Pullulan fermentation in a reciprocating plate bioreactor

Reciprocating plate bioreactors are particularly well suited for conducting fermentations which give rise to highly viscous broth. To evaluate their performance for polysaccharide fermentations, a series of pullulan fermentations were performed with a particular emphasis placed on the influence of aeration on both the quantity and quality of the product. Two experiments were conducted at constant aeration rates and two others with constant dissolved oxygen concentrations. For the latter two experiments, the dissolved oxygen concentration was controlled by manipulating either the aeration flow rate or the reciprocating frequency of the perforated plates.It was found that, in general, a higher dissolved oxygen concentration leads to a higher productivity but the quality of the product, expressed in terms of the viscosity of the fermentation broth, was nevertheless reduced. It appears that the optimum yield, in terms of both quantity and quality, would be achieved at an intermediate dissolved oxygen concentration.List of Symbols DO mg/l Dissolved oxygen concentration - f Hz Agitation frequency - K _L a s^–1 Volumetric mass transfer coefficient - P g/l Pullulan concentration - Q vvm, l/min Volumetric gas flow rate - X g/l Biomass concentration Greek Letters s^–1 Shear rate - Pa.s Apparent viscosity We wish to acknowledge the financial contribution of l'Association des femmes diplômées des Universités (AFDU) and the National Science and Engineering Research Council of Canada (NSERC).     

A mechanistic model for gas–liquid mass transfer prediction in a rocking disposable bioreactor

Rocking disposable bioreactors are a newer approach to smaller-scale cell growth that use a cyclic rocking motion to induce mixing and oxygen transfer from the headspace gas into the liquid. Compared with traditional stirred-tank and pneumatic bioreactors, rocking bioreactors operate in a very different physical mode and in this study the oxygen transfer pathways are reassessed to develop a fundamental mass transfer (k_La) model that is compared with experimental data. The model combines two mechanisms, namely surface aeration and oxygenation via a breaking wave with air entrainment, borrowing concepts from ocean wave models. Experimental data for across the range of possible operating conditions (rocking speed, angle, and liquid volume) confirms the validity of the modeling approach, with most predictions falling within ±20% of the experimental values. At low speeds (up to 20 rpm) the surface aeration mechanism is shown to be dominant with a of around 3.5 hr⁻¹, while at high speeds (40 rpm) and angles the breaking wave mechanism contributes up to 91% of the overall (65 hr⁻¹). This model provides an improved fundamental basis for understanding gas–liquid mass transfer for the operation, scale-up, and potential design improvements for rocking bioreactors.     

Polysaccharide production: Experimental comparison of the performance of four mixing devices

This investigation was undertaken with the objective to compare experimentally the performance of four different mixing devices for the production of the polysaccharide pullulan with Aureobasidium pullulans (2552). Fermentations were performed using identical bioreactors with respectively an assembly of three Rushton turbines (RTB), a helical ribbon impeller (HR) and two different reciprocating plates (RPB1, RPB2). Each mixing vessel had identical geometry and working volume (18 L). These four fermentations were performed with an equal level of power input per unit volume (1000?W/m³) and gas flow rate (0.5 vvm, 9 L/min). For each system, the evolution of biomass, polysaccharide concentration, dissolved oxygen and agitation speed or frequency were recorded as a function of time along with the rheological properties of the culture broths. The type of mixing device used had a significant impact on the rate of biomass production and on polysaccharide physical properties. However, the rate of polysaccharide production appears to be less sensitive to the bioreactor design. The overall productivity, which represents the ability of micro-organisms to convert rapidly substrate into biomass and polysaccharide, was maximised using the RPB2 system. The quality of the synthesised polysaccharide, in terms of viscosity producing power, was highest using the HR system but the overall productivity was very low. Thus, the best compromise between productivity and product quality was achieved with the RPB2.     

