Mycelial pellets formed by Penicillium thomii ZJJ were applied as efficient biosorbents for the removal of polycyclic aromatic hydrocarbons (PAHs), which are a type of ubiquitous harmful hydrophobic pollutants. The live mycelial pellets were able to remove 93.48 % of pyrene at a concentration of 100 mg/L within 48 h, demonstrating a maximum adsorption capacity of 285.63 mg/g. Meanwhile, the heat-killed one also achieved a removal rate of 65.01 %. Among the six typical PAHs (pyrene, phenanthrene, fluorene, anthracene, fluoranthene, benzo[a]pyrene), the mycelial pellets preferentially adsorbed the high molecular weight PAHs, which also have higher toxicity, resulting in higher removal efficiency. The experimental results showed that the biosorption of mycelial pellets was mainly a spontaneous physical adsorption process that occurred as a monolayer on a homogeneous surface, with mass transfer being the key rate-limiting step. The main adsorption sites on the surface of mycelia were carboxyl and N-containing groups. Extracellular polymeric substances (EPS) produced by mycelial pellets could enhance adsorption, and its coupling with dead mycelia could achieve basically the same removal effect to that of living one. It can be concluded that biosorption by mycelial pellets occurred due to the influence of electrostatic and hydrophobic interactions, consisting of five steps. Furthermore, the potential applicability of mycelial pellets has been investigated considering diverse factors. The mycelia showed high environmental tolerance, which could effectively remove pyrene across a wide range of pH and salt concentration. And pellets diameters and humic acid concentration had a significant effect on microbial adsorption effect. Based on a cost-effectiveness analysis, mycelium pellets were found to be a low-cost adsorbent. The research outcomes facilitate a thorough comprehension of the adsorption process of pyrene by mycelial pellets and their relevant applications, proposing a cost-effective method without potential environmental issues (heat-killed mycelial pellets plus EPS) to removal PAHs.
Lignosus rhinocerus (tiger milk mushroom) is an important medicinal mushroom used in Southeast Asia and China, and its sclerotium can be developed into functional food/nutraceuticals. The nutrient composition, antioxidant properties, and anti-proliferative activity of wild type and a cultivated strain of L. rhinocerus sclerotia were investigated.
Natural mycelial exopolysaccharide (EPS) and endopolysaccharide (ENS) extracted from bioreactor-cultivated European Ganoderma applanatum mushrooms are of potential high commercial value for both food and adjacent biopharmaceutical industries. In order to evaluate their potential toxicity for aquaculture application, both EPS (0.01-10 mg/mL) and ENS (0.01-10 mg/mL) extracts were tested for Zebrafish Embryo Toxicity (ZFET); early development effects on Zebrafish Embryos (ZE) were also analyzed between 24 and 120 h post-fertilization (HPF). Both EPS and ENS are considered non-toxic with LC50 of 1.41 mg/mL and 0.87 mg/mL respectively. Both EPS and ENS did not delay hatching and teratogenic defect towards ZE with <1.0 mg/mL, respectively. No significant changes in the ZE heart rate were detected following treatment with the two compounds tested (EPS: 0.01-10 mg/mL: 176.44 ± 0.77 beats/min and ENS: 0.01-10 mg/mL: 148.44 ± 17.75 beats/min) compared to normal ZE (120-180 beats/min). These initial findings support future pre-clinical trials in adult fish models with view to safely using EPS and ENS as potential feed supplements for supplements for development of the aquaculture industry.
The morphology of Ganoderma lucidum BCCM 31549 mycelium in a repeated-batch fermentation (RBF) was studied for exopolysaccharide (EPS) production. RBF was optimised for time to replace and volume to replace. G. lucidum mycelium showed the ability to self-immobilise and exhibited high stability for repeated use in RBF with engulfed pellets. Furthermore, the ovoid and starburst-like pellet morphology was disposed to EPS production in the shake flask and bioreactor, respectively. Seven RBF could be carried out in 500 mL flasks, and five repeated batches were performed in a 2 L bioreactor. Under RBF conditions, autolysis of pellet core in the shake flask and shaving off of the outer hairy region in the bioreactor were observed at the later stages of RBF (R4 for the shake flask and R6 for the bioreactor). The proposed strategy showed that the morphology of G. lucidum mycelium can withstand extended fermentation cycles.
