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.     

Ethanol fermentation by immobilized cells in a trickle bed reactor

A modified discontinuous packed bed reactor with CO₂ ventilation ports, resembling a trickle bed reactor was employed to overcome gas holdup and bed compaction problems which are commonly encountered in cell immobilized packed bed reactors for ethanol fermentation. The reactor consisting of yeast cells entrapped in alginate matrix was operated by varying the substrate concentration, bed volume and inlet flow rates. The number of recirculation cycles (passes) and total stages were dependent upon the liquid flow rate, though the total contact time for complete conversion remains the same for a particular initial substrate level. The total contact time was 1.5, 3 and 4.5 h for initial substrate concentrations of 0.555, 0.933 and 1.3 kmol/m³ respectively. The number of cycles and in turn stages increased with the increase in initial sugar level. A graphical method for predicting the number of stages required for complete conversion was proposed based on material balance equation and evaluated for the operating variables of the present study. The reactor was operated continuously for 30 days producing 1.05– 1.15 kmol/m³.     

Production of ethanol by solid particles of Saccharomyces cerevisiae in a fluidized bed

Summary Carbon dioxide can be used as the fluid continuous phase for the fermentation of 10 to 40 % aqueous solutions of glucose into ethanol with Saccharomyces cerevisiae using a closed circuit consisting of a fluidized bed of small solid yeast particles, a cooled condenser for the sampling of water and ethanol and a blower. At 18°C, a fermentation of 12 moles glucose per min per g dry weight of yeast was achieved.     

Biodiesel production in a magnetically-stabilized, fluidized bed reactor with an immobilized lipase in magnetic chitosan microspheres

Biodiesel production by immobilized Rhizopus oryzae lipase in magnetic chitosan microspheres (MCMs) was carried out using soybean oil and methanol in a magnetically-stabilized, fluidized bed reactor (MSFBR). The maximum content of methyl ester in the reaction mixture reached 91.3 (w/v) at a fluid flow rate of 25 ml/min and a magnetic field intensity of 150 Oe. In addition, the MCMs-immobilized lipase in the reactor showed excellent reusability, retaining 82 % productivity even after six batches, which was much better than that in a conventional fluidized bed reactor. These results suggested that a MSFRB using MCMs-immobilized lipase is a promising method for biodiesel production.     

Ethanol fermentation of acid-hydrolyzed cellulosic pyrolysate with Saccharomyces cerevisiae

The acid hydrolysis of cellulosic pyrolysate to glucose and its fermentation to ethanol were investigated. The maximum glucose yield (17.4%) was obtained by the hydrolysis with 0.2 mol sulfuric acid per liter pyrolysate using autoclaving at 121 degrees C for 20 min. The fermentation by Saccharomyces cerevisiae of a hydrolysate medium containing 31.6 g/l glucose gave 14.2 g/l ethanol in 24 h, whereas the fermentation of the medium containing 31.6 g/l pure glucose gave 13.7 g/l ethanol in 18 h. The results showed that the acid-hydrolyzed pyrolysate could be used for ethanol production. Different nitrogen sources were evaluated and the best ethanol concentration (15.1 g/l) was achieved by single urea. S. cerevisiae (R) was obtained by adaptation of S. cerevisiae to the hydrolysate medium for 12 times, and 40.2 g/l ethanol was produced by S. cerevisiae (R) in the fermentation with the hydrolysate medium containing 95.8 g/l glucose, which was about 47% increase in ethanol production compared to its parent strain.     

Ethanol fermentation of acid-hydrolyzed cellulosic pyrolysate with Saccharomyces cerevisiae

Acid-hydrolysis of cellulosic pyrolysate to glucose and its fermentation to ethanol were investigated. The maximum glucose yield (17.4%) was obtained by the hydrolysis with 0.2 mol/l sulfuric acid using autoclaving at 121 degrees C for 20 min. The fermentation by Saccharomyces cerevisiae of a hydrolysate medium containing 31.6 g/l glucose gave 14.2 g/l ethanol after 24 h, whereas the fermentation of the medium containing 31.6 g/l pure glucose gave 13.7 g/l ethanol after 18 h. The results showed that acid-hydrolyzed pyrolysate could be used for ethanol production. Different nitrogen sources were evaluated and the best ethanol concentration (15.1 g/l) was achieved by single urea. S. cerevisiae (R) was obtained by adaptation of S. cerevisiae to the hydrolysate medium for 12 times, and 40.2 g/l ethanol was produced by it in the fermentation with the hydrolysate medium containing 95.8 g/l glucose, which was about 47% increase in ethanol production compared to its parent strain.     

