Loofa sponge as a scaffold for culture of rat hepatocytes

The dried fruit from Luffa cylindrica (loofa sponge, LS), which represents a new chitinous source material, was used as a 3-D scaffold for the culture of rat hepatocytes. With the macroporous structure and large pore size (ca. 800 microm) of LS, cell loading to the scaffold should be carried out by dynamic seeding with continuous shaking throughout the seeding period. Hepatocytes attach well to the surface of loofa fibers after seeding and maintain their round shapes. The initial ammonia removal and urea-N synthesis rates of hepatocytes immobilized within LS slightly decreased with increasing cell densities, but their metabolic activities were comparable to or better than those in monolayer culture on tissue culture polystyrene control surfaces. Both urea-N synthesis and albumin secretion rates could be maintained up to 7 days for cells immobilized within LS and spheroid-like cell aggregates could be found after the second day.     

Development of a method for immobilization of non-flocculating cells in loofa (Luffa cylindrica) sponge

Both the matrix structure of loofa sponge and the flocculating property of cells were necessary for efficient immobilization. The addition of chitosan to a reactor containing a bed of loofa sponge and a Candida brassicae cell suspension induced cell flocculation and the cells were efficiently immobilized. During ethanol production by the immobilized cells, the free cell concentration in the broth was controlled at the desired level by intermittent addition of chitosan to the reactor. The immobilized cell concentration increased but their specific ethanol productivity decreased with an increase in the chitosan concentration. The maximum ethanol productivity was obtained at a low chitosan concentration of 0·03 g/litre. With this optimal concentration, the cell concentration, ethanol yield and productivity were, respectively, 2, 1·3 and 3 times higher than those of the suspension culture.     

Loofa (Luffa cylindrica) sponge: Review of development of the biomatrix as a tool for biotechnological applications

The review discusses the development of loofa sponge (Luffa cylindrica) as a biotechnological tool and the diversity of applications in which it has been successfully used since it was first reported as a matrix for the immobilization of microbiological cells in 1993. The fibro‐vascular reticulated structure, made up of an open network of random lattices of small cross‐sections coupled with very high porosity (79–93%), having very low density (0.02–0.04 g/cm³), and high specific pore volume (21–29 cm³/g), has the characteristics of a carrier/scaffold well‐suited for cell immobilization. This has been confirmed through the immobilization of cells of diverse types, including filamentous and microalgae, fungi, bacteria, yeasts, higher plants, and human and rat hepatocytes. The cells immobilized in loofa sponge have performed well and better than free suspended cells and those immobilized in conventionally used natural and synthetic polymeric materials for the production of ethanol, organic acids, enzymes, and secondary metabolites. The loofa‐immobilized cell systems have been efficiently used for the treatment of wastewaters containing toxic metals, dyes, and chlorinated compounds, and the technology has been used to develop biofilms for the remediation of domestic and industrial wastewaters rich in inorganic and organic matter. In addition, three‐dimensional loofa sponge scaffolds for hepatocyte culture have been suggested to have the potential for development into a bioartificial liver device. Loofa sponge is a cost‐effective, eco‐friendly, and easy to handle matrix that has been used successfully as a biotechnological tool in a variety of systems, purposes, and applications. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:573–600, 2013     

Scale up of fuel ethanol production from sugar beet juice using loofa sponge immobilized bioreactor

Production of fuel ethanol from sugar beet juice, using cells immobilized on loofa sponge was investigated. Based on ethanol productivity and ease of cell immobilization, a flocculating yeast strain, Saccharomyces cerevisiae IR2 was selected for ethanol production from sugar beet juice. It was found that raw sugar beet juice was an optimal substrate for ethanol production, requiring neither pH adjustment nor nitrogen source supplement. When compared with a 2 l bubble column bioreactor, mixing was not sufficient in an 8 l bioreactor containing a bed of sliced loofa sponges and consequently, the immobilized cells were not uniformly distributed within the bed. Most of the cells were immobilized in the lower part of the bed and this resulted in decreased ethanol productivity. By using an external loop bioreactor, constructing the fixed bed with cylindrical loofa sponges, dividing the bed into upper, middle and lower sections with approximately 1 cm spaces between them and circulating the broth through the loop during the immobilization, uniform cell distribution within the bed was achieved. Using this method, the system was scaled up to 50 l and when compared with the 2 l bubble column bioreactor, there were no significant differences (P > 0.05) in ethanol productivity and yield. By using external loop bioreactor to immobilize the cells uniformly on the loofa sponge beds, efficient large scale ethanol production systems can be constructed.     

