Enzyme activity maintenance in packed-bed reactors via continuous enzyme addition

An operational scheme for using immobilized enzymes in packed-bed reactors that permits operation at a constant throughput rate and constant product quality is described. The scheme used columns operated in series with continuous enzyme addition to compensate for enzyme decay. A mathematical technique was developed to determine the enzyme addition rate, enzyme usage, and enzyme volume in the column system. Operation of columns in series is compared to operation where the flow rate is decreased to compensate for a loss of enzyme activity for both zero-and first-order decay. The analyses indicated that columns in series resulted in better enzyme utilization but larger reactor volumes than parallel reactors with decreasing flow rate.     

Development of a Polyaniline-coated Monolith Reactor for the Synthesis of Cephalexin Using Penicillin G Acylase Aggregates

A monolith reactor for the synthesis of cephalexin was developed using capillary columns. The micro channel in the monolith reactor was coated with polyaniline (PANI), and penicillin G acylase was aggregated with PANI using 0.5% of glutaraldehyde as a cross-linker. The developed monolith reactor exhibited many advantages over other enzyme reactors such as batch and continuous reactors. It showed fast enzyme reaction rates owing to the decrease in external mass transfer and internal diffusion limitations. The reactor can easily be scaled up by bundling together multiple monolith reactors, enabling a corresponding increase in feed rate. Furthermore, the monolith reactor showed good operational stability, with 95% of its original activity maintained after 48 h of continuous operation. The PANI coating on the surface of the capillary column increased the enzyme immobilization capacity and conversion was increased from 15.4% to 70.6% after PANI coating. The conversion ratio increased to approximately 70.6% with an increase in residence time and reactor length.     

Different bioreactor configurations for the decolourisation of the azo dye reactive black 5 by Geotrichum sp. CCMI 1019

Decolourisation of the azo dye Reactive Black 5 by Geotrichum sp. CCMI 1019 was studied using stirred tank reactors (STR) and two types of bubble columns (porous plate (PP) bubble column and aeration tube (AT) bubble column). For the bubble columns, the k_La increased with the gas fractional hold-up (ε_G) and the aeration rate. A linear relationship between ε_G and superficial gas velocity was obtained for all reactors. At same aeration rates, the PP bubble columns showed higher k_La and hold-up values than the AT bubble column. In the STRs, large and dense aggregates were formed which adhered to surfaces whereas bubble columns gave smaller and less compact pellets.

Manganese peroxidase and laccase were detected in the extracellular media in all reactors. However, laccase was only detected after the onset of decolourisation, suggesting that additional enzymes may be involved. Mn peroxidase activity was detected (about 46 U/ml) in both the STRs and AT bubble columns but higher values (110 U/ml) were obtained with the PP bubble columns.

Out of the three reactor systems studied, the AT bubble columns gave the most favourable results for Reactive Black 5 decolourisation. Rapid and complete colour removal was obtained throughout the visible spectrum. Bubble columns are simple in design as well as operation and may be useful for the bioremediation of textile wastewater.     

Analysis of peptide mixtures by capillary high performance liquid chromatography: a practical guide to small-scale separations.

Capillary HPLC is a very effective means of separating small amounts of peptides and proteins. Capillary columns ranging from 0.01 mm to 0.5 mm in diameter can be constructed using recycled supports and inexpensive fused silica capillary tubing. Commercial pumping systems and UV detectors can be readily converted for operation in the flow rate range of 0.5-50 microL/min. Detailed procedures are given for the construction of columns and UV detector flow cells. A mixture of peptides derived from the endo Lys C digest of horse heart cytochrome c was used to illustrate various aspects of capillary chromatography of peptides and compares the performance of various-sized capillary columns and UV detector flow cell types.     

