Enterotoxin production by Vibrio cholerae and Vibrio mimicus grown in continuous culture with microbial cell recycle

We have examined the effect of complete cell recycle on the production of cholera toxin (CT) by Vibrio cholerae and CT-like toxin by Vibrio mimicus in continuous culture fermentations. Complete cell recycle was obtained by filtering culture fluids through Amicon hollow fibers with an exclusion limit of 100,000 daltons (H1P100-20) and returning the concentrated cell slurry to the fermentor. A single 1-liter laboratory fermentor system modified with this recycle loop was capable of producing over 20 liters of cell-free culture filtrate per day. Toxin production in this system was compared with yields obtained in traditional continuous cultures and in shake flask cultures. Yields of CT from V. cholerae 569B in the recycle fermentor were highest at the highest dilution rate employed (1.0 vol/vol per h). The use of complete cell recycle dramatically increased yields over those obtained in continuous culture and equaled those obtained in shake flasks. The concentration of CT in the filtrate was slightly less than half of that measured in culture fluids sampled at the same time. Similarly, V. mimicus 61892 grown in the presence of 50 micrograms of lincomycin per ml produced 280 ng of CT per ml in the recycle fermentor, compared with 210 ng/ml in shake flasks under optimal conditions. The sterile filtrate from this fermentation contained 110 ng/ml.     

Enterotoxin production by Vibrio cholerae and Vibrio mimicus grown in continuous culture with microbial cell recycle.

We have examined the effect of complete cell recycle on the production of cholera toxin (CT) by Vibrio cholerae and CT-like toxin by Vibrio mimicus in continuous culture fermentations. Complete cell recycle was obtained by filtering culture fluids through Amicon hollow fibers with an exclusion limit of 100,000 daltons (H1P100-20) and returning the concentrated cell slurry to the fermentor. A single 1-liter laboratory fermentor system modified with this recycle loop was capable of producing over 20 liters of cell-free culture filtrate per day. Toxin production in this system was compared with yields obtained in traditional continuous cultures and in shake flask cultures. Yields of CT from V. cholerae 569B in the recycle fermentor were highest at the highest dilution rate employed (1.0 vol/vol per h). The use of complete cell recycle dramatically increased yields over those obtained in continuous culture and equaled those obtained in shake flasks. The concentration of CT in the filtrate was slightly less than half of that measured in culture fluids sampled at the same time. Similarly, V. mimicus 61892 grown in the presence of 50 micrograms of lincomycin per ml produced 280 ng of CT per ml in the recycle fermentor, compared with 210 ng/ml in shake flasks under optimal conditions. The sterile filtrate from this fermentation contained 110 ng/ml.     

Influence of Dissolved Oxygen Levels on Production of l-Asparaginase and Prodigiosin by Serratia marcescens

The effect of dissolved oxygen concentrations on the behavior of Serratia marcescens and on yields of asparaginase and prodigiosin produced in shaken cultures and in a 55-liter stainless-steel fermentor was studied. A range of oxygen transfer rates was obtained in 500-ml Erlenmeyer flasks by using internal, stainless-steel baffles and by varying the volume of medium per flask, and in the fermentor by high speed agitation (375 rev/min) or low rates of aeration (1.5 volumes of air per volume of broth per min), or both. Dissolved oxygen levels in the fermentation medium were measured with a membrane-type electrode. Peak yields of asparaginase were obtained in unbaffled flasks (3.0 to 3.8 IU/ml) and in the fermentor (2.7 IU/ml) when the level of dissolved oxygen in the culture medium reached zero. A low rate of oxygen transfer was accomplished by limited aeration. Production of prodigiosin required a supply of dissolved oxygen that was obtainable in baffled flasks with a high rate of oxygen transfer and in the fermentor with a combination of high-speed agitation and low-rate aeration. The fermentation proceeded at a more rapid rate and changes in pH and cell populations were accelerated by maintaining high levels of dissolved oxygen in the growth medium.     

