steady depletion			main sugar consumed			almost exhausted
		→			↓			↓

The Yield of Oxygen Consumption per GA Production, Biomass Formation, and Productivity
As shown in Figures 2 and 3, the ratio of biomass per GA production rose in 216 initial hours; however, it reached its lowest value afterward. Further, between hours 216 and 312 of fermentation, no cell growth is observed despite GA production. It can be argued that in this interval the GA production is partially growth-associated. To obtain more information about this interval, we measured the yield of oxygen consumption per GA biosynthesis (Y_O2/GA), the yield of oxygen consumption per biomass production (Y_O2/X), and the productivity (Y_X/GA) (Eq. 7, 8, and 9).
( ) =                      Equation 7
( ) =                  Equation 8
( ) =                     Equation 9 For this purpose, a more complex medium (i.e. flask number 4) was chosen due to its high cell growth and secondary metabolite production. Based on oxygen consumption, GA production, and biomass formation, desired parameters such as Y_O2/GA, Y_O2/X, and productivity during fermentation were measured at given times (Figure 5).
The maximum Y_O2/X was obtained in 264 h while no increase in cell growth is observed at this time (Figure 3). So, it can be concluded that a large portion of oxygen has been consumed to produce GA. Also, the highest amount of oxygen per GA biosynthesis is seen at 144h in which GA starts to produce (Figure 2). In fact, at the beginning of GA production, the majority of oxygen was consumed. At this time, culturing G. lucidum has the highest productivity. As a consequence, these results could approve OTR trends (Figure 2).
Since most GA was synthesized between hours 192 and 312, this interval was further analyzed to obtain more useful information. Between these ranges, oxygen consumption was not specified, and, this value should be subtracted from the amount of oxygen consumption by biomass formation to determine a better estimation of oxygen uptake by GA production. Therefore, two intervals were chosen; interval one (0-144 h) in which there was no GA biosynthesis, and interval two (192-312 h) with the main GA production. In the first interval, oxygen consumption was calculated to be 0.225 mol/L. This value for the next interval, when GA began to produce, was 0.986 mol/L. Based on biomass analysis (Figure 3), the main mycelia was produced in the first interval (0-144 hr). On the other hand, the cell growth rate decreased in the second interval, suggesting that by secondary metabolites (GA) consumed most of the oxygen. Therefore, to measure GA’s oxygen consumption during the second interval, we should obtain the amount of oxygen consumed by mycelia in interval two. Finally, the actual oxygen consumption by GA production and Y_O2/GA was calculated to be equal with 0.761 mol/L and 3.81 mol/g, respectively. It can be concluded that after reaching maximal biomass accumulation, most of the oxygen has been consumed by secondary metabolite, and consequently GA production was partially growth-associated.

Figure 5. Yield of oxygen consumption per GA production (Y_O2/GA) (■), Yield of oxygen consumption per biomass production (Y_O2/X) (▲), and productivity (Y_X/GA) (●) of culturing G. lucidum under more complex media (flask 4). The error bars show the standard deviation (n=3).

Conclusion

On-line and off-line G. lucidum cultivation monitoring provided insight into its growth kinetics during fermentation. Production of valuable metabolite, i.e, GA in various cultures media indicated that maximum oxygen consumption, cell concentration, and GA production occurred when this mushroom was cultured under a more complex medium (flask 4). High maximum biomass and GA production were obtained at an initial glucose concentration of 35 g/L with the presence of vitamin and KH₂PO₄. Under these conditions, the growth curve was two-phase and oxygen limitation did not occur. A high cell respiratory activity, superior cell growth, and secondary metabolite production were observed under the cultivation of a more complex medium and as a result, this media can be suggested as an optimized media for G. lucidum. The start time of secondary metabolite production can also be estimated by using the OTR evaluation. It was indicated that GA production was partially growth-associated. Additionally, overexpression of hmgr and sqs genes, which are involved in the GA’s biosynthesis pathway, confirmed the results of monitoring. Earlier investigations have tried to examine different factors and conditions to enhance the production of G. lucidum. Despite prior research on G. lucidum, this study has introduced an online monitoring strategy that is more efficient, unsophisticated, and cost-effective than conventional methods. Finally, since finding cell growth behavior during cultivation is always considered a challenging issue, this method can be beneficial for studying the growth kinetics of other mushrooms.

