Continuous fermentation of soy sauce by immobilized cells of Zygosaccharomyces rouxii in an airlift reactor

Summary The optimum conditions for continuous alcohol fermentation of soy sauce with immobilized Zygosaccharomyces rouxii cells were investigated using an airlift reactor. The optimum pH and temperature of the fermentation were 4.5–5.5 and 25°–27.5° C, respectively. Ethanol content in the fermented liquid was increased with increasing height to diameter ratio of the reactor and the ratio of air to nitrogen in the supplied gas (total supplied gas: 0.08 vvm). A notable decrease in ethanol content was observed when only nitrogen gas was supplied. The products fermented by supplying air (0.02 vvm) had a higher conent of aroma components than that by supplying only nitrogen gas, and the aroma of the former products was similar to that of conventional soy sauce. This alcohol fermentation using an airlift reactor was continued for about 50 days without problems even if conditions such as residence time and aeration were altered.     

Continuous production of 4-ethylguaiacol by immobilized cells of salt-tolerant Candida versatilis in an airlift reactor

The effects of pH, temperature, aeration, and residence time on the continuous production of 4-ethyl-guaiacol (4-EG), which is one of the characteristic aroma components in soy sauce, by immobilized cells of the salt-tolerant yeast Candida versatilis were investigated using an airlift reactor. The optimum pH and temperature were about 4.0 and 30–33°C, respectively. The amount of 4-EG in the liquid was constant even during alterations of nitrogen/air ratio in the supplied gas. A large amount of 4-EG (over 20 ppm) was produced at a residence time from 5 to 28 h and 1–3 ppm of 4-EG, which was the optimum concentration in conventional soy souce, was produced at a shorter residence time of 0.5 h. The 4-EG production by immobilized C. versatilis cells using the airlift reactor was stable for 40 d. It was found that the immobilized cell method was effective for the production of 4-EG by C. versatilis cells.     

Degradation of quinoline by immobilized Comamonas acidovorans in a three-phase airlift reactor

Quinolie degradation by Comamonas acidovorans was studied in a continuously operated three-phase airlift reactor. Porous glass beads were applied as support matrix for cell imobilization by colonization. Under steady-state conditions (S approximately 0), cell attachment was poor at low dilution rates but imporved considerably with increasing dilution rate. Conversion of quinoline was investigated below and above the washout for suspended culture (D(crit) = mu(max) = 0.42 h(-1)). With immobilized cells the reactor could be operated at D > mu(max), and complete conversion of quinoline was achieved as long as the specific quinoline feed rate D*S(0)/X did not exceed the maximum specific degradation rate (r(S, max)). The biofilm thickness was about 100 mum, and its efficiency was about 54% compared to suspended organisms. If quinoline overloads were supplied to the reactor, quinoline, as overloads were supplied to the reactor, quinoline, as well as its pathway intermediates, appeared in the reactor and conversion was low. Hence, the immobilized microorganisms remained viable and active. They could survive quinoline overloads. If the quinoline feed rate was reduced agains, complete conversion was reestablished. (c) 1995 John Wiley & Sons, Inc.     

Model for the production of L-tryptophan from L-serine and indole by immobilized cells in a three-phase liquid-impelled loop reactor