Ethanol production from sweet sorghum juice in repeated-batch fermentation by Saccharomyces cerevisiae immobilized on corncob

Ethanol fermentation from sweet sorghum juice containing 240 g/l of total sugar by Saccharomyces cerevisiae TISTR 5048 and S. cerevisiae NP 01 immobilized on low-cost support materials, corncob pieces, was investigated. In batch fermentation, S. cerevisiae TISTR 5048 immobilized on 6 × 6 × 6 mm³ corncobs gave higher ethanol production than those immobilized on 12 × 12 × 12 mm³ corncobs in terms of ethanol concentration (P), yield (Y _p/s ) and productivity (Q _p ) with the values of 102.39 ± 1.11 g/l, 0.48 ± 0.01 and 2.13 ± 0.02 g/l h, respectively. In repeated-batch fermentation, the yeasts immobilized on the 6 × 6 × 6 mm³ corncobs could be used at least eight successive cycles with the average P, Y _p/s and Q _p of 97.19 ± 5.02 g/l, 0.48 ± 0.02 and 2.02 ± 0.11 g/l h, respectively. Under the same immobilization and repeated-batch fermentation conditions, P (90.75 ± 3.05 g/l) and Q _p (1.89 ± 0.06 g/l h) obtained from S. cerevisiae NP 01 were significantly lower than those from S. cerevisiae TISTR 5048 (P < 0.05), while Y _p/s from both strains were not different. S. cerevisiae TISTR 5048 immobilized on the corncobs also gave significantly higher P, Y _p/s and Q _p than those immobilized on calcium alginate beads (P < 0.05).     

Isopropanol biodegradation by immobilized Paracoccus denitrificans in a three-phase fluidized bed reactor

A three-phase bed bioreactor including a mix of immobilized microbes was used to degrade isopropanol (IPA). The immobilization method was studied and cells immobilized with calcium alginate, polyvinyl alcohol, activated carbon, and SiO₂ were demonstrated to be the best immobilization method for the degradation of 90% of 2?g/L IPA in just 4 days, 1 day earlier than with free cells. Acetone was monitored as an indicator of microbial IPA utilization as the major intermediate of aerobic IPA biodegradation. The bioreactor was operated at hydraulic retention time (HRT) values of 32, 24, 16, 12, and 10?hr, which correspond to membrane fluxes of 0.03, 0.04, 0.06, 0.08, and 0.10?L/m²/hr, respectively. The chemical oxygen demand (COD) removal efficiencies were maintained at 98.0, 97.8, 89.1, 80.6, and 71.1% at a HRT of 32, 24, 16, 12, and 10?hr, respectively, while the IPA degradations were 98.6, 98.3, 90.3, 81.6, and 73.3%, respectively. With a comprehensive consideration of COD removal and economy, the optimal HRT was 24?hr. The results demonstrate the potential of immobilized mixed bacterial consortium in a three-phase fluidized bed reactor system for the aerobic treatment of wastewater containing IPA.     

Citric acid production by immobilized Aspergillus niger in a fluidized bed reactor

Summary Citric acid production by immobilized of Aspergillus niger in a fluidized bed reactor was performed, evaluating the productivity and the stability of the process when pulsing device was used. The application of a pulsing flow to fluidized bed reactor and the feed nitrogen limited allow to control of bioparticles morphology avoiding bed compactation. When operated at optimum pulsation frequency (0.3 s^–1) the stability of the bioreactor was maintained for more than 30 days, increasing the citric acid production in more than 52.2%.     

Studies on immobilized Saccharomyces cerevisiae. I. Analysis of continuous rapid ethanol fermentation in immobilized cell reactor

Rapid fermentation of cane molasses into ethanol has been studied in batch, continuous (free-cell and cell-immobilized systems) by a strain of Saccharomyces cerevisiae at temperature 30 degrees C and pH 5.0. The maximum productivity of ethanol obtained in immobilized system was 28.6 g L(-1) h(-1). The cells were immobilized by natural mode on a carrier of natural origin and retention of 0.132 g cells/g carrier was achieved. The immobilized-cell column was operated continuously at steady state over a period of 35 days. Based on the parameter data monitored from the system, mathematical analysis has been made and rate equations proposed, and the values of specific productivity of ethanol and specific growth rate for immobilized cells computed. It has been established that immobilized cells exhibit higher specific rate of ethanol formation compared to free cells but the specific growth rate appears to be comparatively low. The yield of ethanol in the immobilized-cell system is also higher than in the free-cell system.     

Modelling a solid-state fluidized bed fermenter for Ethanol production with S. cerevisiae

A model is proposed to describe the performance of a new type of fermenter for ethanol production, the fluidized bed gas-solid fermenter, with respect to scaling-up effects. Based on the fact that in the fluid bed the substrate is not supplied continuously to each particle, two scale-up parameters are derived, circulation time τ and specific substrate supply Δm _G,P , which are shown to influence reactor efficiency significantly. The validity of the model is checked by comparing the calculated yield coefficients for ethanol, cell mass and carbon dioxide to the results of fermentation experiments performed under aerobic conditions in a laboratory-scale reactor and a semi-technical fermenter.     

