Continuous alcohol fermentation in an immobilized cell rotating disk reactor

The increasing interest in alcohol fermentation over these last years because of the energy crisis has been demonstrated by an increase in scientific research. After a brief analysis of the main results of the literature in the field of alcohol fermentation reactors, the use of a new type of immobilized cell reactor [the rotating biological surface (RBS) reactor] was studied. As is well known, the RBS reactor is a form of fixed-film reactor and can be described as a dynamic trickling filter. Our experimental apparatus employed a spongy material to trap the yeast cells on the disks. The results of fermentations carried out in the RBS reactor working in batch, in continuous with cell support, and in continuous without cell support have been presented in order to compare the different productivities and to assess the performance of the RBS immobilized cell reactor. An ethanol productivity of 7.1 g/L h was achieved in the RBS-ICR at a dilution rate of 0.3 h(-1), 2.5 times higher than the maximum productivity obtained in the RBS reactor without support at a lower dilution rate. The adoption of a spongy material as a cell immobilizer, combined with the use of the RBS reactor, enhances the particular advantages of both systems.     

Synthesis of isomaltooligosaccharides and oligodextrans in a recycle membrane bioreactor by the combined use of dextransucrase and dextranase

A recycle ultrafiltration membrane reactor was used to develop a continuous synthesis process for the production of isomaltooligosaccharides (IMO) from sucrose, using the enzymes dextransucrase and dextranase. A variety of membranes were tested and the parameters affecting reactor stability, productivity, and product molecular weight distribution were investigated. Enzyme inactivation in the reactor was reduced with the use of a non-ionic surfactant but its use had severe adverse effects on the membrane pore size and porosity. During continuous isomaltooligosaccharide synthesis, dextransucrase inactivation was shown to occur as a result of the dextranase activity and it was dependent mainly on the substrate availability in the reactor and the hydrolytic activity of dextranase. Substrate and dextranase concentrations (50-200 mg/mL(-1) and 10-30 U/mL(-1), respectively) affected permeate fluxes, reactor productivity, and product average molecular weight. The oligodextrans and isomaltooligosaccharides formed had molecular weights lower than in batch synthesis reactions but they largely consisted of oligosaccharides with a degree of polymerization (DP) greater than 5, depending on the synthesis conditions. No significant rejection of the sugars formed was shown by the membranes and permeate flux was dependent on tangential flow velocity.     

Continuous two step bioconversion of 4-oxoiso-phorone (OIP) to 4-hydroxy-2,2,6-trimethylcyclo-hexanone (4-HTMCH) by thermophilic bacteria

Summary The continuous two step conversion of 4-oxoisophorone(OIP) to 4-hydroxy-2,2,6-trimethylcyclohexanone(4-HTMCH) via dihydrooxoisophorone(DOIP) was carried out using two types of thermophilic growing cells, Thermomonospora curvata and Bacillus stearothermophilus, in sequential connected continuous stirred tank reactor(CSTR) and hollow fiber reactor system. For more than 80 hours operation, 46% of OIP was converted to 4-HTMCH with productivity of 179 mg/h/l.     

Comparison of culture methods for human-human hybridomas secreting anti-HBsAg human monoclonal antibodies

Human-human hybridomas which secrete a human monoclonal antibody (h-MoAb) against hepatitis B virus surface antigen showed growth associated production kinetics. The rate of h-MoAb production rapidly decreased after cell growth was arrested in a perfusion culture, even if the perfusion rate was increased. A continuous suspended-perfusion culture, in which both culture broth and culture supernatant are continuously harvested and the same volume of fresh medium is continuously fed into the reactor, was developed to maintain continuous growing conditions during cultivation. In this culture system, the production of h-MoAb continued for more than 50 days with an average productivity of 5.0 mg/l of working volume/day. A semicontinuous immobilized-perfusion culture in which parts of the cells are repeatedly removed from the immobilized reactor was another useful technique for the long term cultivation of these h-h hybridomas. As an average h-MoAb production rate, 62 mg/l of immobilized-bed volume/day was achieved for 65 days of cultivation using a ceramic matrix reactor, and 327 mg/l/day was achieved over 47 days of cultivation using a hollow fiber reactor equipped with Cultureflo M^TM Thus, the antibody productivity per reactor volume per day by the semicontinuous immobilized-perfusion culture was much higher than that of the continuous perfusion culture in an agitation reactor.     

