Single cell oil (SCO) production by Mortierella isabellina grown on high-sugar content media

Mortierella isabellina cultivated in nitrogen-limited media presented remarkable cell growth (up to 35.9 g/l) and high glucose uptake even with high initial sugar concentrations (e.g. 100 g/l) in media. After nitrogen depletion, significant fat quantities were accumulated inside the fungal mycelia (50-55%, wt/wt oil in dry biomass), resulting in a notable single cell oil production of 18.1 g/l of culture medium. Total dry biomass and lipid yields presented greatly increased values (0.34 and 0.17 g respectively per gram of glucose consumed). The microbial lipid produced contained gamma-linolenic acid (GLA) at a concentration of 3.5+/-1.0%, wt/wt, which corresponded to 16-19 mg GLA per gram of dry microbial mass and a maximum concentration of 0.801 g GLA per liter of culture medium.     

Optimization of Escherichia coli cultivation methods for high yield neuropeptide Y receptor type 2 production

The recombinant expression of human G protein-coupled receptors usually yields low production levels using commonly available cultivation protocols. Here, we describe the development of a high yield production protocol for the human neuropeptide Y receptor type 2 (Y2R), which provides the determination of expression levels in a time, media composition, and process parameter dependent manner. Protein was produced by Escherichia coli in a defined medium composition suitable for isotopic labeling required for investigations by nuclear magnetic resonance spectroscopy. The Y2 receptor was fused to a C-terminal 8x histidine tag by means of the pET vector system for easy one-step purification via affinity chromatography, yielding a purity of 95-99% for every condition tested, which was determined by SDS-PAGE and Western blot analysis. The Y2 receptor was expressed as inclusion body aggregates in complex media and minimal media, using different carbon sources. We investigated the influences of media composition, temperature, pH, and set specific growth rate on cell behavior, biomass wet weight specific and culture volume specific amounts of the target protein, which had been identified by inclusion body preparation, solubilization, followed by purification and spectrometric determination of the protein concentration. The developed process control strategy led to very high reproducibility of cell growth and protein concentrations with a maximum yield of 800 μg purified Y2 receptor per gram wet biomass when glycerol was used as carbon source in the mineral salt medium composition (at 38 °C, pH 7.0, and a set specific growth rate of 0.14 g/(gh)). The maximum biomass specific amount of purified Y2 receptor enabled the production of 35 mg Y2R per liter culture medium at an optical density (600 nm) of 25.     

Evaluation of Mut+ and MutS Pichia pastoris phenotypes for high level extracellular scFv expression under feedback control of the methanol concentration

Extracellular secretion of over 4 g x L(-1) of the A33 scFv antibody fragment was achieved in Pichia pastoris at the 10 L bioreactor scale using minimal medium and feedback control of the methanol concentration. Since methanol acts as both inducer and carbon source, its close regulation is a crucial factor in achieving optimal fermentation conditions. The antibody fragment production levels of both Mut+ and MutS phenotypes were compared in a bioreactor under closed-loop PID control of the methanol level. As expected, the MutS phenotype has a growth rate lower than that of the Mut+ (0.37 vs 1.05 d(-1)) when growing under methanol. However, protein productivity and cell yield on substrate are almost double that of the Mut+ (18.2 vs 9.3 mg A33 sc per gram of methanol). Induction at wet cell weight of 350 g x L(-1) for the MutS also has a positive effect on the final product concentration. Both Mut+ and MutS phenotypes reach a maximum biomass density around 450 g x L(-1) wet cell weight, independent of methanol concentration, reactor scale, or induction density. This reactor configuration allows for reproducible fermentation schemes with different Pichia pastoris phenotypes with AOX promoters, without prior knowledge of the culture growth parameters.     

