Process control of an ethanol fermentation with an enzyme thermistor as a sucrose sensor

Summary An enzyme thermistor was used to monitor and control the sucrose concentration in a conversion of sucrose to ethanol with immobilized yeast. A continuous stirred tank reactor containing calcium alginate to entrap Saccharomyces cerevisiae was used. The enzyme thermistor was continuously measuring the sucrose concentration in the fermenter with an on-line arrangement giving stable and reproducible heat signals. The control of the sucrose concentration level was performed with an analogue PI-controller.     

Control of an ethanol fermentation carried out with alginate entrapped Saccharomyces cerevisiae

Process control of different reactor models for continuous production of ethanol from sucrose with immobilized yeast has been studied. An enzyme thermistor with immobilized invertase recorded the concentration of sucrose continuously. Ethanol was recorded by a membrane gas sensor with a SnO(2) semiconductor used as detector. A process computer controlled the substrate feed to keep substrate as well as ethanol concentration at preset values by using algorithms of varying complexity. It was thereby demonstrated that PID regulators as well as more advanced algorithms (Otto-Smith regulator, state feedback from a Kalman filter, and cascade control) are useful alternatives to maintain a constant concentration in the fermentor effluents. The time required for the system to return to predetermined conditions after various kinds of disturbances has been especially studied. It was shown that the more advanced regulator used the shorter time.     

A forced flow membrane enzyme reactor for sucrose inversion using molasses

We have developed a bioreactor which uses enzyme immobilized within a ceramic membrane support (1 mm thickness). Substrate is forced through the membrane by cross-flow filtration with the reaction taking place during the process of crossing the membrane. The bioreactor is termed forced-flow membrane enzyme reactor, FFMER. Invertase, which uses sucrose to form glucose and fructose, was tested in this system. The immobilized invertase membrane converted 100% of the sucrose in a feed stream made up of a 50% molasses solution. Because molasses contains many substances besides sucrose, this method is applicable to processes using substrates present in impure feeds.     

Optimization of the multienzyme system for sucrose biosensor by response surface methodology

An immobilized multienzyme- and cathodic amperometry-based biosensor for sucrose was constructed for the analysis of food and fermentation samples. The multienzyme system, comprising invertase, mutarotase and glucose oxidase (GOD), was immobilized by using glutaraldehyde as cross-linking agent. Operating parameters of the biosensor for the estimation of sucrose in the range 1–10% were standardized. Response surface methodology (RSM) based on three-factor, three-variable design was used to evaluate the effect of important variables (concentration of enzymes, (varied in the range invertase (10–50 IU), mutarotase (5–105 IU) and GOD (1–9 IU)) on the response of biosensor. In the range of parameters studied, response time decreased with decrease in the invertase and with increase in mutarotase and GOD. Mutarotase concentration above 75 IU was found to result in an increased response time due to inhibition of mutarotase by its product -D-glucose. The optimal conditions achieved for the analysis of sucrose were: invertase 10 IU, mutarotase 40 IU, and GOD 9 IU. With these conditions, the predicted and actual experimental response time values were 2.26 and 2.35 min respectively, showing good agreement.     

Preparation of some immobilized linked enzyme systems and their use in the automated determination of disaccharides

1. Glucose oxidase (EC 1.1.3.4), amyloglucosidase (EC 3.2.1.3), invertase (EC 3.2.1.26) and beta-galactosidase (EC 3.2.1.23) were covalently attached via glutaraldehyde to the inside surface of nylon tube. 2. The linked enzyme system, comprising invertase immobilized within a nylon tube acting in series with glucose oxidase immobilized in a similar way, was used for the automated determination of sucrose. 3. The linked enzyme system, comprising beta-galactosidase immobilized within a nylon tube acting in series with glucose oxidase immobilized in a similar way, was used for the automated determination of lactose. 4. The linked enzyme system, comprising amyloglucosidase immobilized within a nylon tube acting in series with glucose oxidase immobilized in a similar way, was used for the automated determination of maltose. 5. Mixtures of glucose oxidase and amyloglucosidase were immobilized within the same piece of nylon tube and used for the automated determination of maltose. 6. Mixtures of glucose oxidase and invertase were immobilized within the same piece of nylon tube and used for the automated determination of sucrose.     

Use of an enzyme thermistor in continuous measurements and enzyme reactor control.

