Production of biomass and polysaccharides of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (W.Curt. :Fr.) P. Karst. (higher Basidiomycetes), by submerged cultivation.

Submerged batch and repeated fed-batch cultivation techniques were used for mycelia cultivation and polysaccharide production of the Lingzhi or Reishi medicinal mushroom Ganoderma lucidum. Although most publications use various Asiatic G. lucidum strains, the growth of the strain Ga.l 4 (Biotechnical Faculty Strain Collection, Ljubljana, Slovenia), originally isolated from the Slovenian forest, is much faster. The results between the batch and repeated fed-batch cultivation are compared with the polysaccharide production in batch cultivation. From the aspect of biomass production, the best results were obtained in repeated fed-batch after 44 days, where 12.4 g/L of dry fungal biomass was obtained.

Advances in Pilot-Scale Stirred Bioreactors in Solid-State and Submerged Cultivations of Medicinal Mushrooms.

Great interest for large-scale production of medicinal mushroom biomass and various pharmaceutically active compounds production dictates the development of comprehensive technologies. Solid state and submerged cultivations in bioreactors represent the most promising technologies for fast and large amount production of medicinal fungi biomass and pharmaceutically active products for human and veterinary need. There are many stages from shaking culture studies to large-scale industrial production. Pilot-scale studies represent the bridge and the balance between the gap of laboratory and industrial scale. Therefore it is not a surprise that most of pilot-scale results and experiences remain uncovered industrial secrets. This chapter is an overview of available engineering achievements in submerged and solid-state cultivation experiences in pilot-scale bioreactors.

Advances in Production of Medicinal Mushrooms Biomass in Solid State and Submerged Bioreactors.

Production of mushroom fruit bodies using farming technology could hardly meet the increasing demand of the world market. During the last several decades, there have been various basic and applied studies on fungal physiology, metabolism, process engineering, and (pre)clinical studies. The fundamental aspects of solid-state cultivation of various kinds of medicinal mushroom mycelia in various types of bioreactors were established. Solid-state cultivation of medicinal mushrooms for their biomass and bioactive metabolites production appear very suitable for veterinary use. Development of comprehensive submerged technologies using stirred tank and airlift bioreactors is the most promising technology for fast and large-scale production of medicinal fungi biomass and their pharmaceutically active products for human need. The potentials initiate the development of new drugs and some of the most attractive over-the-counter human and veterinary remedies. This article is to overview the engineering achievements in solid state and submerged cultivations of medicinal mushrooms in bioreactors.

Farming of Medicinal Mushrooms.

Since most of the medicinal mushrooms are rare in nature, production of fungal fruiting bodies is hardly covering the food market and the production of pharmaceutically active products, so artificial cultivation of fruiting bodies in a form of farming has been intensively established during the last 40 years. Various cultivation technologies are presented, including traditional farming of fruiting bodies on wood logs and beds, and also on other substrate-based media, such as cultivation in bags, bottles, and others. The advantage of farming is a cheap but time-consuming large-scale production. Agriculture, wood, and food industry wastes represent the main substrates that are in this process delignified and enriched in proteins and highly valuable pharmaceutically active compounds. The present article presents an overview of achievements in artificial cultivation of fruiting bodies, including the most relevant medicinal mushroom species, such as Ganoderma lucidum, Grifola frondosa, Pleurotus ostreatus, Agaricus brasiliensis, and Lentinula edodes.

Production of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (W.Curt. :Fr.) P. Karst. (higher Basidiomycetes), biomass and polysaccharides by solid state cultivation.

Solid state cultivation of Ganoderma lucidum biomass, strain BFWS Gal 4, originally isolated from the Slovenian forest, was studied in a horizontal stirred tank reactor. Periodic mixing of N = 80 rpm, 2 min/day was used. Production of fungal polysaccharides and fungal biomass on solid substrate based on beech sawdust, olive oil, and mineral salts was studied. Optimal moisture of the solid matrix was in the range of 80% to 74%. When the moisture content dropped below 57%, the growth of the mycelium and polysaccharide production stopped, but it revived when wet air was applied in further processing. Final concentration of biomass was 0.68 mg/g of solid substrate, while proportions of extracellular and intracellular polysaccharides were 4.5 mg/g and 1.05 mg/g, respectively.

