Morphological examination of immobilized Streptomyces aureofaciens during chlortetracycline fermentation

Summary Immobilized spores of Streptomyces aureofaciens ATCC 10762 were grown in situ and formed micropellets under the gel surface. The latter was covered with a membrane-like coat of alginate material, while the bead interior was almost completely free of mycelial growth.High spore concentrations caused a decrease in antibiotic production which might be correlated with the morphological development of cells in the gel.Scanning electron micrographs showed the morphological development of the immobilized cells.     

Production of thermostable and neutral glucoamylase using immobilized <Emphasis Type="Italic">Thermomucor indicae-seudaticae</Emphasis>

Thermomucor indicae-seudaticae was immobilized in alginate, κ-carrageenan, agarose, agar, polyacrylamide and loofah (Luffa cylindrica) sponge (as such or coated with alginate/starch/Emerson YpSs agar), and used for the production of glucoamylase in submerged fermentation. The mycelium developed from alginate-immobilized sporangiospores secreted higher glucoamylase titres (22.7 U ml⁻¹) than those immobilized in other gel matrices and the freely growing mycelial pellets (18.5 U ml⁻¹). Loofah network provided a good support for mycelial growth, but the enzyme production was lower than that attained with alginate beads. Glucoamylase production increased with inoculum density and the optimum levels were achieved when 40 calcium alginate beads (∼5 × 10⁶ immobilized spores) were used to inoculate 50 ml production medium. The alginate bead inoculum displayed high storage stability at 4°C and produced comparable enzyme titres up to 120 days. The glucoamylase production by hyphae emerged from the immobilized sporangiospores was almost stable over eight batches of repeated fermentation. Scanning electron micrographs of alginate beads, after batch fermentation, revealed extensive mycelial growth inside and around the beads.     

Erythromycin production by Streptomyces erythreus entrapped in calcium alginate beads

Summary Mycelia of Streptomyces erythreus were immobilized in calcium alginate beads and employed for production of erythromycin. Compared to conventional and washed mycelial fermentation, the average specific productivity of immobilised mycelia was superior.     

Continuous penicillin production by Penicillium chrysogenum immobilized in calcium alginate beads

Summary A high penicillin-producing Penicillium chrysogenum strain immobilized in calcium alginate beads was used for continuous penicillin fermentation in a bubble column and in a conical bubble fermentor. The fermentation was limited by the growth rate, dilution rates and the stability of the alginate beads. The immobilized cells lost their ability to produce penicillin in the bubble column after 48 h from beginning of the continuous fermentation. In the conical bubble fermentor the immobilized cells remained active for more than 7 days. This bioreactor ensured a good distribution of nutrients and oxygen as well as a higher mechanical stability of the alginate beads.     

Penicillin V production by Penicillium chrysogenum in the presence of Fe3+ and in low-iron culture medium

Late-exponential-phasePenicillium chrysogenum mycelia grown in a complex medium possessed an intracellular iron concentration of 650 μmol/L (2.2±0.6 μmol per g mycelial dry mass). This iron reserve was sufficient to ensure growth and antibiotic production after transferring mycelia into a defined low-iron minimal medium. Although the addition of Fe³⁺ to the Fe-limited cultures increased significantly the intracellular iron levels the surplus iron did not influence the production of penicillin V. Supplements of purified majorP. chrysogenum siderophores (coprogen and ferrichrome) into the fermentation media did not affect the β-lactam production and intracellular iron level. Neither 150 nor 300 μmol/L extracellular Fe³⁺ concentrations disturbed the glutathione metabolism of the fungus, and increased the oxidative stress caused by 700 mmol/L H₂O₂. Nevertheless, when iron was applied in the Fe^II oxidation state the oxidative cell injuries caused by the peroxide were significantly enhanced.     