Acknowledgements

The authors would like to thank the Faculty of New Science and Technologies, University of Tehran. The authors also are so grateful to Mr. Ebrahim Amoabediny for his kindly efforts.

Conflicts of Interest

The authors declared no conflicts of interest

Type of Study: Original Research Article | Subject: Microbial Biotechnology
Received: 2020/02/29 | Accepted: 2020/12/6 | ePublished: 2021/01/10

References

1. Kang Q, Chen S, Li S, Wang B, Liu X, Hao L, Lu J. Comparison on characterization and antioxidant activity of polysaccharides from Ganoderma lucidum by ultrasound and conventional extraction. International Journal of Biological Macromolecules., 2019; 124: 1137-1144. [DOI:10.1016/j.ijbiomac.2018.11.215] [PMID]

2. Sanodiya BS, Thakur GS, Baghel RK. Ganoderma lucidum: a potent pharmacological macrofungus, Curr Pharm Biotechnol., 2009; 10: 717-742. [DOI:10.2174/138920109789978757] [PMID]

3. Frisvad JC, Andersen B, Thrane U. The use of secondary metabolite profiling in chemotaxonomy of filamentous fungi, Mycol Res., 2008; 112:231-240. [DOI:10.1016/j.mycres.2007.08.018] [PMID]

4. Kalantari-Dehaghi S, Hatamian-Zarmi A, Ebrahimi-Hosseinzadeh B, Mokhtari-Hosseini ZB, Nojoki F, Hamedi J, Hosseinkhani S. Effects of microbial volatile organic compounds on Ganoderma lucidum growth and ganoderic acids production in Co-v-cultures (volatile co-cultures). Prep Biochem Biotechnol. 2019; 49(3):286-297. [DOI:10.1080/10826068.2018.1541809] [PMID]

5. Gao JJ, Min BS, Ahn EM. New triterpene aldehydes, lucialdehydes AC, from Ganoderma lucidum and their cytotoxicity against murine and human tumor cells, Chem Pharm Bull., 2002; 50:837-840. [DOI:10.1248/cpb.50.837] [PMID]

6. Wu GS, Lu JJ, Guo JJ. Ganoderic acid DM, a natural triterpenoid, induces DNA damage, G1 cell cycle arrest and apoptosis in human breast cancer cells, Fitoterapia., 2012; 83: 408-414. [DOI:10.1016/j.fitote.2011.12.004] [PMID]

7. Fang, QH, Zhong JJ. Effect of initial pH on production of ganoderic acid and polysaccharide by submerged fermentation of Ganoderma lucidum, Process Biochem., 2002; 37:769-774. [DOI:10.1016/S0032-9592(01)00278-3]

8. Papinutti L. Effects of nutrients, pH and water potential on exopolysaccharides production by a fungal strain belonging to Ganoderma lucidum complex, Bioresour Technol., 2010; 101:1941-1946. [DOI:10.1016/j.biortech.2009.09.076] [PMID]

9. Zhao W, Xu J, Zhong J. Bioresource Technology Enhanced production of ganoderic acids in static liquid culture of Ganoderma lucidum under nitrogen-limiting conditions, Bioresour Technol., 2011; 102:8185-8190. [DOI:10.1016/j.biortech.2011.06.043] [PMID]

10. Xu P, Ding ZY, Qian Z. Improved production of mycelial biomass and ganoderic acid by submerged culture of Ganoderma lucidum SB97 using complex media, Enzyme Microb Technol., 2008; 42:325-331. [DOI:10.1016/j.enzmictec.2007.10.016]