Production of lactic acid with loofa sponge immobilized Rhizopus oryzae RBU2-10

Lactic acid production by Rhizopus oryzae RBU2-10 immobilized in loofa sponge was evaluated. Shape and texture of loofa sponge, which was obtained from the mature dried fruit of Luffa cylindrica, remained intact after its treatment with buffers of varying pH and following its repeated autoclaving for up to four cycles (121 degrees C, 20 min per cycle). The medium having four pieces of loofa sponge (1.008 cm(3)) per 100 ml medium and inoculated with 3 x1 0(6) spores ml(-1) resulted maximum production (80.75 g l(-1)) of lactic acid in 48 h of fermentation. Repeated batch fermentation for lactic acid production could be carried out for 10 cycles. Remarkably higher levels of productivity (1.66-1.84 g l(-1)h(-1)) was obtained during first five cycles of fermentation with a maximum productivity (1.84 g l(-1)h(-1)) obtained during third cycle of fermentation.     

Polyurethane membrane as an efficient immobilization carrier for high-density culture of rat hepatocytes in the fixed-bed reactor

A fixed-bed bioreactor with a polyurethane membrane (PUM) as a cell-supporting material was developed for high-density culture of rat hepatocytes. The PUM has a heterogeneous porous structure of micropores (pore size <100 microm) and macropores (pore size >100 microm) with a porosity of 90%. One important feature of a PUM is that the macropores have finger-like structures and their diameters gradually decrease from the upper to the lower layer of the PUM. Most rat hepatocytes were readily immobilized in the micropores of PUM. Immobilized cell densities of 1-3 x 10(7) cells/cm(3) PUM were achieved within 5 min by natural downflow of cell suspension and their immobilization efficiencies were more than 99%. Using a syringe pump, a cell density of 5 x 10(7) cells/cm(3) PUM was achieved with more than 96% immobilization efficiency. Perfusion cultures using this reactor were performed for 7 days without cell leakage. The optimal cell density for albumin secretion was between 2 x 10(7) and 3 x 10(7) cells/cm(3) PUM. Albumin secretion in the perfusion culture was maintained for a relatively long period of time when compared to that in the monolayer culture. The rate of albumin secretion in the perfusion culture was about 50% of that in monolayer culture. Hepatocytes immobilized in PUM were slightly aggregated, but they maintained spherical form individually even after 7 days of cultivation. The above results show that PUM is a promising cell-supporting material for efficient immobilization of high cell density of hepatocytes.     

Long-Term continuous culture of hepatocytes in a packed-bed reactor utilizing porous resin

As part of our attempt to develop a hybrid artificial liver support system using cultured hepatocytes, we investigated the long-term metabolic function of hepatocytes incubated in a packed-bed type reactor using reticulated polyvinyl formal (PVF) resin as a supporting material. Long-term (up to 1 week) perfusion culture experiments using the packed-bed reactor (20 mm i.d.) loaded with 500 PVF resin cubes (mean pore size 250 mum, 2 x 2 x 2 mm), together with conventional monolayer culture experiments as controls, were performed in serum-free or serum-containing medium. Ammonium metabolism and urea synthesis activities were evaluated quantitatively based on reaction kinetic analyses. Initial rates of ammonium metabolism and urea-N synthesis, as well as GPT enzyme activities, were adopted as indexes of the metabolic performance of the reactor and activities of the cultured hepatocytes.When serum-free medium was used in the perfusion cultures, ammonium metabolic and urea-N synthetic rates showed significant decay with elapse of the culture period, being less than 10% of those measured on day 1. This loss of activity was more prominent in the perfusion culture than in the monolayer cultures using this medium. In contrast, when serum-containing medium was used, approximately 50% of these activities obtained on day 1 were maintained even at the end of the cultures both in the perfusion and monolayer culture experiments.We concluded that the packed-bed reactor using PVF resin enabled high-density culture of hepatocytes, and showed a satisfactory ability to maintain the metabolic function of immobilized hepatocytes for relatively long periods of up to 1 week. This type of reactor is thus considered to represent a breakthrough in overcoming the difficulties involved in the development of a hybridtype artificial liver support system. (c) 1994 John Wiley & Sons, Inc.     