Continuos IBE fermentation by immobilized growing Clostridium beijerinckii cells in a stirred-tank fermentor

The potential of continuous isopropanol-butanol-ethanol (IBE) fermentation by Ca-alginate-immobilized Clostridium beijerinckii cells in a continuous stirred-tank reactor is investigated. A mathematical model is presented to describe steady-state reactor performance. It appeared to be possible to use the biocatalyst particles repeatedly for successive fermentations (at least three times for a total duration of two months). Reactor productivity was 6-16 times higher than that of a batch fermentation (free cells), while the solvents yield was also increased. Measurements of substrate, product, and biomass concentrations were only partially in agreement with the model; however, a solid basis for further technological developement of the process has been laid.     

Comparative study of reactor performance for the resolution of d,l-amino acids

The racemic resolution of l-valine and l-serine by fungal aminoacylase has been evaluated by comparing the performance of various reactor configurations including an anion exchange nylon tangential flow membrane reactor, a tubular reactor with aminoacylase adsorbed onto DEAE-Sephadex as support and a continuous stirred tank reactor with enzyme recycling using a flat ultrafiltration module (CSTR/UF). Among the substrates tested, the N-chloroacetyl-d,l-amino acids were the preferred substrates, showing the highest catalytic efficiency (V_m/K_m).Optimum reactor operational conditions obtained in discontinuous assays were selected to study the behaviour of the reactors in a continuous mode. DEAE-Sephadex loaded six-fold more enzyme than anion exchange nylon (60 and 10 gE/litre, respectively, related to reactor volume), whereas enzyme concentration within the CSTR/UF reactor was limited only by enzyme solubility.The tangential flow membrane reactor configuration with a 10 g/litre enzyme concentration produced higher productivity values (0·35 kg l-valine/litre per day, and 80% conversion degree) and operational stability (t = 161 days) than the CSTR/UF reactor (0·24 kg l-valine/litre per day, and 80% conversion degree) performing with the same enzyme concentration. The tubular reactor with the enzyme adsorbed onto DEAE-Sephadex (60 g/litre enzyme load) showed higher productivity values (1·9 kg l-valine/litre per day, and 80% conversion degree) and operational stability (t = 70 days) than the CSTR/UF reactor (1·05 kg l-valine/litre per day, and 80% conversion degree). However, the CSTR/UF reactor was the preferred configuration, as it had the highest enzyme load and productivity (1·95 kg l-valine/litre per day of reactor volume, and 80% conversion degree), a half-life of 55 days at 50°C, and the possibility of easy continuous enzyme addition.     

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.     

Genetically engineered Escherichia coli FBR5: Part I. Comparison of high cell density bioreactors for enhanced ethanol production from xylose

Five reactor systems (free cell batch, free cell continuous, entrapped cell immobilized, adsorbed cell packed bed, and cell recycle membrane reactors) were compared for ethanol production from xylose using Escherichia coli FBR5. In the free cell batch and free cell continuous reactors (continuous stirred tank reactor‐CSTR) productivities of 0.84 gL^?1 h^?1 and 1.77 gL^?1 h^?1 were achieved, respectively. A cell recycle membrane reactor resulted in the highest productivity of 55.56 gL^?1 h^?1, which is an increase of 66‐fold (e.g., 6614%) over the batch reactor. Calcium alginate gel CSTR resulted in a productivity of 2.04 gL^?1 h^?1 whereas adsorbed cell packed bed reactor resulted in a productivity of 4.39 gL^?1 h^?1. In the five reactor systems, ethanol concentrations ranged from 18.9 to 40.30 gL^?1 with metabolic yields from 0.44 to 0.51. Published 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Continuous production of erythrulose using transketolase in a membrane reactor

Transketolase can be used for synthesis of chiral intermediates and carbohydrates. However the enzyme is strongly deactivated by the educts. This deactivation depends on the reactor employed. An enzyme membrane reactor allows the continuous production of L-erythrulose with high conversion and stable operational points. A productivity (space-time yield) of 45g L d was reached.     