Effect of Oxygen-Supply Rates on Growth of Escherichia coli: II. Comparison of Results in Shake Flasks and 50-Liter Fermentor

Growth of Escherichia coli and chemical changes in the medium were very similar in highly baffled flasks and in a 50-liter fermentor run under the same oxygen-supply conditions, based on sulfite-oxidation rates. Flasks with stainless-steel baffles (Biotech) gave growth patterns and rates of glucose and NH₄-N utilization almost identical to those of the fermentor; results with Bellco 598 flasks (with 6 to 7 mm deep indentations) were quite similar. Unbaffled and Bellco 600 flasks (3 to 4 mm indentations) were similar to the fermentor at very high and very low oxygen-transfer rates, but gave much less growth than the fermentor at intermediate levels. Maximal oxygen-uptake rates occurred in the fermentor at the end of the logarithmic-growth phase when growth was 40 to 75% of maximum. In the fermentor, both sulfite-oxidation rates and rates of oxygen uptake correlated reasonably well with the total amount of growth produced.     

Rapid large-scale growth of Helicobacter pylori in flasks and fermentors.

We developed procedures for large-scale cultivation of Helicobacter pylori in flasks and fermentors. Flasks incubated closed under a microaerophilic gas phase with a cotton plug covered by a plastic bag, followed by removal of the bag after 8 h, gave excellent growth. Growth in a 10-liter fermentor led to excessive foaming if the medium was sparged with gas; silicone- or polyglycol-based antifoaming agents were severely inhibitory. Use of fermentor surface gassing, first with a microaerophilic 6% oxygen gas mixture, then with air, and then with 95% oxygen, allowed the culture to grow to an A600 of 2.5 in < 24 h. This method was modified for scale-up to a 100-liter fermentor.     

Mixed cultures of different yeasts species and yeasts with filamentous fungi in the SCP production. I. Production of single cell protein by mixed cultures Candida lipolytica and Candida tropicalis.

The aim of this study was to determine the application of mixed cultures Candida lipolytica and Candida tropicalis in the SCP production. N-paraffin fraction of crude oil and individual n-alkanes C:7--C:17 and glucose were used as carbon sources. The cultures were grown on laboratory scale in shaking flasks and in a 7 1 fermentor. It was found that the mixed cultures gave about 18% higher yield of biomass than the individual cultures.     

Reproducing shake flasks performance in stirred fermentors: production of alginates by Azotobacter vinelandii

Keeping equal the initial power drawn (0.27 W l(-1)) in shake flasks and in a stirred fermentor did not reproduce the behaviour of alginate production by Azotobacter vinelandii. A lower mean molecular weight (1.1x10(6) Da) of the polymer was obtained in the bioreactor as compared to that obtained in shake flasks (1.9x10(6) Da). The reasons for this can reside in the fact that the evolution of the power drawn in the shake flasks could be considerably different to that observed in the stirred bioreactor. A drastic drop in the specific power drawn is expected in the shake flasks as a consequence of the increased viscosity, which caused the liquid not following the movement of the shaker. This was supported by the fact that cultures developed in the fermentor at lower initial power drawn (as low as 0.027-0.056 W l(-1)) or in a culture in which the power drawn was deliberately reduced along cultivation, produced alginates with similar molecular characteristics as that obtained in shake flasks.     

Technological problems in cultivation of plant cells at high density. Reprinted from Biotechnology and Bioengineering, Vol. XXII, No. 6, Pages 1203-1218 (1981)

Using Cudrania tricuspidata cells as model plant cells which have high sensitivity to hydrodynamic stress, technological problems in the cultivation of the plant cells at high density were investigated. Using "shake" flasks on a reciprocal shaker and Erlenmeyer flasks on a rotary shaker and with a high supply of oxygen in order to obtain high cell densities in shaken cultures, particle breakdown and damage to the largest cell aggregate group (above 1981 microm in diameter) occurred and normal cell growth became impeded. The mass-transfer coefficient (K) for a model solid-liquid system (beta-naphthol particles and water) in place of a system of plant cells and a liquid medium was proposed as an intensity index of hydrodynamic stress effects on plant cells in suspension cultures under various conditions in the bioreactor systems. Normal cell growth was obtained under culture conditions for K values less than about 4.4 x 10(-3) cm/sec. The characteristics of various bioreactors used until now were investigated by considering the three main technological factors (capacity of oxygen supply, intensity of hydrodynamic stress effects on plant cells, and intensity of culture broth mixing and air-bubble dispersion). The most suitable bioreactor for culturing plant cells at high density was a jar fermentor with a modified paddle-type impeller (J-M). The yield of cell mass in the 10-liter J-M (working volume 5 liter) was about 30 g dry weight per liter of medium.     

