Growth model and metabolic activity of brewing yeast biofilm on the surface of spent grains: a biocatalyst for continuous beer fermentation

In the continuous systems, such as continuous beer fermentation, immobilized cells are kept inside the bioreactor for long periods of time. Thus an important factor in the design and performance of the immobilized yeast reactor is immobilized cell viability and physiology. Both the decreasing specific glucose consumption rate (q(im)) and intracellular redox potential of the cells immobilized to spent grains during continuous cultivation in bubble-column reactor implied alterations in cell physiology. It was hypothesized that the changes of the physiological state of the immobilized brewing yeast were due to the aging process to which the immobilized yeast are exposed in the continuous reactor. The amount of an actively growing fraction (X(im)act) of the total immobilized biomass (X(im)) was subsequently estimated at approximately X(im)act = 0.12 g(IB) g(C)(-1) (IB = dry immobilized biomass, C = dry carrier). A mathematical model of the immobilized yeast biofilm growth on the surface of spent grain particles based on cell deposition (cell-to-carrier adhesion and cell-to-cell attachment), immobilized cell growth, and immobilized biomass detachment (cell outgrowth, biofilm abrasion) was formulated. The concept of the active fraction of immobilized biomass (X(im)act) and the maximum attainable biomass load (X(im)max) was included into the model. Since the average biofilm thickness was estimated at ca. 10 microm, the limitation of the diffusion of substrates inside the yeast biofilm could be neglected. The model successfully predicted the dynamics of the immobilized cell growth, maximum biomass load, free cell growth, and glucose consumption under constant hydrodynamic conditions in a bubble-column reactor. Good agreement between model simulations and experimental data was achieved.     

In vivo nuclear magnetic resonance analysis of immobilization effects on glucose metabolism of yeast Saccharomyces cerevisiae

Fermentation rates and intracellular compositions have been determined for alginate-entrapped Saccharomyces cerevisiae and for identical cells in suspension. Glucose uptake and ethanol and glycerol production are approximately two times faster in immobilized cells than in suspended cells. Phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy of fermenting immobilized and suspended cells shows differences in intermediate metabolite levels such as fructose-1,6 diphosphate, glucose-6-phosphate, and 3-phosphoglycerate and in internal pH. Carbon-13 NMR shows an increase in polysaccharide production. These data suggest that immobilization has accelerated the rate of glucose transport or of glucose phosphorylation. These effects of immobilization upon cell metabolism are observed in a very short period of time under conditions in which negligible DNA, RNA, or protein synthesis takes place.     

Scale up of fuel ethanol production from sugar beet juice using loofa sponge immobilized bioreactor

Production of fuel ethanol from sugar beet juice, using cells immobilized on loofa sponge was investigated. Based on ethanol productivity and ease of cell immobilization, a flocculating yeast strain, Saccharomyces cerevisiae IR2 was selected for ethanol production from sugar beet juice. It was found that raw sugar beet juice was an optimal substrate for ethanol production, requiring neither pH adjustment nor nitrogen source supplement. When compared with a 2 l bubble column bioreactor, mixing was not sufficient in an 8 l bioreactor containing a bed of sliced loofa sponges and consequently, the immobilized cells were not uniformly distributed within the bed. Most of the cells were immobilized in the lower part of the bed and this resulted in decreased ethanol productivity. By using an external loop bioreactor, constructing the fixed bed with cylindrical loofa sponges, dividing the bed into upper, middle and lower sections with approximately 1 cm spaces between them and circulating the broth through the loop during the immobilization, uniform cell distribution within the bed was achieved. Using this method, the system was scaled up to 50 l and when compared with the 2 l bubble column bioreactor, there were no significant differences (P > 0.05) in ethanol productivity and yield. By using external loop bioreactor to immobilize the cells uniformly on the loofa sponge beds, efficient large scale ethanol production systems can be constructed.     

Ethanol fermentation in an immobilized cell reactor using Saccharomyces cerevisiae