Different bioreactor configurations for the decolourisation of the azo dye reactive black 5 by Geotrichum sp. CCMI 1019

Decolourisation of the azo dye Reactive Black 5 by Geotrichum sp. CCMI 1019 was studied using stirred tank reactors (STR) and two types of bubble columns (porous plate (PP) bubble column and aeration tube (AT) bubble column). For the bubble columns, the k_La increased with the gas fractional hold-up (ε_G) and the aeration rate. A linear relationship between ε_G and superficial gas velocity was obtained for all reactors. At same aeration rates, the PP bubble columns showed higher k_La and hold-up values than the AT bubble column. In the STRs, large and dense aggregates were formed which adhered to surfaces whereas bubble columns gave smaller and less compact pellets.

Manganese peroxidase and laccase were detected in the extracellular media in all reactors. However, laccase was only detected after the onset of decolourisation, suggesting that additional enzymes may be involved. Mn peroxidase activity was detected (about 46 U/ml) in both the STRs and AT bubble columns but higher values (110 U/ml) were obtained with the PP bubble columns.

Out of the three reactor systems studied, the AT bubble columns gave the most favourable results for Reactive Black 5 decolourisation. Rapid and complete colour removal was obtained throughout the visible spectrum. Bubble columns are simple in design as well as operation and may be useful for the bioremediation of textile wastewater.     

Enzymatic ester synthesis with continuous measurement of water activity

Ester synthesis from aliphatic monoalcohols and organic acids was investigated by using a microbial lipase. The reaction medium only contained the substrates and the enzyme without addition of water or organic solvent. During the reaction, water was produced and the water activity (aw) increased. Batch reactors and continuous-flow reactors were used. In batch, the aw was 0.13 at the beginning of the reaction and increased to reach a plateau at 0.77, after which ester synthesis continued without modification of the aw. Different alcohols and acids were tried in solid-liquid reactors, and all cases synthesis occurred, leading to a significant increase in the water activity. For continuous-flow reactors, the use of silica beads retaining water inside the reactor where the enzymatic reaction took place resulted in some control of the enzymatic reaction by changing the aw.     

Continuous removal of Cr(VI) from aqueous solution catalysed by palladised biomass of Desulfovibrio vulgaris

Growth-decoupled cells of Desulfovibrio vulgaris NCIMB 8303 can be used to reduce Pd(II) to cell-bound Pd(0) (Bio-Pd(0)), a bioinorganic catalyst capable of reducing hexavalent chromium to less toxic Cr(III), using formate as the electron donor. Magnetic resonance imaging showed that Bio-Pd(0), immobilized in chitosan and agar beads, is distinguishable from the surrounding gel and is evenly dispersed within the immobilization matrix. Agar-immobilized Bio-Pd(0) and 'chemical Pd(0)' were packed into continuous-flow reactors, and challenged with a solution containing 100 microM Cr(VI) (pH 7) at a flow rate of 2.4 ml h(-1). Agar-immobilized chemical Pd(0) columns lost Cr(VI) reducing ability by 160 h, whereas columns containing immobilized Bio-Pd(0) maintained 90% reduction until 680 h, after which reduction efficiency was gradually lost.     

Stability of biocatalysts on the basis of carrageenan-immobilized Escherichia coli during continuous synthesis of L-malic acid

Continuous enzymatic synthesis of L-malic acid from potassium fumarate in packed-bed flow reactors was investigated. Carrageenan-immobilized Escherichia coli cells were used as a biocatalyst. The operational stability of the biocatalyst fumarase activity was studied, and conditions for preserving high activity of the biocatalyst were determined.     

Transport phenomena, reactor design and scale-up

Mixing in biological reactors is used to improve mass transfer and provide proper micro-scale and macro-scale shear rates for effective process results. Reactors may be mixed by impellers on rotating shafts, or may be of the flow contactor type such as packed columns, bubble columns or airlift circulators.

In addition, review of kinetics can tell the process performance based on various kinds of mixing conditions. Several interesting and unique mixing studies are also included where appropriate.     

