Mathematical modeling of maltose hydrolysis in different types of reactor

A commercial enzyme Dextrozyme was tested as catalyst for maltose hydrolysis at two different temperatures: 40 and 65 °C at pH 5.5. Its operational stability was studied in different reactor types: batch, repetitive batch, fed-batch and continuously operated enzyme membrane reactor. Dextrozyme was more active at 65 °C, but operational stability decay was observed during the prolonged use in the reactor at this temperature. The reactor efficiencies were compared according to the volumetric productivity, biocatalyst productivity and enzyme consumption. The best reactor type according to the volumetric productivity for maltose hydrolysis is batch and the best reactor type according to the biocatalyst productivity and enzyme consumption is continuously operated enzyme membrane reactor. The mathematical model developed for the maltose hydrolysis in the different reactors was validated by the experiments at both temperatures. The Michaelis–Menten kinetics describing maltose hydrolysis was used.     

High-performance recombinant protein production with <Emphasis Type="Italic">Escherichia coli</Emphasis> in continuously operated cascades of stirred-tank reactors

The microbial expression of intracellular, recombinant proteins in continuous bioprocesses suffers from low product concentrations. Hence, a process for the intracellular production of photoactivatable mCherry with Escherichia coli in a continuously operated cascade of two stirred-tank reactors was established to separate biomass formation (first reactor) and protein expression (second reactor) spatially. Cascades of miniaturized stirred-tank reactors were implemented, which enable the 24-fold parallel characterization of cascade processes and the direct scale-up of results to the liter scale. With PAmCherry concentrations of 1.15 g L^?1 cascades of stirred-tank reactors improved the process performance significantly compared to production processes in chemostats. In addition, an optimized fed-batch process was outperformed regarding space–time yield (149 mg L^?1 h^?1). This study implicates continuous cascade processes to be a promising alternative to fed-batch processes for microbial protein production and demonstrates that miniaturized stirred-tank reactors can reduce the timeline and costs for cascade process characterization.     

Novel,linked bioreactor system for continuous production of biologics

A novel, alternative intensified cell culture process comprised of a linked bioreactor system is presented. An N-1 perfusion bioreactor maintained cells in a highly proliferative state and provided a continuous inoculum source to a second bioreactor operating as a continuous-flow stirred-tank reactor (CSTR). An initial study evaluated multiple system steady-states by varying N-1 steady-state viable cell densities, N-1 to CSTR working volume ratios, and CSTR dilution rates. After identifying near optimum system steady-state parameters yielding a relatively high volumetric productivity while efficiently consuming media, a subsequent lab-scale experiment demonstrated the startup and long-term operation of the envisioned manufacturing process for 83 days. Additionally, to compensate for the cell-specific productivity loss over time due to cell line instability, the N-1 culture was also replaced with younger generation cells, without disturbing the steady-state of the system. Using the model cell line, the system demonstrated a two-fold volumetric productivity increase over the commercial-ready, optimized fed-batch process.     

Fed-batch cultivation for the production of clavulanic acid by an immobilized Streptomyces clavuligerus mutant

In order to obtain high productivity of clavulanic acid, a newly-introduced carrier, polyurethane pellet (PUP) Z97-020 was used for the immobilization process. In a stirred-tank bioreactor, batch cultivation by Streptomyces clavuligerus KK immobilized on PUP Z97-020 gave about 3100 mg of clavulanic acid per litre, representing an increase of 200% in productivity compared with that by fed-batch cultivation of free cells (1500 mg/l). However, the clavulanic acid produced rapidly decomposed due to the pH change during batch cultivation. Fed-batch cultivation by immobilized S. clavuligerus KK gave an excellent level of clavulanic acid up to 3250 mg/l, a productivity increase of 220% compared with that by fed-batch cultivation of free cells. These results suggest that immobilization with PUP Z97-020 is a more effective process for the production of clavulanic acid and that the maintenance of pH by fed-batch cultivation with glycerol as a limiting substrate prevents the clavulanic acid from decomposing during the fermentation.     

