Archives of Microbiology (2023) 205:34
https://doi.org/10.1007/s00203-022-03297-8
ORIGINAL PAPER
The earth‑star basidiomycetous mushroom Astraeus odoratus
produces polyhydroxyalkanoates during cultivation on malt extract
Dau Hung Anh1,2 · Kanchana Dumri1,2,3 · Le Thi Hoang Yen4 · Winita Punyodom1,3
Received: 15 December 2021 / Revised: 20 May 2022 / Accepted: 19 October 2022 / Published online: 21 December 2022
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022
Abstract
Polyhydroxyalkanoates (PHAs) including poly-3-hydroxybutyrate (P3HB) as secondary metabolisms were in vitro produced
by the edible basidiomycetous mushroom Astraeus odoratus during its growth on malt agar extract. Various carbon and
nitrogen sources containing cellulose, glucose, glycerol, rice straw powder, soybean meal and peptone were investigated for
the growth of basidiomycetous mushrooms. During cultivation, the A. odoratus culture exudated the considerably extracel-
lular fluid up to approx. 2.3 ml on 2% malt extract agar plate within 7 days. The chemical compounds of the exudated fluid
were further investigated by Fourier-transform infrared spectroscopy (FTIR) and gas chromatography/mass spectrometry
(GC/MS); and its morphology of the lyophilized sample was observed by scanning electron microscope (SEM). FTIR results
showed the characteristic bands of OH at 3445 cm−1, CH/CH2/symmetric CH3 (stretch) at 2923 and 2852 cm−1, C=O at
1730 cm−1, asymmetric CH3 (bend) at 1454 and 1414 cm−1, C−O of COO– at 1396 cm−1 and C–O–C at 1223, 1160, 1116,
1058 and 1019 cm−1 which were similar to the absorptive characteristics of P3HB. Methyl ester derivatives of GC/MS results
identified 7 compounds including: 3-hydroxybutanoic (monomer of PHB), aminobenzoic, salicylic, hexadecenoic, octadeca-
dienoic, octadecenoic and octadecanoic acids. SEM images revealed a fibriform and porous materials. Hence, the occurrence
of PHAs was first described in a basidiomycetous mushroom A. odoratus. Thus, PHAs could be found not only in bacteria
and but also in basidiomycetous mushroom, which can be promising target for bioplastics and green environmental studies.
Keywords Basidiomycetes · Astraeus odoratus · Poly-3-hydroxybutyrate · Malt extract · Extracellular fluid
Introduction resource” by Rudin and Choi 2013. Bioplastics or biode-
gradable materials can be classified into agriculture-derived
Bioplastics was early defined in last decade as “A bioplas- polymers e.g. cellulose, starch, chitin and bio-polyesters, e.g.
tic can be defined as a polymer that is manufactured into polyhydroxyalkanoates (PHAs) and polylactic acid (Dilshad
a commercial product from a natural source or renewable et al. 2021). Among these compounds, PHAs have found
many applications in pharmaceutics (Williams and Martin
2005), agriculture (Amelia et al. 2019), bio-refinery (Riaz
Communicated by Erko Stackebrandt. et al. 2021), construction industry and packaging indus-
try (Poltronieri and Kumar 2017). The PHA polymers in
* Winita Punyodom nature consist of poly(3-hydroxybutyrate) (PHB), poly(3-
hydroxyvalerate) (PHV) and poly(3-hydroxybutyrate-co-
1
Center of Excellence in Materials Science and Technology, 3-hydroxyvalerate) (PHBV). The finding and production of
Chiang Mai University, Chiang Mai 50200, Thailand those compounds to synthesise massive PHAs have been
2
Biogreen Material Research & Service Part., Ltd., carried out worldwide on bacteria (Poirier et al. 1995; Zinn
Chiang Mai 50140, Thailand et al. 2001), recombinant bacteria (Khanna and Srivastava
3
Department of Chemistry, Faculty of Science, Chiang 2005; Suriyamongkol et al. 2007), transgenic plants (Dobro-
Mai University, 239 Huay Keaw Road, Suthep, gojski et al. 2018) and insects (Williams et al. 1998).