Examination of immobilized fungal cells by phase-contrast and scanning electron microscopy

Conidia of Penicillium urticae were immobilized in kappa-carrageenan beads and then shaken, in a growth-supporting medium to yield an in situ grown population of mycelia. The physical stability of these beads and the degree of mycelial growth inside the beads were significantly affected by the concentrations of kappa-carrageenan and locust bean gum (LBG) in the bead matrix and by the porous or nonporous nature of the interior. Thus 16-h-old porous and nonporous beads, prepared from 1.25% kappa-carrageenan, 0.5% LBG, and conidia, possessed a very dense mycelial mass at the surface. Only the porous beads possessed a moderately dense mycelial mass at the centre. The conidia at the centre of nonporous beads either failed to germinate or formed very small germ tubes. When washed, 36-h-old porous beads were repeatedly (i.e., 48 h) transferred into nitrogen-free medium, the density of mycelia at the centre increased to equal that at the surface after three transfers or 8 days. Mycelia at the surface exhibited signs of physical damage, while those in the centre did not. The addition of 100 micrograms/mL of cycloheximide to these replacement cultures was reflected by the distortion of interior mycelia.     

Physiological and Morphological Modifications in Immobilized Gibberella fujikuroi Mycelia

Constraints created by immobilization conditions modified the physiological behavior and morphological characteristics of Gibberella fujikuroi mycelia in comparison with their development in free-cell conditions. G. fujikuroi mycelia were immobilized in different support matrices (polyurethane, carrageenan, and alginate) and showed a variety of reactions in response to the different microenvironmental factors encountered during and after immobilization. The best support with respect to gibberellic acid yield and biocatalyst stability was found to be an alginate with a high degree of polymerization. The most visible effects of immobilization included changes in growth development, morphological appearance, metabolite production, mycelial pigmentation, mycelial viability under starvation conditions, and induction of resting forms when previously immobilized mycelia were subcultured.     

Reduced utilization of side-chain precursor for penicillin biosynthesis by mutants of Penicillium chrysogenum

Summary A high yielding strain of Penicillium chrysogenum was mutated with EMS and investigated for selective effects of semi-continuous fermentations. Preferential growth of a class of mutants with different colony type and having reduced ability to utilize side-chain precursor led to reduced Pen V synthesis by the heterogeneous mycelial population.     

Semicontinuous penicillin production by two Penicillium chrysogenum strains immobilized in calcium alginate beads

Summary The growth rates of immobilized Penicillium chrysogenum strains are important in their application to semicontinuous penicillin production. Immobilized P. chrysogenum strains produced about 10–15% less biomass but about 1–2 times more penicillin than free suspended mycelia.In a chemically defined medium an industrial P. chrysogenum strain, S₁, produced about 10–12 times more penicillin than strain ATCC 12690. In a complex medium the immobilized P. chrysogenum S₁ produced about 12% penicillin more than in shaken cultures. In bubble column fermentations, penicillin production was 163% higher in the complex medium than in the chemically defined medium.     

产糖化酶黑曲霉固定化方法比较的研究

采用海藻酸钙凝胶电埋法、以沸石、多孔聚酯等材料为固定化载体的吸附法固定黑曲霉（Aspergillus niger AS3.4309)菌丝细胞,以游离菌丝体作为对照,进行发酵产糖化酶的比较,结果表明：以聚酯泡沫作为固定化载体吸附固定化菌丝细胞产糖化酶活力最高。在产糖化酶的发酵过程中,与游离菌丝体细胞相比,固定化黑曲霉持续产酶时间有一定程度的延长。     

Elucidation of optimum conditions for immobilization of viable cells by using calcium alginate

Different factors which affect the stability of calcium alginate gel beads entrapping viable cells during fermentation were investigated. It was found that among others, the initial population of cells per ml of gel beads, the length of period of incubation in CaCl₂ solution, and the concentration of sodium alginate used for the immobilization were the most important factors affecting the stability of the gel beads during fermentation. By using an initial cell population of about 10⁵ cells per ml of 2.0% sodium alginate, and incubating the beads for at least 22 h in a CaCl₂ solution after immobilization, the percentage of beads which developed cracks during fermentation was highly reduced. Also, without the addition of CaCl₂ into the fermenting broth, the gel beads were stable for nine consecutive batch fermentations.     