Effect of Growth Rate and Nutrient Limitation on the Composition and Biomass Yield of Acinetobacter calcoaceticus

Acinetobacter calcoaceticus was grown on ethanol in a chemostat as a model system for single-cell protein production. The substrate yield coefficient (Y(s), grams of biomass/gram of ethanol), protein yield coefficient (Y(p), grams of protein/gram of ethanol), and biomass composition were measured as a function of the specific growth rate. Nucleic acid, protein, Y(p), and Y(s) all increased at higher growth rates. Although protein content increased only 14% (from 53 to 67%), Y(p) almost doubled over the same range of growth rates. The increase in Y(p) was due to the higher protein content of the biomass and to higher values of Y(s). The higher values of Y(s) were attributed to maintenance metabolism, and the value of the maintenance coefficient was found to be 0.11 g of ethanol per g of cell per h. When A. calcoaceticus was cultivated under a phosphorus limitation protein content, Y(p) and Y(s) were lower than in carbon-limited cultures. It was concluded that a single-cell protein fermentation using A. calcoaceticus should be operated at a high growth rate under ethanol-limiting conditions in order to maximize both the protein content of the biomass and the amount of biomass and/or protein made from the substrate.     

Impact of methanol concentration on secreted protein production in oxygen-limited cultures of recombinant Pichia pastoris

The methylotrophic yeast Pichia pastoris is a powerful system for production of recombinant proteins, showing high ability to secrete properly folded proteins. A major plus is the strong AOX1 promoter highly induced by methanol. During growth on methanol, however, oxygen readily becomes limiting. In oxygen-limited cultivations of recombinant Pichia pastoris, the methanol concentration had a strong impact on the production of a single-chain antibody fragment (scFv). High methanol concentrations were required to compensate the lack of oxygen and fully induce recombinant protein production, at the same time reducing gratuitous biomass formation due to a lower biomass yield. Product concentrations of 60, 150, and 350 mg/L were obtained with methanol concentrations of 0.3, 1, and 3% (v/v). Moreover, accumulation of a putative product fragment that cannot be removed during affinity purification was prevented at high methanol concentrations. Cell vitality after 100 h was maintained above 98% and 96% of the culture with 0.3% and 3% methanol, respectively. In cultivations supplemented with oxygen, in contrast, methanol concentration between 0.3% and 3% did not influence the product yield of 300-400 mg/L. Thus, efficient recombinant protein production under oxygen-limitation seems to require high methanol concentrations, enabling product concentration as high as otherwise obtained only with expensive supply of pure oxygen.     

Growth and cyclosporin a production by an indigenously isolated strain ofTolypocladium inflatum

A fungal culture isolated from a local soil sample which showed antifungal activity and produced cyclosporin A, was identified asTolypocladium inflatum. The culture grew best in a medium containing 1% maltose (pH 5–6) when inoculated with a one-day-old inoculum at 2% (V/V) concentration. Under batch fermentation conditions, growth and cyclosporin A production were better in complex media (24.6 g biomass and 205 mg cyclosporin A per liter) in comparison with synthetic media (6.84 g biomass and 35 mg cyclosporin A per liter). While addition of peptone increased culture growth (high biomass yield), supplementation with casein acid hydrolyzate favored cyclosporin A production.     

Relationship between substrate concentration,growth rate,and respiration rate of Escherichia coli in continuous culture

Summary Using a continuous flow technique the relationship between growth rate and substrate concentration was investigated with glucose as the limiting factor of a culture of Escherichia coli. Graphical and numerical analysis of the experimental data demonstrated that the application of the Michaelis-Menten equation produced erroneous results, whereas, the constants obtained from the Teissier equation were in agreement with the experimental data. On this basis, new equations defining the steady state cell and substrate concentration in continuous flow cultures were developed and tested against experimental data.Comparison of the specific growth rates, substrate uptake rates and oxygen consumption rates demonstrated that all were directly proportional to each other and could be related to each other by mathematical equations. Specifically it was shown that as the growth rate increased from 0.06 to k _m =0.76 the substrate uptake rate increased from 134 to 1420 mg glucose per gram cell weight per hour and the oxygen consumption rate increased from 48.6 to 505 mg O₂ per gram cell weight per hour. Independent of the growth rate 37% of the carbohydrate consumed were oxidized. The yield factor varied from 0.44 at low growth rates to 0.54 at high growth rates. Analysis of the growth rate-substrate uptake rate relationship indicated that a minimum substrate uptake rate of 55 mg glucose per gram cell weight per hour existed below which cell reproduction would cease. This was supported by the fact that steady state conditions could not be maintained in the culture at D values below 0.02 when the substrate supply rate decreased below 45 mg glucose per gram cell weight per hour.Material contained in this paper was submitted as a thesis in partial fulfillment of the requirements for the Ph. D. degree of Dr. R. S. Lipe.     