Chiang Mai 50200, Thailand Astraeus odoratus (hed-Thawp in Thai), an ectomycor-
4
Laboratory of Fungi Technology, Institute of Microbiology rhizal fungi and a wild-edible basidiomycetous mushroom in
and Biotechnology, Vietnam National University, Hanoi, Chiang Mai is found in areas where sal tree Shorea robusta
Vietnam
13
Vol.:(0123456789)
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
, 34 Page 2 of 8 Archives of Microbiology (2023) 205:34
grows. Local farmers used to burn the forest to collect A.
odoratus from March to May. And that leads to forest fire
and smoking season in Chiang Mai every year. Therefore,
this work was established to study the growth of A. odo-
ratus indoor. During the growth of A. odoratus on malt
extract agar, an extreme extracellular guttation occurred
quite extremely in comparison to other experimental mush-
room strains. We characterised this biological behaviour
and the chemical composition of the secrete and detected
the presence of P3HB—the most significant composition
for bioplastics. Herein, we described the investigation and
characterisation of P3HB from A. odoratus as the first eukar-
yotic basidiomycetous fungi which produces P3HB under
laboratorial cultivation.
Fig. 1 The earth-star mushroom Astraeus odoratus (Hed-thawp in
Thai) collected in Hod district, Chiang Mai province, Thailand. (Col-
lected and photographed by Dau Hung Anh & Kanchana Dumri in
Materials and methods May 2018)
Chemicals and fungal media components
glycerol, 2% cellulose, 2% glucose, 2% rice straw for the
The 3-hydroxybutyric acid (3-HB) and poly(3-hydroxy- fungal growth depending selected carbon sources cultures.
butyrate) (PHB) were obtained from Sigma-Aldrich (Ger- The 3% soybean meal, 3% glucose and 1% peptone from
many). Other chemicals were of analytical grade and for soybean meal agar as different nitrogen-providing sources
biological uses. Malt extractagar–agar (MEA) and cellulose were also made for A. odoratus cultivation. All cultures
powder were purchased from Merck (Germany). Rice straw were kept at room temperature and observed by naked eye.
(straw) of jasmine rice field in Chiang Mai was milled to The fungal mycelium development was observed under
powder prior to use. Soybean meal and peptone were bio- light microscope (CX23, Olympus, Japan).
logical grade products of Thailand.
Collection and isolation of edible earth‑star Characterisation of chemical compounds of fungal
mushroom A. odoratus extracellular fluid and mycelium extract using
Fourier Transform Infrared (FTIR) spectroscopy
The living unopened gasterocarp (description following
Nouhra and De Toledo 1998) of the mushroom (∅ 4–6 cm Preparation of liquid samples from fungal extracellular
and weight from 7 to 12 gr, white colour) was excavated in fluid and mycelial extract
a familial succeeding “hidden” mountainous areas in Hod
district of Chiang Mai city (North of Thailand) in May 2018 The fungal extracellular fluid was collected by glassy pipet
(Fig. 1) during annual harvesting season. Specimens were and filled in 2 ml glass vial. FTIR sample was prepared in
sliced into 1 mm thin pad and washed with distilled water liquid form. Approx. 0.5 ml of the fluid was mixed in 1 ml
and 70% ethanol, and then placed on 2% (w/v) malt extract of 70% chloroform, vortexed gently and heated at 70 °C for
agar plate (1 litter of liquid medium contained 50 mg chlo- 15 min. Afterwards, sample was vortexed vigorously for
ramphenicol, 50 mg penicillium, 50 mg streptomycin, 40 mg 5 min and centrifuged at 4000 rpm for 10 min at room tem-
nystatin and 50 mg benomyl, pH 5.5) for isolation. Growing perature and supernatant was collected for FTIR analysis.
mycelium on isolating agar plate was picked and inoculated Each 1 cm2 of 7 days of mycelium was collected by pin-
on 2% malt extract agar (MEA) at 25 °C in culture slant tube cer, sprayed and rinsed in distilled H2O, 70% ethanol and
for 1 week and stored at 4 °C in the dark as stock culture. saline solution to remove cultural medium debris. After-
The stock isolated A. odoratus strain was precultured on wards, it was frozen and ground in liquid nitrogen to mild
MEA for 7 days prior to use. powder using pre-cooled mortar and pestle and dried at
room temperature for 20 min. The latter was subsequently
The establishment of A. odoratus mycelium treated with hot chloroform in similar protocol for the fun-
gal extracellular fluid sample as described above.
