International Journal of Medicinal Mushrooms, 21(1):59–69 (2019)

Effect of Environmental Conditions on Synnema

Formation and Nucleoside Production in Cicada
Flower, Isaria cicadae (Ascomycetes)
Kuanbo Liu,a Fen Wang,a Guijun Liu,b & Caihong Donga,*
a
State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China; bBeijing
Radiation Center, Beijing, China

*Address all correspondence to: C.-H. Dong, State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing
100101, China; dongch@im.ac.cn

ABSTRACT: Isaria cicadae (syn. Cordyceps cicadae) is one of the most valued edible and medicinal fungi and has
been used in Asia as a substitute for Ophiocordyceps sinensis. Wild I. cicadae is limited and seasonal, and its cultivation
is deserved. In this investigation we studied synnema formation by and nucleoside production in cicada flower under dif-
ferent environmental conditions. I. cicadae produced an asexual structure and mitospores instead of meiotic ascospores;
this indicates that the term “synnema” is more suitable than “fruiting body” for this species. The optimal temperature
was 25°C for growth of I. cicadae mycelia on potato dextrose agar plates but was 20°C for synnema formation on wheat
medium. Synnemata can grow well under blue, green, and white light, and the dry weight of samples grown under these
3 light wavelengths is not significantly different. However, neither primordia nor synnemata formed under red light. Blue
light promotes conidia production and white light promotes N6-(2-hydroxyethyl)-adenosine (HEA) production. Weak
white light at 50 and 150 lux was more suitable for synnema production than strong-intensity light at 850 lux. The growth
curve showed that HEA content has the same trend as synnema production over the entire cultivation period. The optimal
harvesting time for I. cicadae cultivated on wheat medium is 35 days after inoculation. HEA content in the synnemata
cultivated on wheat medium under the optimal conditions was significantly higher than that of the wild species and of
synnemata cultivated on pupae, suggesting that synnemata cultivated on wheat medium may have potential as a substi-
tute for wild resources. The results presented herein provide a new strategy for producing superior-quality synnemata of
I. cicadae and further elucidate the effects of environmental conditions on metabolite accumulation in fungi.

KEY WORDS: Isaria cicadae, light, medicinal mushrooms, N6-(2-hydroxyethyl)-adenosine, nucleoside, synnema,
temperature

ABBREVIATIONS: HEA, N6-(2-hydroxyethyl)-adenosine; LED, light-emitting diode; PDA, potato dextrose agar

I. INTRODUCTION

Isaria cicadae Miq. (syn. Cordyceps cicadae; Cordycipitaceae, Hypocreales, Ascomycetes), also known as
cicada flower, Chanhua, or Sandwhe,1 parasitizes cicada nymphs and larvae.2 The medicinal value of cicada
flower was first recorded in a Chinese medicinal text, “Lei Gong Pao Zhi Lun” (Lei’s Treatise on Preparation
of Materia Medica) around 1600 years ago,3 800 years earlier than the first mention of Ophiocordyceps
sinensis (syn. Cordyceps sinensis). It is considered to be the most ancient medicinal material.
As a traditional Chinese medicine, cicada flower has been widely used for thousands of years to treat
fever, improve vision, protect renal function, and treat infantile convulsions, palpitations, and dizziness. 4
Pharmacological studies have demonstrated that cicada flower possesses antitumor,5 renal function–amelia-
tory,6 antifatigue,7 antibacterial,8 immunomodulatory,9 and sedative effects.10 Also, extensive studies have
confirmed the safety of cicada flower.11,12
A series of active ingredients has been isolated from Cordyceps. Nucleosides, including adenosine,
guanosine, and N6-(2-hydroxyethyl)-adenosine (HEA), represent the main active components of Cordyceps

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60 Liu et al.

