Reactor design for minimizing product inhibition during enzymatic lignocellulose hydrolysis: II. Quantification of inhibition and suitability of membrane reactors

Product inhibition of cellulolytic enzymes affects the efficiency of the biocatalytic conversion of lignocellulosic biomass to ethanol and other valuable products. New strategies that focus on reactor designs encompassing product removal, notably glucose removal, during enzymatic cellulose conversion are required for alleviation of glucose product inhibition. Supported by numerous calculations this review assesses the quantitative aspects of glucose product inhibition on enzyme-catalyzed cellulose degradation rates. The significance of glucose product inhibition on dimensioning of different ideal reactor types, i.e. batch, continuous stirred, and plug-flow, is illustrated quantitatively by modeling different extents of cellulose conversion at different reaction conditions. The main operational challenges of membrane reactors for lignocellulose conversion are highlighted. Key membrane reactor features, including system set-up, dilution rate, glucose output profile, and the problem of cellobiose are examined to illustrate the quantitative significance of the glucose product inhibition and the total glucose concentration on the cellulolytic conversion rate. Comprehensive overviews of the available literature data for glucose removal by membranes and for cellulose enzyme stability in membrane reactors are given. The treatise clearly shows that membrane reactors allowing continuous, complete, glucose removal during enzymatic cellulose hydrolysis, can provide for both higher cellulose hydrolysis rates and higher enzyme usage efficiency (kg_product/kg_enzyme). Current membrane reactor designs are however not feasible for large scale operations. The report emphasizes that the industrial realization of cellulosic ethanol requires more focus on the operational feasibility within the different hydrolysis reactor designs, notably for membrane reactors, to achieve efficient enzyme-catalyzed cellulose degradation.     

Some aspects of hybridoma cell cultivation

Summary Two hybridoma cell lines were cultivated in an indirectly aerated 10-1 reactor in batch, fed-batch and continuous (perfusion) operations and in spinner flasks. The medium in the reactor was sampled either by an aseptic cross-flow filtration module integrated into a loop or by an in-situ tubular filter. The glucose concentration was monitored by an on-line flow injection analyzer and the ammonia concentration by an ion-selective electrode. Since the membrane transmission of the high-molecular components decreased during cultivation, the product, a monoclonal antibody, was enriched in the reactor. During cultivation, the concentrations of cells, viable cells, glucose, lactase, acetate, citrate, ammonia, urea, amino acids, proteins, and monoclonal antibodies were determined off-line. The specific growth rate, specific production, and consumption rates of the medium components were influenced considerably by the medium composition, especially by the type and amount of serum used.Offprint requests to: K. Schügerl     

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.     

Kinetics of ethanol fermentations in membrane cell recycle fermentors

Ethanol fermentation by yeast was carried out in a cell filtration recycle system with a hollow-fiber membrane filter. Maximum biomass concentrations up to 210 g dry wt/L were obtained, but in normal operation concentrations they were between 100 and 150 g/L. The ethanol productivity using 14% glucose feed was 85 g/L h, with an ethanol concentration of 65 g/L and an ethanol yield of over 90%. The ethanol productivity and yeast growth rate decreased as the cell concentration increased beyond a certain level. The cell mass in the reactor was maintained by a proper manipulation of diluticn rate and bleed ratio depending on the growth rate.     

Biodiesel production using a membrane reactor

The immiscibility of canola oil in methanol provides a mass-transfer challenge in the early stages of the transesterification of canola oil in the production of fatty acid methyl esters (FAME or biodiesel). To overcome or rather, exploit this situation, a two-phase membrane reactor was developed to produce FAME from canola oil and methanol. The transesterification of canola oil was performed via both acid- or base-catalysis. Runs were performed in the membrane reactor in semi-batch mode at 60, 65 and 70 degrees C and at different catalyst concentrations and feed flow rates. Increases in temperature, catalyst concentration and feedstock (methanol/oil) flow rate significantly increased the conversion of oil to biodiesel. The novel reactor enabled the separation of reaction products (FAME/glycerol in methanol) from the original canola oil feed. The two-phase membrane reactor was particularly useful in removing unreacted canola oil from the FAME product yielding high purity biodiesel and shifting the reaction equilibrium to the product side.     