Methods for the Large-Scale Cultivation of an Oxytricha (Ciliophora: Hypotrichida)1

ABSTRACT. Oxytricha strains used in biochemical studies have traditionally been grown in unaerated, unagitated culture tubes or Fernbach flasks. These cultures are limited in volume to about one liter and have a very nonuniform distribution of cells, with the majority of the cells at the very top or bottom of the medium. We have found conditions in which Oxytricha can be grown in 50-liter fermentation vats. The cultures grow to a uniform density of about 6000 cells/ml.     

Technological problems in cultivation of plant cells at high density

Using Cudrania tricuspidata cells as model plant cells which have high sensitivity to hydrodynamic stress, technology problems in the cultivation of the plant cells at high density were investigated. Using “shake” flasks on a reciprocal shaker and Erlenmeyer flasks on a rotary shaker and with a high supply of oxygen on order to obtain high cell densities in shaken cultures, particles breakdown and damage to the largest cell aggregate group (above 1981 μm in diameter) occurred and normal cell growth became impeded. The mass-transfer coefficient (K)for a model solid–liquid system (β-naphthol particles and water) in place of a system of plant cells and a liquid medium was proposed as an intensity index of hydrodynamic stress effects on plant cells in subsequent cultures under various conditions in the bioreactor systems. Normal cell growth was obtained under culture conditions for K values less than about 4.4 × 10^?3 cm/sec. The characteristics of various bioreactors used until now were investigated by considering the three main technological factors (capacity of oxygen supply, intensity of hydrodynamic stress effects on plant cells, and intensity of culture broth mixing and air-bubble desperation). The most suitable bioreactor for culturing plant cells at high density was ajar fermentor with a modified paddle-type impeller (J-M). The yield of cell mass in the 10-liter J-M (working volume 5 liter) was about 30 g dry weight per liter of medium.     

Isolation and Characterization of a Freeze-Tolerant Diploid Derivative of an Industrial Baker's Yeast Strain and Its Use in Frozen Doughs

The routine production and storage of frozen doughs are still problematic. Although commercial baker's yeast is highly resistant to environmental stress conditions, it rapidly loses stress resistance during dough preparation due to the initiation of fermentation. As a result, the yeast loses gassing power significantly during storage of frozen doughs. We obtained freeze-tolerant mutants of polyploid industrial strains following screening for survival in doughs prepared with UV-mutagenized yeast and subjected to 200 freeze-thaw cycles. Two strains in the S47 background with a normal growth rate and the best freeze tolerance under laboratory conditions were selected for production in a 20-liter pilot fermentor. Before frozen storage, the AT25 mutant produced on the 20-liter pilot scale had a 10% higher gassing power capacity than the S47 strain, while the opposite was observed for cells produced under laboratory conditions. AT25 also retained more freeze tolerance during the initiation of fermentation in liquid cultures and more gassing power during storage of frozen doughs. Other industrially important properties (yield, growth rate, nitrogen assimilation, and phosphorus content) were very similar. AT25 had only half of the DNA content of S47, and its cell size was much smaller. Several diploid segregants of S47 had freeze tolerances similar to that of AT25 but inferior performance for other properties, while an AT25-derived tetraploid, TAT25, showed only slightly improved freeze tolerance compared to S47. When AT25 was cultured in a 20,000-liter fermentor under industrial conditions, it retained its superior performance and thus appears to be promising for use in frozen dough production. Our results also show that a diploid strain can perform at least as well as a tetraploid strain for commercial baker's yeast production and usage.     

Isolation and characterization of a freeze-tolerant diploid derivative of an industrial baker's yeast strain and its use in frozen doughs

