Optimization of culture conditions for mycoepoxydiene production by <Emphasis Type="Italic">Phomopsis</Emphasis> sp. Hant25

Culture media and fermentation conditions for cultivation of an endophytic fungus Phomopsis sp. Hant25 were investigated in order to improve the yield of mycoepoxydiene, a novel fungal metabolite having potent cytotoxic activity against many cancer cell lines. Mycoepoxydiene accumulated in the culture broth during the stationary phase of fungal growth. Modified M1D medium was superior to malt Czapek, and Czapek yeast autolysate broths in supporting mycoepoxydiene production. Pellet growth was the morphological form that favored biosynthesis of mycoepoxydiene. This could be achieved by incubating the culture statically for 6 days before shaking at 120 rpm. Incorporation of a cellulose paper disc into the culture flask promoted fungal growth at the liquid surface, which accelerated mycoepoxydiene production and maximized the final yield to a level of 354 mg l⁻¹, though fungal attachment to the solid support was not required. Since the peak concentration of mycoepoxydiene in the culture broth was followed by a steeply declining phase, the harvest time had to be precisely determined for maximum product yield. Understanding the factor(s) involved in rapid degradation of mycoepoxydiene could lead to improved final yields.     

Development of a stirred tank reactor system for the production of lignin peroxidases (ligninases) byPhanerochaete chrysosporium BKM-F-1767

Summary Lignin peroxidases produced byPhanerochaete chrysosporium have several important potential industrial applications based on their ability to degrade lignin and lignin-like compounds. A stirred tank reactor system for the production of lignin peroxidases is described here. Included in this study is an examination of the mechanics of pellet biocatalyst formation and the optimization of an acetate buffered medium. Higher levels of lignin peroxidase were obtained with acetate buffer compared to the other buffer systems tested. Concentrations of 0.05% (w/v) Tween 80 and 0.4 mM veratryl alcohol gave optimal lignin peroxidase activity in acetate buffered medium. In shake flask cultures, mycelial fragments in the inoculum aggregated into pellets during the first eight hours of incubation and thereafter increased in size through the eighth day. The agitation rate in shake flask cultures affected pellet size, the number of pellets formed, and lignin peroxidase activity. Transfer of fungal pellets from shake flask culture to a continuously oxygenated baffled stirred tank reactor (STR) resulted in production of high lignin peroxidase titres comparable to those of shake flask cultures when the agitation rate, oxygen dispersion and foaming were closely controlled.     

Extracellular alkaline protease of newly isolatedRhizopus oryzae

Summary A fungal isolate identified asRhizopus oryzae, produces an extracellular alkaline serine protease. Maximum protease formation was after six days in shake flask culture at two different conditions of pH and temperature optimum (pH 5 at 30°C and pH 10 at 37°C). AgNO₃ and Tween 80 increased protease synthesis. The enzyme is stable between pH 3 and pH 11 and has a temperature optimum of 60°C.     

Economical parallel protein expression screening and scale-up in Escherichia coli

A novel microfermentation and scale-up platform for parallel protein production in Escherichia coli is described. The vertical shaker device Vertiga, which generates low-volume high density (A₆₀₀ ∼ 20) Escherichia coli cultures in 96-position deep-well plates without auxiliary oxygen supplementation, has been coupled to a new disposable shake flask design, the Ultra Yield^TM flask, that allows for equally high cell culture densities to be obtained. The Ultra Yield^TM flask, which accommodates up to 1 l in culture volume, has a baffled base and a more vertical wall construction compared to traditional shake flask designs. Experimental data is presented demonstrating that the Ultra Yield^TM flask generates, on average, an equivalent amount of recombinant protein per unit cell culture density as do traditional shake flask designs but at a substantially greater amount per unit volume. The combination of Vertiga and the Ultra Yield^TM flask provides a convenient and scalable low-cost solution to parallel protein production in Escherichia coli.     