Process-engineering characterization of small-scale bubble columns for microbial process development

Volumetric oxygen transfer rates and power inputs were estimated by a model of the formation of primary gas bubbles at the static sparger (sinter plate) of small-scale bubble columns and a common mass-transfer correlation for bubbles rising in a non-coalescent Newtonian electrolyte solution of low viscosity. Estimations were used to assess the dimensioning and possibilities of small-scale bubble column application with an height/diameter ratio of about 1. Estimations of volumetric oxygen transfer rates (<0.16 s^-1) and power inputs (<100 W m^-3) with a mean pore diameter of the static sparger of 13 µm were confirmed as function of the superficial air velocity (<0.6 cm s^-1) by measurements using an Escherichia coli fermentation medium. Small-scale bubble columns are thus to be classified between shaking flasks and stirred-tank reactors with respect to the oxygen transfer rate, but the maximum volumetric power input is more than one magnitude below the power input in shaking flasks, which is of the same order of magnitude as in stirred-tank reactors. A small-scale bubble columns system was developed for microbial process development, which is characterized by handling in analogy to shaking flasks, high oxygen transfer rates and simultaneous operation of up to 16 small-scale reactors with individual gas supply in an incubation chamber.     

Disposable microbore high-pressure liquid chromatography columns for protein and peptide separations.

A range of high-performance liquid chromatography (HPLC) columns with internal diameters of 0.25 to 1.8 mm have been constructed by securing glass or plastic tubing into standard HPLC fittings. These were packed with chromatographic materials chosen for operation at moderate pressures with high flow rates. These columns were shown to be effective in a conventional HPLC instrument for peptide and protein separations in reverse-phase mode and for proteins in ion-exchange and size-exclusion modes. The simple construction and low cost of these microbore columns allow them to be considered as disposable. Using only small amounts of any type of packing material, they have the flexibility to be adapted to a wide range of analytical and micropreparative separations.     

Monitoring of alpha-ketoglutarate in a fermentation process using expanded bed enzyme reactors.

A bienzyme flow injection system is presented for the monitoring of alpha-ketoglutarate produced in a fermentation process, using glutamate dehydrogenase (GDH) and glutamate oxidase (GlOx) immobilised in two serially connected expanded bed reactors. The use of expanded bed resulted in unhindered passage of the bacterial cells through the columns, and thereby the need of a separate filtering step (e.g. microdialysis) was avoided. In the first reactor, alpha-ketoglutarate was converted to L-glutamate by GDH in the presence of ammonia and NADH. In the following reactor, L-glutamate was converted by GlOx to alpha-ketoglutarate, ammonia and hydrogen peroxide, which was detected in an electrochemical flow-through cell at +650 mV vs. Pt/(0.1 M KCl). The detection limit of alpha-ketoglutarate in the coupled packed bed reactors was 1 microM (defined as 3 S/N), the linear range 0-100 microM, and the sensitivity 0.80 nA/microM (R(2) 0.99). In the coupled expanded bed reactors, the detection limit of alpha-ketoglutarate was 7 microM (defined as 3 S/N), the linear range and the sensitivity being 0-500 microM and 0.11 nA/microM (R(2) 1.00), respectively. The response time (defined as the time between peak rise and return to baseline) was 5 min for coupled packed beds (injection of supernatant), and 12 min for coupled expanded beds (injection of sample containing cellular and particulate matter). Several other parameters, such as reactor stability, flow rate dependency, bed expansion, glutamate interference, etc. were investigated and characterised. When analysing real samples from a fermentation broth, the same results were obtained independent of the nature of the reactor system (packed or expanded bed). The hereby described system can easily be automatised and controlled from a personal computer.     

Continuous flow peptide synthesis on aminopolyoxyethylene-polystyrene graft copolymer using Fmoc-strategy