Performance of airlift bioreactors in the cultivation of some antibiotic producing microorganisms

The specific aspects of airlift reactors emphasizing their function relevance to particular application as bioreactors are presented. The two main groups of airlift reactors – external-loop and concentric-tube reactors – were investigated on a pilot-plant scale with regard to their performance during the cultivation of unicellular and filamentous microorganisms which produce Bacitracin, Cephalosporin C and Nystatin. Some results were compared to those obtained in conventional stirred tank bioreactors. The comparison was carried out based on physical properties (oxygen transfer rate (OTR), volumetric mass transfer coefficient (k_La) and efficiency of oxygen transfer (E)), cell mass, productivity and substrate consumption, secondary metabolite production, and efficiency of the product formation with regard to the specific power input. It was shown that B. licheniformis, C. acremonium and S. noursei fermentations occurred similarly to those performed in stirred vessels, proving that the capacity of the airlift bioreactors surpassed the problems which arise from the morphology and rheology of the broths. From the chemical engineering point of view, it was obvious that the primary tasks of a bioreactor (uniform distribution of microorganisms and nutrients over the entire fermenter volume, appropriate supply of biomass with nutrients and oxygen) were fulfilled by the airlift bioreactors tested. In addition, the efficiency of oxygen transfer (OTR referred to power input) in the airlift fermenters proved to be about 38% higher than in the stirred tank bioreactors (expressed as average values), while the sorption efficiency (OTR referred to antibiotic production) was found to be 22% greater in the airlift system than in an STR. Therefore, the biosyntheses were performed with about a 30–40% increase in energy efficiency and energy savings compared to the conventional system. Moreover, the lack of mechanical devices in the airlift system provides greater safety and a gentler environment for the cultivation of microorganisms.     

Correlation between mass transfer coefficient k<Subscript>L</Subscript>a and relevant operating parameters in cylindrical disposable shaken bioreactors on a bench-to-pilot scale

Background

Among disposable bioreactor systems, cylindrical orbitally shaken bioreactors show important advantages. They provide a well-defined hydrodynamic flow combined with excellent mixing and oxygen transfer for mammalian and plant cell cultivations. Since there is no known universal correlation between the volumetric mass transfer coefficient for oxygen k_La and relevant operating parameters in such bioreactor systems, the aim of this current study is to experimentally determine a universal k_La correlation.

Results

A Respiration Activity Monitoring System (RAMOS) was used to measure k_La values in cylindrical disposable shaken bioreactors and Buckingham’s π-Theorem was applied to define a dimensionless equation for k_La. In this way, a scale- and volume-independent k_La correlation was developed and validated in bioreactors with volumes from 2 L to 200 L. The final correlation was used to calculate cultivation parameters at different scales to allow a sufficient oxygen supply of tobacco BY-2 cell suspension cultures.

Conclusion

The resulting equation can be universally applied to calculate the mass transfer coefficient for any of seven relevant cultivation parameters such as the reactor diameter, the shaking frequency, the filling volume, the viscosity, the oxygen diffusion coefficient, the gravitational acceleration or the shaking diameter within an accuracy range of +/? 30%. To our knowledge, this is the first k_La correlation that has been defined and validated for the cited bioreactor system on a bench-to-pilot scale.

     

Repeated elicitation enhances taxane production in suspension cultures of Taxus chinensis in bioreactors

Methyl jasmonate added at 100 M on days 8, 14 and 20 to cultures of Taxus chinensis growing in flasks and 1 l airlift bioreactors improved taxuyunnanine C (TC) production by 50–60% in flasks and by about 80% in airlift bioreactors compared with single addition of 100 M methyl jasmonate on day 8. The final TC production in the airlift bioreactors reached 565±47 mg l^–1, which is the highest reported for taxoid production in a bioreactor.     