Production of L-tryptophan from L-serine and indole catalyzed by Escherichia coli, immobilized in k-carrageenan gel beads, is technically feasible in the liquidimpelled loop reactor (LLR), using an organic solvent, e.g. n-dodecane.With L-serine in large excess intrinsic reaction kinetics is approximately first order with respect to indole, with a reaction constant of 8.5×10^–5 m³ kg _dw ^–1 s^–1.The overall process kinetics is jointly controlled by intrinsic kinetics and by intraparticle mass transfer resistance, which can be quantified using an effectiveness factor.Mass transfer of indole from the organic to the aqueous phase and from the aqueous to the gel phase are relatively fast and thus have negligible influence in the overall process kinetics, under the operational conditions tested. However, they may become important if the process is intensified by increasing the cell concentration in the gel and/or the gel hold-up in the reactor.A simple model which includes indole mass balances over the aqueous and organic phases, mass transfer and reaction kinetics, with parameters experimentally determined in independent experiments, was successful in simulating L-tryptophan production in the LLR.List of Symbols a, b, c coefficients of the equilibrium curve for indole between organic and aqueous phases - A, B, C, D, E, F auxiliary variables used in liquid-liquid mass transfer studies - a _x specific interfacial area referred to the volume of the aqueous phase (m^–1) - A _x interfacial area (m²) - a _Y specific interfacial area referred to the volume of the organic phase (m^–1) - A _Y interfacial area (m²) - C _b substrate concentration in the bulk of the aqueous phase (kg m^–3) - C _e substrate concentration in exit stream (kg m^–3) - C _E biocatalyst concentration referred to the aqueous phase (kg m^–3) - C _{E s} biocatalyst concentration referred to the volume of gel (kg m^–3) - C _s substrate concentration at the gel surface (kgm^–3) - d, e, f coefficients of the equilibrium curve for indole between aqueous and organic phases - dp particle diameter (m) - K ₂ kinetic constant (s^–1) - K ₁ kinetic constant K₂/K_M (kg^–1 m³ s^–1) - K _M Michaälis-Menten constant (kgm^–3) - K _X mass transfer coefficient referred to the aqueous phase (ms^–1) - K _Xa_X volumetric mass transfer coefficient based on the volume of the aqueous phase (s^–1) - k _Y mass transfer coefficient referred to the organic phase (ms^–1) - K _Ya_Y volumetric mass transfer coefficient based on the volume of the organic phase (s^–1) - N _X mass flux of indole from organic to aqueous Phase (kg m^–2s^–1) - N _Y mass flux of indole from aqueous to organic phase (kg m^–2s^–1) - Q _e volumetric flow rate in exit stream (m³s^–1) - Q _f volumetric flow rate in feed stream (m³s^–1) - _obs observed reaction rate (kg s^–1 m^–3) - intrinsic reaction rate (kg s^–1 m^–3) - Re Reynolds number - Sc Schmidt number - Sh Sherwood number - t time (s) - u superficial velocity (m s^–1) - V _max maximum reaction rate (kg s^–1m^–3) - V _S volume of the support (m³) - V _X volume of aqueous phase (m³) - V _Y volume of the organic phase (m³) - X indole concentration in the aqueous phase (kgm^–3) - Y indole concentration in the organic phase (kg m^–3 Greek Letters overall effectiveness factor - _e external effectiveness factor - _i internal effectiveness factor - Thiele module A fellowship awarded to one of us (D.M.R.)by INICT is gratefuly acknowledged.     

Investigations of cephalosporin C production in an airlift tower loop reactor

Summary Cephalosporin C was produced by Cephalosporium acremonium in a 60 l airlift loop reactor on complex medium (with 30 kg/m³ peanut flour) in fed-batch operation. A final product concentration of 5 kg/m³ and a maximum productivity of 45 g/m³ h were attained. On-line analysis was used to determine ammonia, methionine, phosphate, reducing sugar and cephalosporin C by an autoanalyser, glucose by a flow injection analyser and cephalosporin C, penicillin N, deacetoxycephalosporin C, deacetylce-phalosporin C and methionine by HPLC. The volumetric productivity of the stirred tank reactor was higher than that of the airlift reactor because of differences in cell concentration. Specific productivities in relative to cell mass were similar in the two reactors. The substrate yield coefficient in the airlift reactor was twice that in the stirred tank reactor.Nomenclature E _o2 efficiency of oxygen transfer with regard to the specific power input - K _La volumetric mass transfer coefficient - OTR oxygen transfer rate - P power input - PR volumetric productivity of CPC - q _a volumetric aeration rate/broth volume (vvm) - SPR specific productivity with regard to RNA - V _L broth volume in reactor - z relative height of the aerated reactor     

