Combination of extractive solvent addition and immobilization culture for continuous production of scopoletin by tobacco cells

Extractive solvent addition was combined with immobilization cultures of Nicotiana tabacum cells to produce scopoletin. Using various solvents, the partition coefficients of scopoletin between the solvent and water phases and the solvent toxicity to the cell viability were investigated. The effect of the solvent addition on cell growth and scopoletin production was elucidated in the suspension cultures. Coconut oil, one of the natural vegetable oils, was selected as the most suitable extractive solvent. The cells were immobilized in the calcium alginate gel bead coated with a cell-free gel film and then the batch cultures with the addition of various volumes of the coconut oil were performed. The total scopoletin production increased with the solvent volume according to the amount of scopoletin transferred from the medium to the solvent. The maximum productivity obtained in the batch immobilization cultures was about 16 times larger than that in the suspension culture without solvent. A continuous production system, in which the fresh solvent was supplied to the culture system and the solvent containing scopoletin was recovered from it, was constructed. The integrated scopoletin production in the effluent oil attained 2.21 mg/gDCW for 30 days at 100 cm(3)/day without cell leakage.     

Enhanced production of plumbagin in immobilized cells of Plumbago rosea by elicitation and in situ adsorption

Cell cultures of Plumbago rosea were immobilized in calcium alginate and cultured in Murashige and Skoog's basal medium containing 10 mM CaCl(2) for the production of plumbagin, an important medicinal compound. Studies were carried to find out the impact of immobilization on the increased accumulation of this secondary metabolite. Immobilization in calcium alginate enhanced the production of plumbagin by three, two and one folds compared to that of control, un-crosslinked alginate and CaCl(2) treated cells respectively. Cell loading at a level of 20% to the polymer volume (Na-alginate) was optimal and maximum plumbagin was obtained. At higher cell loading (40-50%), lower plumbagin accumulation was noticed. Addition of 200 mg l(-1) chitosan as an elicitor to the immobilized cells resulted in eight and two folds higher accumulation of plumbagin over control and immobilized cells. Also, more than 70% of the plumbagin was released into the medium, which is highly desirable for easy recovery of the product. Sucrose utilization rate of the cells was higher when cells were subjected to in situ product removal using Amberlite XAD-7. This may indicate that the toxicity of plumbagin was reduced on cells when it was removed from the medium. Cells subjected to combined treatments of chitosan, immobilization and in situ extraction showed a synergistic effect and yielded 92.13 mg g(-1) DCW of plumbagin which is 21, 5.7, 2.5 times higher than control, immobilized, immobilized and elicited cells respectively.     

用吸附法固定化培养紫草细胞

采用生物活性载体 ,通过吸附固定化方式 ,结合液体石蜡原位萃取技术 ,培养紫草细胞。测定了细胞生长、底物消耗和产物合成的动力学 ,紫草宁产率为 0 .916 g/g干重细胞和 0 .95 3g/g干重接种细胞 ,分别为悬浮培养的 12 .7倍和 6 .3倍。同时 ,对吸附与包埋固定化方法进行了综合比较 ,探讨了吸附固定化方法的应用前景。     

Alumina-doped alginate gel as a cell carrier for ethanol production in a packed-bed bioreactor

Alumina-doped alginate gel (AEC) was developed as a new type of cell carrier to be used in ethanol fermentation. The presence of the alumina particles in alginate gel not only improved the porous structure of the carrier, but also provided many advantageous characteristics including good mechanical strength, high stability, and high immobilization yield. The attachment of alumina particles and yeast cells by electrostatic attraction was shown to promote cell growth and increase ethanol productivity. The AEC carrier was found to be more effective for the immobilization of Saccharomyces cerevisiae M30 than the conventional Ca-alginate bead. Ethanol productivities of 1.4 and 7.9 ∼ 12.6 g/(L/h) were obtained using the AEC cultures in batch and continuous modes of operation, respectively, with an ethanol yield of 43.9 ∼ 46.7% and an immobilized yield of 81.4 ∼ 84.5%. Ethanol fermentation in a continuous packed-bed reactor using the AEC carrier was stable for > 30 days.     

