Industrial production of fructooligosaccharides by immobilized cells of <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> in a packed bed reactor

Continuous production of fructooligosaccharides (FOS) by Aureobasidium pullulans immobilized on calcium alginate beads with a packed bed was investigated at a plant scale reactor. Optimum conditions were with 770 g sucrose/l, being fed at 200 l/h at 50°C which gave a productivity of 180 g FOS/l h. Initial activity was maintained for more than 100 days. The reactor was successfully scaled up to a production scale of 1.2 m³.     

Continuous production of fructooligosaccharides using fructosyltransferase immobilized on ion exchange resin

A continuous production of fructooligosaccharides from sucrose was investigated by fructosyltransferase immobilized on a high porous resin, Diaion HPA 25. The optimum pH (5.5) and temperature (55°C) of the enzyme for activity was unaltered by immobilization, and the immobilized enzyme became less sensitive to the pH change. The optimal operation conditions of the immobilized enzyme column for maximizing the productivity were as follows: 600 g/L of sucrose feed concentration, flow rate of superficial space velocity 2.7 h^?1. When the enzyme column was run at 50°C, about 8% loss of the initial activity of immobilized enzyme was observed after 30 days of continuous operation, during which high productivity of 1174 g/L·h was achieved. The kinds of products obtained using the immobilized enzyme were almost the same as those using soluble enzymes or free cells.     

Continuous sucrose hydrolysis by yeast cells immobilized to wool

A novel immobilized biocatalyst with invertase activity was prepared by adhesion of yeast cells to wool using glutaraldehyde. Yeast cells could be immobilized onto wool by treating either the yeast cells or wool or both with glutaraldehyde. Immobilized cells were not desorbed by washing with 1 M KCl or 0.1 M buffers, pH 3.5–7.5. The biocatalyst shows a maximum enzyme activity when immobilized at pH 4.2–4.6 and 7.5–8.0. The immobilized biocatalyst was tested in a tubular fixed-bed reactor to investigate its possible application for continuous full-scale sucrose hydrolysis. The influence of temperature, sugar concentration and flow rate on the productivity of the reactor and on the specific productivity of the biocatalyst was studied. The system demonstrates a very good productivity at a temperature of 70 °C and a sugar concentration of 2.0 M. The increase of the volume of the biocatalyst layer exponentially increases the productivity. The productivity of the immobilized biocatalyst decreases no more than 50% during 60 days of continuous work at 70 °C and 2.0 M sucrose, but during the first 30 days it remains constant. The cumulative biocatalyst productivity for 60 days was 4.8 × 10³kg inverted sucrose/kg biocatalyst. The biocatalyst was proved to be fully capable of continuous sucrose hydrolysis in fixed-bed reactors. Received: 8 November 1996 / Received revision: 31 January 1997 / Accepted: 31 January 1997     

Continuous acetone-butanol-ethanol fermentation using SO2-ethanol-water spent liquor from spruce

SO₂–ethanol–water (SEW) spent liquor from spruce chips was successfully used for batch and continuous production of acetone, butanol and ethanol (ABE). Initially, batch experiments were performed using spent liquor to check the suitability for production of ABE. Maximum concentration of total ABE was found to be 8.79 g/l using 4-fold diluted SEW liquor supplemented with 35 g/l of glucose. The effect of dilution rate on solvent production, productivity and yield was studied in column reactor consisting of immobilized Clostridium acetobutylicum DSM 792 on wood pulp. Total solvent concentration of 12 g/l was obtained at a dilution rate of 0.21 h⁻¹. The maximum solvent productivity (4.86 g/l h) with yield of 0.27 g/g was obtained at dilution rate of 0.64 h⁻¹. Further, to increase the solvent yield, the unutilized sugars were subjected to batch fermentation.     

Continuous pullulan fermentation in a biofilm reactor

Biofilm is a natural form of cell immobilization in which microorganisms attach onto solid support. In this study, a pigment-reduced pullulan-producing strain, Aureobasidium pullulans (ATCC 201253), was used for continuous pullulan fermentation in a plastic composite support (PCS) biofilm reactor. Optimal conditions for the continuous pullulan production were determined by evaluating the effects of the feeding medium with various concentrations of ammonium sulfate and sucrose and dilution rate. Pullulan concentration and production rate reached maximum (8.3 g/l and 1.33 g/l/h) when 15 g/l of sucrose, 0.9 g/l of ammonium sulfate, and 0.4 g/l of yeast extract were applied in the medium, and the dilution rate was at 0.16 h⁻¹. The purity of produced pullulan was 93.0%. The ratio of hyphal cells of A. pullulans increased when it was grown on the PCS shaft. Overall, the increased pullulan productivity can be achieved through biomass retention by using PCS biofilm reactor.     