An airlift biofilm reactor for the biodegradation of phenol by Pseudomonas stutzeri OX1

Phenol bioconversion by Pseudomonas stutzeri OX1 using either free or immobilized cells was investigated with the aim of searching for optimal operating conditions of a continuous bioconversion process. The study was developed by analyzing: (a) free-cell growth and products of phenol bioconversion by batch cultures of P. stutzeri; (b) growth of P. stutzeri cells immobilized on carrier particles; (c) bioconversion of phenol-bearing liquid streams and the establishment and growth of an active bacterial biofilm during continuous operation of an internal-loop airlift bioreactor. We have confirmed that free Pseudomonas cultures are able to transform phenol through the classical meta pathway for the degradation of aromatic molecules. Data indicate that bacterial growth is substrate-inhibited, with a limiting phenol concentration of about 600 mg/L. Immobilization tests revealed that a stable bacterial biofilm can be formed on various types of solid carriers (silica sand, tuff, and activated carbon), but not on alumina. Entrapment in alginate beads also proved to be effective for P. stutzeri immobilization. Continuous bioconversion of phenol-bearing liquid streams was successfully obtained in a biofilm reactor operated in the internal-circulation airlift mode. Phenol conversion exceeded 95%. Biofilm formation and growth during continuous operation of the airlift bioreactor were quantitatively and qualitatively assessed.     

An immobilized enzyme reactor for the detoxification of bilirubin

An immobilized enzyme reactor has been developed for the degradation of bilirubin as a potential treatment for neonatal jaundice. It utilizes the enzyme bilirubin oxidase from Myrothecium verrucaria, which in the presence of molecular oxygen converts bilirubin to biliverdin and other products that are much less toxic than bilirubin. Bilirubin oxidase was covalently attached to agarose beads using cyano transfer activation. Forty percent of the specific activity of bilirubin oxidase was retained after immmobilization, and preparations with 20 units of enzymatic activity per gram of drained wet weight of gel were obtained. The stability of bilirubin oxidase at pH 7.4 and 37 degrees C was improved fivefold by immobilization. A 15-mL column containing immobilized bilirubin oxidase, through which a 37 degrees C solution of 332muM bilirubin and 450muM human serum albumin in 0.05M phosphate buffer (pH 7.4) was passed at 1 mL/min, converted more than 60 percent of the bilirubin per pass. The substrate specificity of the enzyme and the small volume of the reactor are important characteristics for this clinical application where it is desirable to remove only one compound from the blood and to minimize the volume of blood in the extracorporeal circuit. This reactor, by detoxifying the jaundiced infant's blood of bilirubin, would eliminate the risks associated with the use of donor blood as is done currently in treating severe neonatal jaundice.     

Hydrogen production by immobilized cells in the nozzle loop bioreactor

Summary The continuous production of hydrogen in a Nozzle Loop Bioreactor was investigated using immobilized Rhodospirillum rubum KS-301 with glucose as the growth-limiting substrate. The maximum hydrogen production rate in the experimental range was 91mL/h at dilution rate 0.4h^-1, initial glucose concentration 5.4g/L, and circulation rate 70h^-1 .     

Optimal preparation of immobilized liposome-bound cellulase for hydrolysis of insoluble cellulose in an external loop airlift bioreactor