Tissue plasminogen activator (t-PA) production by a high density culture of weakly adherent human embryonic kidney cells using a polyurethane-foam packed-bed culture system

Weakly adherent cells of the 293 line attached well to the internal surface of polyurethane foam (PUF) and grew to the high density of 6.83 × 10⁷ cells/cm³ PUF in stationary culture. The maximum productivity of tissue plasminogen activator (t-PA) was 0.158 IU/10⁶ cells per day. The productivity decreased at the stationary phase of cell growth, so we designed a PUF-plate packed-bed culture system for high density culture and continuous production of t-PA. A maximal cell density of 3.24 × 10⁷ cells/cm³ PUF and a t-PA productivity of 0.326 IU/10⁶cells per day were obtained in 25-day perfusion cultures. Although the cell density decreased to half that in PUF stationary culture, the t-PA productivity increased twofold and was maintained for 25 culture days.     

Effect of pre-immobilization conditions on phosphate uptake by immobilized Chlorella

Summary Batch culture studies of phosphate uptake by non-immobilized (free) and immobilized (Ca-alginate-entrapped) Chlorella emersonii have shown that exponentially growing free cells remove phosphate from the medium five times more rapidly than cells in late stationary phase. Culture age is also shown to be an important factor in determining the uptake abilities of cells when in their immobilized state. When cells of different ages are immobilized in Ca-alginate and placed in a small-scale packed-bed reactors the effects of culture age are sufficient to produce significant differences in reactor performance lasting in the order of five days.     

Recellularization via the bile duct supports functional allogenic and xenogenic cell growth on a decellularized rat liver scaffold

Recent years have seen a proliferation of methods leading to successful organ decellularization. In this experiment we examine the feasibility of a decellularized liver construct to support growth of functional multilineage cells. Bio-chamber systems were used to perfuse adult rat livers with 0.1% SDS for 24 hours yielding decellularized liver scaffolds. Initially, we recellularized liver scaffolds using a human tumor cell line (HepG2, introduced via the bile duct). Subsequent studies were performed using either human tumor cells co-cultured with human umbilical vein endothelial cells (HUVECs, introduced via the portal vein) or rat neonatal cell slurry (introduced via the bile duct). Bio-chambers were used to circulate oxygenated growth medium via the portal vein at 37C for 5-7 days. Human HepG2 cells grew readily on the scaffold (n = 20). HepG2 cells co-cultured with HUVECs demonstrated viable human endothelial lining with concurrent hepatocyte growth (n = 10). In the series of neonatal cell slurry infusion (n = 10), distinct foci of neonatal hepatocytes were observed to repopulate the parenchyma of the scaffold. The presence of cholangiocytes was verified by CK-7 positivity. Quantitative albumin measurement from the grafts showed increasing albumin levels after seven days of perfusion. Graft albumin production was higher than that observed in traditional cell culture. This data shows that rat liver scaffolds support human cell ingrowth. The scaffold likewise supported the engraftment and survival of neonatal rat liver cell slurry. Recellularization of liver scaffolds thus presents a promising model for functional liver engineering.     

Biosynthesis of gibberellic acid from milk permeate in repeated batch operation by a mutant Fusarium moniliforme cells immobilized on loofa sponge

Gibberellic acid (GA) production from milk permeate was studied by 28 mutants of Fusarium moniliforme, among which mutant gamma-14 was selected as the best producer. Experiments were carried out in shaker flasks and fermentative process was analyzed with free and immobilized cells. Immobilization of mutant gamma-14 cells onto loofa sponge discs was studied with respect to the optimization of the incubation temperature, initial pH, inoculum size (number of discs) and its reusability for GA production. Best yield of GA (2.40 gl(-1)) was recorded by immobilized cells under optimized cultural conditions (4 immobilized discs, 30 degrees C and pH 5). Data obtained during four reusable cycles showed high stability of GA production and reduction in the initiation time of acid production, resulting in higher levels of GA in shorter time duration. Immobilization of mutant gamma-14 cells onto loofa sponge discs, permitted repeated reuse under the specified fermentation conditions for GA production from milk permeate.     