High cell density cultivation and recombinant protein production with <Emphasis Type="Italic">Escherichia coli</Emphasis> in a rocking-motion-type bioreactor

Background

Single-use rocking-motion-type bag bioreactors provide advantages compared to standard stirred tank bioreactors by decreased contamination risks, reduction of cleaning and sterilization time, lower investment costs, and simple and cheaper validation. Currently, they are widely used for cell cultures although their use for small and medium scale production of recombinant proteins with microbial hosts might be very attractive. However, the utilization of rocking- or wave-induced motion-type bioreactors for fast growing aerobic microbes is limited because of their lower oxygen mass transfer rate. A conventional approach to reduce the oxygen demand of a culture is the fed-batch technology. New developments, such as the BIOSTAT^® CultiBag RM system pave the way for applying advanced fed-batch control strategies also in rocking-motion-type bioreactors. Alternatively, internal substrate delivery systems such as EnBase^® Flo provide an opportunity for adopting simple to use fed-batch-type strategies to shaken cultures. Here, we investigate the possibilities which both strategies offer in view of high cell density cultivation of E. coli and recombinant protein production.

Results

Cultivation of E. coli in the BIOSTAT^® CultiBag RM system in a conventional batch mode without control yielded an optical density (OD₆₀₀) of 3 to 4 which is comparable to shake flasks. The culture runs into oxygen limitation. In a glucose limited fed-batch culture with an exponential feed and oxygen pulsing, the culture grew fully aerobically to an OD₆₀₀ of 60 (20 g L^-1 cell dry weight). By the use of an internal controlled glucose delivery system, EnBase^® Flo, OD₆₀₀ of 30 (10 g L^-1 cell dry weight) is obtained without the demand of computer controlled external nutrient supply. EnBase^® Flo also worked well in the CultiBag RM system with a recombinant E. coli RB791 strain expressing a heterologous alcohol dehydrogenase (ADH) to very high levels, indicating that the enzyme based feed supply strategy functions well for recombinant protein production also in a rocking-motion-type bioreactor.

Conclusions

Rocking-motion-type bioreactors may provide an interesting alternative to standard cultivation in bioreactors for cultivation of bacteria and recombinant protein production. The BIOSTAT^® Cultibag RM system with the single-use sensors and advanced control system paves the way for the fed-batch technology also to rocking-motion-type bioreactors. It is possible to reach cell densities which are far above shake flasks and typical for stirred tank reactors with the improved oxygen transfer rate. For more simple applications the EnBase^® Flo method offers an easy and robust solution for rocking-motion-systems which do not have such advanced control possibilities.

     

Growth of Trichoderma reesei RUT C-30 in stirred tank and reciprocating plate bioreactors

A series of fed-batch experiments at different agitation speeds were performed using the industrially important strain Trichoderma reesei RUT C-30 in two different bioreactors to understand the close relationship that exists between the shear field within a bioreactor, the morphology of the microorganism, the rheology of cultivation broth, and the process performance. The two bioreactors, stirred tank bioreactor (STB) and reciprocating plate bioreactor (RPB), are characterized by a significantly different shear field to which microorganisms are exposed. Highest biomass concentration (ca. 15 g l⁻¹) was obtained at higher agitation rates in both bioreactors due to better oxygen supply. However, better filter paper activities per mg of protein were obtained at lower agitation in both bioreactors. In both bioreactors, young and healthier fungi in the batch phase were not affected by shear even at higher agitation rates. However, during the fed-batch phase, higher degree of fragmentation of clump morphology at high agitation intensity was confirmed by image analysis. Also, the rheological analysis showed an increase in apparent viscosity during the batch phase and early fed-batch phase due to the increase in the biomass concentration. During the late stages of cultivation, the apparent viscosity decreased due to cell lysis and spore formation.     