The effect of pH,temperature and sucrose concentration on high productivity continuous ethanol fermentation using Zymomonas mobilis

Summary A flocculent strain of Zymomonas mobilis was used for ethanol production from sucrose. Using a fermentor with cell recycle (internal and external settler) high sugar conversion and ethanol productivity were obtained. At a dilution rate of 0.5 h^-1 (giving 96% sugar conversion) the ethanol productivity, yield and concentrations respectively were 20 g/l/h, 0.45 g/g and 40 g/l using a medium containing 100 g/l sucrose. At a sucrose concentration of 150 g/l, the ethanol concentration reached 60 g/l. The ethanol yield was 80% theoretical due to levan and fructo-oligomer formation. No sorbitol was detected. This fermentation was conducted at a range of conditions from 30 to 36°C and from pH 4.0 to 5.5.     

Itaconic acid production by immobilizedAspergillus terreus from xylose and glucose

Summary Aspergillus terreus NRRC 1960 spores were entrapped in calcium alginate gel beads or alternotely the fungal mycelium was immobilized either on Celite R-626 or in agar gel cubes, and the biocatalyst was employed both in repeated batch and in continuous column reactors to produce itaconic acid from D-xylose or D-glucose. The highest itaconic acid yield obtained in a submerged culture batch fermentation was 54.5% based on total initial glucose (55 g/l) with a volumetric productivity of 0.32 g/l h, and 44.8% from xylose (67 g/l) with a productivity of 0.20 g/l h. In a repeated batch fermentation mycelium immobilized in agar gel had a productivity of 0.112 g/l h, and mycelium grown from spores immobilized in calcium alginate gel 0.06 g/l h, both from xylose (60 g/l). With the best immobilized biocatalyst system used employing Celite R-626 as a carrier, volumetric productivities of 1.2 g/l h from glucose and 0.56 g/l h from xylose (both at 60 g/l) were obtained in continuous column operation for more than 2 weeks.     

Microencapsulation of recombinant Saccharomyces cerevisiae cells with invertase activity in liquid-core alginate capsules

As a means of integrating cell growth and immobilization, recombinant Saccharomyces cerevisiae cells with invertase activity were immobilized in liquid-core alginate capsules and cultured to a high density. S. cerevisiae cells of SEY 2102 (MAT alpha ura3-52 leu2-3, 112 his4-519) harboring plasmid pRB58 with the SUC2 gene coding for invertase were grown to 83 g/L of liquid-core volume inside the capsule on a dry weight basis. The cloned invertase was expressed well in the immobilized cells with slightly higher activity than the free cells in a batch culture. Invertase in the immobilized cells showed slightly more improved thermal stability than in the free cells. Storage in a Na-acetate buffer at 4 degrees C and 10 degrees C for 1 month resulted in 7% and 8% loss in activity, respectively. The sucrose hydrolysis reaction was stably maintained for 25 repeated batches for 7 days at 30 degrees C. Continuous hydrolysis of 0.3 M sucrose was carried out in a packed bed reactor with a conversion of more than 90% at a maximum productivity of 55.5 g glucose/L per hour for 7 days. In a continuous stirred tank reactor, the maximum productivity of 80.8 g glucose/L per hour was achieved at a conversion of 59.1% using 1.0 M sucrose solution, and 0.5 M sucrose solution was hydrolyzed for 1 week with a 95% conversion at a productivity of 48.8 g/L per hour. (c) 1996 John Wiley & Sons, Inc.     

Alcoholic fermentation by agar-immobilized yeast cells

Immobilized yeast cells in agar gel beads were used in a packed bed reactor for the production of ethanol from cane molasses at 30°C, pH 4.5. The maximum productivity, 79.5g ethanol/l.h was obtained with 195g/l reducing sugar as feed. Substrate (64.2%) was utilized at a dilution of 1.33h^-1. The immobilized cell reactor was operated continuously at a constant dilution rate of 0.67h^-1 for 100 days. The maximum specific ethanol productivity and specific sugar uptake rate were 0.610g ethanol/g cell.h and 1.275g sugar/g cell.h, respectively.     