Fermentation of sugar by Saccharomyces cerevisiae, for production of ethanol in an immobilized cell reactor (ICR) was successfully carried out to improve the performance of the fermentation process. The fermentation set-up was comprised of a column packed with beads of immobilized cells. The immobilization of S. cerevisiae was simply performed by the enriched cells cultured media harvested at exponential growth phase. The fixed cell loaded ICR was carried out at initial stage of operation and the cell was entrapped by calcium alginate. The production of ethanol was steady after 24 h of operation. The concentration of ethanol was affected by the media flow rates and residence time distribution from 2 to 7 h. In addition, batch fermentation was carried out with 50 g/l glucose concentration. Subsequently, the ethanol productions and the reactor productivities of batch fermentation and immobilized cells were compared. In batch fermentation, sugar consumption and ethanol production obtained were 99.6% and 12.5% v/v after 27 h while in the ICR, 88.2% and 16.7% v/v were obtained with 6 h retention time. Nearly 5% ethanol production was achieved with high glucose concentration (150 g/l) at 6 h retention time. A yield of 38% was obtained with 150 g/l glucose. The yield was improved approximately 27% on ICR and a 24 h fermentation time was reduced to 7 h. The cell growth rate was based on the Monod rate equation. The kinetic constants (K(s) and mu(m)) of batch fermentation were 2.3 g/l and 0.35 g/lh, respectively. The maximum yield of biomass on substrate (Y(X-S)) and the maximum yield of product on substrate (Y(P-S)) in batch fermentations were 50.8% and 31.2% respectively. Productivity of the ICR were 1.3, 2.3, and 2.8 g/lh for 25, 35, 50 g/l of glucose concentration, respectively. The productivity of ethanol in batch fermentation with 50 g/l glucose was calculated as 0.29 g/lh. Maximum production of ethanol in ICR when compared to batch reactor has shown to increase approximately 10-fold. The performance of the two reactors was compared and a respective rate model was proposed. The present research has shown that high sugar concentration (150 g/l) in the ICR column was successfully converted to ethanol. The achieved results in ICR with high substrate concentration are promising for scale up operation. The proposed model can be used to design a lager scale ICR column for production of high ethanol concentration.     

Practical reactor systems for yeast cell immobilization using biomass support particles

The technique of cell immobilization using porous support particles (biomass support particles) has been successfully applied to yeast cells. Two reactor configurations exploiting the use of these particles have been developed and assessed for use in aseptic yeast fermentations. A liquid-fluidized bed fermenter has been devised for use with particles denser than the fermentation liquor whilst a gas-stirred circulating bed fermenter proved suitable for particles of essentially neutral buoyancy. Both systems have been operated successfully for extended periods of continuous operation. The utilization of biomass support particle technology in such reactors provides a practical and robust system for immobilized cell reactors. This technology offers significant opportunities for further development.     

Parameter oscillation attenuation and mechanism exploration for continuous VHG ethanol fermentation

A bioreactor system composed of a stirred tank and three tubular bioreactors in series was established, and continuous ethanol fermentation was carried out using a general Saccharomyces cerevisiae strain and a very high gravity medium containing 280 g L(-1) glucose, supplemented with 5 g L(-1) yeast extract and 3 g L(-1) peptone. Sustainable oscillations of glucose, ethanol, and biomass were observed when the tank was operated at the dilution rate of 0.027 h(-1), which significantly affected ethanol fermentation performance of the system. After the tubular bioreactors were packed with 1/2' Intalox ceramic saddles, the oscillations were attenuated and quasi-steady states were achieved. Residence time distributions were studied for the packed bioreactors by the step input response technique using xylose as a tracer, which was added into the medium at a concentration of 20 g L(-1), indicating that the backmixing alleviation assumed for the packed tubular bioreactors could not be established, and its contribution to the oscillation attenuation could not be verified. Furthermore, the role of the packing's yeast cell immobilization in the oscillation attenuation was investigated by packing the tubular bioreactors with packings with significant difference in yeast cell immobilization effects, and the experimental results revealed that only the Intalox ceramic saddles and wood chips with moderate yeast cell immobilization effects could attenuate the oscillations, and correspondingly, improved the ethanol fermentation performance of the system, while the porous polyurethane particles with good yeast cell immobilization effect could not. And the viability analysis for the immobilized yeast cells illustrated that the extremely lower yeast cell viability within the tubular bioreactors packed with the porous polyurethane particles could be the reason for their inefficiency, while the yeast cells loosely immobilized onto the surfaces of the Intalox ceramic saddles and wood chips could be renewed during the fermentation, guaranteeing their viability and making them more efficient in attenuating the oscillations. The packing Raschig rings without yeast cell immobilization effect did not affect the oscillatory behavior of the tubular bioreactors, further supporting the role of the yeast cell immobilization in the oscillation attenuation.     

