A novel Ganoderma lucidum G0119 fermentation strategy for enhanced triterpenes production by statistical process optimization and addition of oleic acid

A novel enhanced triterpenes fermentation production process by Ganoderma lucidum G0119 with the addition of oleic acid in the medium has been developed and optimized. All of the six exogenous additives tested were found to exhibit stimulatory effect on mycelial growth and triterpenes biosynthesis by G. lucidum. The results show that oleic acid addition had significant role in promoting triterpenes production. The optimal concentration and time of oleic acid addition were determined to be 30 mL/L and 0 h, respectively. Furthermore, three significant factors influencing triterpenes production were identified as glucose, magnesium sulfate and temperature using the Plackett–Burman design. The optimized conditions by central composite design were 27.83 g/L glucose, 1.32 g/L magnesium sulfate, 26.2°C temperature. The triterpenes fermentation yield with the optimized medium based on actual confirmatory experimental data in 6 L fermentor was 1.076 g/L versus the statistical model predicted value of 1.080 g/L. Our innovatively developed triterpenes fermentation production technology and process has been proven to produce high triterpenes productivity and yield conceivably useful for industrial production.     

Optimization of culture conditions and continuous production of chitosan by the fungi,Absidia coerulea

The production of chitosan from the mycelia ofAbsidia coerulea was studied to improve cell growth and chitosan productivity. Culture conditions were optimized in batch cultivation (pH 4.5 agitator speed of 250 rpm, and aeration rate of, 2 vvm) and the maximum chitosan concentration achieved was 2.3 g/L under optimized conditions. Continuous culture was carried out successfully by the formation of new growth spots under optimized conditions, with a chitosan productivity of 0.052 gL⁻¹ h⁻¹, which is the highest value to date, and was obtained at a dilution rate of 0.05 h⁻¹. Cell chitosan concentrations reached about 14% in the steady state, which is similar to that achieved in batch culture. This study shows that for the continuous culture ofAbsidia coerulea it is vital to control the medium composition.     

Optimization and characterization of pullulan production by a newly isolated high-yielding strain Aureobasidium melanogenum

Abstract

Pullulan is an extracellular water-soluble polysaccharide with wide applications. In this study, we screened strains that could selectively produce high molecular weight pullulan for application in industrial pullulan production. A new fungus strain A4 was isolated from soil and identified as Aureobasidium melanogenum based on colony characteristics, morphology, and internally transcribed spacer analysis. Thin-layer chromatography, Fourier-transform infrared spectroscopy, and nuclear magnetic resonance analysis suggested that the dominant exopolysaccharide produced by this strain, which presented a molecular weight of 1.384?×?10⁶ Dalton in in-gel permeation chromatography, was pullulan. The culture conditions for A. melanogenum A4 were optimized at 30?°C and 180?rpm: carbon source, 50?g/L maltose; initial pH 7; and 8?g/L Tween 80. Subsequently, batch fermentation was performed under the optimized conditions in a 5-L stirred-tank fermentor with a working volume of 3?L. The fermentation broth contained 303?g/L maltose, which produced 122.34?g/L pullulan with an average productivity of 1.0195?g/L/h and 82.32?g/L dry biomass within 120?h. The conversion efficiency of maltose to pullulan (Y%) and specific production rate (g/h/g dry cells) (Qs) reached 40.3% and 0.0251?g/L/g dry cells, respectively. The results showed strain A4 could be a good candidate for industrial production.     

