Continuous production of ethanol using immobilized growing yeast cells

Summary Immobilized growing yeast cells were prepared in kappa-carra-geenan gel. Gel beads containing a small number of cells were incubated in a complete medium. The cells grew very well in the gel and the number of living cells per ml of gel increased to over 10 times that of free cells per ml of culture medium. After growing in the gel, the cells formed a dense layer of cells near the gel surface and produced large amounts of ethanol. The conditions for continuous production of ethanol using immobilized growing yeast cells were investigated. The supply of appropriate nutrients for growth was essential for the continuous production. The living cells in the gel were maintained at the high level of 10⁹ per ml of gel and continuous production of ethanol using the complete medium containing 10% glucose was carried out with a retention time of 1 h. In this operation, a stable steady state was maintained for longer than 3 months. The ethanol concentration was 50 mg/ml and the conversion of glucose utilized to ethanol produced was almost 100% of the theoretical yield.     

Reaction characteristics of an immobilized yeast producing ethanol

The reaction behavior of Saccharomyces formaosensis imobilized by polyacrylamide gel is presented. Two types of the immobilized yeast are studied, i. e. the immobilized resting yeast and the immobilized growing yeast. For both of the yeast, reaction retes are expressed by the Michaelis-Menten type equation with a linear ethanol inhibition factor. The Michaelis constants aere close each other, but considerably larger that of native S. cerevisiae. Distribution of the growing yeast cell inside the carrier gel is presented. It is found that the cell density is somewhat higher near the surface of the carrier.     

Biosensing and monitoring of cell populations using the hydrogel bacterial microchip

Advanced development of the hydrogel bacterial microchip (HBMChip) technique is proposed. The microchip represents an array of hemispherical gel elements 0.3-60 nl in volume attached to hydrophobic glass surface and containing live immobilized microbial cells. Separate gel elements contain each up to 10(5) cells and retain them inside even while the cells are dividing. Porous structure of the gel provides easy access of nutrients and tested substances to the immobilized cells. Optical signals from the cells are easily measurable and allow monitoring of intracellular metabolism using vital fluorescent stains or engineered constructs encoding bioluminescent or fluorescent reporters. Two possible application modes of the HBMChip have been investigated, i.e. the observation of bacteria and biosensing. The dynamics of nucleic acids synthesis in growing E. coli cells has been analyzed using vital fluorescent stain SYTO 9. A special function has been suggested for evaluation of the cell growth parameters. Biosensing properties of the HBMChip have been illustrated by quantitative analysis of antibiotics and the detection of sodium meta-arsenite.     

Continuous production ofL-isoleucine using immobilized growing Serratia marcescens cells

An immobilized growing cell system was applied to the continuous L -isoleucine production by Serratia marcescens. In the new immobilized-cell systems using the carrageenan gel method. S. marcescens cells in the gel required nutrients and oxygen for growth, and the numbers of living cells per milliliter of gel increased to the levels of that of free cells in the liquid medium. This immobilized growing cell system exhibited high and stable activity for isoleucine production under steady-state conditions. Continuous isoleucine production was carried out by feeding the nutrient medium under aeration into a fluidized bed reactor containing the immobilized cells. In the continuous operation, an efficient production was maintained by automatically controlling the pH of the reaction mixture at 7.5. The productivity of isoleucine increased using multibed reactors. In a two-bed reactor system, the effluent L -isoleucine concentration reached 4.5 mg/ml at a retention time of 10 hr, and a steady state was maintained for longer than 30 days.     

Production of alkaline phosphatase by immobilized growing cells of Escherichia coli excretory mutants

Summary In continuous cultures, alkaline phosphatase was synthesised and excreted for more than 250 h by immobilized growing cells in contrast to free cells for which the excretion decreased after 150 h of culture. This observed increase in alkaline phosphatase synthesis and excretion by immobilized cells may have resulted from growing conditions within the gel beads.Offprint requests to: C. Manin     

Protection of bacteria against toxicity of phenol by immobilization in calcium alginate

Summary The antibacterial activity of phenol was determined by measuring inhibition of exponentially growing free and immobilized cells of Escherichia coli, Pseudomonas putida and Staphylococcus aureus. Immobilization of microorganisms in calcium alginate beads reduced the growth inhibition caused by bacteriostatic concentrations of phenol. The increase in phenol tolerance occurred at different culture conditions and growth rates of the cells. The strength of the effect, however, was found to correlate with the formation of colonies in the gel matrix. Dissolution of gel beads led to a substantial loss of the protection against phenol of immobilized-grown cells.     