Fungal cultures were grown on MEA (1, 2, 4 and 6%);
2% MEA with additional 2% (w/v) of glycerol, 2% (w/v)
13
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
https://doi.org/10.1007/s00203-022-03297-8
ORIGINAL PAPER
The earth‑star basidiomycetous mushroom Astraeus odoratus
produces polyhydroxyalkanoates during cultivation on malt extract
Dau Hung Anh1,2 · Kanchana Dumri1,2,3 · Le Thi Hoang Yen4 · Winita Punyodom1,3
Received: 15 December 2021 / Revised: 20 May 2022 / Accepted: 19 October 2022 / Published online: 21 December 2022
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022
Abstract
Polyhydroxyalkanoates (PHAs) including poly-3-hydroxybutyrate (P3HB) as secondary metabolisms were in vitro produced
by the edible basidiomycetous mushroom Astraeus odoratus during its growth on malt agar extract. Various carbon and
nitrogen sources containing cellulose, glucose, glycerol, rice straw powder, soybean meal and peptone were investigated for
the growth of basidiomycetous mushrooms. During cultivation, the A. odoratus culture exudated the considerably extracel-
lular fluid up to approx. 2.3 ml on 2% malt extract agar plate within 7 days. The chemical compounds of the exudated fluid
were further investigated by Fourier-transform infrared spectroscopy (FTIR) and gas chromatography/mass spectrometry
(GC/MS); and its morphology of the lyophilized sample was observed by scanning electron microscope (SEM). FTIR results
showed the characteristic bands of OH at 3445 cm−1, CH/CH2/symmetric CH3 (stretch) at 2923 and 2852 cm−1, C=O at
1730 cm−1, asymmetric CH3 (bend) at 1454 and 1414 cm−1, C−O of COO– at 1396 cm−1 and C–O–C at 1223, 1160, 1116,
1058 and 1019 cm−1 which were similar to the absorptive characteristics of P3HB. Methyl ester derivatives of GC/MS results
identified 7 compounds including: 3-hydroxybutanoic (monomer of PHB), aminobenzoic, salicylic, hexadecenoic, octadeca-
dienoic, octadecenoic and octadecanoic acids. SEM images revealed a fibriform and porous materials. Hence, the occurrence
of PHAs was first described in a basidiomycetous mushroom A. odoratus. Thus, PHAs could be found not only in bacteria
and but also in basidiomycetous mushroom, which can be promising target for bioplastics and green environmental studies.
Keywords Basidiomycetes · Astraeus odoratus · Poly-3-hydroxybutyrate · Malt extract · Extracellular fluid
Introduction resource” by Rudin and Choi 2013. Bioplastics or biode-
gradable materials can be classified into agriculture-derived
Bioplastics was early defined in last decade as “A bioplas- polymers e.g. cellulose, starch, chitin and bio-polyesters, e.g.
tic can be defined as a polymer that is manufactured into polyhydroxyalkanoates (PHAs) and polylactic acid (Dilshad
a commercial product from a natural source or renewable et al. 2021). Among these compounds, PHAs have found
many applications in pharmaceutics (Williams and Martin
2005), agriculture (Amelia et al. 2019), bio-refinery (Riaz
Communicated by Erko Stackebrandt. et al. 2021), construction industry and packaging indus-
try (Poltronieri and Kumar 2017). The PHA polymers in
* Winita Punyodom nature consist of poly(3-hydroxybutyrate) (PHB), poly(3-
hydroxyvalerate) (PHV) and poly(3-hydroxybutyrate-co-
1
Center of Excellence in Materials Science and Technology, 3-hydroxyvalerate) (PHBV). The finding and production of
Chiang Mai University, Chiang Mai 50200, Thailand those compounds to synthesise massive PHAs have been
2
Biogreen Material Research & Service Part., Ltd., carried out worldwide on bacteria (Poirier et al. 1995; Zinn
Chiang Mai 50140, Thailand et al. 2001), recombinant bacteria (Khanna and Srivastava
3
Department of Chemistry, Faculty of Science, Chiang 2005; Suriyamongkol et al. 2007), transgenic plants (Dobro-
Mai University, 239 Huay Keaw Road, Suthep, gojski et al. 2018) and insects (Williams et al. 1998).