Fermentation of Insoluble Cellulose by Continuous Cultures of Ruminococcus albus

The hydrolysis and fermentation of insoluble cellulose (Avicel) by continuous cultures of Ruminococcus albus 7 was studied. An anaerobic continuous culture apparatus was designed which permitted gas collection, continuous feeding, and wasting at different retention times. The operation of the apparatus was controlled by a personal computer. Cellulose destruction ranged from ca. 30 to 70% for hydraulic retention times of 0.5 to 2.0 days. Carbon recovery in products was 92 to 97%, and the oxidation-reduction ratios ranged from 0.91 to 1.15. The total product yield (biomass not included) per gram of cellulose (expressed as glucose) was 0.83 g g⁻¹, and the ethanol yield was 0.41 g g⁻¹. The product yield was constant, indicating that product formation was growth linked.     

Control of culture environment for improved polyethylenimine-mediated transient production of recombinant monoclonal antibodies by CHO cells

In this study we describe optimization of polyethylenimine (PEI)-mediated transient production of recombinant protein by CHO cells by facile manipulation of a chemically defined culture environment to limit accumulation of nonproductive cell biomass, increase the duration of recombinant protein production from transfected plasmid DNA, and increase cell-specific production. The optimal conditions for transient transfection of suspension-adapted CHO cells using branched, 25 kDa PEI as a gene delivery vehicle were experimentally determined by production of secreted alkaline phosphatase reporter in static cultures and recombinant IgG4 monoclonal antibody (Mab) production in agitated shake flask cultures to be a DNA concentration of 1.25 microg 10(6) cells(-1) mL(-1) at a PEI nitrogen:DNA phosphate ratio of 20:1. These conditions represented the optimal compromise between PEI cytotoxicity and product yield with most efficient recombinant DNA utilization. Separately, both addition of recombinant insulin-like growth factor (LR3-IGF) and a reduction in culture temperature to 32 degrees C were found to increase product titer 2- and 3-fold, respectively. However, mild hypothermia and LR3-IGF acted synergistically to increase product titer 11-fold. Although increased product titer in the presence of LR3-IGF alone was solely a consequence of increased culture duration, a reduction in culture temperature post-transfection increased both the integral of viable cell concentration (IVC) and cell-specific Mab production rate. For cultures maintained at 32 degrees C in the presence of LR3-IGF, IVC and qMab were increased 4- and 2.5-fold, respectively. To further increase product yield from transfected DNA, the duration of transgene expression in cell populations maintained at 32 degrees C in the presence of LR3-IGF was doubled by periodic resuspension of transfected cells in fresh media, leading to a 3-fold increase in accumulated Mab titer from approximately 13 to approximately 39 mg L(-1). Under these conditions, Mab glycosylation at Asn297 remained essentially constant and similar to that of the same Mab produced by stably transfected GS-CHO cells. From these data we suggest that the efficiency of transient production processes (protein output per rDNA input) can be significantly improved using a combination of mild hypothermia and growth factor(s) to yield an extended "activated hypothermic synthesis".     

Effect of amino acid supplement on cell yield and gene product in Escherichia coli harboring plasmid

Escherichia coli harboring a recombinant plasmid was cultivated in fed-batch culture to enhance production of a gene product. Expression of the leucine gene from Thermus thermophilus in the recombinant plasmid was examined by the assay of beta-isopropylmalate dehydrogenase activity at 75 degrees C. When E. coli was cultivated in medium without leucine, biomass concentration reached 15 g/L and the specific activity became 0.082 U/mg protein. When leucine was fed in the medium throughout cultivation, although biomass concentration reached 63 g/L, the specific activity decreased to 0.016 U/mg protein. When E. coli was cultivated in medium containing 1 g leucine/L, the specific activity remained virtually constant (about 0.13 U/mg protein) and biomass concentration reached 32 g dry cells/L. In these cultivations, growth yields of several amino acids and glucose were examined. When leucine was not added to the medium, growth yields except for histidine were lowest. When leucine was fed throughout the cultivation, growth yields of glucose and tryptophan were highest. The pH-stat was useful for feeding amino acids.     