Continuous ethanol fermentation at 45 °C using Kluyveromyces marxianus IMB3 immobilized in Calcium alginate and kissiris

The thermotolerant ethanol-producing yeast strain Kluyveromyces marxianus IMB3 was immobilized in calcium alginate and a 1:1 mixture of calcium alginate and the porous volcanic mineral, kissiris. Immobilized preparations were placed in fixed-bed column bioreactors and continuous ethanol production by systems containing both immobilized preparations was examined at 45?°C with a 100?g/l glucose feed. The effect of residence time on product concentration, bioreactor efficiency and volumetric productivities have been examined and these were all higher in systems containing the alginate/kissiris mixed immobilization matrix. Maximum ethanol concentrations produced by the continuous system ranged between 46 and 48?g/l representing efficiencies of 90–94%.     

Enhanced production of the polysaccharide arabinogalactan using immobilized cultures of Tinospora cordifolia by elicitation and in situ adsorption

Immobilized callus cultures of Tinospora cordifolia (Willd) Miers ex Hooks and Thoms were investigated to find out the combined effect of elicitation, cell permeabilization with chitosan and in situ product recovery by polymeric neutral resin-like Diaion HP 20. In this study, callus cultures of T. cordifolia were immobilized using sodium alginate and calcium chloride and the beads were cultured in Murashige and Skoog's basal medium along with benzyl adenine (BA), 2,4-dichlorophenoxy acetic acid (2,4-D) and 3% sucrose. The immobilized cultures, when subjected to elicitation and cell permeabilization with chitosan and in situ removal of the secondary metabolites by addition of resin, showed a 10-fold increase in production of arabinogalactan (0.490% dry weight) as compared to respective controls devoid of resin and chitosan. This indicates that in situ adsorption may have reduced the feedback inhibition caused by accumulation of secondary metabolites in the media, while the dual effect of elicitation and cell permeabilization by chitosan may have released the intracellular (secreted) berberine and the polysaccharide arabinogalactan, respectively.     

Effect of calcium on immobilization of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) peroxidase for bioassays in sodium alginate and Agarose gel

The direct immobilization of soluble peroxidase isolated and partially purified from shoots of rice seedlings in calcium alginate beads and in calcium agarose gel was carried out. Peroxidase was assayed for guaiacol oxidation products in presence of hydrogen peroxide. The maximum specific activity and immobilization yield of the calcium agarose immobilized peroxidase reached 2,200 U mg⁻¹ protein (540 mU cm⁻³ gel) and 82%, respectively. In calcium alginate the maximum activity of peroxidase upon immobilization was 210 mU g⁻¹ bead with 46% yield. The optimal pH for agarose immobilized peroxidase was 7.0 which differed from the pH 6.0 for soluble peroxidase. The optimum temperature for the agarose immobilized peroxidase however was 30°C, which was similar to that of soluble peroxidase. The thermal stability of calcium agarose immobilized peroxidase significantly enhanced over a temperature range of 30∼60°C upon immobilization. The operational stability of peroxidase was examined with repeated hydrogen peroxide oxidation at varying time intervals. Based on 50% conversion of hydrogen peroxide and four times reuse of immobilized gel, the specific degradation of guaiacol for the agarose immobilized peroxidase increased three folds compared to that of soluble peroxidase. Nearly 165% increase in the enzyme protein binding to agarose in presence of calcium was noted. The results suggest that the presence of calcium, ions help in the immobilization process of peroxidase from rice shoots and mediates the direct binding of the enzyme to the agarose gel and that agarose seems to be a better immobilization matrix for peroxidase compared to sodium alginate.     

Fermentation kinetics of free and immobilized Penicillium raistrickii able perform the 15α-hydroxylation of 13-ethyl-gon-4-ene-3,17-dione

Growing Penicillium raistrickii i 477 cells immobilized by microencapsulation, entrapment in calcium alginate beads and photopolymerization were used for the 15α-hydroxylation of 13-ethyl-gon-4-en-3,17-dione (I) to 15α-hydroxy-13-ethyl-gon-4-en-3,17-dione (II). The immobilized cells had lower maximum specific growth rates and yield coefficients when cultivated on the carbon source glucose than the non-immobilized cells, which leads to lower volumetric productivities than the use of nonimmobilized cells. However, the cells immobilized by microencapsulation and calcium alginate entrapment showed a specific productivity equal to that of the respective non-immobilized cells based on product formation per dry biomass and time. Photommobilized cells were not able to grow in the presence of the steroid because the substrate concentrations within the polymer reached inhibiting amounts for growth and product formation. In the absence of the steroid, the growing photoimmobilized cells showed a prolonged lag-phase in comparison with the free cells.     