Synthesis of galacto-oligosaccharide from lactose using beta-galactosidase from Kluyveromyces lactis: Studies on batch and continuous UF membrane-fitted bioreactors

A study of galacto-oligosaccharides (GOS) synthesis from lactose with beta-galactosidase from Kluyveromyces lactis (Maxilact L2000) was carried out. The synthesis was performed using various initial lactose concentrations ranging from 220 to 400 mg/mL and enzyme concentrations ranging from 3 to 9 U/mL, and was investigated at 40 degrees C and pH 7, in a stirred-tank reactor. In the experimental range examined, the results showed the amount of GOS formed depended on lactose concentration but not on enzyme concentration. Galactose was a competitive inhibitor, while glucose was a non-competitive inhibitor. In a further study, a laboratory-scale reactor system, fitted with a 10-kDa NMWCO composite regenerated cellulose membrane, was used in a continuous process. The reactor was operated in cross-flow mode. The effect of operating pressures on flux and productivity was investigated by applying different transmembrane pressures to the system. The continuous process showed better production performance compared to the batch synthesis with the same lactose and enzyme concentrations at 40 degrees C, pH 7. Comparison of product structures from batch and continuous processes, analyzed by HPAE-PAD and methylation analysis, showed similarities but differed from the structures found in a commercial GOS product (Vivinal GOS).     

Production of teicoplanin by Actinoplanes teichomyceticus in continuous fermentation.

Production of the potent antibiotic teicoplanin by Actinoplanes teichomyceticus was studied in batch and in chemostat cultures. It is found that the producing strain deactivates to a non-producing strain named NP-12. This strain is used to find the growth kinetics of the A. teichomyceticus without interference from the product teicoplanin. In batch experiments with NP-12 grown on glucose at different initial concentrations and with different added amounts of teicoplanin, the strong inhibitory effect of teicoplanin was determined. These results obtained on NP-12 were validated in a series of chemostat experiments with the processing strain. All experiments in batch and in chemostat cultures were well represented by Monod kinetics with respect to the carbon and energy source (glucose) and with a substantial inhibitory effect of teicoplanin. Further experiments were made with the producing strain in a continuous reactor coupled to a microfilter that delivers a cell-free permeate. It was found that the derived kinetics almost exactly simulated the behavior of the cell recirculation reactor in addition to when the cell concentration in the reactor was more than four times higher than in the chemostat. For industrial production of teicoplanin, a continuous reactor with cell recirculation and working with a low effluent glucose concentration was by far the best mode of operation. Finally, the deactivation of the producing strain to NP-12 was modeled by a two-step deactivation mechanism. Deactivation was independent of dilution rate but dependent on the inoculum preparation and on the previous history of the inoculum.     

Kinetics of olive oil hydrolysis in an enzyme membrane reactor. Bond graph network model of reactor performance (part 1)

Hydrolyses of olive oil were performed in a reactor with lipase immobilized on a laboratory ultrafiltration poliamide-6 membrane. The reactor consisted of two circulating phases of olive oil and buffer solution. For the characterization of the reactor performance, a model of the hydrolysis process was developed. It was created by means of thermodynamic network representation of both the chemical processes and the transport of the reactants. According to an estimated bond graph network, the model is represented quantatively by a set of thirty-three differential equations representing the time derivatives of the particular species concentration. The parameters of the model were estimated based on experimental data and/or literature notations. Close agreement of numerical estimations of the product concentrations with experimental data was gained. The model enabled an extended analysis of the influence of different reaction parameters, enzyme inhibition and concentration of the reactants on reactor performance.     