Hydrophobins are small secreted fungal proteins that play roles in growth and development of filamentous fungi, i.e. in the formation of aerial structures and the attachment of hyphae to hydrophobic surfaces. In Botrytis cinerea, three hydrophobin genes have been identified. Studies by Mosbach et al. (2011) showed that hydrophobins are neither involved in conferring surface hydrophobicity to conidia and aerial hyphae of B. cinerea, nor are they required for virulence. The present study investigated the role of hydrophobins in sclerotium and apothecium development. Expression analysis revealed high expression of the Bhp1 gene during different stages of apothecium development. Two Bhp1 splice variants were detected that differ by an internal stretch of 13 amino acid residues. Seven different mutants in which either a single, two or three hydrophobin genes were knocked out, as well as two wild type strains of opposite mating types, were characterized for sclerotium and apothecium development. No aberrant morphology was observed in sclerotium development when single deletion mutants in hydrophobin genes were analyzed. Sclerotia of double knock out mutant ΔBhp1/ΔBhp3 and the triple knock out mutant, however, showed easily wettable phenotypes. For analyzing apothecium development, a reciprocal crossing scheme was setup. Morphological aberrations were observed in crosses with two hydrophobin mutants. When the double knock out mutant ΔBhp1/ΔBhp2 and the triple knock out mutant were used as the maternal parent (sclerotia), and fertilized with wild type microconidia, the resulting apothecia were swollen, dark brown in color and had a blotched surface. After initially growing upwards toward the light source, the apothecia in many cases collapsed due to loss of structural integrity. Aberrant apothecium development was not observed in the reciprocal cross, when these same mutants were used as the paternal parent (microconidia). These results indicate that the presence of hydrophobins in maternal tissue is important for normal development of apothecia of B. cinerea.
Termitomyces are delicious edible mushrooms found in Africa and South-East Asia including Malaysia. These mushrooms were found to grow symbiotically with termites around termite nests. Numerous efforts have been made worldwide to develop a cultivation method for these mushrooms. Unfortunately, none of those attempts were successful. The main obstacles encountered were the difficulty to identify and isolate pure termitomyces culture. The problem became prevalent as the culture gets contaminated by other fungi. Termitomyces can easily be identified by its mushroom fruiting body eventually but certainly not at the mycelium and hyphea stages. In this study a simple PCR-based genetic marker detection method for confirmation of termitomyces at any culture stage was developed. Using this method, four distinctive PCR assays
were developed using specific PCR primers designed based on the DNA sequence of the termitomyces mushroom. The PCR results showed that the PCR assays using intact termitomyces
DNA as template was not suitable for this purpose. However, PCR using BamHI and EcoRI predigested termitomyces DNA as template showed identical polymorphism pattern for both
termitomyces mushroom DNA and termitomyces culture DNA. Thus, the method reported here can be used for the identific.
Mushroom cultivation benefits humankind as it deliberately encourages wild mushrooms to be commercially propagated while recycling agricultural wastes. Ganoderma neo-japonicum is a rare polypore mushroom found growing on decaying Schizostachyum brachycladium (a tropical bamboo) clumps in Malaysia. The Malaysian indigenous tribes including the Temuans and Temiars use the basidiocarps of G. neo-japonicum to treat various ailments including diabetes. In this study, the domestication of G. neo-japonicum in artificial logs of different agricultural residues was investigated. Sawdust promoted the mycelia spawn colonisation in the shortest period of 38 ± 0.5 days. However, only sawdust and bamboo dust supported the primodia formation. Complex medium supported mycelium growth in submerged cultures and 27.11 ± 0.43 g/L of mycelia was obtained after 2 weeks of cultivation at 28 °C and 200 rpm. Antioxidant potential in mushroom may be influenced by different cultivation and extraction methods. The different extracts from the wild and cultivated basidiocarps as well as mycelia were then tested for their antioxidant properties. Aqueous and ethanol extracts of mycelia and basidiocarps tested had varying levels of antioxidant activities. To conclude, domestication of wild G. neo-japonicum using agroresidues may ensure a continuous supply of G. neo-japonicum for its medicinal use while ensuring the conservation of this rare species.