Continuous ethanol production from Jerusalem artichoke tubers. II. Use of immobilized cells of Kluyveromyces marxianus

Kluyveromyces marxianus UCD (FST) 55-82 cells were immobilized in Na alginate beads and used in a packed-bed bioreactor system for the continuous production of ethanol from the extract of Jerusalem artichoke tubers. Volumetric ethanol productivities of 104 and 80 g ethanol/ L/h were obtained at 80 and 92% sugar utilization, respectively. The maximum volumetric ethanol productivity of the immobilized cell bioreactor system was found to be 15 times higher than that of an ordinary-stirred-tank (CST) bioreactor using cells of K. marxianus. The immobilized cell bioreactor system was operated continuously at a constant dilution rate of 0.66 h(-1) for 12 days resulting in only an 8% loss of the original immobilized cell activity, which corresponds to an estimated half-life of ca. 72 days. The maximum specific ethanol productivity and maximum specific sugar uptake rate of the immobilized cells were found to be 0.55 g ethanol/g/biomass/h and 1.21 g sugars/g biomass/h, respectively.     

Comparative study of production of dextransucrase and dextran by cells of Leuconostoc mesenteroides immobilized on Celite and in calcium alginate beads

In fed-batch fermentation, cells of L. mesenteroides immobilized on three types of Celite were used to produce dextransucrase (DS) followed by production of dextran. A layer of calcium alginate on the porous Celite R630 particles improved their mechanical stability, increased the amount of soluble DS produced and decreased the cell leakage from the highly porous support. Enzyme production with the immobilized cell cultures was significantly affected by both pore and particle size. Immobilized cultures using Celite R648 (average particle radius of 200 mum and pore size of 0.14 mum) produced the highest total enzymatic activity, followed by Celite R633, alginate-coated Celite R630, Celite R630, and then calcium alginate beads. Culture of free cells produced about 18% more total enzymatic activity than immobilized cells in calcium alginate beads, but about 64% less than immobilized cells on Celite R630. It is expected that larger amounts of enzymatic activity than measured are immobilized inside the alginate-coated Celite R630 and calcium alginate beads due to the mass transfer limitation conferred by the dextran product formed therein. The dextran yield from conversion of sucrose to dextran and fructose with all such enzyme-enriched, immobilized-cell cultures was higher than that obtained from free-cell culture under similar conditions.     

Production of propene oxide in an organic liquid-phase immobilized cell reactor

Some major restrictions of the production of propene oxide in an organic liquid-phase immobilized cell packed-bed reactor were quantified, and techniques were investigated to enhance the epoxide production rates. Propene-epoxidizing Mycobacterium cells were entrapped in calcium alginate gel and contacted with the substrates, propene and oxygen, which were dissolved in a continuous organic phase, n-hexadecane. The effects of product inhibition by the toxic epoxide—microbial consumption of propene oxide and immobilized cell deactivation—restricted severely the accumulation of the epoxide in the recirculation reactor system and could be predicted using a simple mathematical model. Epoxide inhibition was reduced by absorbing the product in the gas phase in old di-n-octyl phthalate. The resulting increase in propene oxide production agreed with model calculations. An alternating supply of propene and a co-substrate (ethene) prolonged the half-life of the immobilized cells. Using 50 g dry weight of cells, 1.5 g stereospecific propene oxide was produced in two days, of which 1.0 g was absorbed in the di-n-octyl phthalate phase.     