Effects of microbial volatile organic compounds on Ganoderma lucidum growth and ganoderic acids production in Co-v-cultures (volatile co-cultures)

Abstract

Co-v-culture (co-cultivations of physically separated microbes that only interact through the air) systems were designed to investigate the effects of microbial volatile organic compounds (mVOCs) from about 20 different microbes, on a medicinal fungus, Ganoderma lucidum. For more accuracy in co-cultivations, a novel synchronized cultivation approach was tested for culturing G. lucidum. The hyphal growth of G. lucidum and the content of its ganoderic acids (GAs) were measured. In almost all of the co-v-cultures, there was an inhibiting effect on hyphal growth and a promoting effect on GAs contents. In inducing GAs production, Bacillus cereus PTCC 1247 and Pseudomonas aeruginosa UTMC 1404 were the most effective ones, as, compared to control cultures, GAs content increased 2.8 fold. Comparing different co-v-cultivations demonstrated that the concentrations of mVOCs, oxygen, and carbon dioxide were the main players in co-v-cultures. No correlation was found between hyphal growth and GAs production. Strains of the same species imposed totally different effects on hyphal growth or GAs production. This study has investigated the effects of mVOCs on G. lucidum for the first time. Moreover, it suggests that co-v-cultivation may be a promising biotechnological approach to improve the production in G. lucidum.     

Optimization of the fermentation parameters for the production of Ganoderma lucidum immunomodulatory protein by Pichia pastoris

Abstract

In order to obtain a better fermentation parameter for the production of recombinant Ganoderma lucidum immunomodulatory protein (rFIP-glu), an engineered Pichia pastoris GS115 was investigated on the fermentation time, temperature, methanol concentration and initial pH of media, while immunomodulatory activities of the rFIP-glu was confirmed. L₉(3³) orthogonal experiment were firstly employed to optimize various fermentation parameters in the shake-flask level. The optimized fermentation parameters were subsequently verified in a 5?L fermenter. Biological activities including cell viability and tumor necrosis factor-alpha (TNF-α) mRNA of the rFIP-glu were evaluated on murine macrophage RAW264.7 cells. The results showed that the yield of rFIP-glu was up to 368.71?μg/ml in the shake-flask, and 613.47?μg/ml in the 5?L fermenter, when the Pichia pastoris was incubated in basic media with the methanol concentration 1.0% and initial pH 6.5, and with constant shaking at 280?rpm for 4?days at 26?°C. In vitro assays of biological activity indicated that rFIP-glu had significant toxicity against RAW264.7 cells, and possessed the ability to induce TNF-α mRNA expression in macrophage RAW264.7 cells. In conclusion, engineered P. pastoris showed a good fermentation property under the optimum fermentation parameters. It could be a candidate industrial strain for further study.     

Significance of protein elicitor isolated from Tuber melanosporum on the production of ganoderic acid and Ganoderma polysaccharides during the fermentation of Ganoderma lucidum

Under the elicitation of protein elicitor isolated from the culture mycelia of Tuber melanosporum, the biosynthesis of ganoderic acids (GA) was significantly stimulated during Ganoderma lucidum fermentation. Compared with our previous results that, GA content was inhibited by polysaccharide elicitor isolated from T. melanosporum, while improved by the elicitor of polysaccharide and protein, protein was identified to be the exact component inducing GA biosynthesis in this work. G. lucidum cell growth was significantly inhibited by elicitor of polysaccharide and protein, and polysaccharide elicitor did not inhibit the cell growth. In this work, the remarkable inhibition on the cell growth was considerably eliminated under the elicitation of protein elicitor isolated from T. melanosporum. These suggested maybe the interaction of polysaccharide and protein components existed in the inhibition on the cell growth of G. lucidum. Not only GA content but also total GA accumulation obtained the highest values after the elicitation of protein elicitor. The maximal GA production of 260.5 ± 5.6 mg/L was 31.2% higher than the control. Under the elicitation of protein elicitor, the production of extracellular polysaccharide (EPS) and the content of intracellular polysaccharide (IPS) were also enhanced; however, total IPS accumulation was lower. GA biosynthesis was also significantly affected by the addition time of protein elicitor, whose optimal value was the culture of day 4.     