Operational optimization of a turbine blade reactor using macroporous support and its application to rice callus regeneration

Optimal operation condition was investigated for immobilized rice callus culture using a turbine blade reactor (TBR²) with polyurethane foam supports. By using polyurethane foam block as immobilization support, the inhibition of cell growth at a high agitation speed was avoided because the hydrodynamic stress against immobilized cell was probably reduced. Experimental results in each operational condition were assessed by means of rice callus growth, immobilization ratio in TBR and those regeneration frequencies in regeneration culture using solid medium. Concerning with pore size of polyurethane foam and support size, three-millimeter cube support of polyurethane foam with an average pore size of 1.3 mm was the most suitable support. The maximum immobilization ratio was 50% under 5% support volume by volume of growth medium. For improving the immobilization ratio of rice callus in the TBR, the optimum TBR operation and modification were investigated further. By repeating a periodic operation 3 times (agitating at 300 rpm for 5 min and then 50 rpm for 2 min, and then 200 rpm of constant agitation speed during the remaining time), almost all supports could entrap rice callus and homogeneous immobilization was attained. The immobilization ratio was improved as compared with that using a constant operation at 200 rpm. Next, the TBR was modified by setting an air sparger inside the stainless mesh cylinder. In the modified TBR, the floating support by air bubbles was reduced, and the immobilization ratio increased further and reached 86.3% when we increased the support volume to 15% under periodic operation on a daily basis. The regeneration frequency of immobilized callus was also slightly increased by periodic operation and modification of the TBR.     

Cell immobilization in kappa-carrageenan for ethanol production

A combination of extended Monod kinetics and the diffusional equation was used for evaluating the effectiveness factor of entrapped immobilized cells. Based on the kinetics of Zymomonas mobilis reported in the literature, the numerical results have revealed that the problem of mass transfer diffusional restrictions can be neglected by using small beads (1 mm in diameter) with a corresponding cell loading up to 276 g/L gel. On the basis of the numerical results obtained, the application of immobilized cells for continuous ethanol production was investigated. The kappa-carrageenan method was utilized to entrap Z. mobilis CP4, a potential ethanol producer. A two stage fermentation process has also been developed for ethanol production by the Z. mobilis carrageenan-bound cells. About 90 g/L ethanol was produced by immobilized cells at a total residence time of 1.56 h. The ethanol yield was estimated to be 93% of theoretical. The results obtained in this study also indicated that the control of optimum pH in an immobilized cell column is necessary to enhance the rate of ethanol production.     

Effects of immobilization on growth, fermentation properties, and macromolecular composition of Saccharomyces cerevisiae attached to gelatin

The kinetic properties of Saccharomyces cerevisiae immobilized on crosslinked gelatin were found to be substantially different from those of the suspended yeast. Batch fermentation experiments conducted in a gradientless reaction system allowed comparison of immobilized cell and suspended cell performance. The specific rate of ethanol production by the immobilized cell was 40-50% greater than for the suspended yeast. The immobilized cells consumed glucose twice as fast as the suspended cells, but their specific growth rate was reduced by 45%. Yields of biomass from the immobilized cell population were lower at one-third the value for the suspended cells. Cellular composition was also affected by immobilization. Measurements of intracellular polysaccharide levels showed that the immobilized yeast stored larger quantities of reserve carbohydrates and contained more structural polysaccharide than did suspended cells. Flow cytometry was used to obtain. DNA, RNA, and protein frequency functions for immobilized and suspended cell populations. These data showed that the immobilized cells have higher ploidy than cells in suspension. The observed changes in immobilized cell metabolism and composition may have arisen from disturbance to the yeast cell cycle by the cell attachment, causing alterations in the normal pattern of yeast bud development, DNA replication, and synthesis of cell wall components.     

Continuous production of glucose-6-phosphate by immobilized Achromobacter butyri cells

Summary Whole cells of Achromobacter butyri OUT 8004 having polyphosphate glucokinase activity were immobilized in polyacrylamide gel. The immobilized cells were activated by organic solvents, especially acetone. The immobilization resulted in increased stability of polyphosphate glucokinase. Continuous high yield production of G-6-P from glucose and metaphosphate was performed with an immobilized cell column, which had a half-life of approximately 20 days.Abbreviations G-6-P glucose-6-phosphate - G-1-P glucose-1-phosphate - Cation-S stearyl trimethyl ammonium chloride - SDS sodium dodecyl sulfate - Tris tris(hydroxymethyl)-aminomethane; p-NPP, p-nitrophenyl phosphate - S.V. space velocity     

A batch reactor mass transfer kinetic model for immobilized biomass biosorption

Inactive cells of Rhizopus arrhizus have been immobilized into the form of particles of desirable particle size using a proprietary immobilization technique. The immobilized biomass particles are porous and are members of a new generation of biological origin adsorbents. The uranium adsorptive behavior of the biosorbent particles was modeled using a batch reactor mass transfer kinetic model of the biosorption process. The model successfully predicts the batch reactor adsorbate (uranium) concentration profiles and has provided significant insights on the way biosorbents function.     