Production of acid proteinases byHumicola lutea immobilized in polyacrylamide gel

Summary Fungal spores ofHumicola lutea 120–5 were entrapped in 5% polyacrylamide gel and were cultivated for 44–48 h to form a mycelial network inside the beads. A dense mycelial growth also occurred on the surface of the beads. It was possible to reuse the immobilized mycelium for production of acid proteinases in 12 different batches without loss of mechanical stability. The inoculum size should be controlled prior to its transfer into fresh production medium. Maximal enzyme production exceeding the level of free cell fermentation was registered in the fourth to seventh cycles. According to the size of the inoculum, half of the initial production rate was reached after 7–14 batches.     

Production of conidia of Alternaria cassiae with alginate pellets

Formulations of alginate-encapsulated mycelia are used to generate spores for mycoherbicidal application to weed-infested fields and for bulk production of spore-based products. Spore yield of such formulations is a primary determinant of product efficacy. A number of parameters of the alginate process were studied to develop an optimal alginate formulation for field application of Alternaria cassiae, a mycoherbicide for sicklepod (Cassia obtusifolia). The composition of the fermentation medium and of the filler used in formulation and the fermentation time were important variables. The addition of nutrients to the mycelial homogenate after fermentation increased sporulation but the amount and ratio of nutrients in the fermentation medium had a greater influence on spore yield from pellets. Optimal sporulation resulted from mycelia produced during a 60- to 70-h fermentation in 2.4% dehydrated potato dextrose broth and 14% V-8 vegetable juice and entrapped in pellets containing corn cob grits as the filler.     

Covalent stabilization of alginate gel for the entrapment of living whole cells

Summary Three methods were developed for preparing alginate gels containing cells that are stable in phosphate containing media. In Method I preformed alginate beads containing entrapped cells were treated with polyethyl eneimine followed by glutaraldehyde. In Method II alginate sol was treated with a carbodiimide and N-hydroxysuccinimide (to form active esters), mixed with cells and extruded into calcium chloride solution. The beads were subsequently cross-linked with polyethyleneimine. In Method III alginate so] was treated with periodate (to form aldehyde groups), mixed with cells and extruded into calcium chloride solution. The beads were subsequently cross-linked with polyethyleneimine. Saccharomyces cerevisiae cells, immobilized in such stabilized gels, exhibited almost the same fermentation activity as the standard preparation. The viability of the immobilized cells was retained during the stabilization procedure as judged from their ability to multiply in the presence of nutrients.The preparations remained stable in phosphate buffer for at least ten days without substantial release of cells. The extent of cross-linking was controlled by varying the time and the concentration of reactants, thus giving preparations ranging from beads with a thin stabilized shell to beads homogeneously stabilized.     

Scanning electron microscopic examination of calcium alginate beads immobilizing growing mycelia of Aspergillus phoenicus

Calcium alginate beads inoculated with conidia of Aspergillus phoenicus have been incubated in various culture vessels for 120 h. Scanning electron microscopy revealed that the degree of agitation was a factor in surface stability of the beads. Highly significant was the successful restriction of mycelial growth to the subsurface, a condition required if the full advantages of immobilized fungi are to be realized.     

Controlled mycelial growth for kojic acid production using Ca-alginate-immobilized fungal cells

Summary Conidia of Aspergillus oryzae were immobilized in Ca-alginate beads and then incubated in a nutrient medium to yield an immobilized biocatalyst producing kojic acid. The immobilized cell cultures produced kojic acid linearly during cultivation. Regardless of the size of the immobilized particles, there existed an optimal nitrogen concentration for the maximum production rate of kojic acid, at which smaller bead sizes resulted in a higher production rate. When the growth of mycelia were confined within the bead surface and segregated from each other by gel material, they produced kojic acid with maximal catalytic activity and exhibited the highest conversion yield of glucose. The extent of mycelial segregation was especially higher in cultures of smaller bead particles, and the depth of mycelial growth was 150 to 250 m from the gel bead surface in all cultures of different nitrogen concentrations and bead sizes. Therefore, for the maximum expression of catalytic activities of immobilized mycelial cultures, it was found very critical to optimally control the mycelial distribution in gel beads by the culture conditions affecting mycelial growth.     

