An ultrafiltration membrane bioreactor for the lipolysis of olive oil in reversed micellar media

The enzymatic hydrolysis of olive oil using Chromobacterium viscosum lipase B encapsulated in reversed micelles of dioctyl sodium sulfosuccinate (AOT) in isooctane was investigated in an ultrafiltration ceramic membrane reactor of tubular type, operating in a batch mode. Water concentration was found to be a critical parameter in the enzyme kinetics and hydrolysis yield of the reaction. The size of micelles, recirculation rate, and substrate concentration were found to be the major factors affecting the separation process. A correlation that enables the prediction of final conversion degrees in this bioreactor from the initial reaction conditions was established. (c) 1993 Wiley & Sons, Inc.     

Enzymatic hydrolysis of cellulose in a membrane bioreactor: assessment of operating conditions

The optimization of operating conditions for cellulose hydrolysis was systemically undertaken using an ultra-scaled down membrane bioreactor based on the parameter scanning ultrafiltration apparatus. The bioconversion of cellulose saccharification was carried out with freely suspended cellulase from Aspergillus niger as the biocatalyst. The polyethersulfone ultrafiltration membranes with a molecular weight cutoff of 10 kDa were used to construct the enzymatic membrane bioreactor, with the membrane showing a complete retaining of cellulase and cellobiase. The influence of solution pH, temperature, salt (NaCl) concentration, presence of cellobiase, cellulose-to-enzyme ratio and stirring speed on reducing sugar production was examined. The results showed that the addition of an appropriate amount of NaCl or cellobiase had a positive effect on reducing sugar formation. Under the identified optimal conditions, cellulose hydrolysis in the enzymatic membrane bioreactor was tested for a long period of time up to 75 h, and both enzymes and operation conditions demonstrated good stability. Also, the activation energy (E _a) of the enzymatic hydrolysis, with a value of 34.11 ± 1.03 kJ mol⁻¹, was estimated in this study. The operational and physicochemical conditions identified can help guide the design and operation of enzymatic membrane bioreactors at the industrial scale for cellulose hydrolysis.     

Enzymatic hydrolysis of sodium-hydroxide-pretreated sallow in an ultrafiltration membrane reactor

An ultrafiltration membrane reactor was used to investigate the recovery of biocatalysts during enzymatic hydrolysis of pretreated sallow. Product inhibition could be eliminated by continuous removal of products through the ultrafiltration membrane, thus retaining the macromolecular substrate and enzymes. In this way, the degree of conversion was improved from 40% in a batch hydrolysis to 95% (within 20 h), and the initial hydrolysis rate was increased up to seven times. The recovery studies were focused on mechanical deactivation and irreversible adsorption on to the nonconvertible fraction of the substrate. Cellulase deactivation during mechanical agitation was not significant, and the loss of activity was attributed mainly to strong adsorption of the enzymes onto undigested material. This process was studied in semicontinuous hydrolyses, where fresh substrate was added intermittently. The amount of reducing sugars produced in this experiment was 25.7 g/g enzyme, compared to 4.7 g/g enzyme in a batch hydrolysis.     

Application of ultrafiltration for enzyme retention during continuous enzymatic reaction

In most enzymatic reactions, batch or continuous, separation of the enzyme for reuse is difficult if not impossible. A process will be presented in which an Ultrafiltration membrane serves to separate the reaction products from the enzyme and the substrate. In this manner the enzyme may be retained and re-used. Furthermore, under these conditions, the enzyme need only be present in catalytic amounts regardless of the amount of product produced. Under proper operating conditions and proper ultrafiltration membrane selection, a pure solution of α-amylase from Bacillus subtilis may be retained with no loss in enzyme activity over a test period of 30 hr after steadystate has been achieved. In the presence of substrate, the membrane support and ultrafiltration cell serve as the reaction vessel for the hydrolysis of starch. The substrate is continuously pumped into the cell under constant ultrafiltration pressure. The di-, oligo-, and polysaccharides formed from the enzyme reaction then either pass through the membrane as products or are retained. The molecular weight distribution of the products is dependent on the nominal molecular weight cut-off of the membrane, absolute ultrafiltration pressure, enzyme-to-substrate ratio, temperature, and residence time of the substrate in the reactor. In addition to the partial hydrolysis of starch by α-amylase, some preliminary findings on the complete hydrolysis of starch by glucoamylase will also be presented. In these latter studies, the substrate may be completely hydrolyzed to glucose units.     