Effect of periodate oxidation on the polysaccharide content and microaggregate stability of rhizosphere and non-rhizosphere soils

Summary The relationship between the stability of soil microaggregates in water and the polysaccharide content was examined in rhizosphere and non-rhizosphere samples from a pot experiment using three soils that had grown peas, barley or grasses. The polysaccharide was oxidised and removed using 168h treatment with 0.02M periodate followed by 6h with 0.1M tetraborate. The decrease in polysaccharide content, measured as change in residual reducing sugars, was compared with the stability of soil microaggregates (ca 45m) in water, determined by a turbidimetric method.Total C, N and polysaccharide contents of rhizosphere soils were greater than those for the bulk soil, but the water stability of aggregates was not increased compared to unplanted controls. Periodate oxidation removed a large proportion (59–95%) of the polysaccharide and increased aggregated disruption, but there was no clear relationship between the two measurements. In rhizosphere soil, polysaccharides appreared to make less contribution to aggregate stability than polysaccharide in the bulk soil. The relatively small effect of rhizosphere polysaccharides is probably related to their presence as comparatively massive plant remains and debris; this contrasts with the decomposed and transformed material in the bulk soil.     

Apparent and true longitudinal concentration profiles in reciprocating plate column

Mass transfer for kerosene-water system with benzoic acid and n-butyric acid as solutes has been studied in a reciprocating plate column at different operating conditions including the agitation rate of the plate stack, the plate geometry, the flow rates of the phases and the direction of mass transfer. Apparent and true concentration profiles for the dispersed and the continuous phase along the length of the column were generated, taking axial mixing of the phase into consideration. It is noticed that high agitation rate for the plate stack and low free area for the plate give rise to large difference between the apparent and true values. These observations also agree with the data obtained for carbon dioxide — water system studied in a reciprocating plate column.     

Drag-reducing polysaccharides from marine microalgae: species productivity and drag reduction effectiveness

We have conducted a study of the potential use of drag-reducing biopolymers produced by marine microalgae for engineering applications. Several marine microalgae species were tested for their production of drag-reducing polysaccharides in large custom-designed plate bioreactors. Promising species (such as Porphyridium cruentum, Rhodella maculata, Schizochlamydella capsulata and Chlorella stigmatophora) were cultured for periods of time ranging from a few weeks to over 6 months. The basic drag-reducing ability of the polysaccharides was established by comparing their drag reduction effectiveness at various concentrations in water. The algal polysaccharide mass productivity was also measured per unit area of bioreactor’s illuminated surface. Finally, an all-inclusive criterion, the volumetric production of drag-reducing water giving a set level of drag reduction was quantified, and led us to a ranking of the tested species in order of productivity relevant to implementation. Some aspects of polysaccharide production by aged cultures were investigated as well. We also quantified the drag-reducing effectiveness of intracellular polysaccharides, and visualized the presence of exopolymer particles in the medium.     

Antiviral effect of red microalgal polysaccharides on Herpes simplex and Varicella zoster viruses

The cell-wall sulphated polysaccharide of the red microalga Porphyridium sp. has impressive antiviral activity against Herpessimplex viruses types 1 and 2 (HSV 1, 2) and Varicella zoster virus(VZV). Treatment of cells with 1 g mL^-1 polysaccharideresulted in 50% inhibition of HSV-infection as measured by the plaqueassay. Inhibition of the production of new virus particles was also shownwhen pre-infected cell cultures were treated with the polysaccharide. Inaddition, there was indirect evidence for a strong interaction between thepolysaccharide and HSV and a weak interaction with the cell surface.Depending on the concentration, the polysaccharide completely inhibitedor slowed down the development of the cytopathic effect in HSV or VZVpreinfected cells, but did not show any cytotoxic effects on Vero cells evenwhen a concentration as high as 250 g mL^-1 was used. Itseems therefore that the polysaccharide is able to inhibit viral infection bypreventing adsorption of virus into the host cells and/or by inhibiting theproduction of new viral particles inside the host cells. Thus, this alga seems tobe a good candidate for the development of an antiviral drug.     