An insoluble graft copolymer consisting of the covalently bound polyoxyethylene to cross-linked polystyrene (HO-POE-PS) was prepared by anionic polymerization of ethylene oxide on the resin. The copolymer was then converted to the corresponding amino-polymer (H2N-POE-PS) and the latter was employed as the solid carrier for peptide synthesis. Although HO-POE-PS has successfully been employed as a carrier for peptide synthesis by the standard shaking procedure using t-butoxycarbonyl-amino acids, now we deemed it of interest to test its suitability for the continuous flow synthesis. Thus, the C-terminal octapeptide of the porcine insulin B chain (B23-30) was prepared by this procedure using a photolabile anchoring group and fluoren-9-ylmethoxycarbonyl-amino acids. All the reactions were carried out in a continuous flow manner in a steel column under pressure using a high-performance liquid chromatography (HPLC) system. At the end of the synthesis, a sample of the protected peptide was cleaved from the support by photolysis. For the cleavage of another sample, an aqueous solution of sodium carbonate was employed. The protected peptide was purified on silica gel and Sephadex-LH 20. All the protecting groups of a sample of the octapeptide were removed with piperidine/dimethylformamide and trifluoroacetic acid and the deblocked peptide was purified by ion-exchange chromatography. The free peptide was shown to be homogeneous by thin-layer chromatography, HPLC, and electrophoresis. The identify of the free octapeptide was confirmed by amino-acid analysis, 13C-nuclear magnetic resonance measurement and field-desorption mass spectrometry. The peptide was also shown to be free of racemization.     

A new fast method for nanoLC-MALDI-TOF/TOF-MS analysis using monolithic columns for peptide preconcentration and separation in proteomic studies

A new fast method for identification and characterization of proteolytic digests of proteins by monolithic liquid chromatography coupled with mass spectrometry has been developed. The advantages of the monolithic columns are a high-pressure stability and low back pressure resulting in higher flow rates for capillary or nanosize columns simplifying the system handling. As was shown in several publications, such monolithic stationary phases are highly qualified for the analysis of peptides and proteins, but so far, only small volumes could be injected into the system, which might hamper the sample preparation leading to protein precipitation and partial loss of sample. To overcome the problem of small injection volumes, we established a system including a short monolithic trap column to allow preconcentration of the peptides. The injected sample is flushed at higher flow rates onto the trap column, bound to the stationary phase, and in this way concentrated in a few nanoliters before starting the separation. The expanded system was optimized and tested using different reference protein samples. Eluting peptides were detected by MALDI-TOF/TOF-MS and identified by database searching. The system is now a permanent part for proteome analysis in our lab, and as such, it was successfully applied for the detection of post-translational modifications and the analysis of membrane proteins. One example for these analyses is also included in this paper.     

Intensification of biodiesel synthesis using zigzag micro-channel reactors

Zigzag micro-channel reactors have been fabricated and used for continuous alkali-catalyzed biodiesel synthesis. The influences of the main geometric parameters on the performance of the micro-channel reactors were experimentally studied. It has been found that the zigzag micro-channel reactor with smaller channel size and more turns produces smaller droplets which result in higher efficiency of biodiesel synthesis. Compared to conventional stirred reactors, the time for high methyl ester conversion can be shortened significantly with the methyl ester yield of 99.5% at the residence time of only 28 s by using the optimized zigzag micro-channel reactor, which also exhibits less energy consumption for the same amount of biodiesel during biodiesel synthesis. The results indicate that zigzag micro-channel reactors can be designed as compact and mini-fuel processing plant for distributive applications.     

Preparation of template plasmids for cell-free protein synthesis using a regenerated anion-exchange column

In this report, we describe how reactions of cell-free protein synthesis can be successfully conducted using plasmids prepared with regenerated anion-exchange columns. When washed, stripped, and equilibrated with appropriated buffers, regenerated columns were able to be used repeatedly to prepare plasmids with consistent yield and purity. The regenerated columns exhibited comparable performance to a fresh column with respect to the efficiency of protein synthesis using the plasmids prepared from them. Overall, we expect that the presented results will contribute significantly to economizing the technology of cell-free protein synthesis as a practical method for protein production in preparative scales.     

Preparative peptide purification by cation-exchange and reversed-phase perfusion chromatography.