Production of glycerol by immobilizedPichia farinosa

Summary Cells of the osmophilic yeastPichia farinosa were immobilized in sintered glass Raschig rings for the production of glycerol. The kinetics of production were observed under different conditions in batch, fed-batch and semicontinuous fermentations in fixed-bed column reactors and compared with those of free cells. 2.6 × 10⁹ cells/g sintered glass were adsorbed. The glycerol productivity amounted to 8.1 g/l per day. The highest concentration reached in batch culture was 86 g/l with immobilized cells. Fermentations using immobilized cells were accelerated compared to fermentations using free cells and maximum yield and productivity were reached at lower initial sugar concentrations. Using scanning electron microscopy it was observed that the shape of the cells was related to the sugar concentration in the medium. The experiments show thatP. farinosa produces glycerol with a high and constant productivity over long periods of time.     

Ethanol production by immobilized whole cells ofZymomonas mobilis in a continuous flow expanded bed bioreactor and a continuous flow stirred tank bioreactor

Continuous ethanol fermentation by immobilized whole cells ofZymomonas mobilis was investigated in an expanded bed bioreactor and in a continuous stirred tank reactor at glucose concentrations of 100, 150 and 200 g L^–1. The effect of different dilution rates on ethanol production by immobilized whole cells ofZymomonas mobilis was studied in both reactors. The maximum ethanol productivity attained was 21 g L^–1 h^–1 at a dilution rate of 0.36 h^–1 with 150 g glucose L^–1 in the continuous expanded bed bioreactor. The conversion of glucose to ethanol was independent of the glucose concentration in both reactors.     

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.     

Ethanol production from concentrated food waste hydrolysates with yeast cells immobilized on corn stalk

The aim of the present study was to examine ethanol production from concentrated food waste hydrolysates using whole cells of S. cerevisiae immobilized on corn stalks. In order to improve cell immobilization efficiency, biological modification of the carrier was carried out by cellulase hydrolysis. The results show that proper modification of the carrier with cellulase hydrolysis was suitable for cell immobilization. The mechanism proposed, cellulase hydrolysis, not only increased the immobilized cell concentration, but also disrupted the sleek surface to become rough and porous, which enhanced ethanol production. In batch fermentation with an initial reducing sugar concentration of 202.64 ± 1.86 g/l, an optimal ethanol concentration of 87.91 ± 1.98 g/l was obtained using a modified corn stalk-immobilized cell system. The ethanol concentration produced by the immobilized cells was 6.9% higher than that produced by the free cells. Ethanol production in the 14th cycle repeated batch fermentation demonstrated the enhanced stability of the immobilized yeast cells. Under continuous fermentation in an immobilized cell reactor, the maximum ethanol concentration of 84.85 g/l, and the highest ethanol yield of 0.43 g/g (of reducing sugar) were achieved at hydraulic retention time (HRT) of 3.10 h, whereas the maximum volumetric ethanol productivity of 43.54 g/l/h was observed at a HRT of 1.55 h.     

Continuous production of itaconic acid by Aspergillus terreus immobilized in a porous disk bioreactor

Summary Aspergillus terreus NRRL 1960 was grown on porous disks rotating intermittently in and out of the liquid phase. This immobilized fungal cell bioreactor was used to produce itaconic acid from glucose in a continuous operation. The effect of temperature, pH, disk rotation speed, and feed rate on the itaconic acid concentration and volumetric productivity were studied. The highest itaconic acid concentration and volumetric productivity obtained were 18.2 g/l and 0.73 g/l·h, respectively, under the following conditions: temperature at 36°C, pH 3.0, disk rotation speed at 8 rpm, and feed rate at 60 ml/h. These results are better than those by conventional fermentation or by other immobilized method.Nomenclature F feed rate (l/h) - K _1s saturation constant for immobilized cells (g/l) - K _2s saturation constant for suspended cells (g/l) - M ₁ increased mass of immobilized cells (g) - M ₂ total mass of immobilized cells (g) - P concentration of itaconic acid (g/l) - S substrate concentration in and out of the reactor (g/l) - S ₀ substrate concentration in the feed (g/l) - V liquid volume of the reactor (1) - X concentration of the suspended cells (g/l) - Y ₁ apparent yield of the immobilized cells (g cells/g substrate) - Y ₂ apparent yield of the suspended cells (g cell/g substrate) - Y ₃ apparent yield of itaconic acid (g itaconic acid/g substrate) - m ₁ maintenance and by-products coefficient of the immobilized cells (g substrate/g cell·h) - m ₂ maintenance and by-products coefficient of the suspended cells (g substrate/g cell·h) - µ_1max maximum specific growth rate of the immobilized cells (h^-1) - µ_2max maximum specific growth rate of the suspended cells (h^-1)     

