Effect of Aeration on Production of Anticancer Lignans by Cell Suspension Cultures of Linum album

The effects of aeration within the range of 0.2-0.5 vvm on transformed and high yielding cell cultures of Linum album were investigated in a 5-L stirred tank bioreactor equipped with low shear Setric impeller. The kinetics of cell growth, substrate utilization, and production of lignans, namely, podophyllotoxin and 6-methoxypodophyllotoxin, were established. Maximum biomass of 23.2 g/L and lignan accumulation levels of 176.3 mg/L podophyllotoxin and 10.86 mg/L 6-methoxypodophyllotoxin were obtained with initial air flow rate of 0.3 vvm. Specified oxygen demand of cells was estimated to be 1.35 g O(2)/g biomass. The optimum oxygen transfer coefficient was found to be 16.7 h(-1) (,) which corresponded to aeration rate of 0.3 vvm. The effect of minimum dissolved oxygen (DO) concentration was investigated with respect to biomass and lignan production by comparing identically aerated and agitated bioreactor cultivations at dissolved oxygen concentrations of 10%, 30%, and 50%. Cell growth and podophyllotoxin accumulation were not affected significantly at these DO levels, but 6-methoxypodophyllotoxin production was enhanced when cells were cultivated at 30% DO level. The maximum volumetric productivities of 18.2 mg/L day and 3.2 mg/L day for podophyllotoxin and 6-methoxypodophyllotoxin, respectively, were obtained. These results establish the key role of oxygen on mass scale production of anticancer lignans by cell cultures of L. album. It may serve as a suitable parameter for scale-up.     

Immobilization of <Emphasis Type="Italic">Escherichia coli</Emphasis> novablue γ-glutamyltranspeptidase in Ca-alginate-<Emphasis Type="Italic">k</Emphasis>-carrageenan beads

The recombinant Escherichia coli gamma-glutamyltranspeptidase (EcGGT) was immobilized in Ca-alginate-kappa-carrageenan beads. Effects of alginate concentration, amount of loading enzyme, and bead size on the entrapped activity were investigated. Optimum alginate concentration for EcGGT immobilization was found to be 2% (w/v). Using a loading enzyme concentration of 1.5 mg/g alginate, maximum enzyme activity was observed. With increase in bead size from 1.9 to 3.1 mm, the immobilization efficiency was decreased significantly because of mass transfer resistance. Thermal stability of the free EcGGT was increased as a result of the immobilization. Ca-alginate-kappa-carrageenan-EcGGT beads were suitable for up to six repeated uses, losing only 45% of their initial activity. Upon 30 days of storage the preserved activity of free and immobilized enzyme were found as 4% and 68%, respectively. The synthesis of L: -theanine was performed in 50 mM Tris-HCl buffer (pH 10) containing 25 mM L: -glutamine, 40 mM ethylamine, and 1.5 mg EcGGT/g alginate at 40 degrees C for 12 h, and a conversion rate of 27% was achieved.     

Inhibition of microbial xylitol production by acetic acid and its relation with fermentative parameters

Precipitated sugarcane bagasse hemicellulosic hydrolysate containing acetic acid was fermented by Candida guilliermondii FTI 20037 under different operational conditions (pH 4.0 and 7.0, three aeration rates). At pH 7.0 and k _L a of 10 (0.75 vvm) and 22.5/h (3.0 vvm) the acetic acid had not been consumed until the end of the fermentations, whereas at the same pH and k _L a of 35/h (4.5 vvm) the acid was rapidly consumed and acetic acid inhibition was not important. On the other hand, fermentations at an initial pH of 4.0 and k _L a of 22.5 and 35/h required less time for the acid uptake than fermentations at k _L a of 10/h. The acetic acid assimilation by the yeast indicates the ability of this strain to ferment in partially detoxified medium, making possible the utilization of the sugarcane bagasse hydrolysate in this bioprocess. The effects on xylitol yield and production are reported.     