The enzyme thermistor measures the heat produced by the action of an immobilized enzyme on a substrate present in the sample. Its application in analysis of discrete samples, e.g., in clinical chemistry, is well documented, but it has not been used so far for continuous measurements. We decribe here the application of the enzyme thermistor for continuous monitoring and control of enzyme reactors. An enzyme thermistor filled with coimmobilized glucose oxidase and catalase was used to measure the amount of glucose in the outflow from a column reactor containing immobilized lactase acting on a lactose solution pumped through the reactor. The lactose conversion was kept on a constant level, irrespective of the actual enzymatic activity in the reactor, by regulating the flow through the reactor. The experiments were carried out with aqueous solutions of lactose as well as with whey from cow's milk.     

Active protein and substrate flow effects in a tubular immobilized invertase reactor

Investigations of invertase (EC 3.2.1.26) immobilized inside modified nylon tubes showed that between 4% and 20% (w/w) of the protein exposed to binding sites on the tube was immobilized. An enhanced activity consistent with enzyme purification during immobilization was also evident, suggesting that, in scaled-up commercial applications, nylon tube invertase would be a more economical converter of sucrose than the free enzyme. The quantity and specific activity of the immobilized protein were not stochiometrical with the amount used in the coupling solution and, in the system studied, a concentration of 2 mg ml^?1 was optimal. K_m and V_max values confirmed higher rates of immobilized invertase catalysis when the rates of substrate flow through the reactor were higher. Higher rates of substrate flow imply a shortened residence time in the reactor and would lower the fractional conversion per pass of the substrate, reducing the efficiency of the reactor in flow-through situations. Thus, these higher catalysis rates, attributable at the higher flow rates to a reduction of the diffusion barrier between enzyme and substrate, would not translate into improved economy in the commercial flow-through processes at which the reactor is aimed.     

Immobilization ofβ-fructofuranosidase fromAureobasidium sp. ATCC 20524 on porous silica

Summary -Fructofuranosidase P-1 fromAureobasidium sp. ATCC 20524, which produces a fructo-oligosaccharide (1-kestose) from sucrose, was immobilized covalently onto alkylamine porous silica with glutaraldehyde at high efficiency (44.4%). Optimum pore diameter of porous silica for immobilization of the enzyme was 91.7 nm. The enzymatic profiles of immobilized enzyme were almost identical to the native one except its stabilities to temperature and metal ions were improved. 1-Kestose was produced continuously and selectively from 40% (w/v) sucrose at fast flow rates by a column packed with the immobilized enzyme for up to 26 days, and the effluent concentration of 1-kestose remained in the range 113–135 mg ml^–1.     

Application of the enzyme thermistor to the direct estimation of intrinsic kinetics using the saccharose-immobilized invertase system.

The possibility of using the enzyme thermistor (ET) for the direct determination of kinetic parameters (Km, Ki, Vm) of immobilized enzyme (IME) was evaluated using different preparations of invertase conjugated to bead celluloses. Two different ET columns packed with IME were operated in the mode of a differential enzyme reactor (short length, low substrate conversion). Kinetic parameters of the above IME reactor were computed by a nonlinear curve-fitting procedure. The obtained kinetic parameters were superverified by means of an independent differential reactor (DR) system. This system utilized an indirect postcolumn analytical method based on determination of glucose concentration in the stirred reservoir. Best agreement between the data acquired by direct (ET) and indirect (DR) methods was obtained if the ET column was operated at flow rates within the range of 1.0-1.5 ml min-1 using invertase-cellulose chlorotriazine conjugate. Influence of heat loss and flow nonideality is discussed. The proposed ET method offers a rapid, convenient, and general approach to determination of kinetic constants of IME preparations by omitting postcolumn analytical methods.     

Kinetics of invertase immobilised on poly(phe-lys) coated polystyrene beads

Summary Surface of polystyrene beads was modified by poly(phe-lys) for invertase immobilisation. The optimum immobilisation conditions of invertase were; 0.01% (w/v) poly(phe-lys), 2% (v/v) glutaraldehyde at 25 °C and pH 4.5. The kinetics of sucrose hydrolysis by free and immobilised invertase in a batch reactor at pH 4.5 and 55 °C gave K_m and V_max values for sucrose with free and immobilised invertase of 81, 114 mM and 10.1, 9.2 mol glucose/min.mg enzyme, respectively. The deactivation rate constants of free and immobilised invertase were 0.0347 and 0.0098 min^–1, respectively.     