Ganoderma neo-japonicum is an annual polypore that grows on decaying bamboo in the forests of Malaysia. The indigenous Temuan tribe uses this species as a medicinal mushroom to cure fever and epilepsy and to improve body strength. The potential use of G. neo-japonicum in genoprotection and DNA repair was established using a single-cell gel electrophoresis (comet) assay. The effects of the ethanol and hot aqueous extracts from wild and cultivated basidiocarps, solid substrate-fermented (SSF) wheat grains, and mycelia via submerged culture on H2O2-damaged murine RAW264.7 macrophages were investigated. An ethanol extract from wild basidiocarps showed the most significant protective effect on murine RAW264.7 macrophages, followed by ethanol and hot water extracts of cultivated basidiocarps, and this effect was dose dependent. However, only the ethanol extracts from SSF and submerged culture showed significant protective effects compared with the control. As for DNA repair ability, only the ethanol extract from wild and cultivated basidiocarps showed significant results when compared with the negative control. The findings suggest the potential therapeutic use of G. neo-japonicum in genome protection and as a DNA repair stimulator.
Rigorous research has been carried out regarding the cultivation of Ganoderma lucidum using different agricultural residues. Nevertheless, large-scale cultivation and the separation of active compounds of G. lucidum are still challenges for local farmers. The objective of this study was to evaluate the use of oil palm waste fibers such as empty fruit bunch fibers and mesocarp fibers as effective substrates for the growth of G. lucidum mycelia to study the possibility of solid-state cultivation and to determine the optimum conditions necessary for the growth of mycelia of this mushroom on these waste fibers. Various parameters such as temperature, pH, humidity, and carbon and nitrogen compositions required for the optimum growth of mycelia have been determined. Oil palm fibers are a vivid source of lignocellulose, and their availability in Malaysia is high compared to that of sawdust. G. lucidum is a wood-rotting fungi that can easily decay and utilize this lignocellulose biomass, a major agricultural waste in Malaysia.
Species of the genus Ganoderma are a cosmopolitan wood decaying white rot fungi, which has been used by the Asians for therapeutic purposes for centuries. In the present study, solid-substrate fermentation (SSF) of wheat grains (Triticum aestivum L.) was carried out with indigenous Ganoderma australe (KUM60813) and G. neo-japonicum (KUM61076) selected based on ethnomycological knowledge. G. lucidum (VITA GL) (a commercial strain) was also included in the study. Antioxidant activities of the crude ethanol and aqueous extracts of the fermented and unfermented wheat grains were investigated by ferric reducing antioxidant power (FRAP), Trolox equivalent antioxidant capacity (TEAC), diphenyl-1-picryl-hydrazyl (DPPH) free radical scavenging ability, and lipid peroxidation assay. Among the six mycelia extracts tested, the ethanol extract from wheat fermented with KUM61076 mycelia showed the most potent antioxidant activities, whereas the ethanol extract of wheat grains fermented with KUM60813 mycelia has a good potential in protecting frying oils against oxidation. Total phenolic content (TPC) in the ethanol extracts were higher than that in the aqueous extract. The wheat grains fermented with G. australe (KUM60813) and G. neo-japonicum KUM61076 have greater antioxidant potential compared to the commercially available G. lucidum (VITA GL). The antioxidant activities of the mycelia extracts had a positive correlation with their phenolic contents. Thus phenolic compounds may play a vital role in the antioxidant activities of the selected Ganoderma spp.
Isozyme and protein electrophoresis data from mycelial extracts of 27 isolates of Trichoderma harzianum, 10 isolates of T. aureoviride and 10 isolates of T. longibrachiatum from Southern Peninsular Malaysia were investigated. The eight enzyme and a single protein pattern systems were analyzed. Three isozyme and total protein patterns were shown to be useful for the detection of three Trichoderma species. The isozyme and protein data were analyzed using the Nei and Li Dice similarity coefficient for pairwise comparison between individual isolates, species isolate group, and for generating a distance matrix. The UPGMA cluster analysis showed a higher degree of relationship between T. harzianum and T. aureoviride than to T. longibrachiatum. These results suggested that the T. harzianum isolates had high levels of genetic variation compared to the other isolates of Trichoderma species.