L-Lactic acid production from raw cassava starch in a circulating loop bioreactor with cells immobilized in loofa (Luffa cylindrica)

l-Lactic acid was produced from raw cassava starch, by simultaneous enzyme production, starch saccharification and fermentation in a circulating loop bioreactor with Aspergillus awamori and Lactococcus lactis spp. lactis immobilized in loofa sponge. A. awamori was immobilized directly in cylindrical loofa sponge while the L. lactis was immobilized in a loofa sponge alginate gel cube. In the loofa sponge alginate gel cube, the sponge serves as skeletal support for the gel with the cells. The alginate gel formed a hard outer layer covering the soft porous gel inside. By controlling the rate and frequency of broth circulation between the riser and downcomer columns, the riser could be maintained under aerobic condition while the downcomer was under anaerobic condition. Repeated fed-batch l-lactic acid production was performed for more than 400 h and the average lactic acid yield and productivity from raw cassava starch were 0.76 g lactic acid g^–1 starch and 1.6 g lactic acid l^–1 h^–1, respectively.     

Oxygen supply for CHO cells immobilized on a packed-bed of Fibra-Cel disks

Packed-bed bioreactors (PBR) have proven to be efficient systems to culture mammalian cells at very high cell density in perfusion mode, thus leading to very high volumetric productivity. However, the immobilized cells must be continuously supplied with all nutrients in sufficient quantities to remain viable and productive over the full duration of the perfusion culture. Among all nutrients, oxygen is the most critical since it is present at very low concentration due to its low solubility in cell culture medium. This work presents the development of a model for oxygenation in a packed-bed bioreactor system. The experimental system used to develop the model was a packed-bed of Fibra-Cel disk carriers used to cultivate Chinese Hamster Ovary cells at high density ( approximately 6.1 x 10(7) cell/mL) in perfusion mode. With the help of this model, it was possible to identify if a PBR system is operated in optimal or sub-optimal conditions. Using the model, two options were proposed, which could improve the performance of the basal system by about twofold, that is, by increasing the density of immobilized cells per carrier volume from 6.1 x 10(7) to 1.2 x 10(8) cell/mL, or by increasing the packed-bed height from 0.2 to 0.4 m. Both strategies would be rather simple to test and implement in the packed-bed bioreactor system used for this study. As a result, it would be possible to achieve a substantial improvement of about twofold higher productivity as compared with the basal conditions.     

Three-dimensional cell seeding and growth in radial-flow perfusion bioreactor for in vitro tissue reconstruction

Radial-flow perfusion bioreactor systems have been designed and evaluated to enable direct cell seeding into a three-dimensional (3-D) porous scaffold and subsequent cell culture for in vitro tissue reconstruction. However, one of the limitations of in vitro regeneration is the tissue necrosis that occurs at the central part of the 3-D scaffold. In the present study, tubular poly-L-lactic acid (PLLA) porous scaffolds with an optimized pore size and porosity were prepared by the lyophilization method, and the effect of different perfusion conditions on cell seeding and growth were compared with those of the conventional static culture. The medium flowed radially from the lumen toward the periphery of the tubular scaffolds. It was found that cell seeding under a radial-flow perfusion condition of 1.1 mL/cm2 x min was effective, and that the optimal flow rate for cell growth was 4.0 mL/cm2 x min. At this optimal rate, the increase in seeded cells in the perfusion culture over a period of 5 days was 7.3-fold greater than that by static culture over the same period. The perfusion cell seeding resulted in a uniform distribution of cells throughout the scaffold. Subsequently, the perfusion of medium and hence the provision of nutrients and oxygen permitted growth and maintenance of the tissue throughout the scaffold. The perfusion seeding/culture system was a much more effective strategy than the conventional system in which cells are seeded under a static condition and cultured in a bioreactor such as a spinner flask.     

A cytokine-selective defect in interleukin-1 beta-mediated acute phase gene expression in a subclone of the human hepatoma cell line (HEPG2)

