Cultures In Erlenmeyer Flasks Research Articles

Endochitinase and Chitobiosidase Production by Marine Aeromonas caviae CHZ306: Establishment of Nitrogen Supplementation.

Aeromonas caviae CHZ306, a marine-derived bacterium isolated from zooplankton, can use chitin (a polymer of a β-(1,4)-linked N-acetyl-D-glucosamine) as a carbon source. The chitin is hydrolyzed by chitinolytic enzymes, namely endochitinases and exochitinases (chitobiosidase and N-acetyl-glucosaminidase). Indeed, the chitinolytic pathway is initiated by the coexpression of the enzymes endochitinase (EnCh) and chitobiosidase (ChB); however, few studies, including biotechnological production of these enzymes, have been reported, although chitosaccharide are helpful in several industries, such as cosmetics. This study demonstrates the potential to maximize the simultaneous EnCh and ChB production by nitrogen supplementation on culture media. Twelve different nitrogen supplementation sources (inorganic and organic) previously analyzed in elemental composition (carbon and nitrogen) were tested and evaluated in the Erlenmeyer flask culture of A. caviae CHZ306 for EnCh and ChB expression. None of the nutrients inhibited bacterial growth, and the maximum activity in both EnCh and ChB was observed at 12 h, using corn-steep solids and peptone A. Corn-steep solids and peptone A were then combined at three ratios (1:1, 1:2, and 2:1) to maximize the production. The high activities for EnCh (30.1 U.L-1) and ChB (21.3 U.L-1) were obtained with 2:1 corn-steep solids and peptone A, corresponding to more than 5- and 3-fold enhancement, respectively, compared to the control condition.

Plant growth promoting endophytic Pseudomonas aeruginosa RD10 isolated from water hyacinth for phenol degradation

A total of twelve endophytic bacteria were isolated from the root of E. crassipes. All these bacterial strains were analyzed for phenol degradation potential. Twelve endophytic bacteria were subjected to polyphenol resistance analysis and all showed phenol tolerance up to 300 ppm. Among the twelve bacterial strains, the strain RD10 was resistant up to 800 ppm, and reduced growth were observed at higher concentrations. The plant growth-promoting properties of endophytic bacterial strains were analyzed. Among the isolated bacterial strain, only five strains produced ACC deaminase. IAA production was detected in seven bacterial isolates, while siderophore production was determined from only five bacterial strains. Among the isolated endophytes, the strain RD10 exhibited ACC deaminase, IAA, and siderophore activity in submerged fermentation. ACC deaminase production of 125.2 ± 4.8 m/ml was shown by strain RD10 and IAA production was 89.7 ± 5.1 μg/ml. Moreover, maximum siderophore production was observed (53.9 ± 4.9% siderophore units) in strain RD3. Catechol 2,3-dioxygenase activity of the bacterial strains was analyzed. Catechol 2,3 dioxygenase activity ranges from 0.03 ± 0.01 U/mg to 35.2 ± 1.1 U/mg. Phenol degradation by the isolated endophytes was studied and the initial phenol concentration was 500 ppm. The strain RD1 degraded 1.2 ± 0.2% phenol and more than 50% phenol degradation was observed in strains RD3, RD4, RD6, RD7, RD9, RD10, and RD11, respectively. The endophytic bacterial strain was Gram-negative, rodshaped and motile. It was non-spore-forming bacteria, capsulated and, showed a positive reaction to oxidase and catalase. In MacConkey agar medium, strain RD10 forms smooth and flat colonies, and the diameter of the colonies ranged from 2 to 3 mm. The colonies have an alligator skin-like appearance on top view and have regular margins. The strain RD10 was streaked on blood agar medium and the strain RD10 produced strong pigmentation on blood agar. Antibiotic sensitivity of strain RD10 against various antibiotics in terms of zone of inhibition was analyzed. The endophytic strain RD10 was sensitive to all selected antibiotics. Based on biochemical characters, pigment analysis and, molecular characterization, the strain was confirmed as P. aeruginosa RD10. In this study, P. aeruginosa RD10 was cultured at 400, 500 and, 600 mg/l phenol concentration for 96 h. Optimum growth was achieved at 500 mg/l concentration and an inhibitory effect was observed at 600 mg/l phenol concentration. The results confirmed that the selected strain can withstand and grow well at 500 mg/l phenol concentration. The strain RD10 was cultured at various incubation temperatures with 500 mg/l phenol concentration. Optimization of environmental factors can lead to maximum degradation of phenol. The results revealed that phenol degradation was influenced by temperature. The highest phenol degradation was achieved at 30 °C in Erlenmeyer flask culture and decreased at high temperatures. P. aeruginosa RD10 could degrade phenol at pH 5.5 (10.2 ± 0.5% removal), at pH 6.0, 15.4 ± 1.1% of the phenol was degraded after incubation for 72 h at 30 °C. Phenol removal (%) declined to 70.4 ± 2.9% at higher initial pH value (9.0).