固定化培养和产物释放促进剂对硬紫草细胞代谢的影响

固定化培养的硬紫草细胞生长缓慢,仅包埋球外层的细胞生长明显。其蛋白质合成的量也低。培养３０ｄ的细胞色素产量达到４．２ｍｇ／ｇＦＷ,相对色素分泌量达到７０％,而色素的组成成分及各组分的比例也与悬浮细胞的不同。以正十六烷处理固定化细胞可促进产物释放,其不同的处理时间对细胞没有显著影响。连续培养的固定细胞保持其色素形成能力达８０ｄ之久,色素总产量达２０ｍｇ／ｇＦＷ。     

Immobilization of Bacillus amyloliquefaciens MBL27 cells for enhanced antimicrobial protein production using calcium alginate beads

Cell immobilization is one of the common techniques for increasing the overall cell concentration and productivity. Bacillus amyloliquefaciens MBL27 cells were immobilized in calcium alginate beads and it is a promising method for repeated AMP (antimicrobial protein) production. The present study aimed at determining the optimal conditions for immobilization of B. amyloliquefaciens MBL27 cells in calcium alginate beads and the operational stability for enhanced production of the AMP. AMP production with free and immobilized cells was also done. In batch fermentation, maximum AMP production (7300 AU (arbitrary units)/ml against Staphylococcus aureus) was obtained with immobilized cells in shake flasks under optimized parameters such as 3% (w/v) sodium alginate, 136?mM CaCl2 with 350 alginate beads/flask of 2.7-3.0?mm diameter. In repeated cultivation, the highest activity was obtained after the second cycle of use and approx. 94% production was noted up to the fifth cycle. The immobilized cells of B. amyloliquefaciens MBL27 in alginate beads are more efficient for the production of AMP and had good stability. The potential application of AMP as a wound healant and the need for development of economical methods for improved production make whole cell immobilization an excellent alternative method for enhanced AMP production.     

Investigations on alkaline protease production with B. subtilis PE-11 immobilized in calcium alginate gel beads

Bacillus subtilis PE-11 cells were immobilized in calcium alginate and used for the production of alkaline protease. The influence of alginate concentration, different cations, concentration of cation, curing time, bead diameter and nutrient strength on alkaline protease production and stability of biocatalyst were investigated. Repeated batch fermentations of immobilized cells in shake flasks were carried out with the optimized parameters such as; 3% alginate, 0.25 M calcium chloride with 1 h curing time, 3.24 mm bead diameter and 0.75% glucose and 0.75% peptone as nutrients. The results indicated that, a good level of enzyme was maintained for a period of about 9 days. The immobilized cells of B. subtilis PE-11 in calcium alginate are more efficient for the production of alkaline protease with repeated batch fermentation.     

Oxygen uptake by entrapped hybridoma cells

Oxygen consumption by hybridoma cells immobilized in 1- and 3.9-mm-diameter calcium alginate beads was measured. The entrapped cells consumed oxygen at about 10 mumol/min per 10(9) cells, regardless of the bead size and cell loading. In contrast, the same cells in suspension culture respire at specific rates of 3-8 mumol/min per 10(9) cells (depending on the cell density). The growth rate of the immobilized cells was significantly reduced, while specific antibody production was comparable to that of free cells.     

Cell Culture conditions determine the enhancement of specific monoclonal antibody productivity of calcium alginate-entrapped S3H5/gamma2bA2 hybridoma cells

Immobilization offers several intrinsic advantages over free suspension cultures for the production of monoclonal antibodies. An important advantage of immobilization is the improved specific monoclonal antibody (MAb) productivity (q(MAb)) that can be obtained. However, there are conflicting reports in the literature on the enhancement of the q(MAb) with immobilization. The discrepancies between these reports can be attributed to the different to either the cultivation methods used for immobilized cell or to difference between the cell lines used in the various studies. We show that these differences may be attributed to the different cultivation methods used for one model hybridoma cell line. S3H5/Upsilon2bA2 hybridoma cells entrapped in different sizes of calcium alginate beads were cultivated in both T- and spinner flasks in order to determine whether cultivation methods (T- and spinner flasks) and bead size influence the q(MAb) Free-suspended cell cultures inoculated with cells recovered from alginate beads were also carried out in order to determine whether changes in the q(Mab) of the entrapped cells are reversible.The cultivation methods was found to influence significantly the q(MAb) of the entrapped cells. When the entrapped cells in 1-mn diameter beads were cultivated in T-flasks, the q(MAb) was not increased by 200% as previously observed in an entrapped cell culture using 1-mm-diameter alginate beads in spinner flasks. The q(MAb) of the entrapped cell was approximately 58% higher than that of the free-suspended cells in a control experiment. Unlike the cultivation method, the bead size in the range of 1- to 3-mm diameter did not significantly influence the q(MAb), regardless of cultivations methods. The changes in q(MAb) of an entrapped cells were reversible. When the free-suspended cells recovered from the T- and spinner flasks were sub-cultured in T- and spinner flasks enhanced q(MAb) of the entrapped cells in both cases decreased to the level of the free-suspended cell in a control experiments. Taken together, these results shows that the method of cultivation of hybridoma cells immobilized in alginate beads determines the extent of enhancement of the q(MAb). (c) 1993 John Wiley & Sons, Inc.     