Process Characterization of hCTLA4Ig Production in Transgenic Rice Cell Cultures Using a 3-L Bioreactor

Most of the technical know-how and experience of bioreactor engineering is applicable to plant cell cultures. In this study, transgenic rice cell cultures using RAmy3D promoter were used for the production of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig). In process aspect, the rice cells during production phase are strongly influenced by hydrodynamic stresses, such as shear stress and bubble burst. Therefore, the effects of agitation and aeration rates on cell growth and hCTLA4Ig production were investigated in a 3-L multi-bioreactor. By increasing over 240 rpm, the detrimental effects on cell growth and hCTLA4Ig production were observed. At an aeration rate of 0.3 vvm, relative cell viability sharply decreased 2 days earlier than those of lower aeration rates. In addition, it was confirmed that the specific yields and the specific productivity at 0.3 vvm were superior to those values at 0.05 vvm. Overall, higher aeration rate showed the improved hCTLA4Ig production in combination experiment. High aeration rates in general, however, have an undesired effect as excessive aeration was found to negatively affect the quality of hCTLA4Ig. Consequently, the hydrodynamic conditions must be tightly regulated during bioreactor operation in order to enhance hCTLA4Ig productivity and quality in transgenic rice cell cultures.     

Removal of Toxic As (V) Ions by Adsorption onto Alginate and Carboxymethyl Cellulose Beads

The removal of arsenic ions from dilute aqueous solutions using biopolymeric beads of crosslinked sodium alginate and carboxymethyl cellulose (CMC) as the adsorbent is reported in this paper. The biopolymeric alginate and carboxymethyl cellulose beads were prepared and characterized by FTIR spectra. On the surfaces of the prepared beads were performed static and dynamic adsorption studies of arsenic ions at fixed pH and ionic strength of the aqueous metal ion solutions. The adsorption data were applied to Langmuir and Freundlich isotherm equations and various static parameters were calculated. The dynamic nature of adsorption was quantified in terms of several kinetic constants such as rate constant for adsorption (k₁) and Lagergreen rate constant (K_ad). The influence of various experimental parameters such as solid to liquid ratio, pH, temperature, presence of salts and chemical composition of biopolymeric beads were investigated on the adsorption of arsenic ions.     

Improved oxygen delivery in a continuous-roller-bottle reactor

Variations to the original aeration system in a continuous roller bottle reactor of novel design have been tested and compared for optimal oxygen (O) delivery. Reactor operating parameters that affect O transfer are rotation rate, liquid-volume level, fresh-feed rate, and supplementary-aeration rate. Design modifications to enhance gas-liquid O transfer include the addition of wall baffles and center baffles. The number and location of each of these baffles are compared for their effect on k_La values in the reaction chamber. The liquid feed into the system has been modified to improve the axial liquid mixing and O transfer.     

Influence of a new axial impeller on K L a and xylanase production by Penicillium canescens 10-10c

The effects of a new axial impeller (HTPG4) on oxygen volumetric transfer coefficient, K _L a, and xylanase production by Penicillium canescens 10-10c were studied and compared for dual-impeller systems, one with one DT4 impeller below and one HTPG4 above (DT4-HTPG4) and one with two DT4 (DT4-DT4) impellers, in a 5-L bioreactor. The volumetric coefficient of oxygen transfer was measured in culture medium using a gassing-out method at different gassing rates and agitation speeds. We observed that the DT4-HTPG4 combination provided better K _L a performance than the DT4-DT4 combination. The two combinations were also tested for their influence on xylanase production by a filamentous microorganism; P. canescens 10-10c. These experiments demonstrated that the DT4-HTPG4 combination impeller enhanced enzyme production up to 23% compared with the DT4-DT4 combination at an aeration rate of 1 vvm and an agitation speed of 600 rpm. The main cause for this difference is thought to be a higher shear stress generated by the DT4-DT4 combination, which damages the mycelium of P. canescens and decreases xylanase production.     

Comparison of different technologies for alginate beads production

This paper describes the results of the round robin experiment “Bead production technologies” carried out during the COST 840 action “Bioencapsulation Innovation and Technologies” within the 5th Framework Program of the European Community. In this round robin experiment, calcium alginate hydrogel beads with the diameter of (800 ± 100) μm were produced by the most common bead production technologies using 0.5–4 mass % sodium alginate solutions as starting material. Dynamic viscosity of the alginate solutions ranged from less than 50 mPa s up to more than 10000 mPa s. With the coaxial air-flow and electrostatic enhanced dropping technologies as well as with the JetCutter technology in the soft-landing mode, beads were produced from all alginate solutions, whereas the vibration technology was not capable to process the high-viscosity 3 % and 4 % alginate solutions. Spherical beads were generated by the electrostatic and the JetCutter technologies. Slightly deformed beads were obtained from high-viscosity alginate solutions using the coaxial airflow and from the 0.5 % and 2 % alginate solutions using the vibration technology. The rate of bead production using the JetCutter was about 10 times higher than with the vibration technology and more than 10000 times higher than with the coaxial air-flow and electrostatic technology. In memory of our colleague Stefan Rosinski     