Characterization of an immobilized cell, trickle bed reactor during long term butanol (ABE) fermentation

Acetone-butanol-ethanol (ABE) fermentation was performed continuously in an immobilized cell, trickle bed reactor for 54 days without, degeneration by maintaining the pH above 4.3. Column clogging was minimized by structured packing of immobilization matrix. The reactor contained two serial glass columns packed with Clostridium acetobutylicum adsorbed on 12- and 20-in.-long polyester sponge strips at total flow rates between 38 and 98.7 mL/h. Cells were initially grown at 20 g/L glucose resulting in low butanol (1.15 g/L) production encouraging cell growth. After the initial cell growth phase a higher glucose concentration (38.7 g/L) improved solvent yield from 13.2 to 24.1 wt%, and butanol production rate was the best. Further improvement in solvent yield and butanol production rate was not observed with 60 g/L of glucose. However, when the fresh nutrient supply was limited to only the first column, solvent yield increased to 27.3 wt% and butanol selectivity was improved to 0.592 as compared to 0.541 when fresh feed was fed to both columns. The highest butanol concentration of 5.2 g/L occurred at 55% conversion of the feed with 60 g/L glucose. Liquid product yield of immobilized cells approached the theoretical value reported in the literature. Glucose and product concentration profiles along the column showed that the columns can be divided into production and inhibition regions. The length of each zone was dependent upon the feed glucose concentration and feed pattern. Unlike batch fermentation, there was no clear distinction between acid and solvent production regions. The pH dropped, from 6.18-6.43 to 4.50-4.90 in the first inch of the reactor. The pH dropped further to 4.36-4.65 by the exit of the column. The results indicate that the strategy for long term stable operation with high solvent yield requires a structured packing of biologically stable porous matrix such as polyester sponge, a pH maintenance above 4.3, glucose concentrations up to 60 g/L and nutrient supply only to the inlet of the reactor.     

Hydrolysis of liquefied corn starch in a membrane reactor

The objective of this study was to develop a continuous hydrolysis process for the enzymatic saccharification of liquefied corn starch using a membrane reactor. A residence time distribution study confirmed that the membrane reactor could be modeled as a simple continuous stirred tank reactor (CSTR). Kinetic studies indicated that the continuous reactor operated in the first-order region with respect to substrate concentration at substrate concentrations greater than 200 g/L. At a residence time of 1 h and an enzyme concentration of 1 g/L, the maximum reaction velocity (V(m)) was 3.86 g glucose/L min and the apparent Michaelis constant (K(m) (')) was 562 g/L. The K(m) (') value for the continuous reactor was 2-7 times greater than that obtained in a batch reactor.Kinetic data were fit to a model based on the Michaelis-Menten rate expression and the design equation for a CSTR. Application of the model at low reactor space times was successful. At space times of 6 min or less, the model predicted the reactor's performance reasonably well. Additional work on the detection and quantitation of reversion products formed by glucoamylase is required. Isolation, detection, and quantitation of reversion products by HPLC was difficult. Detailed analysis on the formation of these reversion products could lead to better reactor designs in the future.     

Influence of medium composition on the cephalosporin C production with a highly productive strain Cephalosporium acremonium.

Cephalosporin production by a highly productive Cephalosporium acremonium strain was carried out and optimized by fed-batch operation in a 40 l stirred tank reactor using a complex medium containing 30-120 g l-1 peanut flour. The concentrations of cephalosporin C (CPC) and its precursors: penicillin N (PEN N), deacetoxy cephalosporin C (DAOC), and deacetyl cephalosporin C (DAC) were monitored with an on-line HPLC. The concentrations of amino acids valine (VAL), cysteine (CYS), alpha-amino adipic acid (alpha-AAA), the dipeptide alpha-amino-adipyl-cysteine (AC), and the tripeptide alpha-amino-adipyl-cysteinyl-valine (ACV), were determined off-line by HPLC. The RNA content and dry weight of the sediment as well as the oxygen transfer rate (OTR) and the CO2 production rate (CPR) were used to calculate the cell mass concentration (X). The influences of peanut flour (PF) and the on-line monitored and controlled medium components: glucose (GLU), phosphate, methionine (MET) as well as the dissolved oxygen (DOC) on the cell growth, the product formation, and the pathway of cephalosporin C biosynthesis were investigated and evaluated. When the glucose fed-batch cycle was optimized and oxygen transfer limitation was avoided (DOC greater than 20% of the saturation value), high process performance (103.5 g l-1 X, 11.84 g l-1 CPC, a maximum CPC productivity of 118 mg l-1 h-1, and the whole concentration of the beta-lactam antibiotics CPC, DAC, DAOC, PEN N 17.34 g l-1) was achieved by using 100 g l-1 PF in the medium with the optimum concentration of phosphate (260-270 mg l-1) and a low glucose concentration (less than 0.5 g l-1). The cultivations with different medium concentrations demonstrated that the product formation was directly proportional to the cell mass concentration. On the average, the cell mass-based yield coefficient of CPC: YCPC/X amounted to 0.115 g CPC per g cell mass.     