Improving the bioremediation of phenolic wastewaters by Trametes versicolor

The successful bioremediation of a phenolic wastewater by Trametes versicolor was found to be dependent on a range of factors including: fungal growth, culture age and activity and enzyme (laccase) production. These aspects were enhanced by the optimisation of the growth medium used and time of addition of the pollutant to the fungal cultures. Different media containing 'high' (20 g/L), 'low' (2 g/L) and 'sufficient' (10 g/L) concentrations of carbon and nitrogen sources were investigated. The medium containing both glucose and peptone at 10 g/L resulted in the highest Growth Related Productivity (the product of specific yield and micro) of laccase (1.46 Units of laccase activity)/gram biomass/day and was used in all further experiments. The use of the guaiacol as an inducer further increased laccase activity 780% without inhibiting growth; similarly the phenolic effluent studied boosted activity almost 5 times. The timing of the addition of the phenolic effluent was found to have important consequences in its removal and at least 8 days of prior growth was required. Under these conditions, 0.125 g phenol/g biomass and 0.231 g o-cresol/g biomass were removed from solution per day.     

Perfusion culture of hybridoma cells for hyperproduction of IgG(2a) monoclonal antibody in a wave bioreactor-perfusion culture system

A novel wave bioreactor-perfusion culture system was developed for highly efficient production of monoclonal antibody IgG2a (mAb) by hybridoma cells. The system consists of a wave bioreactor, a floating membrane cell-retention filter, and a weight-based perfusion controller. A polyethylene membrane filter with a pore size of 7 microm was floating on the surface of the culture broth for cell retention, eliminating the need for traditional pump around flow loops and external cell separators. A weight-based perfusion controller was designed to balance the medium renewal rate and the harvest rate during perfusion culture. BD Cell mAb Medium (BD Biosciences, CA) was identified to be the optimal basal medium for mAb production during batch culture. A control strategy for perfusion rate (volume of fresh medium/working volume of reactor/day, vvd) was identified as a key factor affecting cell growth and mAb accumulation during perfusion culture, and the optimal control strategy was increasing perfusion rate by 0.15 vvd per day. Average specific mAb production rate was linearly corrected with increasing perfusion rate within the range of investigation. The maximum viable cell density reached 22.3 x 105 and 200.5 x 105 cells/mL in the batch and perfusion culture, respectively, while the corresponding maximum mAb concentration reached 182.4 and 463.6 mg/L and the corresponding maximum total mAb amount was 182.4 and 1406.5 mg, respectively. Not only the yield of viable cell per liter of medium (32.9 x 105 cells/mL per liter medium) and the mAb yield per liter of medium (230.6 mg/L medium) but also the mAb volumetric productivity (33.1 mg/L.day) in perfusion culture were much higher than those (i.e., 22.3 x 105 cells/mL per liter medium, 182.4 mg/L medium, and 20.3 mg/L.day) in batch culture. Relatively fast cell growth and the perfusion culture approach warrant that high biomass and mAb productivity may be obtained in such a novel perfusion culture system (1 L working volume), which offers an alternative approach for producing gram quantity of proteins from industrial cell lines in a liter-size cell culture. The fundamental information obtained in this study may be useful for perfusion culture of hybridoma cells on a large scale.     

Empirical model for the autotrophic biodegradation of thiocyanate in an activated sludge reactor

AIMS: The aim of this investigation was to develop an empirical model for the autotrophic biodegradation of thiocyanate using an activated sludge reactor. METHODS AND RESULTS: The methods used for this purpose included the use of a laboratory scale activated sludge reactor unit using thiocyante feed concentrations from 200 to 550 mg x l(-1). Reactor effluent concentrations of <1 mg x l(-1) thiocyanate were consistently achieved for the entire duration of the investigation at a hydraulic retention time of 8 h, solids (biomass) retention of 18 h and biomass (dry weight) concentrations ranging from 2 to 4 g x l(-1). A biomass specific degradation rate factor was used to relate thiocyanate degradation in the reactor to the prevailing biomass and thiocyanate feed concentrations. A maximum biomass specific degradation rate of 16 mg(-1) x g(-1) x h(-1) (mg thiocyanate consumed per gram biomass per hour) was achieved at a thiocyanate feed concentration of 550 mg x l(-1). The overall yield coefficient was found to be 0.086 (biomass dry weight produced per mass of thiocyanate consumed). CONCLUSION: Using the results generated by this investigation, an empirical model was developed, based on thiocyanate feed concentration and reactor biomass concentration, to calculate the required absolute hydraulic retention time at which a single-stage continuously stirred tank activated sludge reactor could be operated in order to achieve an effluent concentration of <1 mg x l(-1). The use of an empirical model rather than a mechanistic-based kinetic model was proposed due to the low prevailing thiocyanate concentrations in the reactor. SIGNIFICANCE AND IMPACT OF THE STUDY: These results represent the first empirical model, based on a comprehensive data set, that could be used for the design of thiocyanate-degrading activated sludge systems.     