Plant cell bioreactor for the production of protoberberine alkaloids from immobilized Thalictrum rugosum cultures

Cultured Thalictrum rugosum cells were immobilized using a glass fiber substratum previously shown to provide optimum immobilization efficiency based on spontaneous adhesion mechanisms. When cultivated in shake flasks, immobilized cells exhibited decreased growth and protoberberine alkaloid production rates in comparison to freely suspended cells. Since alkaloid production is growth associated in T. rugosum, the decreased specific production rate was a function of the slower growth rate. Cells immobilized on glass fiber mats appear to be amenable for extended culture periods. Maximum biomass and protoberberine alkaloid levels were maintained for at least 14 days in immobilized cultures. In contrast, fresh weight, dry weight, and total alkaloid content decreased in suspension cultures following the linear growth phase.Glass fiber mats were incorporated in to a 4.5-L plant cell bioreactor as horizontal disks supported on a central rod. Mixing in the reactor was provided by the combined actions of a magnetic impeller and a cylindrical sparging colum. fThe magnetic impeller and a cylindrical sparging column. The entire inoculum biomass of T. rougosum, introduced as suspension, was spontaneously immobilized with in 8h. During liner phase, the growth rate of bioreactor cultivated immobilized cells (mu = 0.06 day(-1)) was 50% that immobilized cell viability in both systems was determined to be similar. The increase in specific production of protoberberine alklodis was initially similar in bioreactor-and culture period. The increase in specific production of protoberberine alkaloids was initially similar in bioreactor-and shake-flask-cultivated immobilized cells. However, the maximum specific production of bioreactor grown cultures was lower. The scale up potential of an immobilization strategy based on the spontaneous adhesion of immobilization strategy based on the spontaneous adhesion of cultured plant cells to glass fiber is demonstrated.     

Immobilization of Saccharomyces cerevisiae for Ethanol Fermentation on γ-Alumina Particles using a Spray-Dryer

Saccharomyces cerevisiae was immobilized on γ-alumina particles with binder polymer by a spray-drying process. Batch fermentations of sucrose to ethanol were performed using the immobilized cells. The optimum pH was 4, and this helped to minimize microbial contamination and facilitate electrostatic attraction between the γ-alumina particles and the yeast cells. Addition of yeast extract resulted in high ethanol conversion. The reasons for differences between cellulose acetate phthalate (CAP) and styrene-maleic acid co-polymer (SMC) as binders on ethanol conversion were not apparent. The release of binder with the SMC from the γ-alumina was less than that from the CAP. Presoaking of γ-alumina particles in resin for binder solution before the immobilization using a spray-dryer was effective in achieving high ethanol conversion.     

Immobilized plant cell reactors

The use of plant cells for the production of biochemicals represents a new area of biotechnological exploration. The techniques envisioned for industrial processes are related to those developed for microorganisms and a strong emphasis should be placed on immobilized cell systems. This review examines the spectrum of products that are synthesized by higher plants and the immobilization techniques that are suited to entrap plant cells from suspension culture. Different reactor configurations are described. Both packed-bed reactors with alginate-entrapped cells and hollow-fibre cartridges with sequestered cells have utility for the continuous production of biochemicals.     

Optimization of accessible catalase activity in polyacrylamide gel-immobilized Saccharomyces cerevisiae

The permeabilization of Saccharomyces cerevisiae (baker's yeast), either before or after immobilization in polyacrylamide gel (PAG), has been examined as a means to increase the catalase activity of PAG-immobilized yeast cells. Prior permeabilization of the cells resulted in large losses of catalase activity during immobilization, but permeabilization after immobilization produced increases in the catalase activity of yeast/PAG particles. A dependence of the accessible catalase activity on the concentration of polyacrylamide in permeabilized yeast/PAG particles, and on the method of permeabilization of the immobilized cells, was observed. Optimal levels of stable catalase activity (1000-2000 IU/g PAG particles; ca. 5%-10% of total available activity) were obtained by immobilizing yeast cells (0.5 g wet cells/mL gel) in 10% (w/v) PAG, followed by permeabilization of the entrapped cells with either cetyltrimethylammonium bromide, Triton X-100 and one freeze-thaw, or five freeze-thaw cycles. (c) 1992 John Wiley & Sons, Inc.     