Bioinert surface of pluronic-immobilized flask for preservation of hematopoietic stem cells

The bioinert materials on which cells do not proliferate, differentiate, nor de-differentiate should be useful for the culture and preservation of stem cells. The Pluronic F127, a triblock copolymer of ethylene oxide, and propylene oxide was activated using carbonyldiimidazole (CDI), and CDI-activated Pluronic was subsequently immobilized on the surface of a lysine-coated polystyrene tissue culture flask. The morphology of fibroblasts (L929 cells) on the Pluronic-immobilized flask was spherical, and did not show spreading behavior. This observation indicates that L929 cells on the Pluronic-immobilized flask were cultured in a bioinert environment. The expression ratio of surface markers on hematopoietic stem cells (CD34 and CD133) cultured in the Pluronic-immobilized flask was significantly higher than that in polystyrene tissue culture flask and commercially available bioinert flask (i.e., low cell binding cultureware). This is caused by the existence of hydrophilic segments of Pluronic F127 on the Pluronic-immobilized flask.     

Culture in the rotating-wall vessel affects recombinant protein production capability of two insect cell lines in different manners

Summary The production of recombinant secreted alkaline phosphatase protein in virally infected insect cells was studied in shaker flask and high aspect rotating-wall vessel (HARV) culture. Two commonly used cell lines, Spodoptera frugiperda Sf-9 (Sf-9) and a nonaggregating isolate of the Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) cell line, Trichoplusia ni Tn-5B1-4-NA (TN-5B1-4-NA), were used and monitored for 120-h postinfection. Different responses to culture in the HARV were seen in the two cell lines. While the Sf-9 cell line was able to produce slightly greater amounts of recombinant protein in the HARV than in shaker flask controls, the Tn-5B1-4-NA cell line produced significantly lesser amounts in the HARV than in the shaker flasks. Both cell lines exhibited longer life spans and longer periods of protein production in HARV culture than in shaker flask culture, presumably due to lower levels of shear encountered in the HARV. The important difference was in the protein production rate responses of the two cell lines. While the protein production rates of Sf-9 cells were comparable in both HARV and shaker flask cultures, the protein production rates of Tn-5B1-4-NA cells were much lower in HARV culture than in shaker flask cultures. The conclusion is drawn that cell line-specific adaptation to the HARV strongly influences recombinant protein production.     

The scale up of mycelial shake flask fermentations: A case study of gamma linolenic acid production byMucor hiemalis IRL 51

Summary Production of gamma linolenic acid (GLA) by the filamentous fungusMucor hiemalis IRL 51 was studied in both shake flask culture and in a 10-L stirred tank fermenter. This study was conducted to assess how the results from shake flask media screening trials compared to those obtained in a 10-L stirred tank fermenter, which is assumed to be more representative of an industrial system. The results show that the biological performance in 10-L fermenters is usually the same as that in shake flask culture. There were some inconsistencies which could possibly be attributed to scale, but no large differences were systematically seen. These results show that for this filamentous fungus, shake flask culture provides a quick and inexpensive way of optimizing medium composition.     

Multiple shoot culture of Dianthus caryophyllus using mist culture system

Summary A mist bioreactor system for the plant tissue cultures was developed. Using this system, the growth of Dianthus caryophyllus multiple shoots was directly measured. Tissue growth in mist bioreactor system was far better than that on agar medium and almost comparable to that in liquid medium. The mass increase (final dry weight/initial dry weight) in the mist culture was 2.85 while 3.28 in the liquid flask culture. Shoots were seriously vitrified in flask culture but these vitrifications could be considerably cured by using the mist culture system.     

Cultivation conditions for extracellular production of penicillinase by Escherichia coli carrying pEAP31 on a semi-large scale

Summary Cultivation conditions for extracellular production of penicillinase on a semi-large scale were established by using Escherichia coli K-12 HB 101 carrying the plasmid pEAP31 with the penicillinase gene from alkalophilic Bacillus sp. no. 170. Extracellular production of the enzyme was affected by several parameters such as concentration of carbohydrates and Nacl, pH value of culture broth, culture temperature, culture volume and shaking speed of the cultivation flask. The organism produced a large amount of the enzyme in culture broth under the optimal conditions established. For example, 180 units/ml of the extracellular enzyme was produced when the organism was inoculated in 300 ml broth in a 500-ml volume cultivation flask and shaken at 30°C on a reciprocal shaker at 172 oscillations/min with 3.2-cm strokes.     