Dynamic modeling of immobilized cell reactor: application to ethanol fermentation

A mathematical model has been developed for the unsteady-state operation of an immobilized cell reactor. The substrate solution flows through a mixed-flow reactor in which cells immobilized in gel beads are retained. The substrate diffuses from the external surface of the gel beads to some internal location where reaction occurs. The product diffuses from the gel beads into liquid medium which flows out of the reactor. The model combines simultaneous diffusion and reaction, as well as cell growth, and it can predict how the rates of substrate consumption, product formation, and cell growth vary with time and with initial conditions. Ethanol fermentation was chosen as a representative reaction in the immobilized cell reactor, and numerical calculations were carried out. Excellent agreement was observed between model predictions and experimental data available in the literature.     

Removal of U and Mo from water by immobilized Desulfovibrio desulfuricans in column reactors

Intact cells of Desulfovibrio desulfuricans were immobilized in polyacrylamide gel and used to remove soluble U and Mo from water by enzymatically mediated reduction reactions in column reactors. Formate or lactate served as the electron donor and oxidized U(VI) and Mo(VI) species served as electron acceptors. Greater than 99% removal efficiencies were achieved for both metals with initial concentrations of 5 mg/L U and 10 mg/L Mo. Hydraulic residence times in the columns were between 24 and 36 h. Sulfate concentrations as high as 2000 mg/L did not inhibit reduction of U or Mo in the columns. However, nitrate inhibited uranium reduction at concentrations near 50 mg/L and inhibited molybdenum reduction at concentrations near 150 mg/L. The results indicate that enzymatic reduction of U and Mo by immobilized cells of D. desulfuricans may be a practical method for removing these contaminants from solution in continuous-flow reactors.     

Product inhibition of immobilized Escherichia coli arising from mass transfer limitation

Mass transfer-limited removal of metabolic products led to product-inhibited growth of Escherichia coli that was immobilized in a model system. Comparison of the growth kinetics of immobilized and free-living cells revealed no further physiological differences between cells in these two modes of existence beyond those manifested in the local concentrations of substrate and product. Bacteria were retained on a microporous membrane in a dense, planar aggregate and were grown anaerobically on a glucose-based minimal medium. Radioisotope labeling of the immobilized cell mass with 35S was used to determine growth kinetic parameters. Growth rates in the immobilized cell layer were measured by an autoradiographic technique which allowed comparison of the size of the growing region with the rate of cell convection caused by growth. Immobilized cell growth rates and growth yields ranged from near maximal (0.56 h-1 and 39 g of dry cell weight/mol of glucose, respectively) to substantially reduced (0.15 h-1 and 15 g/mol). The depression of these kinetic parameters was attributed to product inhibition arising from mass transfer-limited removal of acidic waste products from the cell mass. A simple one-dimensional reaction-diffusion model, which incorporated data on the product-inhibited growth kinetics of free-living cells collected in a product-limited chemostat, satisfactorily predicted product inhibition of immobilized cell growth.     

Product inhibition of immobilized Escherichia coli arising from mass transfer limitation.

Mass transfer-limited removal of metabolic products led to product-inhibited growth of Escherichia coli that was immobilized in a model system. Comparison of the growth kinetics of immobilized and free-living cells revealed no further physiological differences between cells in these two modes of existence beyond those manifested in the local concentrations of substrate and product. Bacteria were retained on a microporous membrane in a dense, planar aggregate and were grown anaerobically on a glucose-based minimal medium. Radioisotope labeling of the immobilized cell mass with 35S was used to determine growth kinetic parameters. Growth rates in the immobilized cell layer were measured by an autoradiographic technique which allowed comparison of the size of the growing region with the rate of cell convection caused by growth. Immobilized cell growth rates and growth yields ranged from near maximal (0.56 h-1 and 39 g of dry cell weight/mol of glucose, respectively) to substantially reduced (0.15 h-1 and 15 g/mol). The depression of these kinetic parameters was attributed to product inhibition arising from mass transfer-limited removal of acidic waste products from the cell mass. A simple one-dimensional reaction-diffusion model, which incorporated data on the product-inhibited growth kinetics of free-living cells collected in a product-limited chemostat, satisfactorily predicted product inhibition of immobilized cell growth.     