Effects of different graphene-based nanomaterials as elicitors on growth and ganoderic acid production by Ganoderma lucidum

Graphene-based nanomaterials (GBNs) have attracted considerable interest nowadays due to their wide range of applications. However, very little attention has been paid to the application of nanomaterials as potential elicitors for production of valuable metabolites. Herein, aiming to earn insight into effects of nanomaterials on secondary metabolite biosynthesis by medicinal fungi, we evaluated the influence of GBNs on growth and production of ganoderic acid (GA) by Ganoderma lucidum in submerged culture. Graphene oxide (GO), reduced graphene oxide (rGO), and rGO/Fe₃O₄ nanocomposite were synthesized successfully and characterized by X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopy analysis. The prepared nanomaterials were added to the culture of G. lucidum at final concentrations of 50, 100, and 150 mg/L on Day 5. The results showed that the elicitation of G. lucidum with GO and rGO decreased the cell dry weight and GA production slightly, especially in higher concentrations. However, rGO/Fe₃O₄ nanocomposite not negatively affected cell growth and improved GA production. G. lucidum growth rate responded to elicitation experiments differently and depended on the type of nanomaterials and their concentrations, but almost all GBNs caused an increase in GA content (mg/100 mg dry weight). Also, field emission scanning electron microscopy morphological study showed that under elicitation, mycelia were more condensed and tightly stacked together. The findings from this study may suggest that GBNs in low concentrations could be applied as elicitors to secondary metabolites production from higher fungus, but further environmental, physiological, and biological studies required.     

Polyhydroxyalkanoate production using waste vegetable oil and filtered digestate liquor of chicken manure

Abstract

The production of polyhydroxyalkanoates (PHA) using digestate of chicken manure combined with waste sunflower oil as no-cost feedstocks in a multi-stage process was investigated. Using Cupriavidus necator H16 in combined culture media, a maximum PHA accumulation of 4.6?±?0.2?g/L at 75.1?±?1.4% of cell dry matter and a residual cell matter yield of 1.5?±?0.1?g/L were obtained after 96?hr of cultivation (30?°C, 160?rpm, pH 7.0) in flask-based experiments. Manure was acidogenically fermented in a continuous stirring tank reactor in fed-batch mode. The bioreactor was operated at varying organic loading rates (OLR) and hydraulic retention times (HRT) ranging from 1–4?g volatile solids (VS)/L/d and 4–8?days, respectively. Optimal operation was observed at an OLR of 4?g VS/L/d and an HRT of 4?days. Analysis showed the presence of significant amounts of ammonia, potassium, magnesium, calcium, and trace elements, i.e. Fe, Cu, Ni, Mn, Co, Zn, Cr in the digestate. The micro-filtered digestate was utilized as a complex culture media base while waste oil served as an additional carbon source and supplemented for effective PHA production. The total volatile fatty acid content of digestate greatly affected the growth performance of the PHA-producing microorganism Cupriavidus necator H16.     

Investigating chitin deacetylation and chitosan hydrolysis during vegetative growth in Magnaporthe oryzae

Chitin deacetylation results in the formation of chitosan, a polymer of β1,4‐linked glucosamine. Chitosan is known to have important functions in the cell walls of a number of fungal species, but its role during hyphal growth has not yet been investigated. In this study, we have characterized the role of chitin deacetylation during vegetative hyphal growth in the filamentous phytopathogen Magnaporthe oryzae. We found that chitosan localizes to the septa and lateral cell walls of vegetative hyphae and identified 2 chitin deacetylases expressed during vegetative growth—CDA1 and CDA4. Deletion strains and fluorescent protein fusions demonstrated that CDA1 is necessary for chitin deacetylation in the septa and lateral cell walls of mature hyphae in colony interiors, whereas CDA4 deacetylates chitin in the hyphae at colony margins. However, although the Δcda1 strain was more resistant to cell wall hydrolysis, growth and pathogenic development were otherwise unaffected in the deletion strains. The role of chitosan hydrolysis was also investigated. A single gene encoding a putative chitosanase (CSN) was discovered in M. oryzae and found to be expressed during vegetative growth. However, chitosan localization, vegetative growth, and pathogenic development were unaffected in a CSN deletion strain, rendering the role of this enzyme unclear.     