Cation exchange was compared to reversed-phase chromatography for the preparative purification of a 28-residue peptide (vasoactive intestinal polypeptide) on the 100-mg scale. Optimized high-speed, high-resolution methods were developed for both chromatographic modes on POROS Perfusion Chromatography flow-through particle chromatography columns. While both methods appeared to provide similar purity, the cation exchange column had approximately ten times the loading capacity per unit column volume as the reversed-phase column. Five-minute methods for desalting the cation exchange-purified peptide and analysis of fractions were developed using small reversed-phase columns. The cation-exchange method was scaled up to process 95 mg of crude peptide in a 12-min run.     

Polymerization of glucans by enzymatically active membranes

Conventional enzyme membrane reactors are not appropriate for a continuous synthesis of macromolecules and simultaneous product release. By immobilizing the enzyme in sufficiently large pores of a membrane an ensemble of miniaturized bioreactors is created. Product molecules are continuously removed from the enzyme by the flow of the reaction mixture across the membrane. Additionally, by varying the flow rate, it ought to be possible to influence the substrate as well as the enzyme-product residence times and thereby the product macromolecule's size. In this paper we present the first results of experiments involving enzymatic 1,4-alpha-glucan synthesis, using sucrose as substrate, maltooligosaccharides (DP 3-6) as primers, and membrane-immobilized amylosucrase. Epoxy groups for a covalent enzyme immobilization were generated on polypropylene microfiltration membranes by heterogeneous photoinitiated graft polymerization of glycidyl methacrylate. The influence of primer concentration and flow rate through the enzyme-membrane on amylosucrase activity, molecule growth, and coupling efficiency for glucose (% of coupled glucose versus free glucose) were investigated. The enzymatically mediated chain elongation of maltooligosaccharides by the successive addition of glucose units was achieved for the first time in a transmembrane process utilizing amylosucrase membranes.     

Bacterial reductive dissolution of crystalline Fe(III) oxide in continuous-flow column reactors

Bacterial reductive dissolution of synthetic crystalline Fe(III) oxide-coated sand was studied in continuous-flow column reactors in comparison with parallel batch cultures. The cumulative amount of aqueous Fe(II) exported from the columns over a 6-month incubation period corresponded to (95.0 +/- 3.7)% (n = 3) of their original Fe(III) content. Wet-chemical analysis revealed that only (6.5 +/- 3.2)% of the initial Fe(III) content remained in the columns at the end of the experiment. The near-quantitative removal of Fe was visibly evidenced by extensive bleaching of color from the sand in the columns. In contrast to the column reactors, Fe(II) production quickly reached an asymptote in batch cultures, and only (13.0 +/- 2.2)% (n = 3) of the Fe(III) oxide content was reduced. Sustained bacterial-cell growth occurred in the column reactors, leading to the production and export of a quantity of cells 100-fold greater than that added during inoculation. Indirect estimates of cell growth, based on the quantity of Fe(III) reduced, suggest that only an approximate doubling of initial cell abundance was likely to have occurred in the batch cultures. Our results indicate that removal of biogenic Fe(II) via aqueous-phase transport in the column reactors decreased the passivating influence of surface-bound Fe(II) on oxide reduction activity, thereby allowing a dramatic increase in the extent of Fe(III) oxide reduction and associated bacterial growth. These findings have important implications for understanding the fate of organic and inorganic contaminants whose geochemical behavior is linked to Fe(III) oxide reduction.     

Growth dynamics of Artemisia annua hairy roots in three culture systems

The transient growth of Artemisia annua hairy roots was compared for cultures grown in shake flasks and in bubble column and mist reactors. Instantaneous growth rates were obtained by numerically differentiating the transient biomass measurements. Specific sugar consumption rates showed good agreement with literature values. From the growth rate and sugar consumption rate, the specific yield and maintenance coefficient for sugar were determined for all three culture systems. These values were statistically indistinguishable for roots grown in shake flasks and bubble columns. In contrast, the values for roots grown in bubble columns and mist reactors were statistically different, suggesting that sugar utilization by roots grown in these two systems may be different. By measuring respiration rates in the bubble column reactor we also determined the actual biomass yield and maintenance coefficient for O(2) and CO(2). Together with an elemental analysis of the roots, this allowed us to obtain a reasonable carbon balance.