Fed-batch culture of Bacillus thuringiensis for thuringiensin production in a tower type bioreactor

Fed-batch culture of Bacillus thuringiensis in a modified airlift reactor has been developed by using adaptive control of glucose concentration in the reactor. The glucose concentration was estimated via a correlation equation between carbon dioxide production rate and glucose consumption rate. The estimated glucose concentration as the output variable was fed back to computer for calculation of substrate addition. The modified reactor was an airlift reactor with a net draft tube. The airlift reactor had high oxygen transfer rate and low shear stress which were important factors for production of thuringiensin. Fed-batch culture of Bacillus thuringiensis in the modified airlift reactor provided significant improvement of thuringiensin production. (c) 1995 John Wiley & Sons, Inc.     

Adaptive fuzzy control of nutrients concentration in fed-batch culture of mammalian cells

An adaptive fuzzy controller was developed to control the glucose and glutamine concentrations in the reactor constant at the desired level. The parameter values of the controller change during the cultivation according to the culture phase which was detected by the lactate concentration. Cultivations with different glucose and glutamine set point concentrations of a recombinant BHK anchorage-dependent cell line were performed in a fed-batch reactor on-line connected with an HPLC system. Glucose and glutamine concentrations were satisfactorily controlled at each set point during all cultivation periods. Ammonia had a determining effect on productivity since it inhibited cell growth and protein specific production. Ammonia production increased with an increase of glutamine or a decrease of glucose set point concentrations, indicating the importance of glucose to glutamine ratio for the optimization of productivity in mammalian cell cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparison of the Baker's yeast process performance in laboratory and production scale

A 215 m³ industrial bubble column reactor for fedbatch production of Baker's yeast was sampled for sugar, to investigate the extent of concentration gradients. The results verify that such gradients exist: the concentration is higher closer to the feeding point. Effects of sugar heterogeneities at different scales were studied by 1)?performing a volumetric scale-down of the industrial process in a laboratory stirred tank reactor (STR); 2) performing the same scaled down process in a Scale-Down Reactor (SDR) with repeated short term exposure of the cells to high sugar concentrations. In this reactor about 10% of the Baker's yeast culture was intermittently exposed to high (0.45–1.9?g?l^?1) concentrations of sugar, for periods of 60 seconds. It was found that physiological parameters of glycolysis and respiration were affected by environmental heterogeneities: 1) A biomass yield reduction of about 6–7% was found, with both the production reactor and the SDR, as compared to the homogeneous reactor. The loss of yield is interpreted in terms of a metabolic by-pass via ethanol, where cells are consuming and producing ethanol with different yields. 2) The maximum respiration rate was higher in cells produced in the production unit and in the SDR. 3) The product quality, expressed as gassing power of the yeast in a dough, was increased for sweet and non-sugar doughs in the SDR, and for sweet doughs in the production reactor. Thus, the SDR, when run with defined glucose gradients, in some aspects resembles the large reactor. It could be regarded as a tool for scale-down and scale-up studies and may be useful in investigations on the scale-up sensitivity of a process.     