Candida tropicalis as a Promising Oleaginous Yeast for Olive Mill Wastewater Bioconversion

Olive mill wastewater (OMW), which is generated during olive oil production, has detrimental effects on the environment due to its high organic load and phenolic compounds content. OMW is difficult to biodegrade, but represents a valuable resource of nutrients for microbial growth. In this study, yeast strains were screened for their growth on phenolic compounds usually found in OMW and responsible for antimicrobial effects. Candida tropicalis ATCC 750 demonstrated an extraordinary capacity to grow in phenolics and was chosen for further experiments with OMW-based medium. The effects of nitrogen supplementation, the pH, and the stirring rate on cellular growth, OMW-components consumption, and added-value compounds production were studied in batch cultures in Erlenmeyer flasks and in a bioreactor. Candida tropicalis was able to reduce 68% of the organic load (chemical oxygen demand) and 39% of the total phenols of OMW in optimized conditions in bioreactor experiments, producing lipase (203 U·L−1) and protease (1105 U·L−1). Moreover, intracellular lipids were accumulated, most significantly under nitrogen-limited conditions, which is common in this type of wastewater. The high potential of C. tropicalis to detoxify OMW and produce added-value compounds from it makes this process an alternative approach to other conventional processes of OMW treatment.

The development of perfusion culture of CHO cells in Erlenmeyer flasks for further transfer of the process to the bioreactor

The article discusses the relevance and problems of perfusion culture of CHO cells, also proposed modeling of perfusion culture in Erlenmeyer flasks.

Evaluation of Neochloris oleoabundans as sustainable source of oil-rich biomass

In microalgae biotechnology, several strategies have been used aiming to increase biomass productivity and oil content, for example, by exploring novel bioreactor design and cultivation techniques. In the present study, the influence of stressing agents (sodium thiosulphate, sodium chloride, and glycerol), under nitrogen starvation, were evaluated for lipid accumulation in Neochloris oleoabundans. Additionally, different light regimes and the use of LED were also evaluated. The results showed that sodium thiosulfate, glycerol, and sodium chloride addition resulted in a reduction in biomass concentration, but, at the same time, an increase in lipid content. 2.5 mM of sodium thiosulfate provided the highest percentage of lipid content up to 44.7%. In a tubular photobioreactor, despite the twofold increase in biomass concentration, there was a decrease in lipid content, compared with cultures in Erlenmeyer flasks. Also, the use of white LED in combination with traditional fluorescent lamp (12 h:12 h) could efficiently replace 24 h light with fluorescent lamp for producing oil-rich N. oleoabundans biomass.

Optimization of cytotoxic activity of Nocardia sp culture broths using a design of experiments.