Immobilization of Bacillus amyloliquefaciens MBL27 cells for enhanced antimicrobial protein production using calcium alginate beads

Cell immobilization is one of the common techniques for increasing the overall cell concentration and productivity. Bacillus amyloliquefaciens MBL27 cells were immobilized in calcium alginate beads and it is a promising method for repeated AMP (antimicrobial protein) production. The present study aimed at determining the optimal conditions for immobilization of B. amyloliquefaciens MBL27 cells in calcium alginate beads and the operational stability for enhanced production of the AMP. AMP production with free and immobilized cells was also done. In batch fermentation, maximum AMP production (7300 AU (arbitrary units)/ml against Staphylococcus aureus) was obtained with immobilized cells in shake flasks under optimized parameters such as 3% (w/v) sodium alginate, 136?mM CaCl2 with 350 alginate beads/flask of 2.7-3.0?mm diameter. In repeated cultivation, the highest activity was obtained after the second cycle of use and approx. 94% production was noted up to the fifth cycle. The immobilized cells of B. amyloliquefaciens MBL27 in alginate beads are more efficient for the production of AMP and had good stability. The potential application of AMP as a wound healant and the need for development of economical methods for improved production make whole cell immobilization an excellent alternative method for enhanced AMP production.     

Immobilization of cultured Catharanthus roseus cells using a fibreglass substratum

Summary Cultured Catharanthus roseus cells were immobilized using geometrically identical needled fibreglass mats prepared with a range of surface coatings. The phenyl (PS), polyglycol (PG), aldehyde (CHO), alkyl (CTMS), and silanol (AW) coatings, along with the untreated glass (HC) surface, produced surfaces with a range of surface tensions. The immobilization efficiency of the substratum, measured as the percentage of cells immobilized, increased with increasing substratum surface tension in the order PS < PG < CHO < CTMS < AW < HC. The dependence of immobilization efficiency on substratum surface tension can be described using a thermodynamic model of adhesion that considers the extent of plant cell adhesion to be a function of the surface tensions of the substratum, the suspending liquid, and the plant cells. In addition, this dependence also demonstrates the fundamental role of adhesion in the immobilization process involving a glass fibre matrix. However, cell entrapment processes are also implicated. The untreated glass fibre substratum (HC), which demonstrated the greatest immobilization efficiency, was used for further characterization of the immobilization strategy. Maximum inoculum biomass was determined to be approximately 1.9 g cells (fresh weight)/g substratum (dry weight) to achieve greater than 90% immobilization efficiency. The growth rate of immobilized cultures was slower than suspension cultures, probably due to mass transfer limitations. Production of the indole alkaloids, tryptamine, catharanthine, and ajmalicine, was also suppressed relative to suspension-cultured cells. These results are considered in relation to other immobilization strategies and their apparent effects on cellular processes. Offprint requests to: F. Dicosmo     

Bioreactors for surface-immobilized cells

Surface immobilization of plant cells avoids the problem of hydrodynamic or shear stress, which tends to be characteristic of suspended cells cultured in typical, mechanically agitated bioreactor systems. Surface immobilization also promotes the natural tendency for plant cells to aggregate, which may improve the synthesis and accumulation of secondary metabolites. In addition, exchange of medium is made simple in surface-immobilized systems, and extracellular secondary products are easily recovered on a continuous basis. However, problems related to regulation of the thickness of the immobilized cell layer, maintenance of the biomass in a productive condition, and vacuolar retention of secondary products have yet to be resolved satisfactorily. This review focusses on two surface-immobilization technologies, differing primarily in the nature and the configuration of the inert support. Prototypes of these designs have been applied to a variety of plant cell systems at bioreactor volumes up to 20 litres. Results obtained with several alternative technologies are also summarized.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - SIPCB surface-immobilized plant cell bioreactor National Research Council of Canada publication no. 38460     

Cell immobilization technique for the enhanced production of alpha-galactosidase by Streptomyces griseoloalbus

Streptomyces griseoloalbus was immobilized in calcium alginate gel and the optimal immobilization parameters (concentrations of sodium alginate and calcium chloride, initial biomass and curing time) for the enhanced production of alpha-galactosidase were determined. The immobilization was most effective with 3% sodium alginate and 0.1M calcium chloride. The optimal initial biomass for immobilization was approximately 2.2g (wet wt.). The alginate-entrapped cells were advantageous because there was a twofold increase in the enzyme yield (55 U/ml) compared to the highest yield obtained with free cells (23.6 U/ml). Moreover, with immobilized cells the maximum yield was reached after 72 h of incubation in batch fermentation under optimal conditions, whereas in the case of free cells the maximum enzyme yield was obtained only after 96 h of incubation. The alginate beads had good stability and also retained 75% ability of enzyme production even after eight cycles of repeated batch fermentation. It is significant that this is the first report on whole-cell immobilization for alpha-galactosidase production.