Mathematical modeling of diffusion and reaction in the hydrolysis of vegetable protein in an immobilized enzyme recycle reactor

Experimental investigation is by far the most effective approach for studying the behavior of physical systems. However, an enzymatic solubilization of vegetable protein is a complex combination of intrinsic problems, of which many are not easily adaptable to experimental investigation. Experimental designs to study enzyme vegetable protein reactions yield data which describe the extramembraneous activity of the immobilized enzyme. In a continuous recycle immobilized enzyme reactor, the microenvironment concentration of the substrate or product in the membrane phase, or the concentrations along the reactor axial length in the bulk phase are not discernible to the experimenter. However, the knowledge of such concentration profiles is important in weighing the significance of such factors as intermembrane diffusion, enzyme loading, wet membrane size, and the mode of operation of the reactor. The simulation of mathematical models, which describe the physical system within the constraints imposed, yields information which is vital to the understanding of the process occurring in the reactor. The kinetics and diffusion of an immobilized thermophilic Penicillium duponti enzyme at pH 3.4-3.7 and 50 degrees C was modeled mathematically. The kinetic parameters were evaluated by fitting a model to experimental data using nonlinear regression analysis. Simulation profiles of the effects of reactor geometry, substrate concentration, membrane thickness, and enzyme leading on the hydrolysis rate are presented. From the profiles generated by the mathematical model, the best operational reactor strategy is recommended.     

Scale down of recombinant protein production: a comparative study of scaling performance

A large bioreactor is heterogeneous with respect to concentration gradients of substrates fed to the reactor such as oxygen and growth limiting carbon source. Gradient formation will highly depend on the fluid dynamics and mass transfer capacity of the reactor, especially in the area in which the substrate is added. In this study, some production-scale (12 m³ bioreactor) conditions of a recombinant Escherichia coli process were imitated on a laboratory scale. From the large-scale cultivations, it was shown that locally high concentration of the limiting substrate fed to the process, in this case glucose, existed at the level of the feedpoint. The large-scale process was scaled down from: (i) mixing time experiments performed in the large-scale bioreactor in order to identify and describe the oscillating environment and (ii) identification of two distinct glucose concentration zones in the reactor. An important parameter obtained from mixing time experiments was the residence time in the feed zone of about 10 seconds. The size of the feed zone was estimated to 10%. Based on these observations the scale-down reactor with two compartments was designed. It was composed of one stirred tank reactor and an aerated plug flow reactor, in which the effect of oscillating glucose concentration on biomass yield and acetate formation was studied. Results from these experiments indicated that the lower biomass yield and higher acetate formation obtained on a large scale compared to homogeneous small-scale cultivations were not directly caused by the cell response to the glucose oscillation. This was concluded since no acetate was accumulated during scale-down experiments. An explanation for the differences in results between the two reactor scales may be a secondary effect of high glucose concentration resulting in an increased glucose metabolism causing an oxygen consumption rate locally exceeding the transfer rate. The results from pulse response experiments and glucose concentration measurements, at different locations in the reactor, showed a great consistency for the two feeding/pulse positions used in the large-scale bioreactor. Furthermore, measured periodicity from mixing data agrees well with expected circulation times for each impeller volume. Conclusions are drawn concerning the design of the scale-down reactor.     

Study of performance of a tubular membrane reactor for an enzyme catalyzed reaction

A tubular membrane reactor offers many advantages over a solid wall reactor to carry out an enzyme catalyzed reaction. With proper membrane selectivity, the product, may be separated from the reacting stream and the enzyme recycled for continuous reuse. In most cases, enzyme reuse contributes to the economic feasibility of a continuous enzyme catalyzed process. Furthermore, the efficiency and performance of a membrane reactor is greater than that of a solid wall reactor. Continuous hydrolysis of starch by the enzyme β-amylase, carried out in a commercially available tubular membrane unit, is studied at different starch and enzyme concentrations for a given system pressure and inlet flow rate. Results show that the performance of the membrane reactor is in all cases greater than that of the solid wall reactor. A steady state in performance of permeation rate is, however, not reached by the membrane reactor, which shows a continuous decline within the periods examined in this study. This decline is caused in part by the aging of the starch solution, but mostly by the formation of a concentrated, or gel, layer at the membrane surface. This appears to be the main limiting factor for this process since the decline in reaction and permeation rate results in a severe decrease in the amount of maltose in the permeate.     