An easy,inexpensive, and sensitive method for the quantification of chitin in insect peritrophic membrane by image processing

ABSTRACT

Chitin, poly (β-(1→4)-N-acetyl-d-glucosamine), is an important biopolymer for insects that is utilized as a major component of peritrophic membrane. The chitin content in peritrophic membrane is of expedient interest from a pest control perspective, although it is hard to quantify chitin. In this study, we establish a facile method for the quantification of chitin in peritrophic membrane by image processing. In this method, chitin was indirectly quantified using chitosan–I₃^? complex, which exhibited a specific red-purple color. A calibration curve using a chitosan solution showed good linearity in a concentration range of 0.05–0.5 μg/μL. We quantified the amount of chitin in peritrophic membrane of Spodoptera litura (Lepidoptera: Noctuidae) larvae using this method. Throughout the study, only common inexpensive regents and easily attainable apparatuses were employed. This method can be easily applied to the sensitive quantification of the amounts of chitin and chitosan in materials by wide range of researchers.

Abbreviations: LOD: limit of detection; LOQ: limit of quantification; ROI: region of interest; RSD: relative standard deviation.     

Optimization of culture conditions and medium components for the production of mycelial biomass and exo-polysaccharides with Cordyceps militaris in liquid culture

Both crude exo-biopolymers and mycelial biomass, produced by liquid culture of Cordyceps species, are believed to possess several potential health benefits. As a result of its known biological activities, Cordyceps militaris has been extensively characterized in regards to potential medicinal applications. However, optimized liquid culture conditions for enhanced polysaccharide productivity have yet to be developed, which is a necessary step for industrial applications. Therefore, in this study, the liquid culture conditions were optimized for maximal production of mycelial biomass and exo-polysaccharide (EPS) by C. militaris. The effects of medium composition, environmental factors, and C/N ratio were investigated. Among these variables 80 g, glucose; 10 g, yeast extract; 0.5 g, MgSO₄·7H₂O; and 0.5 g, KH₂PO₄ in 1 L distilled water were found to be the most suitable carbon, nitrogen, and mineral sources, respectively. The optimal temperature, initial pH, agitation, and aeration were determined to be 24°C, uncontrolled pH, 200 rpm, and 1.5 vvm, respectively. Under these optimal conditions, mycelial growth in shake flask cultures and 5 L jar bioreactors was 29.43 and 40.60 g/L, respectively, and polysaccharide production in shake flask cultures and 5 L jar bioreactors was 2.53 and 6.74 g/L, respectively.     

Enhanced doxorubicin production by Streptomyces peucetius using a combination of classical strain mutation and medium optimization

Abstract

Doxorubicin (DXR), which is produced by Streptomyces peucetius, is an important anthracycline-type antibiotic used for the treatment of various cancers. However, due to the low DXR productivity of wild-type S. peucetius, it is difficult to produce DXR by one-step fermentation. In this study, a DXR-resistance screening method was developed to screen for DXR high-producing mutants. Then, S. peucetius SIPI-11 was treated several times with UV and ARTP (atmospheric and room temperature plasma) to induce mutations. Treated strains were screened by spreading on a DXR-containing plate, isolating a mutant (S. peucetius 33-24) with enhanced DXR yield (570?mg/L vs. 119?mg/L for the original strain). The components of the fermentation medium, including the carbon and nitrogen sources, were optimized to further enhance DXR yield (to 850?mg/L). The pH of the fermentation medium and culture temperature were also optimized for effective DXR production. Finally, DXR production by S. peucetius 33-24 was investigated in flask culture and a fermenter. The yield of DXR was as high as 1100?mg/L in a 5-L fermenter, which is the highest DXR productivity reported thus far, suggesting that S. peucetius 33-24 has the potential to produce DXR by direct fermentation.     