In the context of research for new cytotoxic compounds, obtaining bioactive molecules from renewable sources remain a big challenge. Microorganisms and more specifically Actinobacteria from original sources are well known for their biotechnological potential and are hotspots for the discovery of new bioactive compounds. The strain DP94 studied here had shown an interesting cytotoxic activity of its culture broth (HaCaT: IC50 = 8.0 ± 1.5 μg/mL; B16: IC50 = 4.6 ± 1.8 μg/mL), which could not been explained by the compounds isolated in a previous work. The increase of the cytotoxic activity of extracts was investigated, based on a Taguchi L9 orthogonal array design, after DP94 culture in TY medium using two different vessels (bioreactor or Erlenmeyer flasks). Various culture parameters such as temperature, pH and inoculum ratio (%) were studied. For experiments conducted in a bioreactor, stirring speed was included as an additional parameter. Significant differences in the cytotoxic activities of different extracts on B16 melanoma cancer cell lines, highlighted the influence of culture temperature on the production of cytotoxic compound(s) using a bioreactor. A culture in Erlenmeyer flasks was also performed and afforded an increase of the production of the active compounds. The best conditions for the highest cytotoxicity (IC50 on B16: 6 ± 0.5 μg/mL) and the highest yield (202.0 mg/L) were identified as: pH 6, temperature 37°C and 5% inoculum.

Production of anti-inflammatory compounds in Sphaeralcea angustifolia cell suspension cultivated in stirred tank bioreactor.

Sphaeralcea angustifolia is a plant used for the treatment of inflammatory processes. Scopoletin, tomentin, and sphaeralcic acid were identified as the compounds with anti-inflammatory and immunomodulatory effects. Successful establishment of the cell culture in Erlenmeyer flasks has been reported previously. The aim of this study was to evaluate the ability of cells in suspension from S. angustifolia grown in a stirred tank bioreactor and demonstrate their capacity to produce bioactive compounds. Cells in suspension grown at 200 rpm reached a maximal cell biomass in dry weight at 19.11 g/L and produced 3.47 mg/g of sphaeralcic acid. The mixture of scopoletin and tomentin was only detected at the beginning of the culture (12.13 μg/g). Considering that the profile of dissolved oxygen during the cultures was lesser than 15%, it is possible that the low growth at 100 rpm could be due to oxygen limitations or to cell sedimentation. At 400 rpm, a negative effect on cell viability could be caused by the increase in the hydrodynamic stress, including the impeller tip, average shear rate, and Reynolds number. The sphaeralcic acid content in the cell suspension of S. angustifolia obtained in the bioreactor was two orders of magnitude greater than that reported for the culture grown in Erlenmeyer flasks.

Development of a circulation direct sampling and monitoring system for O2 and CO2 concentrations in the gas\u2013liquid phases of shake-flask systems during microbial cell culture

Monitoring the environmental factors during shake-flask culture of microorganisms can help to optimise the initial steps of bioprocess development. Herein, we developed a circulation direct monitoring and sampling system (CDMSS) that can monitor the behaviour of CO2 and O2 in the gas–liquid phases and obtain a sample without interrupting the shaking of the culture in Erlenmeyer flasks capped with breathable culture plugs. Shake-flask culturing of Escherichia coli using this set-up indicated that a high concentration of CO2 accumulated not only in the headspace (maximum ~100 mg/L) but also in the culture broth (maximum ~85 mg/L) during the logarithmic phase (4.5–9.0 h). By packing a CO2 absorbent in the gas circulation unit of CDMSS, a specialised shake-flask culture was developed to remove CO2 from the headspace. It was posited that removing CO2 from the headspace would suppress increases in the dissolved CO2 concentration in the culture broth (maximum ~15 mg/L). Furthermore, the logarithmic growth phase (4.5–12.0 h) was extended, the U.O.D.580 and pH value increased, and acetic acid concentration was reduced, compared with the control. To our knowledge, this is the first report of a method aimed at improving the growth of E. coli cells without changing the composition of the medium, temperature, and shaking conditions.

Submerged Culture of Grifola gargal and G. sordulenta (Higher Basidiomycetes) from Argentina as a Source of Mycelia with Antioxidant Activity.