In Malaysia and China, the sclerotium of Lignosus rhinocerotis is used by local communities and traditional medicine practitioners as a general tonic and remedy to treat a variety of ailments, including inflammation-associated disorders. In this study, 10 samples from different preparations of L. rhinocerotis sclerotium, including a hot aqueous extract (HAE), an ethanol extract (EE), fractions from the HAE and EE, and crude polysaccharides, were tested for their in vitro cytotoxic and nitric oxide (NO) inhibitory activities in lipopolysaccharide (LPS)--stimulated BV2 microglia. Of the 10 samples tested, HAE was the least cytotoxic toward BV2 microglia, with a half-maximal inhibitory concentration of 176.23 ± 2.64 mg/mL at 24 hours of incubation and 20.01 ± 1.69 mg/ mL at 48 hours of incubation. The inhibition of NO production was explored by pretreatment of BV2 microglia with samples at 2 incubation time points (4 and 24 hours) before the stimulation by LPS for 24 hours. After 24 hours of pretreatment, 8 of the 10 samples inhibited NO production by 50% or more, and cytotoxic effects were not observed. Among the 8 active samples, 500 µg/mL of HAE, 250 µg/mL of an n-butanol fraction of the HAE, and 250 µg/mL of an ethyl acetate fraction of HAE showed maximum inhibition of NO production by 88.95%, 86.50%, and 85.93%, respectively. These results suggest that the L. rhinocerotis sclerotium may contain secondary metabolites that have the potential to inhibit NO production.
Microwave vacuum pyrolysis of palm kernel shell (PKS) was performed to produce biochar, which was then tested as bio-fertilizer in growing Oyster mushroom (Pleurotus ostreatus). The pyrolysis approach produced biochar containing a highly porous structure with a high BET surface area of up to 270m2/g and low moisture content (≤10wt%), exhibiting desirable adsorption properties to be used as bio-fertilizer since it can act as a housing that provides many sites on which living microorganisms (mycelium or plant-growth promoting bacteria) and organic nutrients can be attached or adsorbed onto. This could in turn stimulate plant growth by increasing the availability and supply of nutrients to the targeted host plant. The results from growing Oyster mushroom using the biochar recorded an impressive growth rate and a monthly production of up to about 550g of mushroom. A shorter time for mycelium growth on one whole baglog (21days) and the highest yield of Oyster mushroom (550g) were obtained from cultivation medium added with 20g of biochar. Our results demonstrate that the biochar-based bio-fertilizer produced from microwave vacuum pyrolysis of PKS shows exceptional promise as growth promoting material for mushroom cultivation.
The first and most crucial step of all molecular techniques is to isolate high quality and intact nucleic acids. However, DNA and RNA isolation from fungal samples are usually difficult due to the cell walls that are relatively unsusceptible to lysis and often resistant to traditional extraction procedures. Although there are many extraction protocols for Ganoderma species, different extraction protocols have been applied to different species to obtain high yields of good quality nucleic acids, especially for genome and transcriptome sequencing. Ganoderma species, mainly G. boninense causes the basal stem rot disease, a devastating disease that plagues the oil palm industry. Here, we describe modified DNA extraction protocols for G. boninense, G. miniatocinctum and G. tornatum, and an RNA extraction protocol for G. boninense. The modified salting out DNA extraction protocol is suitable for G. boninense and G. miniatocinctum while the modified high salt and low pH protocol is suitable for G. tornatum. The modified DNA and RNA extraction protocols were able to produce high quality genomic DNA and total RNA of ~ 140 to 160 µg/g and ~ 80 µg/g of mycelia respectively, for Single Molecule Real Time (PacBio Sequel® System) and Illumina sequencing. These protocols will benefit those studying the oil palm pathogens at nucleotide level.
The fluctuation of domestic wastewater characteristic inhibits the current conventional microbial-based treatment. The bioremediation fungi has received attention and reported to be an effective alternative to treat industrial wastewater. Similar efficient performance is envisaged for domestic wastewater whereby assessed performance of fungi for varying carbon-to-nitrogen ratios in domestic wastewater is crucial. Thus, the performance of pre-grown wild-Serbian Ganoderma lucidum mycelial pellets (GLMPs) was evaluated on four different synthetic domestic wastewaters under different conditions of initial pH (pH 4, 5, and 7) and chemical oxygen demand (COD) to nitrogen (COD/N) ratio of 3.6:1, 7.1:1, 14.2:1, and 17.8:1 (C3.6N1, C7.1N1, C14.2N1, and C17.8N1). The COD/N ratios with a constant concentration of ammonia-nitrogen (NH3-N) were chosen on the basis of the urban domestic wastewater characteristics sampled at the inlet basin of a sewage treatment plant (STP). The parameters of pH, COD, and NH3-N were measured periodically during the experiment. The wild-Serbian GLMPs efficiently removed the pollutants from the synthetic sewage. The COD/N ratio of C17.8N1 wastewater had the best COD and NH3-N removal, as compared to the lower COD/N ratio, and the shortest treatment time was obtained in an acidic environment at pH 4. The highest percentage for COD and NH3-N removal achieved was 96.0% and 93.2%, respectively. The results proved that the mycelium of GLMP has high potential in treating domestic wastewater, particularly at high organic content as a naturally sustainable bioremediation system.