Continuous production of L-arginine using immobilized growing Serratia marcescens cells: Effectiveness of supply of oxygen gas

Summary The immobilized growing cell system using Serratia marcescens was applied to continuous L-arginine production. From the determination of oxygen uptake rate, it was shown that the cells entrapped in carrageenan gel were in an oxygen-limited state due to the diffusion barrier to oxygen transport created by the gel layer. This limited state in gel was relieved by supply of oxygen-enriched gas instead of air into the medium. The maximum population of immobilized cells increased to five times that of free cells with the supply of pure oxygen gas. The L-arginine-producing activity of the immobilized growing cells was proportional to the concentration of oxygen gas supplied and was 6 mg/h per millilitre in gel supplied with pure oxyges gas. The continuous L-arginine containing production was constantly maintained by controlling the medium penicillin G at pH 6.5 and more than 10 mg/ml of L-arginine were obtained at 10h of residence time for at least 12 days.     

Continuous production of l-serine by immobilized growing Corynebacterium glycinophilum cells

Summary Auxotrophic mutant cells of Corynebacterium glycinophilum with high l-serine production activity were immobilized by entrapment with various gel materials, such as synthetic prepolymers and natural polysaccharides. The entrapped cells were used for estimation of l-serine productivity in a medium supplemented with glycine as a precursor. Based on the above criteria, including cell growth in gels and cell leakage from gels, calcium alginate was the most suitable gel material. Continuous l-serine fermentation with calcium alginate-entrapped growing cells was successfully achieved in an air-bubbled reactor for at least 13 days.     

Continuous blue pigment formation by gardenia fruit using immobilized growing cells

Bacillus subtilis no. 24 was used as a microorganism which hydrolyzes geniposide and forms a blue pigment. This microorganism possessed β-glucosidase activity during aerobic growth (log phase) and assimilated geniposide as a carbon source. The growth of this cell was depressed by genipin formed by the hydrolysis of geniposide. Blue pigment was formed continuously for 20 d in medium containing geniposide, yeast extract and Polypepton, using growing cells immobilized in calcium-alginate gel.     

A new immobilized cell system with protection against toxic solvents

A new immobilized cell system providing protection against toxic solvents was investigated so that normal fermentations could be carried out in a medium containing toxic solvents. The system consists of immobilized growing cells in Ca-alginate gel beads to which vegetable oils, which are inexpensive absorbents of solvents, had been added. The ethanol fermentation of Saccharomyces cerevisiae ATCC 26603 was used as a model fermentation to study the protection afforded by the system against solvent toxicities. The fermentation was inhibited by solvents such as 2-octanol, benzene, toluene, and phenol. Ethanol production of one batch was not finished even after 35 h using immobilized growing yeast cells in conventional Ca-alginate gel beads in an ethanol production medium (5% glucose) containing 0.1% 2-octanol, which is used as a solvent for liquid-liquid extraction and is one of the most toxic solvents in our experiments. With the new immobilized growing cell system using vegetable oils, however, four repeated batch fermentations were completed in 35 h. Castor oil provided even more protection than soy bean, olive, and tung oils, and it was possible to complete six repeated batches in 35 h. The immobilized cell system with vegetable oils also provided protection against other toxic solvents such as benzene and toluene. A possible mechanism for the protective function of the new immobilized cell system is discussed.     