Submerged culture is an alternative mycelium source for Grifola gargal and G. sordulenta, two rare edible mushrooms related to Grifola frondosa. This work studies their mycelia as a source of antioxidants. The efficient concentrations of methanolic extracts in both radical scavenging (RS) and reducing power (RP) abilities in G. gargal and in G. sordulenta showed a high antioxidant activity. In the experimental design used, the antioxidant activity mainly depended on the culture conditions rather than on the media composition. Irrespective of the basal culture medium, mycelium methanolic extracts of G. sordulenta obtained from culture in Erlenmeyer flasks showed equivalents to ascorbic acid (EQ(AA)) RS-EQ(AA) and RP-EQ(AA) contents higher than the corresponding values obtained with jar cultures. Under stationary cultivation, G. sordulenta produced approximately 50% higher content in both RS-EQ(AA) and RP-EQ(AA) than the medicinal mushroom G. frondosa. Phenolics correlated with RS-EQ(AA) and RP-EQ(AA) in G. gargal and with RP-EQ(AA) in G. sordulenta; besides, thin-layer chromatography showed these compounds to be at least in part related to the RS capacity. It is concluded that G. gargal and G. sordulenta mycelia are excellent sources of antioxidant metabolites.

Obtención de escopolamina en biorreactor a partir de cultivos de raíces de Brugmansia candida

Given the pharmacological importance of tropane alkaloids and the possibilityof producing them from plant tissue cultures of plant species such as Brugmansia candida, the main goal of the present paper was to evaluate the alkaloid production of root cultures developed ina bioreactor. We set up a bioreaction system consisting of two units, one forbiomass growth and the other one for liquid medium conditioning. After 21 days of culture, biomass and scopolamine production were evaluated, resulting in the extraction of 9.5 mg of scopolamine/g of dry roots. This value was higher than the one obtained in a previous culture in Erlenmeyer flasks (6mg/g of dry roots) and higher than the scopolamine extracted from hairy roots cultures using a bioreactor reported in other studies. Regarding biomass, the growth index (GI) was 1.58, similar to the GI obtained with cultures in Erlenmeyer flasks (GI 1.4), which suggests that scaling up the process did not affect biomass productionnegatively.

Metabolic Pathways of Hydrogen Production in Fermentative Acidogenic Microflora

Biohydrogen production from organic wastewater by anaerobically activated sludge fermentation has already been extensively investigated, and it is known that hydrogen can be produced by glucose fermentation through three metabolic pathways, including the oxidative decarboxylation of pyruvic acid to acetyl-CoA, oxidation of NADH to NAD+, and acetogenesis by hydrogen-producing acetogens. However, the exact or dominant pathways of hydrogen production in the anaerobically activated sludge fermentation process have not yet been identified. Thus, a continuous stirred-tank reactor (CSTR) was introduced and a specifically acclimated acidogenic fermentative microflora obtained under certain operation conditions. The hydrogen production activity and potential hydrogen-producing pathways in the acidogenic fermentative microflora were then investigated using batch cultures in Erlenmeyer flasks with a working volume of 500 ml. Based on an initial glucose concentration of 10 g/l, pH 6.0, and a biomass of 1.01 g/l of a mixed liquid volatile suspended solid (MLVSS), 247.7 ml of hydrogen was obtained after a 68 h cultivation period at 35 +/- 1 degrees C. Further tests indicated that 69% of the hydrogen was produced from the oxidative decarboxylation of pyruvic acid, whereas the remaining 31% was from the oxidation of NADH to NAD+. There were no hydrogen-producing acetogens or they were unable to work effectively in the anaerobically activated sludge with a hydraulic retention time (HRT) of less than 8 h.

Recombinant Antibody Production by Perfusion Cultures of rCHO Cells in a Depth Filter Perfusion System

Recombinant Chinese hamster ovary cells, producing recombinant antibody against the human platelet, were cultivated in a depth filter perfusion system (DFPS). When perfusion cultures with working volume of 1 L were operated at perfusion rates of 5/d and 6/d, volumetric antibody productivities reached values 28 and 34 times higher than that of batch suspension culture in Erlenmeyer flasks and 43 and 53 times higher than that of batch culture in a controlled stirred tank reactor, respectively. Perfusion cultures in the DFPS showed stable antibody production over the whole culture period of up to 20 days. In the DFPS, inoculated cells in suspension were entrapped in a few hours within the depth filter matrix by medium circulation and retained there until the void space of the filter matrix was saturated by the cultured cells. After cells in the depth filter matrix reached saturation, overgrown viable cells at a perfusion rate of 5/d or 6/d were continuously collected into waste medium at a density of 2-4 x 10(5) cells/mL, which resulted in stable operation at high perfusion rates, maintaining values of process parameters such as glucose/lactate concentration, pH, and dissolved oxygen concentration. Because the DFPS overcomes most drawbacks observed with conventional perfusion systems, it is preferable to be used as a key culture system to produce monoclonal antibody stably for a long culture period.