Performance analyses of a pH-shift and DOT-shift integrated fed-batch fermentation process for the production of ganoderic acid and Ganoderma polysaccharides by medicinal mushroom Ganoderma lucidum

Investigations on Ganoderma lucidum fermentation suggested that the responses of the cell growth and metabolites biosynthesis to pH and dissolved oxygen tension (DOT) were different. The ganoderic acid (GA) production of 321.6 mg/L was obtained in the pH-shift culture by combining a 4-day culture at pH 3.0 with the following 6-day culture at pH 4.5, which was higher by 45% and 300% compared with the culture at pH 3.0 and 4.5, respectively. The GA production of 487.1 mg/L was achieved in the DOT-shift culture by combining a 6-day culture at 25% of DOT with a following 6-day culture at 10% of DOT, which was higher by 43% and 230% compared with the culture at 25% and 10% of DOT, respectively. A fed-batch fermentation process by combining the above-mentioned pH-shift and DOT-shift strategies resulted in a significant synergistic enhancement of GA accumulation up to 754.6 mg/L, which is the highest reported in the submerged fermentation of G. lucidum in stirred-tank bioreactor.     

Anti-Tumor Activities of the Antlered Form of Ganoderma lucidum in Allogeneic and Syngeneic Tumor-Bearing Mice

We investigated the anti-tumor effects of a dry powder preparation of the antlered form of Ganoderma lucidum (G. lucidum AF, rokkaku-reishi in Japanese), a variant type of G. lucidum, not only in allogeneic Sarcoma 180-bearing ddY mice, but also in syngeneic MM 46-bearing C3H/He mice. G. lucidum AF inhibited tumor growth and elongated the life span when orally administered to mice by free-feeding of a 2.5% G. lucidum AF-containing diet. It also showed anti-tumor activity in spite of post-feeding after tumor inoculation. G. lucidum AF significantly countered the depression of splenic CD8⁺ cells and protected the decrease in interferon-gamma (IFN-γ) production in regional lymph nodes of MM 46-bearing mice, indicating that the anti-tumor activity of G. lucidum AF might be caused by its immunostimulating action. These results suggest that the ingestion of G. lucidum AF can be useful for the prevention and curing of cancer.     

Influence of cultivation conditions on citrate production by Aspergillus niger in a semi-pilot-scale plant

The influence of some fermentation parameters on the semi-pilot scale (alteration of growth conditions,e.g., sugar concentration, incubation temperature and initial pH) on citrate production was demonstrated in parent and mutant strains ofAspergillus niger. Raw material from sugar industry (cane molasses) was examined as basal fermentation medium in a stirred stainless-steel 15-L fermentor. After growth on medium with 150 g/L sugar, the parent strain produced 51.2 g/L citric acid; the mutant strain achieved production maximum of 96.2 g/L. Comparing the growth, kinetic (volumetric substrate uptake rate, rate of substrate consumption and volumetric productivity rate) and production parameters it was found that the mutant strain grows more rapidly, with slightly changed morphology (intermediate, shiny round pellets with diameter 0.6–0.7 mm), and exhibits a higher citrate production and higher efficiency of sugar utilization.     

Catalytic and thermodynamic properties of β-glucosidases produced by Lichtheimia corymbifera and Byssochlamys spectabilis

Abstract

The objective of the present study was to optimize parameters for the cultivation of Lichtheimia corymbifera (mesophilic) and Byssochlamys spectabilis (thermophilic) for the production of β-glucosidases and to compare the catalytic and thermodynamic properties of the partially purified enzymes. The maximum amount of β-glucosidase produced by L. corymbifera was 39?U/g dry substrate (or 3.9?U/mL), and that by B. spectabilis was 77?U/g (or 7.7?U/mL). The optimum pH and temperature were 4.5 and 55?°C and 4.0 and 50?°C for the enzyme from L. corymbifera and B. spectabilis, respectively. β-Glucosidase produced by L. corymbifera was stable at pH 4.0–7.5, whereas the enzyme from B. spectabilis was stable at pH 4.0–6.0. Regarding the thermostability, β-glucosidase produced by B. spectabilis remained stable for 1?h at 50?°C, and that from L. corymbifera was active for 1?h at 45?°C. Determination of thermodynamic parameters confirmed the greater thermostability of the enzyme produced by the thermophilic fungus B. spectabilis, which showed higher values of ΔH, activation energy for denaturation (E_a), and half-life t_(1/2). The enzymes were stable in the presence of ethanol and were competitively inhibited by glucose. These characteristics contribute to their use in the simultaneous saccharification and fermentation of vegetable biomass.     