Immobilized liposome-bound cellulase (ILC) was optimally prepared for the ILC-catalyzed hydrolysis of insoluble cellulose in an external loop airlift bioreactor. The liposomes with mean diameters of 200, 100, and 50 nm were used to prepare three kinds of ILCs, i.e., ILC(200), ILC(100) and ILC(50), respectively. The activity and stability of ILC(100) were examined with soluble cellulose (CMC) in addition to the insoluble substrate of cellulose powder (CC31) in a shaking flask as well as the airlift bioreactors. The experiments were carried out with 45 degrees C and pH 4.8 being found to be optimal for the activity. The activity of ILC(100) was stable in either airlift or shaking flask bioreactor during the five times repeated hydrolyses of CC31 corresponding to a total reaction time of 240 h. This confirmed that the cellulase molecules were covalently bonded to the liposomes covalently bound to the chitosan gel beads. Nevertheless, the activity of ILC(100) with CMC steadily decreased throughout the repeated reactions, suggesting an adverse effect of CMC on the ILC(100) activity. Among the three ILCs, ILC(50) was found to be the most stable and productive biocatalyst during the repeated hydrolyses of insoluble CC31 in the airlift bioreactor. More than 70% of the initial activity of ILC(50) was retained even after the six times repeated reactions for 288 h. Conversely, the ILC(200) was found to be the most unstable catalyst. Such a difference in stability among these ILCs was suggested to be caused by the difference in physical stability of their liposome membranes to the liquid shear stress due to the rising bubbles and circulating liquid as well as that in the amount of the cellulase molecules unstably incorporated in the membranes. ILC(50) was thus shown to have the most potential for an efficient hydrolysis of insoluble cellulose in a practical airlift bioreactor.     

An immobilized alpha-galactosidase continuous flow reactor

An α-galactosidase which will hydrolyze the oligosaccharides melibiose, raffinose, and stachyose has been immobilized on nylon microfibrils suitable for use in large flow-through reactors. This catalyst system is stable for many months, both under use and storage conditions. The immobilized enzyme behaves similarly to the enzyme in solution, characteristically exhibiting both product and substrate inhibition. The catalyst is prepared in situ and a large, 8-liter reactor has been made. The catalyst has been used to reduce the raffinose concentration in beet sugar molasses.     

Selective recycle of viable animal cells by coupling of airlift reactor and cell settler

A new system for the perfusion culture of animal cells in suspension is described. It consists of an airlift loop reactor and a settling tank for cell retention. Insufficient nutrient and oxygen supply of the cells in the settling tank was prevented by cooling the cell suspension before entering the settler. As a result, the catabolic activity of the cells in the settler was reversibly reduced. Furthermore, the density gradient induced by cooling caused a liquid motion through the settler. Thus, it was not necessary to pump medium containing shear, sensitive cells. With this simple system, it was possible to prduce 2 to 5 g of antibodies in a 5.4-L reactor in continuous runs of 400 to 600 h. The productivity was increased by a factor of 17 and the cell density was 4 times higher in comparison with the corresponding batch system. The cell retention system was found to have the property of separating viable and nonviable cells. With the increasing perfusion rate, dead cells and debris were preferably washed out. For perfusion rates up to 1.3 d(-1), the retention efficiency of the settler was nearly 100% for viable cells; hence, this system may show advantages at the industrial scale.     

Steroid transformation by activated living immobilized Arthrobacter simplex cells

A preparation of living Arthrobacter simplex cells immobilized in polyacrylamide gel, which showed steroid-Δ¹-dehydrogenase activity, was studied. The entrapped microorganisms catalyzed the transformation of cortisol to prednisolone and this reaction was followed spectrophotometrically or with the aid of thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC). About 40% of the original activity found with free bacteria was retained after immobilization. The steroid dehydrogenase activity of polyacrylamide-entrapped A. simplex could be raised to a minor extent in alcoholic solvents or by addition of a cofactor such as menadione. On incubation in various nutrient media, on the other hand, the activity could be increased considerablyl, usually 7–10 times. Possible causes for the observed increase in activity have been investigated, and microbial growth of the original entrapped microorganisms appears to be the major reason. Frozen activated preparations of immobilized A. simplex showed only a small loss of activity on storage for at least four months. A semicontinuous batch wise operation with immobilized A. simplex in different nutrient media was carried out. At the end of the experiment the steroid transformation capacity was 0.5 g steroid per day per g gel (wet weight).     