Production of immunoglobulin A in different reactor configurations

A murine hybridoma line (Zac3), secreting an IgA monoclonal antibody, was cultivated in different systems: a BALB/c mouse, a T-flask, a stirred-tank bioreactor and a hollow fiber reactor. These systems were characterized in terms of cell metabolism and performances for IgA production. Cultures in T-flask and batch bioreactor were found to be glutamine-limited. Ammonia and lactate were produced in significant amounts. IgA productivity was found to be constant and growth associated. Final IgA concentration was similar in both systems. In fed-batch cultures, supplemented with glutamine and glucose, maximum viable cell concentration was increased by 60% and final IgA concentration by 155%. The hollow fiber reactor was able to produce very large amounts of IgA at very high concentrations, similar to the value found in ascites fluid. The productivity ofZac3 is similar to the values reported for IgG-producing cell lines.     

Influences of yeast extract on specific cellular yield of Ovine growth hormone during fed-batch fermentation of E. coli

In most cases of E. coli high cell density fermentation process, maximizing cell concentration helps in increasing the volumetric productivity of recombinant proteins usually at the cost of lower specific cellular protein yield. In this report, we describe a process for maintaining the specific cellular yield of Ovine growth hormone (oGH) from E. coli by optimal feeding of yeast extract during high cell density fermentation process. Recombinant oGH was produced as inclusion bodies in Escherichia coli. Specific cellular yield of recombinant oGH was maintained by feeding yeast extract along with glucose during fed-batch fermentation. Glucose to yeast extract ratio of 0.75 was found to be optimum for maintaining the specific cellular oGH yield of 66 mg/g of E. coli cells. Continuous feeding of yeast extract along with glucose helped in reducing acetic acid secretion and promoted higher cell growth during fed-batch fermentation. High cell growth of E. coli and high specific yield of recombinant oGH thus helped in achieving high volumetric productivity of the expressed protein. A maximum of 2 g/l of ovine growth hormone was expressed as inclusion bodies in 12 h of fed-batch fermentation.     

A novel split-cylinder airlift reactor for fed-batch cultures

Conventional airlift reactors are not adequate to carry out variable volume processes since it is not possible to achieve a proper liquid circulation in these reactors until the liquid height is higher than that of the downcomer. To carry out processes of variable volume, the use of a split-cylinder airlift reactor is proposed, in the interior of which two multi-perforated vertical baffles are installed in order to provide several points of communication between the reactor riser and downcomer. This improves the liquid circulation and mixing at any liquid volume. In fed-batch cultures, it is important to know how liquid height affects the hydrodynamic characteristics and the volumetric oxygen transfer coefficient since this impacts on the kinetic behavior of any fermentation. Thus, in the present work, the effect of the liquid height on the mixing time, the overall gas hold-up, and the volumetric oxygen transfer coefficient of the proposed airlift reactor were determined. The mixing time was increased and the volumetric oxygen transfer coefficient decreased due to the increase of the liquid height in the reactor in all the superficial gas velocities tested, which corresponded to a pseudohomogeneous flow regime. The experimental values of the mixing time and the mass-transfer coefficient were properly described through correlations in which the U_GR/H_L ratio was used as the independent variable. Thus, this variable might be used to describe the hydrodynamic behavior and the oxygen transfer coefficient of airlift reactors when such reactors are used in processes where the liquid volume changes with time. However, these correlations are useful for the particular device and for the particular operating conditions at which they were obtained. These empirical correlations are useful to understand some factors that influence the mixing time and volumetric oxygen transfer coefficient, but such correlations do not have a sufficient predictive potential for a satisfactory reactor design. The overall gas hold-up values were not significantly affected when the liquid height was lower than the downcomer height. However, the values decreased abruptly when the reactor was operated with liquid heights over the downcomer height, especially at high superficial gas velocities.     

Multi-stage high cell continuous fermentation for high productivity and titer

We carried out the first simulation on multi-stage continuous high cell density culture (MSC-HCDC) to show that the MSC-HCDC can achieve batch/fed-batch product titer with much higher productivity to the fed-batch productivity using published fermentation kinetics of lactic acid, penicillin and ethanol. The system under consideration consists of n-serially connected continuous stirred-tank reactors (CSTRs) with either hollow fiber cell recycling or cell immobilization for high cell-density culture. In each CSTR substrate supply and product removal are possible. Penicillin production is severely limited by glucose metabolite repression that requires multi-CSTR glucose feeding. An 8-stage C-HCDC lactic acid fermentation resulted in 212.9 g/L of titer and 10.6 g/L/h of productivity, corresponding to 101 and 429% of the comparable lactic acid fed-batch, respectively. The penicillin production model predicted 149% (0.085 g/L/h) of productivity in 8-stage C-HCDC with 40 g/L of cell density and 289% of productivity (0.165 g/L/h) in 7-stage C-HCDC with 60 g/L of cell density compared with referring batch cultivations. A 2-stage C-HCDC ethanol experimental run showed 107% titer and 257% productivity of the batch system having 88.8 g/L of titer and 3.7 g/L/h of productivity. MSC-HCDC can give much higher productivity than batch/fed-batch system, and yield a several percentage higher titer as well. The productivity ratio of MSC-HCDC over batch/fed-batch system is given as a multiplication of system dilution rate of MSC-HCDC and cycle time of batch/fed-batch system. We suggest MSC-HCDC as a new production platform for various fermentation products including monoclonal antibody.     