Ganoderic acid produced from submerged culture of <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> induces cell cycle arrest and cytotoxicity in human hepatoma cell line BEL7402

Ganoderic acid (GA), produced by submerged culture of Ganoderma lucidum, at 500 μg/ml, caused nearly a 70% inhibition of the growth of human hepatoma cell line BEL7402 but not of a normal human liver cell line L02. Flow cytometry analyses showed that GA blocked the BEL7402 cell cycle at the transition from G₁ to S phase.     

Kinetic modeling of Candida shehatae ATCC 22984 on xylose and glucose for ethanol production

Candida shehatae ATCC 22984, a xylose-fermenting yeast, showed an ability to produce ethanol in both glucose and xylose medium. Maximum ethanol produced by the yeast was 48.8?g/L in xylose and 52.6?g/L in glucose medium with ethanol yields that varied between 0.3 and 0.4?g/g depended on initial sugar concentrations. Xylitol was a coproduct of ethanol production using xylose as substrate, and glycerol was detected in both glucose and xylose media. Kinetic model equations indicated that growth, substrate consumption, and product formation of C. shehatae were governed by substrate limitation and inhibition by ethanol. The model suggested that cell growth was totally inhibited at 40?g/L of ethanol and ethanol production capacity of the yeast was 52?g/L, which were in good agreement with experimental results. The developed model could be used to explain C. shehatae fermentation in glucose and xylose media from 20 to 170?g/L sugar concentrations.     

Experimental analysis of the oil addition effect on mycelia and polysaccharide productions in Ganoderma lucidum submerged culture

The effect of corn oil addition on mycelium growth and polysaccharide productions in the medicinal mushroom Ganoderma lucidum was studied. The results showed that when a level of 2% corn oil was added at the beginning of culture, the biomass and polysaccharide productions reached a maximum of 12.9 and 1.038 g/L, respectively, during 13-day cultivation. The pH variation along with morphology observation in culture provided an indirect inference to the promotional effect of oil addition. Moreover, a curve fitting analysis was carried out to assay the elevated effect on biomass and exopolysaccharide productions in oil added culture. The experimental data of substrates consumption and products formation in culture with oil addition were predicted through the fitting equations obtained in single carbon source culture. The numerical results further clarified the stimulatory effects of oil addition in G. lucidum culture.     

Parameter optimization for thermostable lipase production and performance evaluation as prospective detergent additive

Abstract

Lipase based formulations has been a rising interest to laundry detergent industry for their eco-friendly property over phosphate-based counterparts and compatibility with chemical detergents ingredients. A thermo-stable Anoxybacillus sp. ARS-1 isolated from Taptapani Hotspring, India was characterized for optimum lipase production employing statistical model central composite design (CCD) under four independent variables (temperature, pH, % moisture and bio-surfactant) by solid substrate fermentation (SSF) using mustard cake. The output was utilized to find the effect of parameters and their interaction employing response surface methodology (RSM). A quadratic regression with R²?=?0.955 established the model to be statically best fitting and a predicted highest lipase production of 29.4?IU/g at an optimum temperature of 57.5?°C, pH 8.31, moisture 50% and 1.2?mg of bio-surfactant. Experimental production of 30.3?IU/g lipase at above conditions validated the fitness of model. Anoxybacillus sp. ARS-1 produced lipase was found to resist almost all chemical detergents as well as common laundry detergent, proving it to be a prospective additive for incorporation.