Ajmalicine production by cell cultures of Catharanthus roseus: from shake flask to bioreactor

The productivity of a cell culture for the production of a secondary metabolite is defined by three factors: specific growth rate, specific product formation rate, and biomass concentration during production. The effect of scaling-up from shake flask to bioreactor on growth and production and the effect of increasing the biomass concentration were investigated for the production of ajmalicine by Catharanthus roseus cell suspensions. Growth of biomass was not affected by the type of culture vessel. Growth, carbohydrate storage, glucose and oxygen consumption, and the carbon dioxide production could be predicted rather well by a structured model with the internal phosphate and the external glucose concentration as the controlling factors. The production of ajmalicine on production medium in a shake flask was not reproduced in a bioreactor. The production could be restored by creating a gas regime in the bioreactor comparable to that in a shake flask. Increasing the biomass concentration both in a shake flask and in a stirred fermenter decreased the ajmalicine production rate. This effect could be removed partly by controlling the oxygen concentration in the more dense culture at 85% air saturation.     

Biosorption Potency of Aspergillus niger for Removal of Chromium (VI)

Aspergillus niger isolated from soil and effluent of leather tanning mills had higher activity to remove chromium. The potency of Aspergillus niger was evaluated in shake flask culture by absorption of chromium at pH 6 and temperature 30°C. The results of the study indicated removal of more than 75% chromium by Aspergillus niger determined by diphenylcarbazide colorimetric assay and atomic absorption spectrophotometry after 7 days. Study of microbial Cr(VI) reduction and identification of reduction intermediates has been hindered by the lack of analytical techniques that can identify the oxidation state with subcellular spatial resolution. Therefore, removal of chromium was further substantiated by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX), which indicated an accumulation of chromium in the fungal mycelium.     

Scale-up of purine nucleoside fermentation from a shaking flask to a stirred-tank fermentor

Scaling-up purine nucleoside fermentation by a mutant strain of Bacillus subtilis from a shaking flask to a stirred-tank fermentor was attempted. The dimensions and the operating conditions of the stirred tank were determined in order to satisfy the optimum conditions of O₂ transfer and power consumption per unit volume for the shaking flask. When the purine nucleoside fermentation was carried out in the stirred-tank fermentor under these conditions, in which the temperature simulated that in the shaking flask, the total amount of purine nucleosides produced was almost the same as that in the shaking flask, but the accumulation ratio of guanosine to total nucleotides was different from that in the flask. Since urea could not be utilized so efficiently in the stirred-tank fermentor, the NH^p+ _f4 concentration and the pH of the culture broth were lower than those in the shaking-flask culture during fermentation. The activity of inosine monosphosphate dehydrogenase and the accumulation ratio were significantly affected by the NH^p+ _f4 concentration. When the pH of the stirred-tank culture was maintained at 6.9 by ammonia water to keep the NH^p+ _f4 level higher, the ratio was improved to the same level as that observed in the shaking-flask culture. The fermentation heat calculated from the shaking-flask data and its pattern of change were similar to those in the stirred-tank fermentor. Correspondence to: Y. Sumino     

Rational optimization of culture conditions for the most efficient ethanol production in Scheffersomyces stipitis using design of experiments

Optimization of culture parameters for achieving the most efficient ethanol fermentation is challenging due to multiple variables involved. Here we presented a rationalized methodology for multi‐variables optimization through the design of experiments DoE approach. Three critical parameters, pH, temperature, and agitation speed, affecting ethanol fermentation in S. stipitis was investigated. A predictive model showed that agitation speed significantly affected ethanol synthesis. Reducing pH and temperature also improved ethanol production. The model identified the optimum culture conditions for the most efficient ethanol production with the yield and productivity of 0.46 g/g and 0.28 g/l h, respectively, which is consistent with experimental observation. The results also indicated the scalability of the model from shake flask to bioreactor. Thus, DoE is a promising tool permitting the rapid establishment of culture conditions for the most efficient ethanol fermentation in S. stipitis. The approach could be useful to reduce process development time in lignocellulosic ethanol industry. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Comparative study of some microbial arabinan-degrading enzymes