Effect of growth hormone modifications on shikonin production fromLithospermum erythrorhizon cell cultures within situ extraction

Summary The effect of growth hormone modifications on shikonin production was studied with the cell cultures ofLithospermum erythrorhizon. The cells grown in SH–H or SHA medium were effective for shikonin production in M–9 medium and maximum shikonin concentrations reached 43 and 63 mg/L, respectively, within situ extraction. In the case of the cells grown in SHA medium, induction time required for shikonin production was very short and the maximum shikonin concentration was obtained within 6 days.     

Development of hepatocyte spheroids immobilization technique using alternative encapsulation method

Primary hepatocytes of small animals such as rat and rabbit were often used for the study of extracorporeal liver support systems. Freshly isolated rat hepatocytes form spheroids within two days when cultivated as suspension in spinner vessels. These spheroids showed enhanced liver specific functions and more differentiated morphology compared to hepatocytes cultured as monolayers. However, shear stress caused by continuous agitation deteriorated spheroids gradually. In this work we immobilized spheroids to prolong liver specific activities. First, hepatocyte spheroids were suspended in collagen solution containing calcium chloride and then dropped into alginate solution. A thin layer of calcium alginate was formed around the droplet and then was removed after the inner collagen was gelled by treatment of sodium citrate buffer. Spheroids embedded in collagen-gel bead maintained liver specific functions such as albumin secretion rate longer than hepatocyte spheroids exposed to shear stress. Therefore, we suggest that this immobilization technique may offer an effective long-term hepatocyte cultivation and facilitate the development of a bioartificial liver support device.     

Production of alkaline protease by entrapped Bacillus licheniformis cells in repeated batch process

In this study, Bacillus licheniformis cells were immobilized by entrapment in calcium alginate beads and were used for production of alkaline protease by repeated batch process. In order to increase the stability of the beads, the immobilization procedure was optimized by statistical full factorial method, by which three factors including alginate type, calcium chloride concentration, and agitation speed were studied. Optimization of the enzyme production medium, by the Taguchi method, was also studied. The obtained results showed that optimization of the cell immobilization procedure and medium constituents significantly enhanced the production of alkaline protease. In comparison with the free-cell culture in pre-optimized medium, about 7.3-fold higher productivity was resulted after optimization of the overall procedure. Repeated batch mode of operation, using optimized conditions, resulted in continuous production of the alkaline protease for 13 batches in 19 days.     

Protease production by immobilized growing cells of Serratia marcescens and Myxococcus xanthus in calcium alginate gel beads

Summary Serratia marcescens and Myxococcus xanthus cells were immobilized in calcium alginate gel beads. Immobilization under various conditions had no effect on the extracellular proteolytic activity of S. marcescens cells. Protease production seemed rather to depend on the free cells in the medium. However, the stability over time of enzyme production was enhanced, as immobilization increased protease production half-life from 5 to 12 days. On the other hand, Myxococcus xanthus produced proteases inside the gel beads which could diffuse into the medium. The proteolytic activity increased as a function of the initial cell content of the beads and of the bead inoculum. Compared to free cells, immobilized cells of Myxococcus xanthus could produce 8 times more proteolytic activity, with a very low free-cell concentration in the medium.