Theoretical and methodological studies of continuous microbial bioreactors

This article reviews most of the author's studies on process development and reactor design for continuous microbial reactions. (1) Enzyme reactions of growing and non-growing microbial cells immobilized in agar gel beads were analyzed pertaining to the effects of external and internal diffusion of substrate on reaction kinetics. (2) Experimental correlations of production rates of beta-fructosidase and acid phosphatase with dilution rate of continuous culture were simulated based on an operon model for enzyme regulation. (3) Population dynamics of an amylase-producing bacteria and their mutant were discussed in relation to enzyme productivity in a continuous culture of spore-forming bacteria. (4) Plasmid mobilization in a mixed population of donor, recipient, and helper cells was investigated in a continuous culture as a model study of accidental release of a genetically modified plasmid into a natural environment. (5) A production rate increase of up to 100-fold was achieved by cell-recycle culturing of continuous acetic acid fermentation using a filter module with a hollow fiber membrane. (6) The feasibility of a continuous surface culture for the biooxidation of organic substances was ascribed to an enhanced oxygen absorption rate in the presence of a microbial film on a liquid surface. (7) Simultaneous separation of inhibitory products using an electrodialysis module during some organic acid fermentations was effective for increasing production in a continuous culture.     

Continuous production of ethanol in high concentration using immobilized growing yeast cells

Summary Application of an immobilized growing yeast cell system to continuous production of ethanol in high concentration (10%) was investigated using Saccharomyces cerevisiae IFO 2363. When a medium containing 25% glucose was fed, the growth of yeast cells in gel was inhibited. The inhibitory effect was found to be reduced by a stepwise increase in concentration of glucose in the feed medium. The stepwise operation resulted in constant growth of cells in the gel even in the medium containing 25% glucose. By this stepwise feeding system, continuous production of ethanol of 114 mg/ml was maintained at a retention time of 2.6 h for over 2 months and a conversion rate of glucose to ethanol of over 95% of theoretical, was achieved.     

Analysis of Distributed Growth of Saccharomyces cerevisiae Cells Immobilized in Polyacrylamide Gel

A technique is described for the quantitative determination of the distributed growth of Saccharomyces cerevisiae immobilized in polyacrylamide gel. Gel specimens were embedded in paraffin or gelatin and paraffin before sectioning and staining. Photomicrographs of specimen sections were enlarged, and cell microcolony volumes were determined as a function of position in the gel by grid transparency analysis. Overall cell densities within the gel were calculated for a quantitative comparison with values measured by a second spectrophotometric method. The results show good agreement and demonstrate the sigmoidal growth of the immobilized cells, reaching a maximum steady-state value. The technique shows promise as a general method for following the transient growth of organisms immobilized within gel particles.     

Growth behavior of immobilized acetic acid bacteria

Summary Released free cells of immobilized growing acetic acid bacteria during 460 days of long run in a bioreactor were increased at a high dilution rate. This was interpreted by the facts that newly released cells from the carrier showed a synchronized growth and had extremely high growth rates and a respiratory activity. The suitable leakage is effective for high productivity of the immobilized growing cell culture.     

Properties of Trichoderma reesei cells immobilized by the irradiation technique

Aerobic cells of Trichoderma reesei have been immobilized by the radiation polymerization technique using fibrous substances and hydroxyethyl methacrylate. The enzyme [cellulase, 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] productivity and growth of the cells in the immobilized growing cells have been studied. The enzyme (filter paper) activity in the immobilized cells was comparable to that of the intact cells, showing that the cells immobilized with fibrous materials grow and become adhered to the surface of the fibrils. The filter paper activity of the immobilized cells was affected mainly by monomer concentration and the content of the fibrous materials, as well as the irradiation dose. It was demonstrated that in repeated batch culture of the immobilized cells the filter paper activity gave a constant value, and leakage of the cells was not observed.     

New method for the characterization of the microbial growth in carrageenan gel

A method for the analysis of distributed growth of E. coli in k-carrageenan gel is described. The method uses optical and electron microscopy to determine morphometric data of cell microcolonies in the gel, such as average microcolony volume, distance of microcolonies to gel surface and cell density in the microcolonies. Logarithmic equations describing the distribution of authentic values of volume occupied by cells in gels at different times of incubation were determined. With these equations it is possible to predict the quantitative distribution of cells in the gel and to define some parameters. These parameters can characterize the growth behaviour of the microbial strains in any particular culture condition and can be useful in the future search to improve the industrial applications of immobilized cells.