Kinetic studies of constitutive human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression in continuous culture of <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Extracellular human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression was studied under the control of the GAP promoter in recombinant Pichia pastoris in a series of continuous culture runs (dilution rates from 0.025 to 0.2 h⁻¹). The inlet feed concentration was also varied and the steady state biomass concentration increased proportionally demonstrating efficient substrate utilization and constancy of the biomass yield coefficient (Y_x/s) for a given dilution rate. The specific product formation rate (q_P) showed a strong correlation with dilution rates demonstrating growth associated product formation of hGM-CSF. The volumetric product concentration achieved at the highest feed concentration (4×) and a dilution rate of 0.2 h⁻¹ was 82 mg l⁻¹ which was 5-fold higher compared to the continuous culture run with 1× feed concentration at the lowest dilution rate thus translating to a 40 fold increase in the volumetric productivity. The specific product yield (Y_P/X) increased slightly from 2 to 2.5 mg g⁻¹, with increasing dilution rates, while it remained fairly invariant, for all feed concentrations demonstrating negligible product degradation or feed back inhibition. The robust nature of this expression system would make it easily amenable to scale up for industrial production.     

Influence of specific growth rate on biomass yield, productivity, and compostion of Candida utilis in batch and continuous culture.

Candida utilis was grown in batch and continuous culture on prickly pear juice as sole carbon and energy source. In batch culture the maximum specific growth rate (mum) and the substrate yield coefficient (Yps) varied according to sugar concentration. When the fermentation was carried out with 1% sugar, mum and Ys were 0.47/h and 42.6%, respectively. The best yields occurred in a chemostat at the pH range of 3.5 to 4.5 and temperature of 30 C. A beneficial effect on Ys was observed when the dilution rate (D) was increased. At a D of 0.55/h, the productivity was 2.38 g/liter per h. The maintenance coefficient attained a value of 0.09 g of sugar/g of biomass per h. Increases of D produced higher protein contents of the biomass. The information obtained indicates that protein production with Candida utilis, using prickly pear juice, should be carried out a high dilution rates where the Ys and protein content of the cell mass are also higher.     

Influence of specific growth rate on biomass yield, productivity, and compostion of Candida utilis in batch and continuous culture.

Candida utilis was grown in batch and continuous culture on prickly pear juice as sole carbon and energy source. In batch culture the maximum specific growth rate (mum) and the substrate yield coefficient (Yps) varied according to sugar concentration. When the fermentation was carried out with 1% sugar, mum and Ys were 0.47/h and 42.6%, respectively. The best yields occurred in a chemostat at the pH range of 3.5 to 4.5 and temperature of 30 C. A beneficial effect on Ys was observed when the dilution rate (D) was increased. At a D of 0.55/h, the productivity was 2.38 g/liter per h. The maintenance coefficient attained a value of 0.09 g of sugar/g of biomass per h. Increases of D produced higher protein contents of the biomass. The information obtained indicates that protein production with Candida utilis, using prickly pear juice, should be carried out a high dilution rates where the Ys and protein content of the cell mass are also higher.     

Factors affecting the fermentative production of a lysozyme-binding antibody fragment in Escherichia coli

Fed-batch fermentation for production of a single-chain Fv antibody fragment (scFv) expressed as a recombinant periplastic protein from Escherichia coli was investigated. A high cell density of 50 g dry cell weight per liter was routinely achieved in a 14-L vessel by controlled exponential feeding of glucose to impose a constant specific growth rate. Following biomass accumulation, induction of the tac promoter by addition of IPTG was accompaied by a linear feed of yeast extract. The concentration of yeast extract feed was found to be highly influential upon both concentration and location of active product. Although scFv fragments were specifically targeted to the periplasmic space, at yeast extract feed rates of 0.72 g/h the final location was largely extracellular (68% to 79%). Total concentrations (extracellular + periplasmic) were of the order of 5 to 8 mg/L. A ten-fold increase in yeast extract supply increased total scFv concentration to almost 200 mg/L and 78% of this yield was retained in the periplasm. Control of such leakage of the recombinant product is fundamental to process design of downstream operations for product recovery. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 611-622, 1997.     

