Marin Berovič

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.

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.

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.

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.

Biochemical Engineering Aspects of Solid State Bioprocessing

Despite centuries of use and renewed interest over the last 20 years in solid-state fermentation (SSF) technology, and despite its good potential for a range of products, there are currently relatively few large-scale commercial applications. This situation can be attributed to the complexity of the system: Macroscale and microscale heat and mass transfer limitations are intrinsic to the system, and it is only over the last decade or so that we have begun to understand them. This review presents the current state of understanding of biochemical engineering aspects of SSF processing, including not only the fermentation itself, but also the auxiliary steps of substrate and inoculum preparation and downstream processing and waste disposal. The fermentation step has received most research attention. Significant advances have been made over the last decade in understanding how the performance of SSF bioreactors can be controlled either by the intraparticle processes of enzyme and oxygen diffusion or by the macroscale heat transfer processes of conduction, convection, and evaporation. Mathematical modeling has played an important role in suggesting how SSF bioreactors should be designed and operated. However, these models have been developed on the basis of laboratory-scale data and there is an urgent need to test these models with data obtained in large-scale bioreactors.

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.

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 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.

The use of spent brewery grains for Pleurotus ostreatus cultivation and enzyme production.

In the brewing industry, spent brewery grains (SBGs) are byproducts with a low economic value. The potential use of this leftover as a substrate ingredient for Pleurotus ostreatus fruiting body cultivation and enzyme production was evaluated. The best substrate mixture for P. ostreatus mycelium growth comprised 30% wheat bran (WB), 68% beech sawdust (BS) and 2% CaCO3. On the substrates containing SBG, the fastest mycelium growth was observed on the substrate composed of 10% SBG, 20% WB, 68% BS and 2% CaCO3. The highest biological efficiency (51%) of fruiting bodies was determined on the mixtures containing 20% WB, 10% SBG and 2% CaCO3. The SBGs with the addition of WB were also shown to be suitable as a substrate for enzyme production. However, the supplementation levels designate which enzymes are produced and in what amounts.

Dual feeding strategy for the production of α-amylase by Bacillus caldolyticus using complex media

In this study, the objective was to investigate an exponential feeding strategy for fed-batch production of thermostable alpha-amylase (E.C. 3.2.1.1.) from the Bacillus caldolyticus (DSM405). The parameters for establishing compositions of feed media and feeding rate were obtained by statistical analysis of batch and continuous shake flask experiments. These parameters were casitone to starch ratio of 2.67g(casitone)g(starch)(-1), maintenance coefficient 0.174g(casitone)g(DW)(-1)h(-1), cell yield 0.62g(DW)g(casitone)(-1) and mu(opt)=0.2h(-1). The exponentially fed fermentation resulted in yield of 120Uml(-1) alpha-amylase that was thermostable up to 105 degrees C. Results of the exponentially fed fermentation have been discussed in the light of a feed-back controlled fed-batch fermentation reported earlier by the authors. A comparison of the temperature and pH effects on amylase produced by B. caldolyticus and on several other commercially available amylases has also been presented.