Filament formation and ethanol production by Zymomonas mobilis in adsorbed cell bioreactors

Zymomonas mobilis immobilized on microporous ion exchange resins has previously been shown to allow the attainment of high ethanol productivities in packed-bed bioreactors. The formation of bacterial filaments after several days of continuous operation, however, had resulted in excessive pressure increases across the reactor bed. The present work examines techniques for controlling filament formation by Z. mobilis in two reactor sizes (161 mL and 7.85 L) and a feed glucose concentration of 100 g/L. By controlling the fermentation temperature at 20-25 degrees C it has been possible to eliminate filament formation by Z. mobilis and to operate the larger bioreactor for 232 h with an ethanol productivity of 50 g/L h (based on total reactor volume). The rate of ethanol production has been shown to be very sensitive to temperature in the range 20-30 degrees C, and it is likely that slightly higher temperatures than those used in this study will improve ethanol productivity while still permitting long-term operation.     

The long-term effects of ethanol on immobilized cell reactor performance using K. fragilis

The effects of ethanol on reactor performance were studied in a small, 5-cm packed height, "differential" type immobilized cell reactor. Lactose utilizing yeast cells, Kluyveromyces fragilis, were absorbed to a porous adsorbant sponge matrix in a gas continuous reactor. Step changes in the feed ethanol concentration to the column (10-130 g/L) were used to test the reactor response over extended periods of time (about 30-50 h per dosage level) followed by a return to basal zero inlet ethanol feed. Effluent cell density and effluent cell viability were measured at intervals. An inhibitory response in ethanol productivity to feed dosage ethanol levels above 20 g/L was detected almost immediately, with a near steady state response noted within 2.5 h of initiating the dosage. Feed ethanol levels above 50 g/L resulted in a subsequent gradual decrease in reactor productivity over time, which was associated with a decrease in the fraction of viable shed cells in the reactor effluent. The reactor response to a step removal of the ethanol inhibition was also monitored. Quick and complete rebounding of the fermentation rate to the original basal rate was noted following dosage concentrations of under 50 g/L ethanol. Recovery rates slowed following ethanol dosage levels above 50 g/L. Viable shed cell density improved overtime during the slow recovery periods. Growth rates (as determined by shed cell density) were more strongly inhibited than productivity. Growth responded more slowly to changes in ethanol environment as growth rates at 30 h fell to about 40% of the rates measured 7.5 h after initiation of a dosage level. It is concluded that ethanol contributions to cell injury and death (and consequent ICR performance degradation) may be more important than ethanol inhibition of productivity rates in the long-term operation of immobilized cell reactors at ethanol concentrations over 50 g/L.     

Continuous production of bacitracin by immobilized living whole cells of Bacillus sp.

Whole cells of Bacillus sp., a bacitracin-producing bacteria, were immobilized in polyacrylamide gel. The continuous production of bacitracin by an immobilized whole-cell-containing air-bubbled reactor was examined with 0.5% peptone solution. The bacitracin productivity (28 units/ml/hr) obtained with this system was higher than that with a batch system. The effluent bacitracin concentration increased with increasing aeration rate and reached a steady-state maximum above the aeration rate of 3.0 liter/min. A high bacitracin productivity was retained for at least eight days when the gel was washed with sterilized saline at a flow rate of 250 ml/hr for 2 hr once a day. The half-life of the immobilized whole-cell system was about 10 days. Bacitracin productivity by the immobilized whole-cell reactor was higher than that by a conventional continuous fermentation process at high dilution rates.     

Enhanced continuous production of lovastatin using pellets and siran supported growth of Aspergillus terreus in an airlift reactor

Lovastatin, a hypocholesterolemic agent, is a secondary metabolite produced by filamentous microorganism Aspergillus terreus in submerged batch cultivation. Lovastatin production by pellets and immobilized siran cells was investigated in an airlift reactor. The process was carried out by submerged cultivation in continuous mode with the objective of increasing productivity using pellet and siran supported growth of A terreus. The continuous mode of fermentation improves the rate of lovastatin production. The effect of dilution rate and aeration rate were studied in continuous culture. The optimum dilution rate for pellet was 0.02 h⁻¹ and for siran carrier was 0.025 h⁻¹. Lovastatin productivity using immobilized siran carrier (0.0255 g/L/h) was found to be greater than pellets (0.022 g/L/h). The productivity by both modes of fermentation was found higher than that of batch process which suggests that continuous cultivation is a promising strategy for lovastatin production.     