Production of high-fructose syrup from date by-products in a packed bed bioreactor using a novel thermostable invertase from Aspergillus awamori

Abstract

Dates by-products (discarded dates) from the sucrose-rich variety of ‘Deglet Nour’ were used as starting biomass to produce high-fructose syrup (HFS) based on an immobilized invertase process. A novel extracellular thermostable invertase obtained from Aspergillus awamori cultivated in submerged medium was induced with sucrose at 1% and used for this purpose. A zymogram of the crude extract showed the presence of a unique enzyme form that was optimally produced on the 5^th day. This enzyme preparation was biochemically characterized and immobilized on acetic acid-solubilized chitosan by covalent binding using glutaraldehyde (Yi = 88%, Ya = 54% and 15.53 U/g). When deployed in a packed bed reactor (PBR), HFS was efficiently and continuously produced from sucrose derived from aqueous date extracts. Feeding with an extract initially containing 139.2 g/L total sugar with 78.6 g/L sucrose at a flow rate of 17 ml/h, 50°C and pH 6 resulted in a conversion factor of 0.95 and a final fructose content in the syrup of 69 g/L.     

Analysis of various sugars by means of immobilized enzyme coupled flow injection analysis

Glucose, maltose, sucrose, lactose, xylose, sorbose, galactose, fructose and gluconolactone were analyzed by means of immobilized pyranose oxidase as well as by the combination of immobilized glucose oxidase with immobilized glycoamylase, invertase, mutarotase, maltase (α-glucosidase) and glucose isomerase by flow injection analysis (FIA). For the simultaneous analysis of glucose and other sugars three different flow-injection configurations were applied and compared. The average error of prediction of the analyses were better than 3% in model media and better than 6% in yeast extract containing media.     

Immobilization studies of whole microbial cells on transition metal activated inorganic supports

Summary Whole cells of Saccharomyces bayanus, Saccharomyces cerevisiae and Zymomonas mobilis were immobilized by chelation/metal-link processes onto porous inorganic carriers. The immobilized yeast cells displayed much higher sucrose hydrolyzing activities (90–517 U/g) than the bacterial, Z. mobilis, cells (0.76–1.65 U/g). The yeast cells chelated on hydrous metal oxide derivative of pumice stone presented higher initial -d-fructofuranosidase (invertase, EC 3.2.1.26) activity (161–517 U/g) than on other derivatives (90–201 U/g). The introduction of an organic bridge between the cells and the metal activator led to a decrease of the initial activity of the immobilized cells, however S. cerevisiae cells immobilized on the carbonyl derivative of titanium (IV) activated pumice stone, by covalent linkage, displayed a very stable behaviour, which in continuous operation at 30° C show only a slightly decrease on invertase activity for a two month period (half-life=470 days). The continuous hydrolysis of a 2% w/v sucrose solution at 30° C in an immobilized S. cerevisiae packed bed reactor was described by a simple kinetic model developed by the authors (Cabral et al., 1984a), which can also be used to predict the enzyme activity of the immobilized cells from conversion degree data.     

Continuous inversion of sucrose by gel-entrapped yeast cells

The feasibility of immobilizing invertase (β-d-fructofuranosidase; EC 3.2.1.26) from Saccharomyces cerevisiae cells by various methods was examined. The yeast cells were adapted for maximal invertase activity by growth in a medium containing 0.2% glucose and 1% lactate. There was no permeability barrier for the enzyme in the whole cells. Entrapment in acrylamide polymerized by gamma-rays (200 kR) was observed to be most effective, with retention of 85% of the activity. The evaluation of the properties of the immobilized invertase indicated that the kinetic values were not appreciably altered despite a broad pH optimum. The enzyme was more stable to both heat and gamma-radiation. The immobilized cells could be used repeatedly in a packed bed reactor system for inversion of sucrose without observable loss in activity for over one month.     

Kinetic modeling of a multiple immobilized enzyme system. I. Development and testing of the model

A kinetic model for the reaction sequence catalyzed by coimmobilized invertase and glucose oxidase with a sucrose substrate in a tubular reactor has been developed. The computerized mathematical model employs and orthogonal collection technique for solving oxidase were coimmobilized in poly(2-hydroxyethlmethacrylate) gels and used in a continuous flow packed-bed tubular reactor system. In addition to describing the development of the kinetic model, this article compares experimentally determined reactor effluent concentrations for various sucrose feed solutions to those predicted by the model. Variations between experimental and predicted reactor effluent concentrations were found to be on the micromolar level for sucrose feed concentrations as low as 1.38mM.     