11. Zhang WX, Zhong JJ. Effect of oxygen concentration in gas phase on sporulation and individual ganoderic acids accumulation in liquid static culture of Ganoderma lucidum, J Biosci Bioeng., 2010; 109:37-40. [DOI:10.1016/j.jbiosc.2009.06.024] [PMID]

12. Wei Z, Duan Y, Qian Y. Screening of Ganoderma strains with high polysaccharides and ganoderic acid contents and optimization of the fermentation medium by statistical methods, Bioprocess Biosyst Eng., 2014; 37: 1789-1797. [DOI:10.1007/s00449-014-1152-2] [PMID]

13. Kirk TV, Szita N. Oxygen transfer characteristics of miniaturized bioreactor systems, Biotechnol Bioeng., 2013; 110:1005-1019. [DOI:10.1002/bit.24824] [PMID] [PMCID]

14. Wagner R, Mitchell DA, Sassaki GL. Current Techniques for the Cultivation of Ganoderma lucidum for the Production of Biomass, Ganoderic Acid and Polysaccharides, Food Technol. Biotechnol., 2003; 41:371-382.

15. Keypour S, Rafat H, Riahi H. Qualitative analysis of ganoderic acids in Ganoderma lucidum from Iran and China by RP-HPLC and electrospray ionisation-mass spectrometry (ESI-MS, Food Chem., 2010; 119:1704-1708. [DOI:10.1016/j.foodchem.2009.09.058]

16. Heydarian MS, Hatamian-Zarmi A, Amoabediny G, Yazdian F, Doryab A. Synergistic Effect of Elicitors in Enhancement of Ganoderic Acid Production: Optimization and Gene Expression Studies. Applied food biotechnology. 2015; 2(3): 57-62.

17. Anderlei T, Zang W, Papaspyrou M, Büchs J. Online respiration activity measurement (OTR, CTR, RQ) in shake flasks, Biochem Eng J., 2004; 17:187-194. [DOI:10.1016/S1369-703X(03)00181-5]

18. Fang, QH, Zhong JJ. Submerged fermentation of higher fungus Ganoderma lucidum for production of valuable bioactive metabolites - Ganoderic acid and polysaccharide, Biochem Eng J., 2002; 10:61-65. [DOI:10.1016/S1369-703X(01)00158-9]

19. Tang YJ, Zhong JJ. Fed-batch fermentation of Ganoderma lucidum for hyperproduction of polysaccharide and ganoderic acid, Enzyme Microb Technol., 2002; 31:20-28. [DOI:10.1016/S0141-0229(02)00066-2]

20. Amoabediny G, Rezvani M, Rashedi H. Application of a novel method for optimization of bioemulsan production in a miniaturized bioreactor, Bioresour Technol., 2010; 101:9758-9764. [DOI:10.1016/j.biortech.2010.07.009] [PMID]

21. Amoabediny G, Ziaie-Shirkolaee Y, Büchs J. Development of an unsteady-state model for a biological system in miniaturized bioreactors, Biotechnol Appl Biochem., 2009; 54:163-170. [DOI:10.1042/BA20090141] [PMID]

22. Amoabediny G, Büchs J. Modelling and advanced understanding of unsteady-state gas transfer in shaking bioreactors, Biotechnol Appl Biochem., 2007; 46:57-67. [DOI:10.1042/BA20060120] [PMID]

23. Xu JW, Xu YN, Zhong JJ. Production of individual ganoderic acids and expression of biosynthetic genes in liquid static and shaking cultures of Ganoderma lucidum, Appl Microbiol Biotechnol., 2010; 85:941-948. [DOI:10.1007/s00253-009-2106-5] [PMID]

24. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method, Methods., 2001; 25: 402-408. [DOI:10.1006/meth.2001.1262] [PMID]

25. Ren A, Qin L, Shi L. Methyl jasmonate induces ganoderic acid biosynthesis in the basidiomycetous fungus Ganoderma lucidum, Bioresour Technol., 2010; 101:6785-6790. [DOI:10.1016/j.biortech.2010.03.118] [PMID]