Oxygen transfer characteristics of a centrifugal, packed-bed reactor during viscous xanthan fermentation

The oxygen transfer properties of a novel, centrifugal, packed-bed reactor (CPBR) during viscous xanthan fermentation were determined with respect to the effects of the arrangement of the centrifugal, packed bed (CPB) and the recirculation loop (RL). Characterized by the maximum volumetric transfer coefficient (k_La) in xanthan broth, the aeration efficiency of CPBR was compared to those in stirred-tank reactors (STR) equipped with disc turbines (DT) or marine propellers (MP), and to that in a water-in-oil emulsion (WIO). As expected, STR-WIO showed the highest k_La (0.038 s^-1 at 2%) among all systems studied due to reduced broth viscosity; however, practical difficulties exist in product recovery. It was found that, at 3.5% xanthan the k_La in CPBR (0.018 s^-1) was higher than that of STR (0.005 s^-1) and close to that of STR-WIO (0.020 s^-1), indicating improved oxygen transfer at such a xanthan concentration. The exterior baffles along the rotating fibrous matrix offer additional agitation in the viscous broth. A gas-continuous arrangement, in which the CPB was kept above the broth, was able to elevate k_La to 0.023 s^-1, higher than that of STR-WIO. The external RL operated by a peristaltic pump was found to play an important role in CPBR aeration by providing better gas-liquid contact. With the improved oxygen transfer efficiency in CPBR at high xanthan concentrations, the CPBR system is practically the preferred system for xanthan fermentation. The characteristic roles of CPB arrangement and the RL should be considered primarily during scale-up operation.     

Continuous alpha-amylase production using Bacillus amyloliquefaciens adsorbed on an ion exchange resin

Summary A method for the continuous production of extracellular alpha amylase by surface immobilized cells of Bacillus amyloliquefaciens NRC 2147 has been developed. A large-pore, macroreticular anionic exchange resin was capable of initially immobilizing an effective cell concentration of 17.5 g DW/1 (based on a total reactor volume of 160 ml). The reactor was operated continuously with a nutrient medium containing 15 g/l soluble starch, as well as yeast extract and salts. Aeration was achieved by sparging oxygen enriched air into the column inlet. Fermentor plugging by cells was avoided by periodically substituting the nutrient medium with medium lacking in both soluble starch and yeast extract. This fermentor was operated for over 200 h and obtained a steady state enzyme concentration of 18700 amylase activity units per litre (18.7 kU/l), and an enzyme volumetric productivity of 9700 amylase activity units per litre per hour (9.7 kU/l-h). Parallel fermentations were performed using a 2 l stirred vessel fermentor capable of operation in batch and continuous mode. All fermentation conditions employed were identical to those of the immobilized cell experiments in order to assess the performance of the immobilized cell reactor. Batch stirred tank operation yielded a maximum amylase activity of 150 kU/l and a volumetric productivity of 2.45 kU/l-h. The maximum cell concentration obtained was 5.85 g DW/l. Continuous stirred tank fermentation obtained a maximum effluent amylase activity of 6.9 kU/l and a maximum enzyme volumetric productivity of 2.73 kU/l-h. Both of these maximum values were observed at a dilution rate of 0.345 l/h. The immobilized cell reactor was observed to achieve larger volumetric productivities than either mode of stirred tank fermentation, but achieved an enzyme activity concentration lower than that of the batch stirred tank fermentor.     