Immunomodulating activities of cultivated maitake medicinal mushroom Grifola frondosa (Dicks.: Fr.) S.F. Gray (higher Basidiomycetes) on peripheral blood mononuclear cells.

Grifola frondosa is a culinary-medicinal mushroom that contains several physiologically active compounds, of which polysaccharides, specifically ß-glucans, are known to possess immunomodulating properties. Its extracts are studied for application as adjuncts for chemotherapy, and experiments in animal models support the use of this mushroom for cancer treatment. The effect of extracts obtained from mushrooms cultivated on different substrates and their capacity of inducing the secretion of cytokines from human peripheral blood mononuclear cells were studied. The activity of extracts at concentrations 12.5, 100, and 200 µg/mL on induction of TNF-α, IFN-γ, and IL-12 was screened. Two extracts from substrates fortified with olive oil press cakes showed appreciable activity and induced the secretion of TNF-α, IL-12, and INF-γ. The extracts differed from the others in the amount of sugar, protein, and ß-glucans, which can explain their higher activity. Present results show that different substrates and different source materials can reasonably modify the bioactivity of cultivated G. frondosa.

Cultivation Technologies for Production of Medicinal Mushroom Biomass: Review.

Since most medicinal mushrooms are rare in nature, artificial cultivation and farming of their fruiting bodies as well as biotechnological cultivation of their fungal biomass in bioreactors on solid substrates and in liquid media has been established. This review compares the benefits and weaknesses of all three capabilities. Cultivation technologies are presented, including traditional cultivation via farming of fruiting bodies on wood logs and on sawdust-based substrates and modern biotechnological cultivation of mycelia in bioreactors by submerged and solid-state cultivation technologies. Our findings indicate that farming is cost-effective but large-scale production is time-consuming. In addition, solid-state cultivation is a comprehensive well-controlled technology that is close to the natural growth process and is suitable for veterinary use and use in food supplements. Finally, submerged liquid-state cultivation of mushroom mycelia is fast and well controlled.

Heat transfer in citric Acid production with axial and radial flow impellers.

In order to produce fermentation broth for downstream recovery, a total of 15 fermentations were done in a 15 m3 and two 7.5 m3 vessels. Apart from the evaluation of fermentation yield and productivity, information on the heat and mass transfer coefficients were required for design purposes. The focus of the fermentation study was therefore directed to obtain information on broth rheology, heat transfer aspects and considerations. Broth rheology was found to deviate from Newtonian behavior with increasing biomass concentration. Using axial flow impellers, rather than radial flow producing Rushton turbines, significantly improved heat transfer in this study.

Cultivation of Medicinal Mushroom Biomass by Solid-State Bioprocessing in Bioreactors.

Basidiomycetes of various species and their wide range of pharmaceutically interesting products in the last decades represent one of the most attractive groups of natural products in Asia and North America. Production of fungal fruit bodies using farming technology is hardly covering the market. Comprehensive solid-state technologies and bioreactors are the most promising part for fast and large amount of cultivation of medicinal fungi biomass and its pharmaceutically active products. Wood, agriculture, and food industry wastes represent the main substrates that are in this process delignified and enriched in proteins and highly valuable pharmaceutically active compounds. Research in physiology, basic and applied studies in fungal metabolism, process engineering aspects, and clinical studies in the last two decades represent large contribution to the development of these potentials that initiate the development of new drugs and some of the most attractive over-the-counter human and veterinary remedies. Present article is an overview of the achievements in solid-state technology of the most relevant medicinal mushroom species production in bioreactors. Graphical Abstract.

Engineering Aspects in Production of Various Medicinal Mushrooms Biomass in Submerged Bioreactors.

Basidiomycetes of various species and their wide range of pharmaceuticaly interesting products in the past decades represents one of the most attractive groups of natural products in Asia and North America. Production of mushroom fruit bodies using farming technology is hardly covering the market. Development of comprehensive submerged technologies in stirred tank and air lift bioreactors are the most promising technologies for fast and large-amount cultivation of medicinal mushroom biomass and its pharmaceutically active products. Research in physiology, basic and applied studies in mushroom metabolism, process engineering aspects, and clinical studies in the past two decades represent a large cotribution to the development of this potential, which initiates the development of new drugs and some very attractive over-the-counter human and veterinary remedies. The current article is an overview of the most relevant engineering achievements in submerged cultivation of some medicinal mushrooms-Grifola frondosa, Trametes versicolor, Hericium erinaceus, and Cordyceps militaris-and some other species biomass production in bioreactors.