Expression of a human anti-rabies virus monoclonal antibody in tobacco cell culture

A Nicotiana tabacum cv. Xanthi cell culture was initiated from a transgenic plant expressing a human anti-rabies virus monoclonal antibody. Within 3 months, plant cell suspension cultures were established and recombinant protein expression was examined. The antibody was stably produced during culture growth. ELISA, protein G purification, Western blotting, and neutralization assay confirmed that the antibody was fully processed, with association of light and heavy-chains, and that it was able to bind and neutralize rabies virus. Quantification of antibody production in plant cell suspension culture revealed 30 μg/g of cell dry weight for the highest-producing culture (0.5 mg/L), 3 times higher than from the original transgenic plant. The same production level was observed 3 months after cell culture initiation. Plant cell suspension cultures were successfully grown in a new disposable plastic bioreactor, with a growth rate and production level similar to that of cultures in Erlenmeyer flasks.

Bioreactor design for propagation of somatic embryos

Six identical bioreactors were constructed and built at the Agricultural University of Norway to provide optimal conditions for plant cell regeneration from cells into somatic embryos (‘clonal or somatic seeds’). This was made possible through cooperation in COST87 by a European network in a working group on regeneration from plant cell cultures. The bioreactor design provides gentle stirring through a slow-speed stirrer that regularly changes direction of rotation to prevent ‘quiet’ zones in the suspension in which cells can settle and grow. In addition, the oxygen is provided, bubble-free, through thin silicone tubing loops that are hanging loose, moving with the liquid to prevent cell growth on these tubes. We used off-the-shelf components whenever possible, to reduce the costs to a minimum, which was another aim of the construction. The result was a suite of relatively inexpensive computer-controlled bioreactors that could control temperature, oxygen, pH, stirrer speed and stirrer direction. In addition, we have provided different light spectral qualities by simple means of filtering the light. Using the present software, the parameters can be set up to alter every hour during the 24-h day/night cycle. All our cultures have improved growth in the bioreactors compared to identical cultures in Erlenmeyer flasks. The cultures used are: embryogenic cultures of carrot (Daucus carota), Norway spruce (Picea abies), birch (Betula pendula), cyclamen (Cyclamen persicum) and shoot cultures of Christmas begonia (Begonia x cheimantha). The paper also discusses recommendations for improvements of the current system for future revisions.

Optimal conditions for the production of sulfated polysaccharide by marine microalga Gyrodinium impudicum strain KG03

A marine microalga Gyrodinium impudicum strain KG03 produced sulfated exopolysaccharide designated as p-KG03, which showed a strong antiviral activity against encephalomyocarditis virus (EMCV). To optimize culture conditions for the production of p-KG03, mineral salts, vitamins, plant growth hormones, temperature, pH and light conditions were examined. From this study, M-KG03 medium for the maximum production of p-KG03 was suggested as follows; NH 4Cl 75 μM, NaH 3PO 4 200 μM, NaHCO 3 50 μM, Na 2SO 4 10 μM, FeCl 2·6H 2O 10 μM, MnCl 2·4H 2O 0.1 μM, vitamin B 12 0.75 μg, naphthalene acetic acid (NAA) 7.5 μg and myo-inositol 200 mg per liter of aged sea water. The optimal temperature and pH were 22.5 °C and 8.0, respectively. The optimal light conditions of intensity and period were 150 μE m −2 s −1 and 16:8 h light:dark cycle. Finally, the cell growth and p-KG03 production were measured in one liter of M-KG03 medium with 1% CO 2 and 50 ml min −1 of airflow using two liters airlift balloon type photobioreactor (ABTPR). At these optimal conditions, p-KG03 production and cell growth were 134.6±5.9 mg l −1 and 123,076±1,597 cells ml −1, respectively, representing a 7.7 and 5.1 times compared with f/2 medium with Erlenmeyer flask culture (p-KG03 production 17.5±1.3 mg l −1 and cell growth 24,311±1,291 cells ml −1).