An ultrafiltration membrane reactor for obtaining experimental reaction rates at defined concentrations of inhibiting sugars during enzymatic saccharification of alkali-pretreated sallow: Formulation of a simple empirical rate equation

The kinetics of the enzymatic hydrolysis of sodium hydroxide-pretreated sallow were studied in an ultrafiltration membrane reactor in the presence of different concentrations of glucose. In the UF membrane reactor low-molecular-weight products were continuously removed at a low dilution rate and replaced by a buffer solution that contained different concentrations of glucose, which made it possible to keep the inhibiting product concentration constant throughout an experiment. The reaction rate was related to the degree of substrate conversion and a mathematical relationship was formulated that describes the influence of the product concentration on the rate coefficient.     

Enzymatic cellulose hydrolysis in an attrition bioreactor combined with an aqueous two-phase system

Cellulose was hydrolyzed in the attrition bioreactor (ABR) with enzyme recycling by employing an aqueous two-phase system (composed of dextran and polyethylene glycol) and an ultrafiltration unit. The ABR combines wet ball milling and enzymatic hydrolysis in one process step. The cellulase enzymes were more stable in the two-phase system than in the normal buffer solution. With the initial substrate concentration (Solka Floe BW200) of 40 g/L and intermittent addition of cellulose, sugar was semicontinuously produced at dilution rates of 0.06 h(-1) and productivities of 2.1 g/L h, which is approximately a 10-fold increase of the previously reported values performed in a regular stirred reactor with an aqueous two-phase system. The conversion of the substrate was 86%.     

Kinetic analysis of bioconversion of cellulose in attrition bioreactor

The attrition bioreactor (ABR) combines wet ball milling and enzymatic hydrolysis in one process step. It was found that the ABR did not accelerate enzyme deacti-vation. Interfacial forces, not shear forces, caused the most deactivation. Elimination of the air-liquid interface by covering the reactor substantially increased enzyme stability. A simple exponential kinetic model was tested to predict the cellulose conversion in an ABR. Kinetic parameters were estimated from batch runs performed at various enzyme and substrate concentrations.     

Studies on enzymatic continuous production of cyclodextrins in an ultrafiltration membrane bioreactor

Studies on simultaneous hydrolysis of starch and synthesis of cyclodextrins by Thermo-aerobacter cyclodextrin glucosyltransferase were conducted in an ultrafiltration membrane bioreactor, allowing enzyme recovery and reduction of product inhibition. The influence of various reaction parameters like starch concentration, enzyme dosage and residence time on cyclodextrin composition was tested. A comparison of batch and continuous cyclodextrin production indicates that employing an ultrafiltration membrane bioreactor increases process efficiency.     

Enzymatic hydrolysis of sodium caseinate in a continuous ultrafiltration reactor using an inorganic membrane

Continuous hydrolysis of sodium caseinate by alcalase was investigated in a recycle bioreactor coupled to an inorganic M5 membrane module. The effects of various substrate concentrations and the role of an ultrafiltration membrane on conversion rate were reported. Although a high level of conversion was obtained in the retentate side at a steady state, only part of the products formed was transmitted through the inorganic membrane. Degree of hydrolysis and product concentration in the reactor seem to be the main factors limiting product output during the continuous hydrolysis.     