Kanamycin selection in temporary immersion bioreactors allows visual selection of transgenic citrus shoots

In mature citrus transformation, the nptII gene is most commonly used for selection and it is confounded by the high number of non-transformed, escaped shoots that develop on semi-solid kanamycin selection medium, even at high concentrations. Selection in liquid medium with kanamycin in temporary immersion bioreactors might provide a better means of distinguishing between transformed and non-transformed shoots. A dose-response curve was constructed for wild-type Carrizo rootstock in liquid medium to evaluate the effects of kanamycin concentration on the number and the length of microshoots. Kanamycin at 200 mg/l was chosen as the optimal concentration for selection of transgenic mature citrus shoots in bioreactors. At this dose, most non-transgenic microshoots turned yellow and their lengths and numbers were significantly reduced in comparison to the no kanamycin controls. Selection of transgenic shoots in bioreactors was tested after Agrobacterium transformations of mature Carrizo and Valencia using three different binary vectors containing nptII as the selectable marker. Shoots developed on semi-solid medium and were transferred to temporary immersion bioreactors containing liquid MS medium with 200 mg/l kanamycin. After two weeks of culture in bioreactors, 21 dark green shoots were visually selected on the basis of color from a total of 6882 microshoots, and 17 of them (81%) were confirmed as transgenic with either the GUS histochemical assay, GFP fluorescence or PCR. Yellow shoots (5675) to be discarded from pTLAB21 and pCAMBIA2301 transformations were also tested for GUS or GFP expression and only one (0.01%) was positive. Kanamycin selection of mature transgenic shoots in temporary immersion bioreactors permitted transgenics to be visually distinguished on the basis of color, and reduced labor and consumable costs for PCR screening, particularly when reporter genes were not used.     

Calcium-phosphate mediated DNA transfer into HEK-293 cells in suspension: control of physicochemical parameters allows transfection in stirred media. Transfection and protein expression in mammalian cells

This is the first report of transient transfection of suspended cells with purified plasmid DNA in bioreactors or spinner flasks. DNA/calcium phosphate complexes were pumped or injected directly into stirred cultures of the immortalized human embryo kidney cell line 293 (HEK-293) which had been adapted to growth in suspension. We identified culture conditions suitable for this approach and modified the protocol for the generation of precipitate complexes, based on our earlier work. In order to stabilize the DNA-vehicle-complex in the culture medium, we identified pH ranges and ion-concentrations which prevent dissolution or aggregation of the precipitate particles. Such conditions maintained suspended fine particles in spinners or bioreactors for up to 6 hr. During that period, cells and precipitate complexes interacted sufficiently to allow DNA transfer and subsequent expression of recombinant protein. In a simple 5 day batch process, with a starting density of 0.3 × 10⁶ cells mL^-1, about 0.5 mg L^-1 of a recombinant tissue plasminogen activator variant was observed.     

Characterization of heat transfer of large orbitally shaken cylindrical bioreactors

Disposable shaking bioreactors are a promising alternative to other disposable bioreactors owing to their ease of operation, flexibility, defined hydrodynamics and characterization. Shaken bioreactors of sizes 20 L and 50 L are characterized in terms of heat transfer characteristics in this research work. Water and an 80% glycerol–water system were used as fluid. Results indicated large heat generation due to shake mixing which was observed by temperature difference between the fluid inside the vessel and the surrounding air outside the vessel. Maximum temperature difference of ca. 30 K was encountered for a 50 L vessel, at 300 rpm and 20 L filling volume. Outside heat transfer rate was governing the overall heat transfer process. Lateral air flow did increase heat transfer rates to large extent. An empirical correlation of overall heat transfer coefficient was obtained in terms of filling volume, rotational speed and lateral air flow rate. However, as the vessel thickness increased, the overall heat transfer process was limited by vessel wall resistance.     