Improved operational stability of cell-free glucose-fructose oxidoreductase from Zymomonas mobilis for the efficient synthesis of sorbitol and gluconic acid in a continuous ultrafiltration membrane reactor

For the continuous, enzymatic synthesis of sorbitol and gluconic acid by cell-free glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis, the principal determinants of productivity have been identified. Most important, the rapid inactivation of the soluble enzyme during substrate conversion can be avoided almost completely when weak bases such as tris(hydroxymethyl)aminomethan or imidazol are used for the titration of the produced gluconic acid and when 5-10 mM dithiothreitol are added to prevent thiol oxidations. With regard to a long-term operational stability of the enzyme for continuous syntheses, thermal deactivation becomes significant at reaction temperatures above 30 degrees C. Without any additional purification being required, the crude cell extract of Z. mobilis can be employed in a continuous ultrafiltration membrane reactor over a time period of more than 250 h without significant decrease in substrate conversion or enzyme activity. The use of soluble GFOR thus appears to be an interesting alternative to employing permeabilized cells of Zymomonas for the production of sorbitol and gluconic acid and may be superior with regard to reactor productivities, at comparable operational stabilities.     

An immobilized cell reactor with simultaneous product separation. II. Experimental reactor performance

The simultaneous separation of volatile fermentation products from product-inhibited fermentations can greatly increase the productivity of a bioreactor by reducing the product concentration in the bioreactor, as well as concentrating the product in an output stream free of cells, substrate, or other feed impurities. The Immobilized Cell Reactor-Separator (ICRS) consists of two column reactors: a cocurrent gas-liquid "enricher" followed by a countercurrent "stripper" The columns are four-phase tubular reactors consisting of (1) an inert gas phase, (2) the liquid fermentation broth, (3) the solid column internal packing, and (4) the immobilized biological catalyst or cells. The application of the ICRS to the ethanol-from-whey-lactose fermentation system has been investigated. Operation in the liquid continuous or bubble flow regime allows a high liquid holdup in the reactor and consequent long and controllable liquid residence time but results in a high gas phase pressure drop over the length of the reactor and low gas flow rates. Operation in the gas continuous regime gives high gas flow rates and low pressure drop but also results in short liquid residence time and incomplete column wetting at low liquid loading rates using conventional gas-liquid column packings. Using cells absorbed to conventional ceramic column packing (0.25-in. Intalox saddles), it was found that a good reaction could be obtained in the liquid continuous mode, but little separation, while in the gas continuous mode there was little reaction but good separation. Using cells sorbed to an absorbant matrix allowed operation in the gas continuous regime with a liquid holdup of up to 30% of the total reactor volume. Good reaction rates and product separation were obtained using this matrix. High reaction rates were obtained due to high density cell loading in the reactor. A dry cell density of up to 92 g/L reactor was obtained in the enricher. The enricher ethanol productivity ranged from 50 to 160 g/L h while the stripper productivity varied from 0 to 32 g/L h at different feed rates and concentrations. A separation efficiency of as high as 98% was obtained from the system.     

Performance of the continuous glucose isomerase reactor system for the production of fructose syrup