Engineering Aspects of Lingzhi or Reishi Medicinal Mushroom Ganoderma lucidum (Agaricomycetes) Biomass Submerged Cultivation in Bioreactors: A Review.

The Ganoderma lucidum wood-degrading basidiomycete, with its large complex of pharmacological effects, is the most outstanding and influential medicinal mushroom in Far East traditional medicine. In the past 2 decades, the fundamentals of submerged cultivation of G. lucidum mycelia in bioreactors has been established. Development of comprehensive submerged cultivations in stirred tank and air lift bioreactors are the most promising technologies. This article provides an engineering overview of the achievements in submerged technology of G. lucidum biomass production in bioreactors.

Citric acid production.

Citric acid is a commodity chemical produced and consumed throughout The World. It is used mainly in the food and beverage industry, primarily as an acidulant. Although it is one of the oldest industrial fermentations, its World production is still in rapid increasing. Global production of citric acid in 2007 was over 1.6 million tones. Biochemistry of citric acid fermentation, various microbial strains, as well as various substrates, technological processes and product recovery are presented. World production and economics aspects of this strategically product of bulk biotechnology are discussed.

Ganoderma lucidum and its pharmaceutically active compounds.

Ganoderma lucidum is a wood-degrading basidiomycete with numerous pharmacological effects. Since the mushroom is very rare in nature, artificial cultivation of fruiting bodies has been known on wood logs and on sawdust in plastic bags or bottles. Biotechnological cultivation of G. lucidum mycelia in bioreactors has also been established, both on solid substrates and in liquid media by submerged cultivation of fungal biomass. The most important pharmacologically active constituents of G. lucidum are triterpenoids and polysaccharides. Triterpenoids have been reported to possess hepatoprotective, anti-hypertensive, hypocholesterolemic and anti-histaminic effects, anti-tumor and anti-engiogenic activity, effects on platelet aggregation and complement inhibition. Polysaccharides, especially beta-d-glucans, have been known to possess anti-tumor effects through immunomodulation and anti-angiogenesis. In addition, polysaccharides have a protective effect against free radicals and reduce cell damage caused by mutagens.

Influence of heat shock on glycerol production in alcohol fermentation.

The influence of single and double heat shocks induced during the exponential growth phase of the Saccharomyces cerevisiae fermentation of cultivar Sauvignon Blanc grape must was examined. Rapid temperature changes from 18 degrees C to 34 degrees C have been applied. The effect of the duration of exposure to a high temperature has been analyzed. By the applications of a single heat shock and a double heat shock, up to 8.2 g l(-1) and 11.0 g l(-1) glycerol have been produced, respectively. To prevent the evaporation of fine wine bouquet compounds during the temperature changes, reflux coolers on the top of bioreactors have been employed. By using this method, glycerol production was increased by up to 65%.

Sterilisation in biotechnology.

Sterilisation of fermentation media, inlet and outlet gases and maintenance of aseptic conditions during bioprocessing represents one of the most relevant parts of comprehensive upstream processing. The present chapter reviews various sterilisation techniques and methods for sterilisation of inlet gases, liquids, including sterilisation of small and large industrial equipment and validation of sterilisation procedures. Various methods for sterilisation using different kinds of filters, filter cartridges and membrane filters are also presented. Liquid sterilisation methods by filtration as well as heat sterilisation are discussed. Sterilisation of small and industrial scale equipment by chemical agents and physical methods as ionising radiation and dry heat sterilisation including the validation of sterilisation are discussed.

Submerged cultivation of Ganoderma lucidum and the effects of its polysaccharides on the production of human cytokines TNF-α, IL-12, IFN-γ, IL-2, IL-4, IL-10 and IL-17.