Biotechnological aspects of the production of the anticancer drug podophyllotoxin

The natural lignan podophyllotoxin, a dimerized product of two phenylpropanoid moieties which occurs in a few plant species, is a pharmacologically important compound for its anticancer activities. It is used as a precursor for the chemical synthesis of the anticancer drugs etoposide, teniposide and etopophose. The availability of this lignan is becoming increasingly limited because of the scarce occurrence of its natural sources and also because synthetic approaches for its production are still commercially unacceptable. Biotechnological production using cell culture may be considered as an alternative source. Selection of the best performing cell line, its maintenance and stabilization are necessary prerequisites for its production in bioreactors and subsequent scale-up of the cultivation process to the industrial level. Scale-up of growth and product yield depends on a multitude of factors, such as growth medium, physicochemical conditions, seed inoculum, type of reactor and processing conditions. The composition of the growth medium, elicitors and precursors, etc. can markedly influence the production. Optimum levels of parameters that facilitate high growth and product response in cell suspensions of Podophyllum hexandrum have already been determined by statistical design. P. hexandrum cells have successfully been cultivated in a 3-l stirred-tank bioreactor under low shear conditions in batch and fed-batch modes of operation. The batch kinetic data were used to identify the mathematical model which was then used to develop nutrient-feeding strategies for fed-batch cultivation to prolong the productive log phase of cultivation. An improvement in the production of podophyllotoxin to 48.8 mg l^–1 in a cell culture of P. hexandrum was achieved, with a corresponding volumetric productivity of 0.80 mg l^–1 day^–1, when the reactor was operated in continuous cell-retention mode. Efforts are being made to further enhance its production levels by the development of hairy root culture or by varying the channeling of precursors towards the desired biosynthetic pathway by molecular approaches.     

Cultivation of recombinant E. coli and production of fusion protein in 60-1 bubble column and airlift tower loop reactors.

E. coli K12 with multicopy plasmid (lambda PR-promoter and temperature-sensitive lambda cI 857 repressor) was cultivated in 60-l bubble column and airlift tower loop reactors. The medium composition, cell concentration, and intracellulary enzyme activity were monitored on-line during batch, fed-batch, and continuous cultivations. The specific growth rates, cell mass yield coefficients, plasmid stabilities, productivities of the amount of active fusion protein (beta-galactosidase activity), concentrations and yields of acetic acid, and volumetric oxygen transfer coefficient were evaluated for different medium compositions and cultivation conditions. The enzyme activity was also monitored during the temperature induction. The results evaluated in the 60-l bubble column and airlift tower loop reactors are compared with those evaluated in a 1-1 stirred-tank reactor.     

Maximizing productivity of a continuous fermenter using nonlinear adaptive optimizing control

The control of a continuously operated fermenter at its maximum productivity level gives rise to a difficult control problem as the location of the optimum operating point changes due to the disturbances. In addition, the fermenter exhibits a change in the sign of the steady state gain near the optimum operating point. This study is aimed at developing an on-line optimizing control scheme that can track the changing location of the steady state optimum so as to maximize the fermenter productivity. A nonlinear Laguerre model, whose parameters are estimated on-line, is used for tracking the optimum operating point. The control at the optimum point is achieved using an adaptive nonlinear MPC strategy that uses the nonlinear Laguerre model for prediction. The efficiency of the proposed algorithm is demonstrated by simulating the control of a continuous fermenter that exhibits shift in the location of the optimum operating point in response to the changes in the maximum specific growth rate. The proposed on-line optimizing control strategy is shown to result in a considerable improvement in the closed loop performance even in the presence of measurement noise.     

Effect of feeding strategy on the stability of anaerobic sequencing batch reactor responses to organic loading conditions

The goal of this study was to examine the effect of feeding strategy on the capability for treatment and the stability of an anaerobic sequencing batch reactor (ASBR) under increasing organic loading. The lab-scale ASBR systems were operated at 35 degrees C using synthetic organic wastewater under both batch and fed-batch operational modes with different feed to cycle time (F:C) ratios. Experimental studies were conducted over a wide range of volumetric organic loading rates (VOLRs) (1.524 g COD/l/d) by varying the hydraulic retention time (HRT) (1.25, 2.5, and 5d) and the feed wastewater's COD (3750-30,000 mg/l). With an F:C ratio greater than or equal to 0.42, the fed-batch mode operation showed higher system efficiency in COD removal, volumetric methane production rate (VMPR), and specific methane production rate (SMPR) as compared to those in the batch mode with identical VOLR and HRT. In the fed-batch mode, the COD removals reached 86-95% with VOLR up to 12 g COD/l/d. The maximums for VMPR of 3.17 l CH4/l/d and for SMPR of 1.63 g CH4-COD/g VSS/d were achieved with a VOLR of 12 g COD/l/d at HRTs of 2.5 and 1.25 d, respectively. The fed-batch operation presented a lower concentration of volatile fatty acids (VFAs) than those in the batch operation. A lower concentration of VFAs confirmed the stability and efficiency of the fed-batch mode operation. The specific methanogenic activity (SMA) analysis showed that the VFA-degrading activity of the biomass in the fed-batch mode was higher for acetate and butyrate, and lower for propionate. Determined biomass yield and bacterial decay coefficients in the fed-batch operational mode were 0.05 g VSS/g COD rem and 0.001 d(-1), respectively.     