sensu lato.13,14 Adenosine, a natural and ubiquitous nucleoside, can be used as a cardioprotective and
therapeutic agent for treating chronic heart failure15 and as a modulator of homeostasis in the central
nervous system.16 Guanosine exerts important neuroprotective and neuromodulator effects in the central
nervous system and may be used for preventing ischemia and Parkinson and Alzheimer diseases.17 HEA,
the first calcium antagonist derived from a biological source,18 has been intensively investigated because
of its ability to decrease inflammation,19 to protect kidneys and to function as a sedative,20 and to act as
an insecticide.21 I. cicadae is, to our knowledge, the most important biological origin of HEA.22
The production of wild cicada flower is seasonal and thus limited. Many studies have investigated its
cultivation, and now its synnemata can be cultivated. Cicada flower has been regarded as a substitute for
O. sinensis and as a valuable traditional Chinese herbal tonic in China,23,24 and it has shown the potential
for use in edible/medicinal applications.
Environmental conditions play a crucial role in whether a fruiting body can form, and this has been well
studied at the physiological level in economically important fungi, most of which are Basidiomycetes. 25,26
In this work, we evaluated the effects of environmental conditions, including temperature, light wavelength,
and light intensity, on synnema (fruiting body) growth and nucleoside production in cicada flower. Then
we optimized the harvest time of synnemata by plotting a growth curve applying the optimal conditions.
Last, we compared the nucleoside contents of synnemata cultivated under the optimal conditions with
those of wild cicada flower. As a result, we determined optimal environmental conditions for cultivating
cicada flower and producing nucleosides.

II. MATERIALS AND METHODS

A. Fungal Strains

Fungal strain 463, used in this study, was isolated from wild cicada flower collected from Tianmushan,
Zhejiang Province, China, by the corresponding author (CD). The strain was maintained as stock on
potato dextrose agar (PDA) at 4°C. To ensure the correct identification of the I. cicadae strain, its identity
was determined on the basis of DNA sequence analysis and the morphological characteristics observed
in culture. The internal transcribed spacer of nuclear ribosomal DNA was amplified and sequenced. The
sequences were compared with those in a data set that was generated in our laboratory and contains
internal transcribed spacer sequences from dried specimens and living strains of I. cicadae obtained from
various regions.

B. Cultivation of Cicada Flower Synnemata on Wheat Medium and Pupae

The fungus was cultivated on wheat medium in order to produce synnemata. The strain was incubated
on PDA medium in a Petri dish at 25°C for 10 days. Then a 5-mm-diameter circle was punched from
the agar plate culture with a sterilized cutter and transferred to the seed culture medium, potato dextrose
liquid medium. The seed cultures were grown in a 250-mL flask containing 80 mL potato dextrose liquid
medium, which was shaken on a rotary shaker incubator at 150 rpm at 25°C for 3 days. The samples were
cultivated in 250-mL glass bottles, each containing wheat medium, under 12-hour light/12-hour dark
conditions and relative humidity greater than 80%. The harvested synnemata were subsequently air-dried
at 45°C for 48 hours then ground to a fine powder in a laboratory mill (HR2027; Philips, Hong Kong).
The dry ground materials were kept at 4°C in glass jars wrapped with aluminum foil.
Synnemata were also cultured on pupae of Bombyx mori, which were purchased from Yukang Natural
Products Company (Henan, China). The seed cultures were produced as described above, then filtered

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Synnema Formation and Nucleoside Production in Cicada Flower 61

through sterilized gauze to remove entangled hyphae. Seeds (0.2 mL) were injected into each pupa by
using a syringe. After the injection, the inoculated pupae were cultured in containers at 20°C until their
bodies became hard and mummified as a result of fungal proliferation. To induce synnema growth, each
container with the inoculated pupae was kept at 20°C under a 12-hour light/12-hour dark cycle, with light
intensity of 850 lux and relative humidity greater than 80%.

C. Effect of Culture Temperature

First we studied the temperature at which the I. cicadae strain grew on the Petri dishes. A 5-mm-diameter
disc of agar with mycelia was punched out from the inoculum preparation with a sterilized cutter and
transferred to a fresh Petri dish containing PDA medium. The inoculated dishes were sealed with parafilm
and incubated at 15°C, 20°C, 25°C, 27°C, 30°C, and 35°C. The growth rate was determined by measuring
the colony diameter. Then the synnemata were cultivated at 20°C, 25°C, and 27°C under white light at
150 lux. The synnemata were harvested after 30 days.