Summary A variety of thermophilic organisms andBacillus species were screened in shake flask culture for arabanase andp-nitrophenyl--l-arabinosidase activities. Highest arabanase activity was produced by strains ofThielavia terrestris andSporotrichum cellulophilum. Thermoascus aurantiacus and severalBacillus species were most active producers of arabinosidase. Arabinosidases fromBacillus strains had pH optima in the range 5.9–6.7. pH optima of fungal arabinosidases ranged from 2.9 to 6.7.Bacillus arabanases had neutral pH optima, whereas fungal arabanases had pH optima in the range 3.7–5.1. In general, arabinosidases were found to be relatively thermostable, retaining >70% activity for 3 h at 60°C. TheT. aurantiacus enzyme retained 98% activity at 70°C after 3 h.Bacillus arabanases were relatively unstable. All fungal arabanases except theT. aurantiacus enzyme were fully denatured at 70°C after 3 h.     

Factors affecting in vitro formation of cormlets in <Emphasis Type="Italic">Gladiolus hybridus</Emphasis> Hort. and their field performance

The effect of number of important factors on in vitro cormlet formation has been investigated in Gladiolus hybridus Hort. Sucrose concentration of 232 mM was found to be best for producing higher number of cormlets per flask, whereas, the average mass of a cormlet increased with increase in sucrose concentration. Amongst three cultivars (cvs), maximum number of cormlets produced per flask was recorded in cv ‘Her Majesty’, but the average cormlet mass was higher in case of cv ‘Bright Eye’. Although the number of cormlets produced was found to be marginally higher at 30°C when compared with 20°C, the average cormlet mass was higher at the lower temperature. Both the number of cormlets formed per culture flask as well as the average fresh mass of a cormlet increased with increase in the size (volume) of the culture flask used. The known inhibitors of gibberellin biosynthesis used in this study suppressed cormlet formation, and the maximum inhibition was recorded in case of maleic acid hydrazide. Polyamines were found to be beneficial for cormlet formation, and amongst the polyamines used, incorporation of spermidine in the culture medium resulted in maximum number of cormlet formation per culture flask. Field trials indicated that the performance of such in vitro produced cormlets was comparable to that of conventionally produced cormels of the same weight range. The plants raised from in vitro produced cormlets were found to be morphologically similar to the mother plant.     

<Emphasis Type="Italic">Fusarium oxysporum</Emphasis> cell elicitor enhances betalain content in the cell suspension culture of <Emphasis Type="Italic">Celosia cristata</Emphasis>

We started a cell suspension culture from magenta coloured calli of cockscomb to study the effect of biotic and abiotic elicitors on the biosynthesis of betalain pigments. The cultures were grown in a flask containing 30 ml MS media fortified with 13.5 μM 2,4-D and 0.44 μM BAP. These cultures were elicited during its log-phase of growth using fungal elicitors (prepared from mycelia of Fusarium oxysporum), yeast extract, copper sulphate and cobalt chloride. The elicitation reduced the cell count, cell viability and percent pigmented cell in the suspension culture. Similarly, it also resulted in reduced betalain content by all the elicitors except 0.125 × 10^?3% fungal elicitor. Rather, fungal elicitor at this concentration significantly enhanced the amaranthin, betanin, betalamic acid and betaxanthin content in the culture. Besides this, copper sulphate doubled the pigment contribution (ratio of particular pigment content to total pigment content) of betaxanthin at all the concentrations. Therefore, we conclude that fungal elicitor can further be investigated to enhance the content of betalain pigments in suspension culture at a larger scale.     