Chiang Mai 50200, Thailand Astraeus odoratus (hed-Thawp in Thai), an ectomycor-
4
Laboratory of Fungi Technology, Institute of Microbiology rhizal fungi and a wild-edible basidiomycetous mushroom in
and Biotechnology, Vietnam National University, Hanoi, Chiang Mai is found in areas where sal tree Shorea robusta
Vietnam
13
Vol.:(0123456789)
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
, 34 Page 2 of 8 Archives of Microbiology (2023) 205:34
grows. Local farmers used to burn the forest to collect A.
odoratus from March to May. And that leads to forest fire
and smoking season in Chiang Mai every year. Therefore,
this work was established to study the growth of A. odo-
ratus indoor. During the growth of A. odoratus on malt
extract agar, an extreme extracellular guttation occurred
quite extremely in comparison to other experimental mush-
room strains. We characterised this biological behaviour
and the chemical composition of the secrete and detected
the presence of P3HB—the most significant composition
for bioplastics. Herein, we described the investigation and
characterisation of P3HB from A. odoratus as the first eukar-
yotic basidiomycetous fungi which produces P3HB under
laboratorial cultivation.
Fig. 1 The earth-star mushroom Astraeus odoratus (Hed-thawp in
Thai) collected in Hod district, Chiang Mai province, Thailand. (Col-
lected and photographed by Dau Hung Anh & Kanchana Dumri in
Materials and methods May 2018)
Chemicals and fungal media components
glycerol, 2% cellulose, 2% glucose, 2% rice straw for the
The 3-hydroxybutyric acid (3-HB) and poly(3-hydroxy- fungal growth depending selected carbon sources cultures.
butyrate) (PHB) were obtained from Sigma-Aldrich (Ger- The 3% soybean meal, 3% glucose and 1% peptone from
many). Other chemicals were of analytical grade and for soybean meal agar as different nitrogen-providing sources
biological uses. Malt extractagar–agar (MEA) and cellulose were also made for A. odoratus cultivation. All cultures
powder were purchased from Merck (Germany). Rice straw were kept at room temperature and observed by naked eye.
(straw) of jasmine rice field in Chiang Mai was milled to The fungal mycelium development was observed under
powder prior to use. Soybean meal and peptone were bio- light microscope (CX23, Olympus, Japan).
logical grade products of Thailand.
Collection and isolation of edible earth‑star Characterisation of chemical compounds of fungal
mushroom A. odoratus extracellular fluid and mycelium extract using
Fourier Transform Infrared (FTIR) spectroscopy
The living unopened gasterocarp (description following
Nouhra and De Toledo 1998) of the mushroom (∅ 4–6 cm Preparation of liquid samples from fungal extracellular
and weight from 7 to 12 gr, white colour) was excavated in fluid and mycelial extract
a familial succeeding “hidden” mountainous areas in Hod
district of Chiang Mai city (North of Thailand) in May 2018 The fungal extracellular fluid was collected by glassy pipet
(Fig. 1) during annual harvesting season. Specimens were and filled in 2 ml glass vial. FTIR sample was prepared in
sliced into 1 mm thin pad and washed with distilled water liquid form. Approx. 0.5 ml of the fluid was mixed in 1 ml
and 70% ethanol, and then placed on 2% (w/v) malt extract of 70% chloroform, vortexed gently and heated at 70 °C for
agar plate (1 litter of liquid medium contained 50 mg chlo- 15 min. Afterwards, sample was vortexed vigorously for
ramphenicol, 50 mg penicillium, 50 mg streptomycin, 40 mg 5 min and centrifuged at 4000 rpm for 10 min at room tem-
nystatin and 50 mg benomyl, pH 5.5) for isolation. Growing perature and supernatant was collected for FTIR analysis.
mycelium on isolating agar plate was picked and inoculated Each 1 cm2 of 7 days of mycelium was collected by pin-
on 2% malt extract agar (MEA) at 25 °C in culture slant tube cer, sprayed and rinsed in distilled H2O, 70% ethanol and
for 1 week and stored at 4 °C in the dark as stock culture. saline solution to remove cultural medium debris. After-
The stock isolated A. odoratus strain was precultured on wards, it was frozen and ground in liquid nitrogen to mild
MEA for 7 days prior to use. powder using pre-cooled mortar and pestle and dried at
room temperature for 20 min. The latter was subsequently
The establishment of A. odoratus mycelium treated with hot chloroform in similar protocol for the fun-
gal extracellular fluid sample as described above.
Fungal cultures were grown on MEA (1, 2, 4 and 6%);
2% MEA with additional 2% (w/v) of glycerol, 2% (w/v)
13
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