Process modeling of the lipase-catalyzed dynamic kinetic resolution of (<Emphasis Type="Italic">R</Emphasis>, <Emphasis Type="Italic">S</Emphasis>)-suprofen 2,2,2-trifluoroethyl thioester in a hollow-fiber membrane

A Candida rugosa lipase immobilized on polypropylene powder was employed as the biocatalyst for the enantioselective hydrolysis of (R, S)-suprofen 2,2,2-trifluorothioester in cyclohexane, in which trioctylamine was added as the catalyst to perform in situ racemization of the remaining (R)-thioester. A hollow-fiber membrane was also integrated with the dynamic kinetic resolution process in order to continuously extract the desired (S)-suprofen into an aqueous solution containing NaOH. A kinetic model for the whole process (operating in batch and feed-batch modes) was developed, in which enzymatic hydrolysis and deactivation, lipase activation, racemization and non-enantioselective hydrolysis of the substrate by trioctylamine, and reactive extraction of (R)- and (S)-suprofen into the aqueous phase in the membrane were considered. Theoretical predictions from the model for the time-course variations of substrate and product concentrations in each phase were compared with experimental data.     

Hydrolysis and large scale ultrafiltration study of alfalfa protein concentrate enzymatic hydrolysate

Batch enzymatic hydrolysis of insoluble Alfalfa Protein Concentrate by Delvolase was carried out at laboratory and at pilot-plant scale coupled to an ultrafiltration reactor with a mineral tubular membrane. Parametric studies were carried out on the batch system to determine the biochemical and hydrodynamical optimum conditions. The hydrolysis conditions selected were 40 degrees C, pH 9.5, initial substrate level 3 g protein/100 g and the enzyme substrate ratio 152 U/g protein. After 5 h of hydrolysis, 96% of the total amount of initial nitrogen was solubilized. The ultrafiltration conditions selected were a 10 000 Nominal Molecular Weight Cut-Off, a transmembrane pressure of 1.5 bar, a flux velocity of 0.8 m/s. Fifty percent of the initial nitrogen appeared in the permeate.     

Enzyme hydrolysis of plasma proteins in a CSTR ultrafiltration reactor: Performances and modeling

By investigating the effects of four operating variables-volume (V), Ultrafiltration flux (J), enzyme concentration (E), and substrate concentration (S)-on capacity (K) and conversion rate (epsilon) of a hollow fiber CSTR, the performances of the CSTR and the kinetic constants of the reaction were determined. A model which takes into account the course of fractional conversion (X) according to the modified space-time parameter, tau (integrated form of V, J, S, and E), was devised by employing the relationship to integrate the equation for the reaction rate of the CSTR and the expression of the modified space time. Correlation of this model and the experimentally obtained results demonstrates that the characteristics for an ultrafiltration membrane reactor for enzymatic hydrolysis by alcalase of plasma proteins are close to those of an ideal CSTR. Optimal scaling up, however, remains dependent on the compromise which may be obtained between capacity and the conversion rate.     

Performance of a membrane reactor for cellobiose hydrolysis

A continuous enzymatic hollow fiber reactor (HFR), obtained by immobilizing cellobiose active cells into the shell side of hollow-fiber modules, was studied. The HFR yield was monitored by glucose analysis resulting from hydrolysis of cellobiose. The residence time of substrate in the bioreactor to obtain convenient hydrolysis yields was calculated from tests carried out by varying the reactor dilution rate in the range 0.001-0.004 L/min. The glucose yield was measured for 300 h (continuous substrate flux). The yield decreased from 40 to 15%. This decrease was due to the loss of specific activity in the operating conditions and to the pressure drop increase from 0.2 to 1.7 atm. The pressure drop increase is in turn dependent on the cell loading (0.2-2.1 g dry cell) and the substrate flux.     