Semicontinuous cultivation of immobilized Claviceps purpurea

Summary Mycelia of Claviceps purpurea CBS 164.59 were immobilized in 2%, 4%, and 8% calcium alginate. Alkaloid production by free cells declined after 60 days, while immobilized cells retained their activity for 200 days. The cumulative alkaloid production for all fermentation cycles using 8% calcium alginate immobilized mycelia was 25 times higher than that from free cells. The best yields of the ergopeptide ergometrine were reached with 4% gel immobilized mycelia, while higher gel concentrations caused a shift in the alkaloid biosynthesis towards high clavine alkaloid production.Beginning with the third cycle of reincubation the immobilized mycelia showed a marked tendency to fragmentize into vacuolated arthrosporoid-like structures and produced violet-black pigments so that the beads recalled sclerotial structures of parasitically living Claviceps.Dedicated to Prof. Dr. K. Esser to his 60th birthday     

The D-xylose fermenting capacities of immobilizedPichia stipitis andPachysolen tannophilus

Summary The yeastsP. stipitis NRRL Y-7124 andP. tannophilus NRRL Y-2460 were entrapped in -carrageenan beads and used for repeated batch fermentation of D-xylose, in a series of four reactors. The operating conditions finally chosen gave an oxygen coefficient (K_La) of 0.83 min^–1, as measured by the sulphite method. Ethanol yields were 0.40 g/g forP. stipitis and 0.36 g/g forP. tannophilus (respectively 78.4% and 70.5% of the theoretical yields). In spite of its lower retention by the gel,P. stipitis exhibited greater fermenting capacities thanP. tannophilus.     

Adsorption ofCatharanthus roseus cells on various support particles

Summary Adsorption ofC. roseus cells producing indole alkaloids on various support particles were investigated in an attempt to find a suitable support material for surface culture of plant cells. Five different support particles namely gelatin, agar, alginate, polypropylene and glass beads were tested. Gelatin was found to be the most effective adsorbent resulting in nearly 30% adsorption of cells initially present in suspension. Adsorption isotherm of cells on gelatin beads was represented by a three parameter expression due to sigmoidal shape of the isotherm. The constants of the adsorption isotherm were determined using the experimental data.     

Alginate Encapsulation of the White Rot Fungus Phanerochaete chrysosporium

In the laboratory, the white rot fungus Phanerochaete chrysosporium degrades numerous organic pollutants. Lack of a slow-release delivery system to toxic waste sites, for this and other fungi, however, constitutes an important barrier to practical implementation. In this study, the use of calcium alginate as an encapsulant for mycelia was investigated; samples were in the form of pellets 1–3 mm in diameter. When refrigerated, alginate-embedded mycelia of P. chrysosporium were viable for one year, both with and without nutrient supplementation. At room temperature, in the absence of nutrient supplementation, viability decreased sharply within 2 months. Addition of sawdust or corncob grits extended the viability of alginate-embedded mycelia; nevertheless, after 9 months only about 20% of the pellets stored at room temperature yielded fungal growth. Spores of P. chrysosporium, embedded in alginate pellets together with corncob grits, gave 75% viability after 9 months of storage at room temperature. Alginate-embedded mycelia were used in Petri plate toxicity tests with 2,4,6-trinitrotoluene (TNT) and gave more rapid and reproducible results than tests performed with mycelial plugs. These experiments demonstrated the feasibility of encapsulating P. chrysosporium in calcium alginate pellets, thus providing a potential method of delivering white rot fungi to toxic waste sites, as well as for developing a system of standardized toxicity testing in plate assays. Received: 10 July 1996 / Accepted: 13 August 1996