Polysaccharide concentration and molecular weight effects on the oxygen mass transfer in a reciprocating plate bioreactor

In most polysaccharide fermentations, the nature of the fermentation broth changes drastically with time and, as a result, the overall oxygen mass transfer coefficient (K(L)a) can vary by orders of magnitude. To obtain a better understanding of this phenomenon, an experimental program was devised to study the respective influence of molecular weight and concentration of dextran solutions on K(L)a. Experiments were conducted in a reciprocating plate bioreactor. This bioreactor uses a stack of perforated plates that is reciprocated axially in the column and it is therefore well suited for mixing viscous liquid broths and providing uniform overall mass transfer coefficients. The variation of K(L)a with the power input per unit volume and the superficial gas velocity were obtained for three ranges of molecular weights and five concentrations of dextran. In every medium, two regimes of operation were observed as a function of the power input per unit volume: a first regime, at low power inputs per unit volume where K(L)a remains constant until a threshold of power input is attained; and a second regime, which is characterized by a steep increase of K(L)a as a function of the power input per unit volume. The presence of dissolved biological macromolecules, not only because of their effect on the rheology of the medium but also because their effect on the gas-liquid interface, has a significant impact on K(L)a. It was found that, generally, small concentrations of polysaccharide favor oxygen mass transfer despite the increase in medium viscosity. However, the respective influence of polysaccharide concentration and molecular weight was different for the two regimes of operation. (c) 1996 John Wiley & Sons, Inc.     

Structural studies of an extracellular polysaccharide (S-198) elaborated by Alcaligenes ATCC 31853

The structure of an extracellular polysaccharide, S-198, elaborated by Alcaligenes ATCC 31853 has been investigated; methylation analysis, specific degradations, and ¹H-n.m.r. spectroscopy were the main methods used. It is suggested that the polysaccharide is composed of “repeating units” with the structure

A sugar residue in the chain may be either L-rhamnose or L-mannose and only ≈50% of the residues contain the branching α-L-rhamnopyranosyl group. The polysaccharide further contains O-acryl groups. It belongs to a group of polysaccharides, elaborated by Alcaligenes and Pseudomonas species, which all have the same linear backbone (except that some of them do not contain L-mannose) without branching or with branches that differ in their chemical structures and/or positions.     

Galactan sulfate of Grateloupia indica: Isolation, structural features and antiviral activity

Natural compounds offer interesting pharmacological perspectives for antiviral drug development with regard to broad-spectrum antiviral properties and novel modes of action. In this study, we have analyzed polysaccharide fractions isolated from Grateloupia indica. The crude water extract (GiWE) as well as one fraction (F3) obtained by anion exchange chromatography had potent anti-HSV activity. Their inhibitory concentration 50% (IC₅₀) values (0.12-1.06 μg/ml) were much lower than cytotoxic concentration 50% values (>850 μg/ml). These fractions, which were effective antiviral inhibitors if added only during the adsorption period, had very low anticoagulant activity. Furthermore, they had no direct inactivating effect on virions in a virucidal assay. Chemical, chromatographic and spectroscopic methods showed that the active polysaccharide, which has an apparent molecular mass of 60 kDa and negative specific rotation −16° (c 0.2, H₂O), contains α-(1 → 4)- and α-(1 → 3)-linked galactopyranose residues. Sulfate groups, if present, are located mostly at C-2/6 of (1 → 4)- and C-4/6 of (1 → 3)-linked galactopyranosyl units, and are essential for the anti herpetic activity of this polymer.     

Mycelial β-N-Acetylglucosaminidase of Streptomyces sp. Having β-N-Acetylgalactosaminidase Activity

Formation of transfer products from soybean arabinogalactan and glycerol by endo-1,4-β-d-galactanase from Penicillium citrinum was described. The amount of transfer products depended on the glycerol concentration. About 50% of the galactose residues which could be liberated from the polysaccharide by the enzyme were transferred to glycerol at an acceptor concentration of 2.5% (w/v). Transfer products with various polymerization degrees were accumulated at the beginning of the reaction and then those with higher polymerization degrees were degraded gradually. At a final stage of the reaction, two transfer products in addition to two hydrolysis products (galactose and galactobiose) were mainly accumulated. The two transfer products were isolated and their structures were examined. They were 2-O-β-d-galactosyl glycerol and O-β-d-galactosyl-(1 → 4)-O-β-d-galactosyl-(1 → 2)glycerol.