Modeling intrinsic kinetics of enzymatic cellulose hydrolysis

A multistep approach was taken to investigate the intrinsic kinetics of the cellulase enzyme complex as observed with hydrolysis of noncrystalline cellulose (NCC). In the first stage, published initial rate mechanistic models were built and critically evaluated for their performance in predicting time-course kinetics, using the data obtained from enzymatic hydrolysis experiments performed on two substrates: NCC and alpha-cellulose. In the second stage, assessment of the effect of reaction intermediates and products on intrinsic kinetics of enzymatic hydrolysis was performed using NCC hydrolysis experiments, isolating external factors such as mass transfer effects, physical properties of substrate, etc. In the final stage, a comprehensive intrinsic kinetics mechanism was proposed. From batch experiments using NCC, the time-course data on cellulose, cello-oligosaccharides (COS), cellobiose, and glucose were taken and used to estimate the parameters in the kinetic model. The model predictions of NCC, COS, cellobiose, and glucose profiles show a good agreement with experimental data generated from hydrolysis of different initial compositions of substrate (NCC supplemented with COS, cellobiose, and glucose). Finally, sensitivity analysis was performed on each model parameter; this analysis provides some insights into the yield of glucose in the enzymatic hydrolysis. The proposed intrinsic kinetic model parametrized for dilute cellulose systems forms a basis for modeling the complex enzymatic kinetics of cellulose hydrolysis in the presence of limiting factors offered by substrate and enzyme characteristics.     

Kinetic and enzymatic adsorption model in a recirculation hollow-fibre bioreactor

A general procedure has been developed to model the behaviour of enzymatic reactions in a membrane bioreactor. This procedure unifies the kinetics of the reaction and the adsorption of the enzyme or enzymatic complexes on the membrane, enabling the selection of the most appropriate kinetic model. The general procedure proposed has been particularized and applied to experimental results obtained with two enzymatic reactions carried out in a hollow-fibre reactor, enzymatic hydrolysis of lactose by β-galactosidase and glucose–fructose isomerization by glucose isomerase. The application of the general model has allowed us to determine the mechanism of the reaction for both kinetic reactions, assuming the adsorption of the enzymatic complex EGa for lactose hydrolysis and the adsorption of the free enzyme onto the membrane for glucose–fructose isomerization.     

Kinetic studies of fusarium solani pisi cutinase used in a gas/solid system: transesterification and hydrolysis reactions

Fusarium solani cutinase supported onto Chromosorb P was used to catalyze transesterification (alcoholysis) and hydrolysis on short volatile alcohols and esters in a continuous gas/solid bioreactor. In this system, a solid phase composed of a packed enzymatic preparation was continuously percolated with carrier gas which fed substrates and removed reaction products simultaneously. A kinetic study was performed under differential operating conditions in order to get initial reaction rates. The effect of the hydration state of the biocatalyst on the kinetics was studied for 3 conditions of hydration (a(w) = 0.2, a(w) = 0.4 and a(w) = 0.6), the alcoholysis of propionic acid methyl ester with n-propanol, and for 5 hydration levels (from a(w) = 0.2 to a(w) = 0.6) for the hydrolysis of propionic acid methyl, ethyl or propyl esters. F. solani cutinase was found to have an unusual kinetic behavior. A sigmoid relationship between the rate of transesterification and the activity of methyl propionate was observed, suggesting some form of cooperative activation of the enzyme by one of its substrate. For the hydrolysis of short volatile propionic acid alkyl esters, threshold effects on the reaction rate, highly depending on the water activity and the substrate polarity, are reported. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 1-8, 1997.     

膜生物反应器连续发酵法制取甲醇及其发酵动力学初探

采用膜生物反应器系统连续发酵制取甲醇能够及时分离产物,有效抑制甲醇对细胞的毒副作用,因此延长稳定期产甲醇的时间以提高产量。本文对比间歇发酵,研究了不同稀释率0．05h^-1～0．13h^-1下连续发酵的甲醇生成和甲烷氧化菌株Methylomonas．QJ16生长状况,并且初步探讨了该菌株的生长、甲醇形成的动力学特性。结果表明,稀释率为0．1h^-1时,菌体积累和甲醇的体积产率均较高,最长连续发酵持续时间为300h左右;描述连续发酵过程的动力学模型,菌体生长和产物合成的曲线拟合优度分别为0．991、0．994,基本反映了该甲烷氧化菌株连续发酵过程的动力学特征。     