Steroid transformation by living cells immobilized in calcium alginate

Summary Whole cells of Arthrobacter simplex were immobilized in a living state in calcium alginate gel. The bacteria showed steroid-¹-dehydrogenase activity and the production of prednisolone from cortisol was investigated. The ¹-dehydrogenase activity of the immobilized cells could be increased about ten-fold by incubation in nutrient media (e.g., containing 0.5% peptone abd 0.2% glucose). The reason for this activation was examined and it was found that the immobilized cells were capable of multiplying when supplied with nutrients. Furthermore, provided that an inducer, cortisol, was present, the steroid-¹-dehydrogenase activity increased in proportion to the increase in the number of cells and it was thus concluded that microbial growth was the cause of activation.Experiments on repeated, batch-wise pseudocrystallofermentation with immobilized A. simplex cells also showed that immobilized cells could be advantageously used for pseudocrystallofermentation of steroids.     

Ethanol fermentation by immobilized cells in a trickle bed reactor

A modified discontinuous packed bed reactor with CO₂ ventilation ports, resembling a trickle bed reactor was employed to overcome gas holdup and bed compaction problems which are commonly encountered in cell immobilized packed bed reactors for ethanol fermentation. The reactor consisting of yeast cells entrapped in alginate matrix was operated by varying the substrate concentration, bed volume and inlet flow rates. The number of recirculation cycles (passes) and total stages were dependent upon the liquid flow rate, though the total contact time for complete conversion remains the same for a particular initial substrate level. The total contact time was 1.5, 3 and 4.5 h for initial substrate concentrations of 0.555, 0.933 and 1.3 kmol/m³ respectively. The number of cycles and in turn stages increased with the increase in initial sugar level. A graphical method for predicting the number of stages required for complete conversion was proposed based on material balance equation and evaluated for the operating variables of the present study. The reactor was operated continuously for 30 days producing 1.05– 1.15 kmol/m³.     

Continuous neomycin production by immobilized cells of Streptomyces marinensis NUV-5 in an airlift bioreactor

Neomycin production by free and calcium alginate immobilized cells was investigated in an airlift reactor. The average volumetric productivity with continuous fermentation (72.97 mg/l/h) was greater than with free cells (45.05 mg/l/h). The total neomycin produced with continuous fermentation was 62% greater than with that of free cells. Immobilized Streptomyces particles showed a half-life of 42 days during continuous fermentation under airlift conditions.     

Microbial transformation of validamycin A to valienamine by immobilized cells

Immobilized Pseudomonas sp. HZ519 cells have been used for transformation of validamycin A to valienamine and the degradation pathway of validamycin A by Pseudomonas sp. HZ519 has also been studied. Substrate inhibition in immobilized cell system was avoided. An average of 8.6 g L^?1 valienamine concentration was obtained when concentration of validamycin A was increased up to 120 g L^?1. Through a treatment of the immobilized cells with 0.3 mol L^?1 substrate, the activity of the immobilized cells was increased distinctly. Compared with free cells, the productivity of valienamine by CA-immobilized cells was improved about three times. The reusability of the immobilized cells was evaluated with repeated–batch degradation experiments. The Tiele modulus was obtained from the experimental effectiveness factor. The result showed that the degradation process in the immobilized system was governed by intraparticle diffusion and chemical reaction.     

Microbial transformation of validamycin A to valienamine by immobilized cells

Immobilized Pseudomonas sp. HZ519 cells have been used for transformation of validamycin A to valienamine and the degradation pathway of validamycin A by Pseudomonas sp. HZ519 has also been studied. Substrate inhibition in immobilized cell system was avoided. An average of 8.6 g L^-1 valienamine concentration was obtained when concentration of validamycin A was increased up to 120 g L^-1. Through a treatment of the immobilized cells with 0.3 mol L^-1 substrate, the activity of the immobilized cells was increased distinctly. Compared with free cells, the productivity of valienamine by CA-immobilized cells was improved about three times. The reusability of the immobilized cells was evaluated with repeated-batch degradation experiments. The Tiele modulus was obtained from the experimental effectiveness factor. The result showed that the degradation process in the immobilized system was governed by intraparticle diffusion and chemical reaction.