The routine production and storage of frozen doughs are still problematic. Although commercial baker's yeast is highly resistant to environmental stress conditions, it rapidly loses stress resistance during dough preparation due to the initiation of fermentation. As a result, the yeast loses gassing power significantly during storage of frozen doughs. We obtained freeze-tolerant mutants of polyploid industrial strains following screening for survival in doughs prepared with UV-mutagenized yeast and subjected to 200 freeze-thaw cycles. Two strains in the S47 background with a normal growth rate and the best freeze tolerance under laboratory conditions were selected for production in a 20-liter pilot fermentor. Before frozen storage, the AT25 mutant produced on the 20-liter pilot scale had a 10% higher gassing power capacity than the S47 strain, while the opposite was observed for cells produced under laboratory conditions. AT25 also retained more freeze tolerance during the initiation of fermentation in liquid cultures and more gassing power during storage of frozen doughs. Other industrially important properties (yield, growth rate, nitrogen assimilation, and phosphorus content) were very similar. AT25 had only half of the DNA content of S47, and its cell size was much smaller. Several diploid segregants of S47 had freeze tolerances similar to that of AT25 but inferior performance for other properties, while an AT25-derived tetraploid, TAT25, showed only slightly improved freeze tolerance compared to S47. When AT25 was cultured in a 20,000-liter fermentor under industrial conditions, it retained its superior performance and thus appears to be promising for use in frozen dough production. Our results also show that a diploid strain can perform at least as well as a tetraploid strain for commercial baker's yeast production and usage.     

Improved cell growth in tobacco suspension cultures expressing Vitreoscilla hemoglobin

Expression of the gene encoding bacterial hemoglobin (VHb) from Vitreoscilla has been previously used to improve recombinant cell growth and enhance product formation under microaerobic conditions, a common phenomenon in large-scale cultivations of bacteria. This technology has now been applied to tobacco suspension cultures. Tobacco suspension cultures have been generated from VHb-expressing tobacco plants. Cell cultures were capable of producing an active hemoglobin. When grown in shake flasks, the cells did not show any lag-phase and exhibited improved cell growth, compared to controls carrying the parental plasmid.     

Biodegradation of chicken feathers waste directed by Bacillus subtilis recombinant cells: scaling up in a laboratory scale fermentor

Biodegradation of chicken feathers waste directed by Bacillus subtilis DB 100 (p5.2) cells was successfully carried out in 14 L Bio Flo 110 laboratory scale fermentor. Seven liters of feathers-based modified basal medium II, feathers-based tap water and feathers-based distilled water separately in the fermentor were inoculated with activated bacterial cells. The fermentation processes were conducted at 37 °C, 700 rpm agitation speed and 0.7 vvm air flow rate in the absence of kanamycin. Highest net levels of released feathers hydrolysis end products [soluble proteins and NH₂-free amino groups] and keratinolytic alkaline protease activity in the fermentor were greatly comparable to those of shake flasks. Interestingly, the plasmid (p5.2) inside the recombinant B. subtilis cells growing in the fermentor displayed 100% stability till the fifth day of incubation and this presents a great challenge. Data certainly would encourage the transfer to larger scale fermentors to carry out feathers biodegradation process.     

Overexpression and purification of fructose-1-phosphate kinase from Escherichia coli: application to the assay of fructose 1-phosphate

Fructose 1-phosphate is a metabolite that plays a regulatory role in liver and is best measured using an assay based on its conversion to fructose 1,6-bisphosphate by a bacterial fructose-1-phosphate kinase (Fru1PK). The open reading frame encoding Escherichia coli Fru1PK has been introduced in an expression plasmid (pET3a) based on the T7 promoter-driven system, which was used to overexpress the enzyme. The conditions for the production of soluble Fru1PK were optimized. The purification procedure used involved ammonium sulfate precipitation and chromatography on DEAE-Sepharose and was aimed mostly at stabilizing the enzyme and at freeing Fru1PK from bacterial contaminants that could interfere in the fructose 1-phosphate assay. From a 1-liter culture, more than 50 mg protein is obtained. This preparation can be used in an enzymatic assay that measures specifically fructose 1-phosphate in tissue extracts.     

人三叶因子3在毕赤酵母中表达条件的研究

为提高人三叶因子 3 (HumanTrefoilfactor 3 ,hTFF3 )在毕赤酵母中的表达量 ,研究了转化子生长的培养条件 ,包括不同碳源对转化子生长的影响和接种量、甲醇浓度、pH值、摇瓶转速及不同诱导时间对人三叶因子 3表达的影响。结果表明转化子在生长阶段加入葡萄糖生长旺盛 ,培养 14h后OD₆₀₀ 就可达到 50。在 100mL生长培养基上的菌液以 1∶1接入诱导培养基时蛋白表达量最高 ;转化子在 1%的甲醇、pH60、摇瓶转速240r/min的条件下诱导4 8h ,菌体密度OD₆₀₀为 15 ,目的蛋白表达量达到 20mg L。用 5L发酵罐进行了高密度发酵 ,经2%甲醇32h诱导 ,最终菌体密度OD₆₀₀ 达到 120 ,每升发酵液中含目的蛋白100mg。     