The development of a kitten lung cell strain and its chromosomes

Summary The development of a line of epithelial cells derived from lung tissue of a 4-week old kitten (KL strain) with evidence regarding its chromosomal changes in vitro is described. The outgrowing cells from fragments in primary cultures in Eagle's medium plus 10% horse serum were scraped with a rubber policeman and dispersed with a syringe fitted with a No. 15 needle. The cell suspension was transferred into a T 30 flask. The floor of the T 30 flask was covered with avian plasma clot which was allowed to set for 10 minutes before adding the cell suspension. The appearance of the cells was epithelial-like at all stages of cultivation. The most frequent chromosome number in 6-day primary culture preparations was 38 (68%). Counts made from cells in the 4th, 9th and 33rd subcultures (64th, 83rd and 165th days from the date of primary culture) showed a spread of the chromosome numbers. In the latest observation, the 84th subculture (411th day), the most frequent chromosome numbers were 90 (22%) and 92 (26%). In addition, the mitotic activity of cells in the strain cultures was observed by phase microscopy.Tobacco Industry Research Committee Fellow.     

Occurrence of Bursaphelenchus mucronatus (Nematoda: Aphelenchoididae) and the microhabitat distribution of fungi in declining pine trees in a locale in Korea

A total of 33 pine trees with symptoms of decline were collected in Jeonnam Province, South Korea, and were examined for the presence of nematodes. About 20% of the trees sampled were positive with Bursaphelenchus species. All Bursaphelenchus species were found in recently dead or dying trees. Based on morphological observations, the nematode extracted from the declining pine trees was identified as B. mucronatus. The highly pathogenic pine wood nematode B. xylophilus was not found in any pine trees sampled. B. mucronatus was easily reared on fungus Botrytis cinerea. Twenty one fungal isolates were isolated from dead trees, fallen twigs, and healthy pine trees. The fungal isolates belonged to Trichoderma genus and were dominant in the wood of partially declining pines. The blue‐stain fungi transmitted by the Monochamus beetle were not detected. The B. mucronatus population decreased markedly on Auxarthron reticulatum DY‐2 isolated from soils. The number of nematodes also reduced on Verticillium saksenae A‐1, a nematophagous fungus, and Beauveria bassiana, an entomopathogenic fungus. This observation suggested the fungal production of nematicidal activity against B. mucronatus. When the fungal culture filtrates were also used for nematicidal activity on B. mucronatus, the culture filtrates of A‐1, DY‐2 and B. bassiana showed over 50% mortality within 48 h exposure. The fungi BC4, BC5 and BC6 isolated from declining pine trees inhibited the reproduction of B. mucronatus, and their culture filtrates also expressed nematicidal activity, indicating a possible interaction between the fungi in pine trees and nematodes at microhabitat level.     

Conversion of<Emphasis Type="Italic">G. hansenii</Emphasis> PJK into non-cellulose-producing mutants according to the culture condition

The conversion of a cellulose-producing cell (Cel ⁺) fromGluconacetobacter hansenii PJK (KCTC 10505 BP) to a non-cellulose-producing cell (Cel ⁻) was investigated by measuring the colony forming unit (CFU). This was achieved in a shaking flask with three slanted baffles, which exerted a strong shear stress. The addition of organic acid, such as glutamic acid and acetic acid, induced the conversion of microbial cells from a wild type toCel ⁻ mutants in a flask culture. The supplementation of 1% ethanol to the medium containing an organic acid depressed the conversion of the microbial cells toCel ⁻ mutants in a conventional flask without slanted baffles. The addition of ethanol to the medium containing an organic acid; however, accelerated the conversion of microbial cells in the flask with slanted baffles. TheCel ⁺ cells from the agitated culture were not easily converted intoCel ⁻, mutants on the additions of organic acid and ethanol to a flask without slanted baffles, but some portion of theCel ⁺ cells were converted toCel ⁻ mutants in a flask with slanted baffles. The conversion ratio ofCel ⁺ cells toCel ⁻ mutants was strongly related to the production of bacterial cellulose independently from the cell growth.     

Large scale fermentation and alkaloid production of cell suspension cultures of Catharanthus roseus

Cell cultures of Catharanthus roseus were scaled up to volumes of 50001 using conventional reactors equipped with flat-blade impellers. The behavior of the fermenter grown cells was compared with corresponding shake flask experiments with respect to growth and indole alkaloid inducibility and production. The limits and problems of transferring shake flask experiments of culture systems such as Catharanthus, in which alkaloid production depends greatly upon the physiological state of the cells, to large scale multistage processes is discussed.