Reaction and diffusion in a gel membrane reactor containing immobilized cells

To investigate the effect of diffusional limitations and heterogeneous cell distribution in a gel-immobilized cell system, a gel membrane reactor has been constructed. The reactor consists essentially of a gel layer with immobilized cells, flanked by two well-mixed chambers. Through one chamber substrate is pumped, and this chamber is the equivalent of the outside of a spherical gel bead. The second closed measuring chamber contains a small quantity of liquid that can equilibrate with the inside surface of the membrane, eventually after a long transient. Analysis of the liquid in this chamber can give direct information on substrate and product concentrations at the gel surface, and is and indication of the situation in the center of a gel bead. The gel membrane reactor appears to be an excellent tool to study diffusion and reaction in a gel-containing immobilized cells. A mathematical model with time- and position-dependent cell concentration and diffusion coefficient is described. Experimental data show the effective diffusion coefficient of glucose in an alginate gel to decrease with yeast cell concentration. Moreover, kinetic parameters could be determined, using the mathematical model. Microscopic analysis confirmed the proliferation of the gel-entrapped microorganisms in the outer layer of the matrix, as predicted by the model. Potentially, this type of reactor has a clear potential to study the physiology of gel-immobilized cells. (c) 1992 John Wiley & Sons, Inc.     

Kinetic characteristics of chloroplast glucose transport

Influx of labelled D-glucose into isolated spinach (Spinacia oleracea L. cv. Melody hybrid) chloroplasts was initially rapid followed by a period of slower influx. The stroma glucose concentration attained equilibrium rapidly with low external glucose concentrations and the two were linearly proportional. The period of slower influx resulted from conversion of glucose to acidic products that remained trapped in the chloroplast. As the external glucose concentration increased, the stroma glucose concentration increased less and less, attaining a maximal concentration of 72 mol m(-3). The maintenance of an equilibrium stroma glucose concentration lower than that in the external medium is evidence that plastid glucose efflux involves secondary active transport. The equilibrium stroma glucose concentration increased in response to light and protonophoric uncouplers. It is proposed that glucose efflux is coupled with a proton and the stroma glucose concentration equilibrates in response to the proton gradient across the membrane. To determine if glucose is a significant product of starch mobilization, chloroplasts were isolated from spinach leaves labelled with 14CO2 during the preceding light period. Chloroplasts degraded starch at the same rate as the intact leaf. Glucose, maltose, and isomaltose were the principal labelled products that appeared in the medium during starch mobilization. The glucose concentration in the chloroplast was 2 mol m(-3), which is similar to the measured Km for zero trans efflux. The data support the role of the glucose translocator as an important component in the pathway for sucrose synthesis at night.     

Effect of High Product Concentration in a Dual Fed-batch Asymmetric 3-oxo Ester Reduction by Baker's Yeast

Baker's-yeast-mediated asymmetric ethyl 3-oxobutanoate reduction using a fed-batch feeding strategy for both the 3-oxo ester and the electron donor, was explored as potential production system for enantiopure ethyl ( S )-3-hydroxybutanoate. The dual feed strategy was based on kinetic and stoichiometric data. One major aspect is the effect of high product concentrations on the progress of the reduction. According to initial rate experiments, product inhibition occurs at concentrations above 600 mM product causing a 10-fold decrease of the initial biomass-specific reduction rate. By using optimized feed rates and a biomass concentration of 43 g dw l -1 , a product concentration of 350 mM was reached within 80 h with a degree of conversion of 95%. The volumetric productivity was 0.58 g l -1 h -1 , using 2.1 kg pressed yeast kg product -1 and 0.52 kg glucose kg product -1 . During the fed-batch biotransformation the reduction rate continuously decreased and reduction ceased after 80 h, due to biocatalyst inactivation after prolonged use at increasing high product concentrations. The continuous decrease in reducing activity led to very high ethyl 3-oxobutanoate levels in the reactor resulting in an increase of the undesired specific ethyl ( R )-3-hydroxybutanoate production rate. Therefore, the enantiomeric excess of the product decreased, from initially 100 to ~75% at 80 h. It is concluded that the design of processes for efficient asymmetric bioreduction cannot solely be based on initial rate kinetics, but require detailed knowledge of the effects on activity and enantioselectivity upon long-term exposure to process conditions.