Production of mead by immobilized cells of Hansenula anomala

Summary Mead was produced by immobilized cells of Hansenula anomala in calcium alginate gels. The immobilized cell beads of 3 mm diameter packed in column reactors of dimensions 2.2x60, 4x40 and 8x80 cm, produced mead containing maximum concentrations of ethanol and ethyl acetate of 70 g/l and 730 mg/l, respectively at a dilution rate of 0.1 h^–1. The maximum alcohol productivity achieved was 23.1 g/l·h at a dilution rate of 0.33 h^–1. With intermittent regenerations of the cells the reactor operated continuously for 110 days. This process enables the quick production of matured mead by a single culture and the elimination of the traditionally used long aging periods.     

Examination of immobilized cells in a rotating packed drum reactor for the production of ethanol from d-glucose

A rotating packed drum reactor has been proposed as an immobilized whole cell reactor and its performance for ethanol production has been studied with yeast cells immobilized in calcium alginate gel. In a continuous operation with synthetic d-glucose medium containing 125 g d-glucose l^?1, ethanol productivity was 20 g l^?1 h^?1 at a space velocity of 0.38 l (l gel)^?1 h^?1. With intermittent aeration the viability of yeast cells after 270 h of operation remained above 65%. CO₂ removal was easy, but d-glucose conversion was low at a high space velocity.     

Continuous acetone-butanol fermentation with high productivity by cell ultrafiltration and recycling

To increase the productivity of the acetone-butanol fermentation, a hollow-fiber ultrafilter is used to separate and recycle cells in a continuous fermentation ofClostridium acetobutylicum. Under partial cell recycling and at a dilution rate of 0.5 hr^–1, a cellular concentration of 20 g/l and a solvent productivity of 6.5 g/l.hr is maintained for several days at a total solvent concentration of 13 g/l.     

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 production ofl-lactic acid from whey permeate by immobilizedLactobacillus casei subsp.casei

Summary The production of l-lactic acid from whey permeate, a waste product of the dairy industry, by fermentation with the lactic acid bacterium Lactobacillus casei subsp. casei was investigated. A fermentation medium consisting of permeate and supplements, which enables exponential growth of the organisms, was developed. A fast method for determination of free and immobilized biomass in solid-rich media, based on measurement of cellular ATP, was evolved. Continuous fermentations in a stirred tank reactor (STR) and in a fluidized bed reactor (FBR) with immobilized biomass were compared. In the STR a volumetric productivity of 5.5 g/l per hour at 100% substrate conversion [dilution rate (D) = 0.22 h^–1] was determined. In the FBR porous sintered glass beads were used for immobilization and a maximum biomass concentration of 105 g/kg support was measured. A productivity of 10 g/l per hour was obtained at D = 0.4 h^–1 (substrate conversion 93%) and of 13.5 g/l per hour at D = 1.0 h^–1 (substrate conversion 50%). Offprint requests to: W. Krischke     

Mannose production from fructose by free and immobilized <Emphasis Type="SmallCaps">d</Emphasis>-lyxose isomerases from <Emphasis Type="Italic">Providencia stuartii</Emphasis>

A recombinant d-lyxose isomerase from Providencia stuartii was immobilized on Duolite A568 beads which gave the highest conversion of d-fructose to d-mannose among the various immobilization beads evaluated. Maximum activities of both the free and immobilized enzymes for fructose isomerization were at pH 7.5 and 45°C in the presence of 1 mM Mn²⁺. Enzyme half-lives were 14 and 30 h at 35°C and 3.4 and 5.1 h at 45°C, respectively. The immobilized enzyme in 300 g fructose/l (replaced hourly), produced 75 g mannose/l at 35°C = 25% (w/w) yield with a productivity of 75 g mannose l⁻¹ h⁻¹ after 23 cycles.