Glucose isomerase in the form of heat-treated whole-cell enzyme prepared from Streptomyces phaeochromogenus follows the reversible single-substrate reaction kinetics in isomerization of glucose to fructose. Based on the Kinetic constants determined and the mathematical model of the reactor system developed, the preformance of a plug-flow-type continuous-enzyme reactor system was studied experimentally and also simulated with the aid of a computer for the ultimate objective of optimization of the glucose isomerase reactor system. The enzyme decay function for both the enzyme storage and during the use in the continuous reactor, was found to follow the first-order decay kinetics. When the enzyme decay function is taken into consideration, the ideal homogeneous enzyme reactor kinetics provided a satisfactory working model without further complicatin of the mathematical model, and the results of computer simulation were found to be in good agreement with the experimental results. Under a given set of constraints the performance of the continuous glucose isomerase reactor system can be predicted by using the computer simulation method described in this paper. The important parameters studied for the optimization of reactor operation were enzyme loading, mean space time of the reactor, substrate feed concentration, enzyme decay constants, and the fractional conversion, in addition to the kinetic constants. All these parameters have significant effect on the productivity. Some unique properties of the glucose isomerization reaction and its effects on the performance of the continuous glucose isomerase reactor system have been studied and discussed. The reaction kinetics of glucose isomerase and the effects of both the enzyme loading and the changes in reaction rate within a continuous reactor on the productivity are all found to be of particular importance to this enzyme reactor system.     

Comparative study of ethanol production by an immobilized yeast in a tubular reactor and in a multistage reactor

Summary The productivity of continuous ethanol fermentation has been increased using fixed bed reactors where a high density of yeast cells was maintained on a packing of wood chips. Two different systems have been used: 1. A tubular reactor which produced alcohol solutions containing up to 13.5% (V/V) ethanol. High CO₂ retention and a poor mass transfer between bulk medium and immobilized biomass prevented production rates higher than 2.2 g/l·h. 2. A multistage reactor where a better utilisation of the reactor volume led to improved performances. Solutions containing 132 g/l of ethanol (16.5% V/V) were produced with a productivity increased up to 4.8 g/l·h. A better distribution of the active biomass and a lower gradient of alcohol concentration between support and bulk medium are possible reasons for this improvement.     

Increase of substrate conversion by pervaporation in the continuous butanol fermentation

Summary In a continuous fermentation of glucose to butanol and isopropanol by immobilized Clostridium beyerinckii cells the products were removed continuously by pervaporation. Both the glucose conversion and the reactor productivity were 65–70% higher than in a continuous fermentation without product removal.     

Optimization of Operating Temperature for Continuous Immobilized Glucose Isomerase Reactor with Pseudo Linear Kinetics

In this work, the optimal operating temperature for the enzymatic isomerization of glucose to fructose using a continuous immobilized glucose isomerase packed bed reactor is studied. This optimization problem describing the performance of such reactor is based on reversible pseudo linear kinetics and is expressed in terms of a recycle ratio. The thermal deactivation of the enzyme as well as the substrate protection during the reactor operation is considered. The formulation of the problem is expressed in terms of maximization of the productivity of fructose. This constrained nonlinear optimization problem is solved using the disjoint policy of the calculus of variations. Accordingly, this method of solution transforms the nonlinear optimization problem into a system of two coupled nonlinear ordinary differential equations (ODEs) of the initial value type, one equation for the operating temperature profile and the other one for the enzyme activity. The ODE for the operating temperature profile is dependent on the recycle ratio, operating time period, and the reactor residence time as well as the kinetics of the reaction and enzyme deactivation. The optimal initial operating temperature is selected by solving the ODEs system by maximizing the fructose productivity. This results into an unconstrained one‐dimensional optimization problem with simple bounds on the operating temperature. Depending on the limits of the recycle ratio, which represents either a plug flow or a mixed flow reactor, it is found that the optimal temperature of operation is characterized by an increasing temperature profile. For higher residence time and low operating periods the residual enzyme activity in the mixed flow reactor is higher than that for the plug flow reactor, which in turn allows the mixed flow reactor to operate at lower temperature than that of the plug flow reactor. At long operating times and short residence time, the operating temperature profiles are almost the same for both reactors. This could be attributed to the effect of substrate protection on the enzyme stability, which is almost the same for both reactors. Improvement in the fructose productivity for both types of reactors is achieved when compared to the constant optimum temperature of operation. The improvement in the fructose productivity for the plug flow reactor is significant in comparison with the mixed flow reactor.     

The application of cell recycle to continuous fermentative lactic acid production

Summary A continuous stirred tank reactor (CSTR) with cell recycle was used to produce lactic acid from glucose usingLactobacillus delbreuckii NRRLB445. A volumetric productivity of 76 gm/1-h was obtained with an effluent concentration of 35 gm/1 lactic acid and a residual glucose concentration of less than 0.02 gm/l.