Biodegradation of ortho-cresol by a mixed culture of nitrate-reducing bacteria growing on toluene.

A mixed culture of nitrate-reducing bacteria degraded o-cresol in the presence of toulene as a primary growth substrate. No degradation of o-cresol was observed in the absence of toluene or when the culture grew on p-cresol and 2,4-dimethylphenol. In batch cultures, the degradation of o-cresol started after toluene was degraded to below 0.5 to 1.0 mg/liter but continued only for about 3 to 5 days after the depletion of toluene since the culture had a limited capacity for o-cresol degradation once toluene was depleted. The total amount of o-cresol degraded was proportional to the amount of toluene metabolized, with an average yield of 0.47 mg of o-cresol degraded per mg of toluene metabolized. Experiments with [ring-U-14C]o-cresol indicated that about 73% of the carbon from degraded o-cresol was mineralized to CO2 and about 23% was assimilated into biomass after the transient accumulation of unidentified water-soluble intermediates. A mathematical model based on a simplified Monod equation is used to describe the kinetics of o-cresol degradation. In this model, the biomass activity toward o-cresol is assumed to decay according to first-order kinetics once toluene is depleted. On the basis of nonlinear regression of the data, the maximum specific rate of o-cresol degradation was estimated to be 0.4 mg of o-cresol per mg of biomass protein per h, and the first-order decay constant for o-cresol-degrading biomass activity was estimated to be 0.15 h-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Continuous culture of Candida utilis: influence of medium nitrogen concentration

When Candida utilis was grown in continuous culture, decreasing the concentration of N in the medium affected cell composition, biomass yield, biomass productivity, maximal growth rate and cell morphology. When the dilution rate was low (0.1 h^-1), reducing N from 1100 to 100 mg/l led to a 40% decrease in RNA content of the cells. Nitrogen-limited growth, which occurred when N<420 mg/l, was associated with significant changes in cell-wall carbohydrates and a significant reduction in the glycogen content of the cells. A set of culture conditions was established which permitted maximal consumption of the main nutrients in the medium and the production of yeast biomass suitable as a source of single-cell protein.     

Participation of extracellular polysaccharides in detoxication of cadmium ions by cyanobacteria Nostoc muscorum

Using the methods of absorption and fluorescence spectroscopy, histochemistry and micrography, the influence of cadmium in different concentrations on the exudation of exopolysaccharides by Nostoc muscorum was studied to elucidate the possibility of distance detoxication of cadmium ions by the cyanobacterium with the participation of its exometabolites. The toxic action of Cd was controlled by changes in biomass, trichomes morphology, and the photosynthetic apparatus of the cyanobacterium. The content of exopolysaccharides in the extracellular medium was 0.7 +/- 0.01 gram glucose units per gram dry biomass at the culture density of 2 gram dry biomass per liter. In the initial phase of growth, the exopolysaccharide content decreased to 0.4 +/- 0.1 gram glucose units per gram dry biomass, while upon incubation with cadmium ions it increased several times during a week, after a short adaptation period. The kinetics of exopolysaccharide accumulation and their concentration depended on cadmium content in the medium. After the incubation of N. muscorum with Cd2+, the exometabolites and the mucous coat colored with ditizone, which was indicative of cadmium accumulation. The decomposition of the absorption spectra of the hydrolyzed extracellular metabolites showed that in the presence of cadmium ions, the dominant monosaccharide became glucosamine, which readily binds to Cd2+. It was concluded that the system of N. muscorum protection against cadmium includes the binding of the metal by cell structures, primarily, by the mucilaginous sheath and the distance detoxication by exopolysaccharides in the cultural medium. Cadmium induces the activation of the mucilaginous sheath protective function by changing its composition and the rate of renewal. To provide the distance protection of N. muscorum, the rate of polysaccharide release of the altered primary structure increased.