Reactor kinetics for submerged aerobic biofilms

Sclerotium rolfsii ATCC 15205 was cultivated in continuous culture (48 l reactor volume) for scleroglucan production. The maximum volumetric productivity (Q_Pvmax) amounted to 7.2 g/ld and was more than twice as high as in comparable batchwise cultivations. In addition, the relation between (a) volumetric productivity (Q_P [g/ld]) and product yield (Y_{Glucan/Glucose} [1]), (b) volumetric productivity and product quality (M_W [g/mol]), and (c) product quality and impeller tip speed (Nd [m/s]) was studied in continuous culture. It was found, that an increase in volumetric productivity led to a decline in product yield and product quality. Furthermore, an impeller tip speed of >0.7 m/s decreased the attainable product quality considerably. Based on these results, the impact of the operational setpoint of the process in terms of oxygen supply and reactor scale on the economics of scleroglucan production was discussed. In contrast to batchwise cultivation, scleroglucan production in continuous culture proved to be not feasible under non-aseptic conditions.     

High efficiency ethanol fermentation by entrapment of Zymomonas mobilis into LentiKats

AIMS: To examine the potential of Zymomonas mobilis entrapped into polyvinylalcohol (PVA) lens-shaped immobilizates in batch and continuous ethanol production. METHODS AND RESULTS: Cells, free or immobilized in PVA hydrogel-based lens-shaped immobilizates - LentiKats, were cultivated on glucose medium in a 1 l bioreactor. In comparison with free cell cultivation, volumetric productivity of immobilized batch culture was nine times higher (43.6 g l(-1) h(-1)). The continuously operated system did not improve the efficiency (volumetric productivity of the immobilized cells 30.7 g l(-1) h(-1)). CONCLUSIONS: We demonstrated Z. mobilis capability, entrapped into LentiKats, in the cost-efficient batch system of ethanol production. SIGNIFICANCE AND IMPACT OF THE STUDY: The results reported here emphasize the potential of bacteria in combination with suitable fermentation technology in industrial scale. The innovation compared with traditional systems is characterized by excellent long-term stability, high volumetric productivity and other technological advantages.     

A rational approach to improving productivity in recombinant Pichia pastoris fermentation

A Mut(S) Pichia pastoris strain that had been genetically modified to produce and secrete sea raven antifreeze protein was used as a model system to demonstrate the implementation of a rational, model-based approach to improve process productivity. A set of glycerol/methanol mixed-feed continuous stirred-tank reactor (CSTR) experiments was performed at the 5-L scale to characterize the relationship between the specific growth rate and the cell yield on methanol, the specific methanol consumption rate, the specific recombinant protein formation rate, and the productivity based on secreted protein levels. The range of dilution rates studied was 0. 01 to 0.10 h(-1), and the residual methanol concentration was kept constant at approximately 2 g/L (below the inhibitory level). With the assumption that the cell yield on glycerol was constant, the cell yield on methanol increased from approximately 0.5 to 1.5 over the range studied. A maximum specific methanol consumption rate of 20 mg/g. h was achieved at a dilution rate of 0.06 h(-1). The specific product formation rate and the volumetric productivity based on product continued to increase over the range of dilution rates studied, and the maximum values were 0.06 mg/g. h and 1.7 mg/L. h, respectively. Therefore, no evidence of repression by glycerol was observed over this range, and operating at the highest dilution rate studied maximized productivity. Fed-batch mass balance equations, based on Monod-type kinetics and parameters derived from data collected during the CSTR work, were then used to predict cell growth and recombinant protein production and to develop an exponential feeding strategy using two carbon sources. Two exponential fed-batch fermentations were conducted according to the predicted feeding strategy at specific growth rates of 0.03 h(-1) and 0.07 h(-1) to verify the accuracy of the model. Cell growth was accurately predicted in both fed-batch runs; however, the model underestimated recombinant product concentration. The overall volumetric productivity of both runs was approximately 2.2 mg/L. h, representing a tenfold increase in the productivity compared with a heuristic feeding strategy.     