Economic improvement of continuous pharmaceutical production via the optimal control of a multifeed bioreactor

Projections on the profitability of the pharmaceutical industry predict a large amount of growth in the coming years. Stagnation over the last 20 years in product development has led to the search for new processing methods to improve profitability by reducing operating costs or improving process productivity. This work proposes a novel multifeed bioreactor system composed of independently controlled feeds for substrate(s) and media used that allows for the free manipulation of the bioreactor supply rate and substrate concentrations to maximize bioreactor productivity and substrate utilization while reducing operating costs. The optimal operation of the multiple feeds is determined a priori as the solution of a dynamic optimization problem using the kinetic models describing the time‐variant bioreactor concentrations as constraints. This new bioreactor paradigm is exemplified through the intracellular production of beta‐carotene using a three feed bioreactor consisting of separate glucose, ethanol and media feeds. The performance of a traditional bioreator with a single substrate feed is compared to that of a bioreactor with multiple feeds using glucose and/or ethanol as substrate options. Results show up to a 30% reduction in the productivity with the addition of multiple feeds, though all three systems show an improvement in productivity when compared to batch production. Additionally, the breakeven selling price of beta‐carotene is shown to decrease by at least 30% for the multifeed bioreactor when compared to the single feed counterpart, demonstrating the ability of the multifeed reactor to reduce operating costs in bioreactor systems. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:902–912, 2017     

Incomplete mixing in large bioreactors — a study of its role in the fermentative production of streptokinase

The production of streptokinase in a batch fermentation has been analysed for the role of incomplete macromixing of the broth. The analysis is based on a kinetic model exhibiting inhibition by the substrate and a primary metabolite (lactic acid), and a mixing model comprising two continuous flow reactors (CFRs) with closed-loop recycle. The inoculum is introduced into one region (one CFR) and the mixing process determines its distribution, growth and reactivity. By varying the dilution rates of the CFRs, any degree of macromixing can be simulated. For dilution rates larger than 1.0 h^?1 almost complete macromixing is achieved, for which an analogy has been drawn with micromixing. Increasing the volume of the inoculated region relative to the noninoculated region improves the maximum attainable activity of streptokinase and shortens the time for this. In such a situation an imperfectly mixed bioreactor is superior to a perfectly mixed one, implying that good productivity requires a large inoculated region and incomplete macromixing. These inferences are supported by earlier studies of fluid mixing and relaxation times in bioreactors.     

Lipase-mediated hydrolysis of corn DDGS oil: Kinetics of linoleic acid production

In this study, we investigated the kinetics of linoleic acid production via lipase-mediated hydrolysis of corn DDGS oil in a batch reactor with continuous mechanical agitation and developed a kinetic model that incorporated the product inhibition to study the complete hydrolysis. The model agreed very well with observed data; though situations with low enzyme dosage or low stirring rates were modeled successfully without product inhibition, actual product concentration in such situations was too low to exert any inhibitory effects. Increasing the enzyme concentration increased hydrolysis, and beyond certain enzyme concentrations, effects tended to fade away because of excessive enzyme desorption from the interface. An enzyme dosage within the range of 40–60 KLU/L of oil dispersion could be successfully applied for a substrate concentration of 25–50 g/L of DDGS oil. Increasing the agitation rates improved enzymatic hydrolysis, but a higher stirring rate of 1000 rpm moderately improved production of linoleic acid compared with a stirring rate of 750 rpm. Within the range of substrate concentrations studied, enzymatic inhibition was moderate but still evident. The high degree of hydrolysis (i.e., ∼96% of theoretical linoleic acid yield) from DDGS oil suggests this method has potential for commercial production of linoleic acid.