溶氧对Blakeslea trispora分批发酵生产β-胡萝卜素的影响

在摇瓶和5 L发酵罐中研究了溶氧 (DO) 对Blakeslea trispora分批发酵生产β-胡萝卜素的影响,总结了5 L发酵罐中β-胡萝卜素发酵过程中溶氧的变化规律.结果表明,当500 mL摇瓶装液量为50 mL,转速为240 r/min条件下发酵生产β-胡萝卜素产量最大,达到3.416 g/L; 5 L发酵罐中,在搅拌转速为1 000 r/min,通气量为1.5 vvm的条件下,β-胡萝卜素的产量可达到3.712 g/L,略高于摇瓶,这可能是由于5 L发酵罐中的气液传递和混合状况好于摇瓶,促进了产物的合成.     

Orbital shaker technology for the cultivation of mammalian cells in suspension

For large-scale applications in biotechnology, cultivation of mammalian cells in suspension is an essential prerequisite. Typically, suspension cultures are grown in glass spinner flasks filled to less than 50% of the nominal volume. We propose a superior system for suspension cultures of mammalian cells based on orbital shaker technology. We found that "square-shaped" bottles (square bottles) provide an inexpensive but efficient means to grow HEK-293 EBNA and CHO-DG44 cells to high density. Cultures in agitated 1-L square bottles exceeded the performance of cultures in spinner flasks, reaching densities up to 7 x 10(6) cells/mL for HEK-293 EBNA cells and 5 x 10(6) cells/mL for CHO-DG44 cells in comparison to (2.5-4) x 10(6) cells/mL for cultures of the same cells grown in spinner flasks. For 1-L square bottles, optimal cell growth and viability were observed with a filling volume of 30-40% of the nominal volume and an agitation speed of 130 rpm at a rotational diameter of 2.5 cm. Transient reporter gene expression following gene delivery by calcium phosphate-DNA co-precipitation was the same or slightly better for HEK-293 EBNA cells grown in square bottles as compared to spinner flasks. Reductions in cost, simplified handling, and better performance in cell growth and viability make the agitated square bottle a new and very promising tool for the cultivation of mammalian cells in suspension.     

Efficacy of commercial products in enhancing oil biodegradation in closed laboratory reactors

Summary A laboratory screening protocol was designed and conducted to test the efficacy of eight commercial bacterial cultures and two non-bacterial products in enhancing the biodegradation of weathered Alaska North Slope crude oil in closed flasks. Three lines of evidence were used to support the decision to progress to field testing in Prince William Sound: rapid onset and high rate of oxygen uptake, substantial growth of oil degraders, and significant degradation of the aliphatic and aromatic hydrocarbon fractions of the weathered Alaska North Slope crude oil. A product had to enhance biodegradation greater than that achieved with excess mineral nutrients. Experiments were conducted in closed respirometer flasks and shake flasks, using seawater from Prince William Sound and weathered crude oil from a contaminated beach. Analysis of the data resulted in selection of two of the ten products for field testing. Both were bacterial products. Findings suggested that the indigenous Alaskan microorganisms were primarily responsible for the biodegradation in the closed flasks and respirometer vessels.     

High-productivity fermentation process for cultivating industrial microorganisms

Summary High-productivity continuous fermentation processes have been developed for the production of important industrial microorganisms in specially designed fermentors.Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces fragilis, andCandida utilis yeasts have been grown in bench-scale fermentors at cell densities of over 120 g/l, whileEscherichia coli, Bacillus megaterium, Methylomonas sp. andPseudomonas putida bacteria have been cultivated to cell densities of more than 110 g/l. Productivities (g cells per 1 per h) greater than 25 have been achieved in both bench-scale and 1500-liter fermentors with yeasts, and values as high as 55 have been achieved with bacteria in the bench-scale fermentor. The microorganisms were grown on defined media using ammonia for pH control and as nitrogen source. The fermentor, capable of high oxygen and heat transfer rates, was operated at constant volume with continuous feed and product discharge. The high-productivity process reduces fermentor size, media sterilization requirements, and may under some circumstances eliminate waste and recycle streams. It can also be applied to a variety of biological products.