Several well-differentiated human hepatoma cell lines (HepG2, Hep3B) have been used to identify factors which regulate hepatic gene expression during the host response to inflammation/tissue injury (acute phase response). Studies in these cell lines, as well as in primary cultures of rat, rabbit, and mouse hepatocytes, have demonstrated that interleukin-1 beta (IL-1 beta), tumor necrosis factor (TNF-alpha), and interferon-beta 2 (IFN-beta 2) each mediate changes in expression of several hepatic acute phase genes. In this study we identify a subclone of the HepG2 cell line in which there is a selective defect in IL-1 beta-mediated acute phase gene expression. Recombinant human IL-1 beta mediates an increase in synthesis of the positive acute phase complement protein factor B and a decrease in synthesis of negative acute phase protein albumin in the parent uncloned HepG2 cell line (HG2Y), but not in the subclone HG2N. Recombinant human IFN-beta 2 and TNF-alpha, however, regulate acute phase protein synthesis in the subclone HG2N; i.e. IFN-beta 2 and TNF-alpha increase synthesis of factor B and decrease synthesis of albumin in both HG2Y and HG2N cells. Equilibrium binding analysis with 125I-rIL-1 beta at 4 degrees C showed that both HG2N and HG2Y cells bind IL-1 beta specifically and saturably. HG2N and HG2Y possess 3.8 and 4.0 x 10(3) plasma membrane receptors/cell with affinities of 0.96 and 1.07 x 10(-9) M, respectively. Thus, the defect in this subclone of the HepG2 cell line is likely to involve the signal transduction pathway for the biological activity of IL-1 beta and will be useful in elucidation of this signal transduction pathway.     

A novel rotating-shaft bioreactor for two-phase cultivation of tissue-engineered cartilage

A novel rotating-shaft bioreactor (RSB) was developed for two-phase cultivation of tissue-engineered cartilage. The reactor consisted of a rotating shaft on which the chondrocyte/scaffold constructs (7.5 mm diameter x 3.5 mm thickness) were fixed and a reactor vessel half-filled with medium. The horizontal rotation of the shaft resulted in alternating exposure of the constructs to gas and liquid phases, thus leading to efficient oxygen and nutrient transfer, as well as periodically changing, mild shear stress exerting on the construct surfaces (0-0.32 dyn/cm2 at 10 rpm), as revealed by computer simulation. Strategic operation of the RSB (maintaining rotating speed at 10 rpm for 3 weeks and lowering the speed to 2 rpm in week 4) in combination with higher seeding density (6 x 10(6) chondrocytes/scaffold) and medium perfusion resulted in uniform cell distribution and increased glycosaminoglycan (3.1 mg/scaffold) and collagen (7.0 mg/scaffold) deposition. The 4-week constructs resembled native cartilages in terms of not only gross appearance and cell morphology but also distributions of glycosaminoglycan, total collagen, and type II collagen, confirming the maintenance of chondrocyte phenotype and formation of cartilage-like constructs in the RSB cultures. In summary, the novel RSB may be implicated for in vitro study of chondrogenesis and de novo cartilage development under periodic mechanical loading. With proper optimization of the culture conditions, a RSB may be employed for the production of cartilage-like constructs.     

A packed-bed reactor utilizing porous resin enables high density culture of hepatocytes

Summary To enable high density culture of hepatocytes for use as a hybrid artificial liver support system or a bioreactor system, a packed-bed reactor using collagen-coated reticulated polyvinyl formal (PVF) resin was applied to a primary culture of hepatocytes. Cubic PVF resins (2×2×2 mm, mean pore size: 100, 250 or 500 m) were used as supporting substrates to immobilize hepatocytes. Two hundred and fifty cubes were packed in a cylindrical column, and 2.6–11.3×10⁷ hepatocytes were seeded in the column by irrigating with 3 ml of the medium containing hepatocytes. Perfusion culture experiments using this packed-bed reactor, as well as monolayer cultures using conventional collagen-coated petri dishes as control experiments, were performed. Sufficient amounts of hepatocytes were found to be immobilized in the reticulated structure of the PVF resins. The highest density of immobilized hepatocytes attained with PVF resin was 1.2×10⁷ cells/cm³ PVF, which showed levels of ammonium removal and urea-N secretion comparable to those in the monolayer culture. It is concluded that the packed-bed reactor system utilizing PVF resin is a promising process for developing a bioreactor or a bioartificial organ using hepatocytes. Correspondence to: N. Ohshima     

Acetone-butanol-ethanol (ABE) fermentation in an immobilized cell trickle bed reactor