Application of immobilized invertase to continuous hydrolysis of concentrated sucrose solutions

Invertase immobilized onto corn grits was utilized in the hydrolysis of highly concentrated sucrose solutions producting liquid sugar solutions containing glucose and fructose. Comparisons of conversion efficiencies of this immobilized invertase in a continuous stirredtank reactor and a plug-flow reactor indicated that the plug-flow reactor has an higher efficiency. Continuous sucrose hydrolysis was then performed in 0.1- and 1-L tubular reactors. This tenforld scaling-up was achieved without any noticeable loss in efficiency. This process thus was scaled-up to a 17.6-L pilot reactor set in a cane sugar refinery. This reactor was fed with highly concentrated sucrose solutions [71% (w/w)] to produce invert sugar syrup with the desired inversion degree. It allows a productivity equal to 9.1 kg sucrose hydrolyzed/h in the case of a 69% (w/w) sucrose initial concentration with a 72% conversion rate.     

Analysis of micromolar concentrations of glucose by an interference free flow injection based biosensor

A fast, sensitive, interference-free, single enzyme single reagent glucose biosensor, operated in flow injection analysis (FIA) mode, was developed. The method used involved formation of colored complex of titanium sulfate reagent with the peroxide generated by glucose oxidase immobilized in a packed bed reactor. The color developed was detected spectrophotometrically in a flow cuvette. The system could measure down to 0.5 mg glucose l^–1 and the response was reproducible and linear in the range 1 mg l^–1 to 100 mg l^–1. The analysis time for a 500 l sample was 35 s and was free of interference from a number of substances tested. Analysis results using an off-line batch kit were observed to be in agreement with the developed system for determination of glucose in blood plasma samples.     

Immobilization of invertase via carbohydrate moiety on chitosan to enhance its thermal stability

A new technique using chitosan as support for covalent coupling of invertase via carbohydrate moiety improved the activity and thermal stability of immobilized invertase. The best preparation of immobilized invertase retained 91% of original specific activity (412 U mg^–1). The half-life at 60°C was increased from 2.3 h (free invertase) to 7.2 h (immobilized invertase). In contrast, the immobilization of invertase via protein moiety on chitosan or using Sepharose as support resulted in less thermostable preparations. Additionally, immobilization of invertase on both supports caused the optimal reaction pH to shift from 4.5 to 2.5 and the substrate (sucrose) concentration for maximum activity to increase from 0.5 M to 1.0 M.     

Hydrolysis of concentrated sucrose syrups by invertase immobilized on anion exchanger waste cotton thread.

Yeast invertase was immobilized on polyethyleneimine-coated cotton thread by adsorption followed by crosslinking with glutaraldehyde. The thread-bound invertase was used as an easily retrievable system for the hydrolysis of 80% w/v commercial sucrose syrups. The immobilized enzyme was stable for over 90 days to a temperature of 50 degrees C, only when stored in 80% sucrose solution. Above this temperature, inactivation of enzyme was observed. The cotton threads were used in a batch reactor for hydrolysis of sucrose in about 30 batches carried out over a period of 50 days without loss in activity. The threads could also be used in a packed bed reactor (1.51) for 97% hydrolysis of 80% sucrose syrups at 50 degrees C at a rate of about 360 kg per month for a period of 3 months.     

Properties of invertase immobilized on the poly(ethylene-co-vinyl alcohol) hollow fiber membrane

Invertase was ionically immobilized on the poly(ethylene-co-vinyl alcohol) hollow fiber inside surface, which was aminoacetalized with 2-dimethylaminoacetaldehyde dimethyl acetal. Immobilization and enzyme reaction were carried out by letting the respective solutions pass or circulate through the inside of the hollow fiber, and the activity of invertase was determined by the amount of glucose produced enzymatically from sucrose. Immobilization conditions were examined with respect to the enzyme concentration and to the time, and consequently the preferable conditions at room temperature were found to be 5 mug/mL of enzyme concentration and 4 h of immobilization time. Under those conditions the immobilization yield and the ratio of the activity of the immobilized invertase to that of the native one were 89 and 80%, respectively. For both repeating and continuous usages, the activity fell to ca. 60% of the initial activity in the early stage and after that almost kept that value. The apparent Michaelis constant K(m) (') for the immobilized invertase decreased with increasing the flow rate of the substrate solution, to be close to the value for the native one. Furthermore, the possibility of the separation of the enzymatically formed glucose from the reaction mixture through the hollow fiber membrane was preliminarily examined.