An original strain of Ganoderma lucidum (W.Curt.:Fr.) Lloyd, MZKI G97 isolated from Slovenian habitats was grown by a submerged liquid substrate cultivation in a laboratory stirred tank reactor. Five fractions of extracellular and cell-wall polysaccharides were obtained by extraction, ethanol precipitation, and purification by ion-exchange, gel and affinity chromatography. The capacity of isolated polysaccharide fractions to induce innate inflammatory cytokines, and to modulate cytokine responses of activated lymphocytes was investigated. Human peripheral blood mononuclear cells (PBMC) were activated in vitro with polysaccharide fractions, in order to induce innate inflammatory cytokines: tumor necrosis factor alpha (TNF-α), interleukin (IL) 12 and interferon gamma (IFN-γ). For the immunomodulation capacity, polysaccharide fractions were cultured with ionomycine and phorbol myristate acetate (IONO+PMA) activated PBMC, and the concentrations of induced IL-2, IL-4, IFN-γ, IL-10 and IL-17 were measured. The results showed that polysaccharides from G. lucidum induced moderate to high amounts of innate inflammatory cytokines. Fungal cell-wall polysaccharides were stronger innate inflammatory cytokines inducers, while extracellular polysaccharides demonstrated a higher capacity to modulate cytokine responses of IONO+PMA induced production of IL-17. The results indicate that G. lucidum polysaccharides enhance Th1 response with high levels of IFN-γ and IL-2, and display low to no impact on IL-4 production. A similar pattern was observed at regulatory cytokine IL-10. All of the polysaccharide fractions tested induced IL-17 production at different concentration levels.

Overview of solid state bioprocessing.

Solid-state fermentation has centuries of history, but it is only in the last two decades that there has been a concerted effort to understand the bioprocessing issues involved and to apply them to a wide range of new products. This article provides an overview of the knowledge of solid-state bioprocessing that has been gained over this time. It shows that, although significant advances have been achieved in understanding of what controls process performance, much research is still required.

Submerged cultivation of Ganoderma lucidum biomass and immunostimulatory effects of fungal polysaccharides.

Original Ganoderma lucidum strain MZKI G97 isolated from Slovenian forests was cultivated in a liquid substrate based on potato dextrose and olive oil. The influences of inoculum and oxygen partial pressure in batch and fed batch cultivation in a 10-l laboratory stirred tank reactor were studied. Fungal biomass was found to be oxygen and shear sensible. Using a 17% (wet weight) 6 days old vegetative inoculum, 9.6 g l(-1) of dry biomass in batch cultivation and 15.2 g l(-1) in fed batch process were obtained. Extracellular (9.6 g l(-1)) and intracellular (6.3 g l(-1)) polysaccharide fractions were isolated. Extracellular polysaccharide fraction and four intracellular polysaccharide fractions were obtained. Polysaccharides were further separated by ion-exchange, gel and affinity chromatography. The isolated polysaccharides were mainly beta-D-glucanes. Immunostimulatory effects of isolates were tested on induction of cytokine (tumour necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma)) synthesis in primary cultures of human peripheral blood mononuclear cells (PBMC) isolated from a buffy coat. The TNF-alpha inducing activity is comparable with romurtide, which has been used as a supporting therapy in cancer patients treated with radiotherapy and/or chemotherapy.

Characterization of CIM monoliths as enzyme reactors.

The immobilization of the enzymes citrate lyase, malate dehydrogenase, isocitrate dehydrogenase and lactate dehydrogenase to CIM monolithic supports was performed. The long-term stability, reproducibility, and linear response range of the immobilized enzyme reactors were investigated along with the determination of the kinetic behavior of the enzymes immobilized on the CIM monoliths. The Michaelis-Menten constant K(m) and the turnover number k(3) of the immobilized enzymes were found to be flow-unaffected. Furthermore, the K(m) values of the soluble and immobilized enzyme were found to be comparable. Both facts indicate the absence of a diffusional limitation in immobilized CIM enzyme reactors.

Growth, lipid extraction and thermal degradation of the microalga Chlorella vulgaris.

The microalga Chlorella vulgaris was cultured in a combined medium obtained by mixing standard Jaworski medium with a solution from the modified Solvay process that contained only NaHCO(3) and NH(4)Cl. Cell number, pH and nitrogen content were monitored throughout growth. Lipids were extracted from lyophilised biomass using CHCl(3)-MeOH. A combination of grinding, microwave treatment and sonication proved to give the best lipid extract yield. Freeze-dried algal biomass was also utilised for thermal degradation studies. The degradation exhibited three distinct regions - primary cell structure breakage paralleled by evaporation of water, followed by two predominant exothermic degradation processes. The latter were modelled using nth order apparent kinetics. The activation energies of the degradation processes were determined to be 120-126kJ/mol and 122-132kJ/mol, respectively. The degradation model may be readily applied to an assortment of thermal algal processes, especially those relating to renewable energy.