D. Effect of Light Wavelength and Intensity

Light-emitting diode (LED) lamps (XD2121; Xuzhou Gypsy, Xuzhou, China) were used to apply differ-
ent light wavelengths: 620–630 nm (red light), 440–450 nm (blue light), 500–540 nm (green light), and
white light.
We evaluated the effects of white light intensity on synnema growth and nucleoside production. We
used light intensities of 50, 150 and 850 lux, as measured by using a digital lux meter (TES1332A; Taishi,
Taiwan, China). The synnemata were cultivated at 20°C and harvested.

E. Growth Curve of Synnemata Produced on Wheat Medium

Thirty glass bottles were prepared for use in determining the growth curve of the I. cicadae strain during
synnema cultivation. The conditions were set as the optimized outcomes (temperature of 20°C, white
light at an intensity of 150 lux). Synnemata were harvested from 3 bottles every 5 days, and dry weight
and nucleoside content were measured.

F. Comparison of Nucleoside Content between Wild and Cultivated I. cicadae

Synnemata

Wild samples of cicada flower were collected from Tianmushan, Zhejiang Province, China. Both the wild
and cultivated cicada flower grown on pupae were divided into the sclerotium (pupae) and synnemata,
then ground separately into fine powders. The 2 samples are referred to as sclerotium and synnemata on
the pupae. The dry ground materials were kept at 4°C in glass jars were wrapped with aluminum foil.

G. Determination of Nucleoside Content by Using High-Performance Liquid

Chromatography

Samples were extracted for 0.5 hours in deionized water by using ultrasonic extraction, then filtered through
a 0.45-µm filter membrane before being injected into a high-performance liquid chromatography system.
Nucleosides were analyzed by using reversed-phase high-performance liquid chromatography in a
Waters 2695 Separations Module (Waters Corporation, Milford, MA) equipped with a built-in quaternary
pump, a 120-sample autosampler, and a Waters 2998 Photodiode Array Detector. Empower software was

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62 Liu et al.

used to analyze the data. We also used a prepacked Puritex C18 column (4.6 × 250 mm, 5-μm particle
size; Beijing Green Herbs Science and Technology Development Co. Ltd., Beijing, China). Samples
were eluted in the mobile phase, which consisted of acetonitrile and double-distilled water (0.1% acetic
acid) (5:95), at 25°C for 20 minutes. The flow rate was 1 mL/min and the injection volume was 10 μL.
Nucleosides were monitored and quantified at 260 nm. Commercially available adenosine (Sigma, Munich,
Germany), guanosine (Beijing Bio dee Biotechnology Co., Ltd, Beijing, China), and HEA (Shanghai Pure
One Biotechnology, Pudong, Shanghai, China) were dissolved in distilled water and used as calibrators.

H. Statistical Analysis

All trials were performed in triplicate. The data were analyzed by using 1-way analysis of variance.
Significant differences were determined by using the Duncan multiple range test; a P value of 0.05 was
considered to be significant. The results were processed using SPSS software (SPSS Inc., Chicago, IL)
and OriginPro 8.5 (Origin Lab Corp., Northampton, MA).

III. RESULTS

A. Effect of Temperature

The rate of mycelial growth increased as temperature increased from 15°C to 25°C, then it decreased as
temperature increased. As shown in Fig. 1A, weak growth occurred at 15°C and 30°C, and no growth
occurred at 35°C, even after the sample had been cultivated for 8 days. The optimal growth occurred at
25°C, followed by 27°C and 20°C. The growth rate at 20°C was significantly slower than that at 25°C
and 27°C (P < 0.05) (Fig. 1B). After the temperature was reduced to 25°C and the samples were incubated
for another 5 days, the samples that had been at 35°C for 8 days resumed growth, suggesting that 35°C
is not the lethal temperature.
Mycelia of the I. cicadae strain grew well on PDA medium at 20°C, 25°C, and 27°C, and so the
synnemata were cultivated at these 3 temperatures. The appearance of samples cultivated at the various
temperatures was obviously different (Fig. 1C). The synnemata grew fastest at 20°C and reached a length
of 8.00 cm after 30 days of cultivation, appearing light yellow. The synnemata grew relatively slowly at
25°C to about 6.00 cm in length; the samples were slim, with many branches at the top. White powder
full of conidia grew at 27°C, and few synnemata formed. A temperature of 20°C seemed to be suitable
for differentiating synnemata, and 27°C, for producing conidia.