Hybrid Self-Assembled Gel Beads for Tuneable pH-Controlled Rosuvastatin Delivery

This article describes the fabrication of new pH-responsive hybrid gel beads combining the polymer gelator calcium alginate with two different low-molecular-weight gelators (LMWGs) based on 1,3 : 2,4-dibenzylidene-d -sorbitol: pH-responsive DBS-COOH and thermally responsive DBS-CONHNH₂, thus clearly demonstrating that different classes of LMWG can be fabricated into gel beads by using this approach. We also demonstrate that self-assembled multicomponent gel beads can be formed by using different combinations of these gelators. The different gel bead formulations exhibit different responsiveness – the DBS-COOH network can disassemble within those beads in which it is present upon raising the pH. To exemplify preliminary data for a potential application for these hybrid gel beads, we explored aspects of the delivery of the lipid-lowering active pharmaceutical ingredient (API) rosuvastatin. The release profile of this statin from the hybrid gel beads is pH-dependent, with greater release at pH 7.4 than at pH 4.0 – primary control of this process results from the pK_a of the API. The extent of pH-mediated API release is also significantly further modified according to gel bead composition. The DBS-COOH/alginate beads show rapid, highly effective drug release at pH 7.4, whereas the three-component DBS-COOH/DBS-CONHNH₂/alginate system shows controlled slow release of the API under the same conditions. These initial results indicate that such gel beads constitute a promising, versatile and easily tuned platform suitable for further development for controlled drug-delivery applications.     

Diffusion Behavior of Microbial Transglutaminase to Induce Protein Crosslinking in Oil-in-Water Emulsions

Food-grade hydrogel particles composed of sodium alginate were used to investigate the diffusion behavior of microbial transglutaminase (mTG) to induce crosslinking of interfacially adsorbed protein. For this purpose, mTG-loaded hydrogel beads were mixed with caseinate-stabilized oil-in-water emulsions, whereas Bradford assay and ammonia measurements were utilized to monitor the enzyme-induced interfacial protein crosslinking. Different alginate (0.5–1.5%) and gelling concentrations (50–500 mM CaCl₂) were used to modulate the hydrogel mesh size and number of junction zones. The results indicated that mTG was able to diffuse out of alginate beads. However, a decrease in NH₃ concentration with increasing alginate and CaCl₂ levels was observed due to the formation of tight and dense bead structures. These results illustrate that the spatial distribution of molecules in complex matrices plays a key role on the enzyme accessibility     

Effect of aeration on lignin peroxidase production by Streptomyces viridosporus T7A

The effect of aeration on lignin peroxidase production by Streptomyces viridosporus T7A was studied in a bench-scale bioreactor using a previously optimized growth medium (0.65% yeast extract and 0.1% corn oil, pH7.0) at 37°C and natural pH. Airflow rates of 0.3, 1.0, and 1.5 vvm and a fixed agitation of 200 rpm were initially studied followed by 1.0 vvm and 200, 300, 400, and 500 rpm. The use of 1.0 vvm and 400 rpm increased enzyme concentration 1.8-fold (100–180 U/L) and process productivity 4.8-fold (1.4–6.7 U/[L·h]) in comparison with the use of 200 rpm and 0.3 vvm. The inexpensive corn oil, used as carbon source, besides its antifoam properties, proved to be nonrepressive for enzyme production.     

Lactic acid production from cheese whey by immobilized bacteria

The performance of immobilized Bifidobacterium longum in sodium alginate beads and on a spiral-sheet bioreactor for the production of lactic acid from cheese whey was evaluated. Lactose utilization and lactic acid yield of B. longum were compared with those of Lactobacillus helveticus. B. longum immobilized in sodium alginate beads showed better performance in lactose utilization and lactic acid yield than L. helveticus. In the spiral-sheet bioreactor, a lactose conversion ratio of 79% and lactic acid yield of 0.84 g of lactic acid/g of lactose utilized were obtained during the first run with the immobilized L. helveticus. A lactose conversion ratio of 69% and lactic acid yield of 0.51 g of lactic acid/g of lactose utilized were obtained during the first run with immobilized B. longum in the spiral-sheet bioreactor. In producing lactic acid L. helveticus performed better when using the Spiral Sheet Bioreactor and B. longum showed better performance with gel bead immobilization. Because B. longum is a very promising new bacterium for lactic acid production from cheese whey, its optimum fermentation conditions such as pH and metabolic pathway need to be studied further. The ultrafiltration tests have shown that 94% of the cell and cheese whey proteins were retained by membranes with a mol wt cutoff of 5 and 20 KDa.     