Development and optimization of two-stage cyclic fed-batch culture for hG-CSF production using L-arabinose promoter of Escherichia coli

The long-term process for producing human granulocyte-colony stimulating factor (hG-CSF) was developed using two-stage cyclic fed-batch culture, in which hG-CSF expressing-recombinant Escherichia coli was directed by an L-arabinose promoter system. For the optimization, the preinduction growth rate during the growth stage and the feeding strategy during the production stage were investigated. The maximum harvest volume during the production stage was predicted before long-term cyclic operation. Based on those optimized strategies, the two-stage cyclic fed-batch culture was performed for 12 cycles (86 h). The cell growths in both stages were maintained at 45-50 g/L and 71-77 g/L, respectively. hG-CSF was stably produced at a level of 8-9 g/L and the plasmid stability was maintained at more than 90%. Volumetric productivity by the two-stage cyclic fed-batch culture was 0.643 g/L/h, which was about 280% higher than that of conventional DO-stat fed-batch culture.     

Acetate production from whey lactose using co-immobilized cells of homolactic and homoacetic bacteria in a fibrous-bed bioreactor

Acetate was produced from whey lactose in batch and fed-batch fermentations using co-immobilized cells of Clostridium formicoaceticum and Lactococcus lactis. The cells were immobilized in a spirally wound fibrous sheet packed in a 0.45-L column reactor, with liquid circulated through a 5-L stirred-tank fermentor. Industrial-grade nitrogen sources, including corn steep liquor, casein hydrolysate, and yeast hydrolysate, were studied as inexpensive nutrient supplements to whey permeate and acid whey. Supplementation with either 2.5% (v/v) corn steep liquor or 1.5 g/L casein hydrolysate was adequate for the cocultured fermentation. The overall acetic acid yield from lactose was 0.9 g/g, and the productivity was 0.25 g/(L h). Both lactate and acetate at high concentrations inhibited the homoacetic fermentation. To overcome these inhibitions, fed-batch fermentations were used to keep lactate concentration low and to adapt cells to high-concentration acetate. The final acetate concentration obtained in the fed-batch fermentation was 75 g/L, which was the highest acetate concentration ever produced by C. formicoaceticum. Even at this high acetate concentration, the overall productivity was 0.18 g/(L h) based on the total medium volume and 1.23 g/(L h) based on the fibrous-bed reactor volume. The cells isolated from the fibrous-bed bioreactor at the end of this study were more tolerant to acetic acid than the original culture used to seed the bioreactor, indicating that adaptation and natural selection of acetate-tolerant strains occurred. This cocultured fermentation process could be used to produce a low-cost acetate deicer from whey permeate and acid whey.     

Repeated batch and continuous operations for phosphatidylglycerol synthesis from phosphatidylcholine with immobilized phospholipase D

Summary The repeated batch and continuous operations for transphosphatidylation reaction were carried out for phosphatidylglycerol (PG) synthesis from phosphatidylcholine (PC) with the help of immobilized cabbage phospholipase D (PLD) in the presence of glycerol. The biphasic reaction system was used which included the aqueous phase containing immobilized PLD along with high concentrations of glycerol (30%–50%) and buffer, whereas the main part of substrate (PC) and products (mainly PG) formed were in the organic phase (diethyl ether).Octyl-Sepharose CL-4B having a hydrophobic octyl group was chosen for the PLD immobilization. Both immobilization yield and activity yield of immobilized enzyme were 100%. The effects of solvents, temperature and glycerol concentrations on the immobilized PLD were examined. Repeated batch conversion of PC (15 g/l) to PG was examined with the immobilized PLD in 30% glycerol. In all five batch cycles examined, 100% selectivity was obtained and there was no significant decrease in the fractional conversion of PC to PG (98%–99%) in the first three batch cycles. In the cases of a packed-bed reactor (PBR) and a continuous stirred-tank reactor (CSTR) used for continuous synthesis of PG with the immobilized PLD, the operational stabilities of the immobilized enzyme were almost the same (half life=14 h at 30°C) when purified PC was used, while in the case of partially purified PC in CSTR the half life increased more than five times.Abbreviations used PC phosphatidylcholine - PG phosphatidylglycerol - PA phosphatidic acid - PLD phospholipase D - PBR packed bed reactor - CSTR continuous stirred tank reactor Studies on enzymatic conversion of phospholipids (III)