B. Effect of Various Light Wavelengths and Light Intensities

Light wavelengths substantially influenced synnema growth. As shown in Fig. 2, the primordia do not
differentiate and synnemata do not form under red light; only dense mycelia grow. Synnemata can, how-
ever, grow under blue, green, and white light. Dry weight was not significantly different for the samples
grown under the various light wavelengths (Table 1). Much more conidia powder formed under blue light
than under the other lights.
Light wavelength has no effect on the contents of guanosine and adenosine (Table 1). However, the
HEA content in the sample grown under white light was significantly higher than that in samples grown
under the other light conditions (P < 0.05).
The effect of white light on synnema growth is shown in Fig. 3. The synnemata fell down and produced
much white conidia powder under a light intensity of 850 lux. The synnemata grew well at intensities

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Synnema Formation and Nucleoside Production in Cicada Flower 63

B C

FIG. 1: Effect of temperature on the mycelial growth of and synnema production by Isaria cicadae. Morphology
of (A), growth rate of (B), and synnema production by (C) the I. cicadae strain grown at various temperatures.
Different letters above the bars indicate significant differences (P < 0.05).

of 150 and 50 lux. The dry weights of synnemata samples were significantly different depending on the
tested intensity, following the order of 150 lux > 50b lux > 850 lux (P < 0.05) (Table 2). Light intensity
also affected nucleoside production. The HEA content was highest at 850 lux and second highest at 150
lux. However, the guanosine content was highest at 150 lux. White light at 150 lux was used in the sub-
sequent experiments.

C. Growth Curve of the I. cicadae Strain Cultivated under the Optimal Conditions

To study the kinetic mode of synnema growth and nucleoside production, I. cicadae synnemata were
grown in glass bottles under the optimal conditions determined by the aforementioned experiments (at
20°C under white light at an intensity of 150 lux) over a typical time course.
The synnemata began to form after being cultivated for 8 days. The maximal dry weight of
synnemata—3.304 ± 0.032 g/bottle—was achieved at day 35; this then decreased by the end of cultiva-
tion (Fig. 4). The 3 nucleosides showed different kinetics during the entire cultivation period. Guanosine
slightly increased, reached the maximum at day 30, then decreased until the end of the study. Adenosine
content kept decreasing as cultivation time increased (Fig. 4). HEA content showed the same trend as
that seen for the dry weight of synnemata, which increased with incubation time to day 35, achieved a
maximal amount of 3.657 ± 0.018 mg/g, then decreased until the study was terminated. Thus 35 days
after inoculation seems to be the best time to harvest synnemata.

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64 Liu et al.

FIG. 2: Synnema production by Isaria cicadae under light of various wavelengths

D. Comparison of Nucleoside Content between Wild and Cultivated Cicada Flowers

The wild I. cicadae samples and the I. cicadae cultivated on pupae are divided into 2 parts: synnemata and
sclerotium (Fig. 5A–C). Instead of meiotic ascospores, an asexual structure full of conidia was produced
on both the wild cicada flower and the synnemata cultivated on wheat medium (Fig. 5E and F). Nucleoside
content was compared between the wild I. cicadae and those cultivated on pupae and wheat medium.
As shown in Table 3, the amount of HEA in the synnemata was 3 times that in the sclerotium of both
the wild and the artificially cultivated samples. Comparing the 3 kinds of synnemata, HEA was highest
in the synnemata cultivated on wheat medium and was significantly higher than that in the wild cicadae
flower and in synnemata cultivated on pupae (P < 0.05). We found no similar trend for the guanosine and
adenosine contents. Wild synnemata showed the highest guanosine content, but synnemata cultivated on
wheat medium showed the highest adenosine content.