SEM–EDS study of ionically cross-linked alginate and alginic acid bead formation

Alginate is a natural polysaccharide that is widely used in industrial applications. Here, beads of alginic acid, calcium, and copper alginate were prepared by the addition of soluble sodium alginate to a solution of hydrochloric acid or the corresponding cation. The progress of gelation inside the beads was followed by the degree of opacity at different times, which corresponded well to an apparent morphology change observed in freeze-dried bead cross sections analyzed by scanning electron microscopy. Furthermore, diffusion of Cu²⁺ ions across the beads was followed by energy dispersive X-ray spectroscopy, which revealed colocalization of the cross-linking cation and the morphologically distinct bead regions.     

A Study of the Effects of Aeration and Agitation on the Properties and Production of Xanthan Gum from Crude Glycerin Derived from Biodiesel Using the Response Surface Methodology

The effects of aeration and agitation on the properties and production of xanthan gum from crude glycerin biodiesel (CGB) by Xanthomonas campestris mangiferaeindicae 2103 were investigated and optimized using a response surface methodology. The xanthan gum was produced from CGB in a bioreactor at 28 °C for 120 h. Optimization procedures indicated that 0.97 vvm at 497.76 rpm resulted in a xanthan gum production of 5.59 g L^?1 and 1.05 vvm at 484.75 rpm maximized the biomass to 3.26 g L^?1. Moreover, the combination of 1.05 vvm at 499.40 rpm maximized the viscosity of xanthan at 0.5 % (m/v), 25 °C, and 25 s^?1 (255.40 mPa s). The other responses did not generate predictive models. Low agitation contributed to the increase of xanthan gum production, biomass, viscosity, molecular mass, and the pyruvic acid concentration. Increases in the agitation contributed to the formation of xanthan gum with high mannose concentration. Decreases in the aeration contributed to the xanthan gum production and the formation of biopolymer with high mannose and glucose concentrations. Increases in aeration contributed to increased biomass, viscosity, and formation of xanthan gum with greater resistance to thermal degradation. Overall, aeration and agitation of CGB fermentation significantly influenced the production of xanthan gum and its properties.     

Ionically crosslinked alginate/carboxymethyl chitin beads for oral delivery of protein drugs

Complex beads composed of alginate and carboxymethyl chitin (CMCT) were prepared by dropping aqueous alginate-CMCT into an iron(III) solution. The structure and morphology of the beads were characterized by IR spectroscopy and scanning electron microscopy (SEM). IR confirmed electrostatic interactions between iron(III) and the carboxyl groups of alginate as well as CMCT, and the binding model was suggested as a three-dimensional structure. SEM revealed that CMCT had a porous morphology while alginate and their complex beads had a core-layer structure. The swelling behavior, encapsulation efficiency, and release behavior of bovine serum albumin (BSA) from the beads at different pHs were investigated. The BSA encapsulation efficiency was fairly high (>90%). It was found that CMCT disintegrated at pH 1.2 and alginate eroded at pH 7.4 while the complex beads could effectively retain BSA in acid (>85%) and reduce the BSA release at pH 7.4. The results suggested that the iron(III)-alginate-CMCT bead could be a suitable polymeric carrier for site-specific protein drug delivery in the intestine.     

Design of a spray-cycle bioreactor and its application for riboflavin production

A highly efficient spray-cycle reactor for oxygen supply was developed. A typical arrangement of the reactor consists of a spray column fitted with a nozzle and a coaxal tube, and a reservoir vessel. The culture broth was circulated between the column and vessel by a peristaltic pump. The volumetric oxygen-transfer coefficient, k₁a was evaluated as a parameter for oxygen supply. The liquid circulation rate in the spraycycle reactor was represented in terms of the number of circulations. The k₁a value increased as the number of circulations increased, reaching 208 h^-1 at 4.4 min^-1 of circulation numbers. This value was 1.8 times higher than that in a 1500-mL stirred-tank reactor under the agitation of 20.7g and the aeration of 1.0 volume per min. The spray-cycle reactor was applied to riboflavin production by an aerobic microorganism. The riboflavin production increased as k₁a values increased and the maximal riboflavin production was 161 mg/L at 208 h^-1 of k₁a. These results suggest that the spray-cycle reactor is useful to oxygen-demanding fermentation because of the high k₁a value in comparison with the stirred-tank reactor.     