Production of a novel exopolysaccharide by Rahnella aquatilis

A bacterium Tomakomai B-1 that was isolated from the activated sludge of a treatment process for wastewater of the pulp industry was found to produce soluble and insoluble exopolysaccharides (EPSs) when grown aerobically in a medium containing sucrose as the sole carbon source. The sugar component of soluble EPSs consisted of rhamnose, mannose, galactose, glucose, mannuronic acid, galacturonic acid and glucuronic acid, and that of insoluble EPSs consisted of rhamnose, mannose, galactose, glucose, mannuronic acid and glucuronic acid. The concentrations of insoluble and soluble EPS were approximately 1.63 and 2.58 g/ l, respectively, by Erlenmeyer flask culture at 20°C for 3 d with a synthetic medium containing 1% sucrose, 0.05% CaCO 3, 0.05% MgSO 4, 0.1% K 2HPO 4, 0.0001% Na 2MoO 4, 0.01% urea, 1.07% MES and small amounts of trace metals. Tomakomai B-1 was identified as Rahnella aquatilis. To our knowledge the production of EPSs by R. Aquatilis is reported here for the first time.

Construction of catalase deficient Escherichia coli strains for the production of uricase.

To produce catalase-free uricase preparations, we constructed catalase-deficient strains from Escherichai coli MC1000 and MM294 and used them as recombinant host strains. The parent strains and catalase-deficient strains showed no differences in the growth characteristics by shaking culture in Erlenmeyer flasks. The catalase deficient strain derived from MC1000 transformed with the uricase expression plasmid pUT118 (strain SN0037) had growth characteristics and the uricase productivity comparable to those of the parent host strain MC1000 in fed-batch culture in a jar fermentor and no catalase activity was detected in cell-free extracts. However, the katG disrupted strains from MM294 carrying pUT118 had poor growth and their uricase productivities were low compared to those of the parent strain MM294. Using the strain SN0037, a catalase-free uricase preparation was obtained with fewer purification procedures and the final recovery of uricase activity was improved. The catalase-deficient E. coli host strain will be a suitable host for the production of the uricase, free of catalase activity, in high yield.

A novel method of growing fungi for DNA extraction

Preparation of fungi for DNA extraction typically involves growing cultures in liquid culture in Erlenmeyer flasks, Roux bottles or even microfuge tubes (Cenis 1992 Nucl. Acids Res. 20:2380). Growing fungal cultures in liquid may require formulating new media or determining aeration requirements, and there are no rapid means of confirming the identification of the resulting mycelium. Fungi are grown routinely on agar media for identification, but agar complicates DNA extraction.

Effect of oxygen supply on the suspension culture of genetically modified tobacco cells.

The effect of oxygen supply on the cultivation of the genetically modified tobacco cells and the formation of a foreign protein, beta-glucuronidase (GUS), was investigated in 250-mL Erlenmeyer flasks, a 5-L stirred tank fermenter, and a 7-L air-lift fermenter. The oxygen supply was varied by using different volumes of medium in the case of the 250-mL Erlenmeyer flask culture or by the different aeration rate in the case of the two types of fermenters tested. Higher oxygen supply stimulated cell growth and increased oxygen consumption rate, the level of phenolics, and GUS productions.

Cultivation of poliomyelitis virus in tissue culture. V. Observations on virus propagation in certain animal tissues with a synthetic nutrient medium.

Further observations have been made on the propagation of Lansing poliomyelitis virus in tissue cultures. It has been observed that tissues derived from several organs of rhesus monkeys will support virus growth in tissue cultures in Erlenmeyer flasks with a synthetic medium as the source of nutrient. Cultures of tissues from monkey testis, lung, kidney, and gut have survived for long periods, and virus has been regularly recovered even from fluids removed from cultures after as late as 125 days. Cultures of tissues from monkey brain and cord, and muscle, did not survive as long, and less virus was demonstrated in the supernatant fluids. Muscle from the diaphragm did not appear to support growth. Cultures of tissues from the brain, kidney, and lung of beef embryos survived for long periods, but no virus was found in any of the culture fluids.