TABLE 1: Dry Weight and Nucleoside Content in Synnemata Cultivated under Colored Light

Light Dry Weight (g/bottle) Guanosine (mg/g) Adenosine (mg/g) HEA (mg/g)*
White 2.034 ± 0.111 0.479 ± 0.018 1.193 ± 0.016 2.271 ± 0.124 a
Blue 1.942 ± 0.505 0.533 ± 0.130 1.222 ± 0.046 1.368 ± 0.032 b
Green 2.040 ± 0.337 0.434 ± 0.024 1.199 ± 0.020 1.238 ± 0.003 b

Data are the mean ± SD, calculated from 3 measurements. Values within a column followed by the same letter are not sig-
nificantly different (P < 0.05, Duncan multiple range test).

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Synnema Formation and Nucleoside Production in Cicada Flower 65

FIG. 3: Effect of white light intensity on the growth of Isaria cicadae synnemata

IV. DISCUSSION

Some Cordyceps sensu lato species have been used for centuries as traditional Chinese medicines, and
the manufacturing of Cordyceps-derived products, including O. sinensis, C. militaris, and I. cicadae, is
a large industry in China,1,27 as well as in Korea and Japan. As far as we know, the study of I. cicadae
has been more superficial than that of the other 2 developed Cordyceps species. In this study we found
that temperature, light wavelength, and light intensity regulated not only the growth of synnemata of
I. cicadae but also its nucleoside production. Synnemata cultivated on wheat medium may be a potential
substitute for wild cicada flower.
A fruiting body is a multicellular structure bearing spore-producing structures such as basidia or asci.
The fruiting body forms as part of the sexual phase of the life cycle of a fungus.28 A synnema refers to
the erect macroscopic structure formed by fused conidiophores that bear conidia terminally, laterally, or
both, sometimes forming the appearance of a “sheath of wheat.”29 In contrast to other Cordyceps sensu
lato fungi, instead of producing meiotic ascospores, I. cicadae produces an asexual structure that produces

TABLE 2: Effect of White Light Intensity on Synnema Growth and Nucleoside Production

Light Intensity (lux) Dry Weight (g/bottle) Guanosine (mg/g) Adenosine (mg/g) HEA (mg/g)
850 3.095 ± 0.237 c 0.278 ± 0.013 b 0.751 ± 0.011 b 1.633 ± 0.038 a
150 3.452 ± 0.312 a 0.406 ± 0.023 a 0.674 ± 0.036 c 1.512 ± 0.034 b
50 3.304 ± 0.032 b 0.267 ± 0.006 b 0.887 ± 0.019 a 1.299 ± 0.021 c

Data are mean ± SD, calculated from 3 measurements. Values within a column followed by the same letter are not significantly
different (P < 0.05, Duncan multiple range test).

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66 Liu et al.

FIG. 4: Growth curve of Isaria cicadae cultured on wheat medium. HEA, N6-(2-hydroxyethyl)-adenosine.

A B C D

E F

FIG. 5: Synnemata and sclerotium of Isaria cicadae. (A) Wild cicada flower. (B and C) Synnemata (B) and scle-
rotia (C) of I. cicadae cultivated on pupae. (D) Synnemata of I. cicadae cultivated on wheat medium. (E and F)
Conidia on synnemata of the wild cicada flower (E) and cultivated on wheat medium (F). Scale bars = 10 μm.

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Synnema Formation and Nucleoside Production in Cicada Flower 67

TABLE 3: Nucleoside Content Comparison between the Wild and Cultivated Cicada Flower

Sample Guanosine (mg/g) Adenosine (mg/g) HEA (mg/g)

Wild synnemata 0.945 ± 0.040 a 0.781 ± 0.034 b 1.349 ± 0.209 b
Wild sclerotium 0.614 ± 0.019 b 0.674 ± 0.024 c 0.473 ± 0.003 c
Synnemata cultivated on pupae 0.479 ± 0.027 c 0.905 ± 0.038 a 1.309 ± 0.019 b
Cultivated sclerotium 0.235 ± 0.021 e 0.230 ± 0.007 d 0.429 ± 0.007 c
Synnemata cultivated on wheat medium 0.406 ± 0.023 d 0.674 ± 0.036 c 1.512 ± 0.034 a

Data are mean ± SD, calculated from 3 measurements. Values within a column followed by the same letter are not significantly
different (P < 0.05, Duncan multiple range test).