Acetone-butanol-ethanol (ABE) fermentation was successfully carried out in an immobilized cell trickle bed reactor. The reactor was composed of two serial columns packed with Clostridium acetobutylicum ATCC 824 entrapped on the surface of natural sponge segments at a cell loading in the range of 2.03-5.56 g dry cells/g sponge. The average cell loading was 3.58 g dry cells/g sponge. Batch experiments indicated that a critical pH above 4.2 is necessary for the initiation of cell growth. One of the media used during continuous experiments consisted of a salt mixture alone and the other a nutrient medium containing a salt mixture with yeast extract and peptone. Effluent pH was controlled by supplying various fractions of the two different types of media. A nutrient medium fraction above 0.6 was crucial for successful fermentation in a trickle bed reactor. The nutrient medium fraction is the ratio of the volume of the nutrient medium to the total volume of nutrient plus salt medium. Supplying nutrient medium to both columns continuously was an effective way to meet both pH and nutrient requirement. A 257-mL reactor could ferment 45 g/L glucose from an initial concentration of 60 g/L glucose at a rate of 70 mL/h. Butanol, acetone, and ethanol concentrations were 8.82, 5.22, and 1.45 g/L, respectively, with a butanol and total solvent yield of 19.4 and 34.1 wt %. Solvent productivity in an immobilized cell trickle bed reactor was 4.2 g/L h, which was 10 times higher than that obtained in a batch fermentation using free cells and 2.76 times higher than that of an immobilized CSTR. If the nutrient medium fraction was below 0.6 and the pH was below 4.2, the system degenerated. Oxygen also contributed to the system degeneration. Upon degeneration, glucose consumption and solvent yield decreased to 30.9 g/L and 23.0 wt %, respectively. The yield of total liquid product (40.0 wt %) and butanol selectivity (60.0 wt %) remained almost constant. Once the cells were degenerated, they could not be recovered.     

Enhancement of Lead(II) Biosorption by Microalgal Biomass Immobilized onto Loofa (Luffa cylindrica) Sponge

A unicellular green microalga, Chlorella sorokiniana, was immobilized on loofa (Luffa cylindrica) sponge and successfully used as a new biosorption system for the removal of lead(II) ions from aqueous solutions. The biosorption of lead(II) ions on both free and immobilized biomass of C. sorokiniana was investigated using aqueous solutions in the concentration range of 10–300 mg/L. The biosorption of lead(II) ions by C. sorokiniana biomass increased as the initial concentration of lead(II) ions increased in the medium. The maximum biosorption capacity for free and immobilized biomass of C. sorokiniana was found to be 108.04 and 123.67 mg lead(II)/g biomass, respectively. The biosorption kinetics were found to be fast, with 96 % of adsorption within the first 5 min and equilibrium reached at 15 min. The adsorption of lead(II) both by free and immobilized C. sorokiniana biomass followed the Langmuir isotherm. The biosorption capacities were detected to be dependent on the pH of the solution; and the maximum adsorption was obtained at a solution pH of about 5. The effect of light metal ions on lead(II) uptake was also studied and it was shown that the presence of light metal ions did not significantly affect lead(II) uptake. The loofa sponge‐immobilized C. sorokiniana biomass could be regenerated using 0.1 M HCl, with up to 99 % recovery. The desorbed biomass was used in five biosorption‐desorption cycles, and no noticeable loss in the biosorption capacity was observed. In addition, fixed bed breakthrough curves for lead(II) removal were presented. These studies demonstrated that loofa sponge‐immobilized biomass of C. sorokiniana could be used as an efficient biosorbent for the treatment of lead(II) containing wastewater.     

Biodiesel synthesis in a solvent-free system by recombinant Rhizopus oryzae: comparative study between a stirred tank and a packed-bed batch reactor

A simultaneous synthesis of biodiesel, as fatty acid methyl esters, and monoacylglycerols catalysed by the recombinant Rhizopus oryzae lipase immobilized by adsorption on Relizyme OD/403M is presented. The use of this 1(3)-positional specific lipase prevents the formation of glycerol as a by-product, thus avoiding its drawbacks. The synthesis was carried out in a solvent-free system and it has been studied in two different reactor systems: stirred tank and packed-bed reactor. Stirred tank reactor presented a high-initial reaction rate and achieved a 33.6% yield, which corresponds to a value of 50.4% of the maximum yield that can be achieved with a 1(3)-positional specific lipase. In packed-bed reactor there was a smaller initial reaction rate, but it was achieved a 49.1% yield, which corresponds to a 73.6% of the maximum yield. When a second batch is performed, the yield decreased only 4% when packed-bed reactor is employed whereas a drastic decrease is observed in a stirred tank operation. Therefore, packed-bed reactor showed a best performance and minor damage to the biocatalyst.