Preparation and evaluation of berberine alginate beads for stomach-specific delivery

The purpose of this research was to prepare floating calcium alginate beads of berberine for targeting the gastric mucosa and prolonging their gastric residence time. The floating beads were prepared by suspending octodecanol and berberine in sodium alginate (SA) solution. The suspension was then dripped into a solution of calcium chloride. The hydrophobic and low-density octodecanol enhanced the sustained-release properties and floating ability of the beads. The bead formulation was optimized for different weight ratios of octodecanol and SA and evaluated in terms of diameter, floating ability and drug loading, entrapment and release. In vitro release studies showed that the floating and sustained release time were effectively increased in gastric media by addition of octodecanol. In vivo studies with rats showed that a significant increase in gastric residence time of beads had been achieved.     

Effect of operating conditions on biomass growth during citric acid production by solid-state fermentation

The effect of aeration, agitation, and bed loading on biomass and product concentration during citric acid biosynthesis by solid-state fermentation was investigated. For this purpose, Aspergillus niger S was cultivated on sugar beet pulp in a 4.5 dm³ horizontal rotating drum bioreactor. The results suggest that the parameters examined have a remarkable effect on the quantity of biomass being formed and on the product concentration. The maximum citric acid production (about 150 g per kg of the substrate dry matter) was obtained under the following conditions: aeration rate 0.2 dm³ kg⁻¹ min⁻¹, mixing (periodical) 1 min once an hour, and bed loading 30 % of the bioreactor working volume. However, these values did not favour biomass formation. Moreover, it was found that accumulation of the product reached its maximum when the amount of biomass was minimal (approximately 252-29 g per kg of the substrate dry matter) under the conditions involved. Presented at the 34th International Conference of the Slovak Society of Chemical Engineering, Tatranské Matliare, 21–25 May 2007.     

Effect of Aeration and Agitation Regimes on Lipase Production by Newly Isolated Rhodotorula mucilaginosa–MTCC 8737 in Stirred Tank Reactor Using Molasses as Sole Production Medium

The influence of media and process parameters (aeration and agitation) on fermentation broth rheology and biomass formation has been studied in 1.5-l stirred tank reactor for lipase production using Rhodotorula mucilaginosa MTCC 8737. Molasses, as sole production medium, is used for lipase production by varying aeration (1, 2, and 3 vvm) and agitation speeds (100, 200, and 300 rpm). Maximum lipase activity of 72 U/ml was obtained during 96 h of fermentation at 2 vvm, 200 rpm, pH 7, and 25 +/- 2 degrees C temperature. Lipase production kinetics with respect to dry cell weight of biomass showed Y (P/S) of 25.71 U/mg, specific product formation of 10.9 U/mg DC, and Y (X/S) 2.35 mg/mg. Maximum lipase activity (MC 2) of 56 U/ml was observed at 1% molasses, and a further increase in the molasses concentration of (%) 1.5 and 2 inhibited the product formation of lipase with 15 and 8.5 U/ml, respectively. The production kinetics of molasses media showed Y (P/X) was 14 U/mg DC, Y (P/S) 16 U/mg, and Y (X/S) 1.14 mg/mg during 96 h of bioreactor operation. The k(L)a values for all batches (MC 1-MC 4) at 96 h of fermentation were 32, 28, 21, and 19/h, and the |oxygen transfer rate were 54.4, 56, 35.7, and 17.29 mg/l h, respectively. Increase in molasses concentration resulted in decreased lipase activity by increase in viscosity of the fermentation broth.     

Effect of process parameters on production of a biopolymer by Rhizobium sp.

The production of biopolymers by a Rhizobium strain was studied under batch and bioreactor conditions. The best viscosity levels were obtained under low mannitol concentrations as well as low agitation and aeration conditions. Infrared spectra indicated the presence of chemical groups characteristic of microbially produced biopolymers, including C=O and O-acetyl groups. Thermogravimetric analysis showed the characteristic degradation profiles of the exopolysaccharide produced (T _onset=290°C). The experimental design showed that a low substrate concentration (10.0 g/L), and low aeration (0.2 vvm) and agitation (200 rpm) levels should be used. The maximum yield of the process was a Yp/s (g/g) of 0.19±0.1, obtained under optimized conditions.