conidia (Fig. 5E and F). The sexuality of I. cicadae is still enigmatic,30 and in fact, no one has observed its
ascospore until now. Thus the term “synnema” is more suitable than “fruiting body” for this species. The
word “synnema” also has been used for other Cordyceps sensu lato species (e.g., Hirsutella gigantea31
and Paecilomyces tenuipes32).
Most studies have focused on the effect of temperature on vegetative growth and have indicated that
the optimal temperature for the growth of Cordyceps sensu lato mycelia depends on the species and strain
being grown.33–36 The optimal temperature was 15–18°C for the growth of O. sinensis mycelia,36 20°C for C.
militaris mycelia,34,37 26°C for O. jiangxiensis JXPJ0109 mycelia,35 and 28°C for P. tenuipes C240 mycelia
(an anamorph of C. takaomontana Yakush. and Kumaz.).38 The optimal growth of I. cicadae mycelia on
PDA plates occurred at 25°C, and the growth rate at 20°C was significantly lower than that at 25°C and 27°C
(P < 0.05) (Fig. 1B). A temperature of 20°C was the optimal temperature for synnema growth (Fig. 1C);
however, 25°C and 27°C were apparently not suitable for forming synnema. I. cicadae produces conidia
powder at 27°C. We found that 20°C was the optimal temperature for C. militaris—both mycelial growth
on PDA medium and fruiting body production on wheat medium. The optimal temperatures for mycelial
growth and synnema formation are very different, and the developmental mechanism is fascinating.
Another possible environmental factor regulating synnema production is light. Light has been shown
to influence fruiting body formation in many Ascomycetes from various phylogenetic groups. In this work,
light wavelength significantly influenced synnema production. Under red light, only dense mycelia accu-
mulated and neither primordia nor synnemata formed. Another study found that the fewest C. militaris
fruiting bodies formed under red light.39 Synnemata can grow under blue, green, and white light, and the
dry weight of samples grown under the various light wavelengths was not significantly different. Blue
light seems to promote conidia production and white light, HEA production.
Not only light wavelength but also light intensity affected growth and nucleoside production.
C. militaris fruiting bodies have been induced and produced at light intensities between 500 and
1000 lux,40 which are obviously different from those under which I. cicadae synnemata grow. Weak light
at 150 and 50 lux was more suitable for producing synnemata than a strong intensity of 850 lux. Under
850 lux, the synnemata produced more conidia.
HEA content showed as the same trend as the dry weight of synnemata during the entire cultivation
period and is similar to HEA produced in Beauveria bassiana under submerged culture.22 The optimal
harvesting time for I. cicadae cultivated on wheat medium is 35 days after inoculation, according to
the growth curve. Fruiting bodies of phylogenically related C. militaris usually grow in 40 days.41 The
amounts of the 3 nucleosides showed different trends during the growth period, suggesting that each has
a different biosynthesis pathway.

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68 Liu et al.

HEA was the most important nucleoside in Cordyceps,22 and HEA content in the synnemata cultivated
on wheat medium was significantly higher than that in wild samples and in synnemata cultivated on
pupae (P < 0.05). The synnemata of wild cicada flower and the mycosed cicada cadaver have been used
for many years in Asia as medicinal herbs for treating chronic kidney disease.6,42 Our results suggest that
synnemata cultivated on wheat medium might have the potential to act as a substitute for wild samples.

V. CONCLUSIONS

Environmental conditions play a crucial role in synnema formation and nucleoside production in cicada
flower, a well-known traditional medicinal mushroom. The optimal temperature for synnema formation
on wheat medium was 20°C. Synnemata can grow well under blue, green, and white light, but not under
red light. Weak white light was more suitable for producing synnemata. The optimal harvesting time for
I. cicadae cultivated on wheat medium is 35 days after inoculation. Synnemata cultivated on wheat medium
under the optimized conditions may have the potential to substitute for wild resources.

ACKNOWLEDGMENTS

This study was funded by the National Natural Science Foundation of China (Grant Nos. 31572179 and
31600054), the Coal-Based Key Scientific and Technological Project from Shanxi Province (Grant No. FT2014-
03-01), and the Key Research and Development Program from Guangxi Province (Grant No. 2016AB05317).

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Volume 21, Issue 1, 2019