Ganoderma lucidum has been purported as a potent remedy in the treatment and prevention of several ailments, including hypertension. This study aimed to explore the anti-ACE potential of protein fractions from the mycelia of G. lucidum.
Mycelium-bound lipase (MBL), from a locally isolated Geotrichum candidum strain, was produced and characterized as a natural immobilized lipase. A time course study of its lipolytic activity in 1 L liquid broth revealed the maximum MBL activity at 4 h for mycelium cells harvested after 54 h. The yield and specific activity of MBL were 3.87 g/L dry weight and 508.33 U/g protein, respectively, while less than 0.2 U/mL lipase activity was detected in the culture supernatant. Prolonged incubation caused release of the bound lipase into the growth medium. The growth pattern of G. candidum, and production and properties of MBL were not affected by the scale. The stability of mycelia harboring lipase (MBL), harvested and lyophilized after 54 h, studied at 4 °C depicted a loss of 4.3% and 30% in MBL activity after 1 and 8 months, while the activity of free lipase was totally lost after 14 days of storage. The MBL from G. candidum displayed high substrate selectivity for unsaturated fatty acids containing a cis-9 double bond, even in crude form. This unique specificity of MBL could be a direct, simple and inexpensive way in the fats and oil industry for the selective hydrolysis or transesterification of cis-9 fatty acid residues in natural triacylglycerols.
Mycelium-bound lipase (MBL) was prepared using a strain of Geotrichum candidum isolated from local soil. At the time of maximum lipase activity (54 h), the mycelia to which the lipase was bound were harvested by filtration and centrifugation. Dry MBL was prepared by lyophilizing the mycelia obtained. The yield of MBL was 3.66 g/l with a protein content of 44.11 mg/g. The lipase activity and specific lipase activity were 22.59 and 510 U/g protein, respectively. The moisture content of the MBL was 3.85%. The activity of free (extracellular) lipase in the culture supernatant (after removal of mycelia) was less than 0.2 U/ml. The MBL showed selectivity for oleic acid over palmitic acid during hydrolysis of palm olein, indicating that the lipase from G. candidum displayed high substrate selectivity for unsaturated fatty acid containing a cis-9 double bond, even in crude form. This unique specificity of MBL could be a direct, simple and inexpensive way in the fats and oil industry for the selective hydrolysis or transesterification of cis-9 fatty acid residues in natural triacylglycerols.
The sclerotium of Lignosus rhinocerotis (Cooke) Ryvarden (Tiger Milk mushroom) is used as a traditional medicine to relieve cough, asthma and chronic hepatitis. The traditional uses of the sclerotium are presumably related to its anti-inflammatory effect. The present study was carried out to evaluate the anti-inflammatory activity of the sclerotial powder of L. rhinocerotis (Cooke) Ryvarden (Tiger Milk mushroom) cultivar TM02.
Previous studies on the nutritional and nutraceutical properties of Lignosus rhinocerotis focused mainly on the sclerotium; however, the supply of wild sclerotium is limited. In this investigation, the antioxidant capacity and cytotoxic effect of L. rhinocerotis cultured under different conditions of liquid fermentation (shaken and static) were compared to the sclerotium produced by solid-substrate fermentation. Aqueous methanol extracts of the mycelium (LR-MH, LR-MT) and culture broth (LR-BH, LR-BT) demonstrated either higher or comparable antioxidant capacities to the sclerotium extract (LR-SC) based on their radical scavenging abilities, reducing properties, metal chelating activities, and inhibitory effects on lipid peroxidation. All extracts exerted low cytotoxicity (IC50>200 µg/ml, 72 h) against selected mammalian cell lines. Several low-molecular-weight compounds, including sugars, fatty acids, methyl esters, sterols, amides, amino acids, phenolics, and triterpenoids, were identified using GC-MS and UHPLC-ESI-MS/MS. The presence of proteins (<40 kDa) in the extracts was confirmed by SDS-PAGE and SELDI-TOF-MS. Principal component analysis revealed that the chemical profiles of the mycelial extracts under shaken and static conditions were distinct from those of the sclerotium. Results from bioactivity evaluation and chemical profiling showed that L. rhinocerotis from liquid fermentation merits consideration as an alternative source of functional ingredients and potential substitute for the sclerotium.