Ethanol fermentation by nystatin-resistant strains of Saccharomyces cerevisiae

Nystatin-resistant mutants of haploid and polyploid strains of Saccharomyces cerevisiae were isolated by plating on gradient plates with increasing nystatin concentrations (60-3000 U/ml). Some of the mutants were defective in ergosterol biosynthesis, and produced zymosterol and cholestatetraenol-like sterols. Those mutants which do not form ergosterol produce less ethanol than the parent strains. They also had lower viability during fermentation of glucose solutions (8-13% vs. 33-47%). This became more pronounced in fermentations of higher concentrations of glucose. A nystatin-resistant but ergosterol-forming mutant had a similar fermentation capacity to the parent strain.     

Ethanol fermentation by nystatin-resistant strains of Saccharomyces cerevisiae

Nystatin-resistant mutants of haploid and polyploid strains of Saccharomyces cerevisiae were isolated by plating on gradient plates with increasing nystatin concentrations (60–3000 U/ml). Some of the mutants were defective in ergosterol biosynthesis, and produced zymosterol and cholestatetraenol-like sterols. Those mutants which do not form ergosterol produce less ethanol than the parent strains. They also had lower viability during fermentation of glucose solutions (8–13% vs. 33–47%). This became more pronounced in fermentations of higher concentrations of glucose. A nystatin-resistant but ergosterol-forming mutant had a similar fermentation capacity to the parent strain.     

A novel magnetic adsorbent for immunoglobulin-g purification in a magnetically stabilized fluidized bed

A novel magnetic poly(ethylene glycol dimethacrylate-N-methacryloly-L-histidinemethylester) [m-poly(EGDMA-(MAH)] support was prepared for purification of immunoglobulin G (IgG) in a magnetically stabilized fluidized bed by suspension polymerization. Elemental analysis of the magnetic beads for nitrogen was estimated as 70 micromol MAH/g polymer. Magnetic poly(EGDMA-MAH) beads were used in the separation of immunoglobulin-G (IgG) from aqueous solutions and/or human plasma in a magnetically stabilized fluidized bed system. IgG adsorption capacity of the beads decreased with an increase in the flow rate. The maximum IgG adsorption was observed at pH 6.0 for MES buffer. IgG adsorption onto the m-poly(EGDMA) was negligible. Higher adsorption values (up to 262 mg/g) were obtained in which the m-poly(EGDMA-MAH) sorbents were used from aqueous solutions. Higher amounts of IgG were adsorbed from human plasma (up to 320 mg/g) with a purity of 87%. IgG molecules could be repeatedly adsorbed and desorbed with these sorbents without noticeable loss in their IgG adsorption capacity.     

Antibiotics production in a fluidized bed reactor utilizing a transverse magnetic field

A mathematical model is developed to describe the performance of a three-phase fluidized bed reactor utilizing a transverse magnetic field. The model is based on the axially dispersed plug flow model for the bulk of liquid phase and on the Michaelis-Menten kinetics. The model equations are solved by the explicit finite difference method from transient to steady state conditions. The results of the numerical simulation indicate that the magnetic field increases the degree of bioconversion. The mathematical model is experimentally verified in a three-phase fluidized bed reactor with Penicillium chrysogenum immobilized on magnetic beads. The experimental results are well described by the developed model when the reactor operates in the stabilized regime. At low and relatively high magnetic field intensities certain discrepancy in the model solution is observed when the model over estimates the product concentration.     

Countercurrent multistage fluidized bed reactor for immobilized biocatalysts: II. Operation of a laboratory-scale reactor

In Part I of this series,(1) we derived a model and made simulations for a multistage fluidized bed reactor (MFBR). It was concluded that the MFBR can be an attractive alternative for a fixed bed reactor when operated with a deactivating biocatalyst. In Part II of this series, the design of a laboratory-scale MFBR and its evaluation to investigate the practical feasibility of this reactor type, will be described. Experiments with a duration as long as 10 days were carried out successfully using immobilized glucose isomerase as a model reaction system. The results predicted by the model are in good agreement with the measured glucose concentration and biocatalyst activity gradients, indicating perfect mixing of the particles in the reactor compartments.The diameters of the biocatalyst particles used in the experiments showed a large spread, with the largest being 1.7 times the smallest. Therefore, an additional check was carried out, to make sure that the particles were not segregating according to size. Particles withdrawn from the reactor compartments were investigated using an image analyzer. Histograms of particle size distribution do not indicate segregation and it is concluded that the particles used have been mixed completely within the compartments. As a result, transport of biocatalyst is nearly plug flow.     

Ethanol production by immobilized Saccharomyces cerevisiae, Saccharomyces uvarum, and Zymomonas mobilis

Saccharomyces cerevisiae NRRL Y-2034, S, uvarum NRRL Y-1347, and Zymomonas mobilis NRRL B-806 each were separately immobilized in a Ca-alginate matrix and incubated in the presence of a free-flowing and continuous 1, 3, 5, 10, or 20% (w/w) glucose solution. In general, the yeast cells, converted 100percnt; of the 1, 3, and 5% glucose to alcohol within 48 h and maintained such a conversion rate for at least two weeks. The bacterium converted ca. 90% (w/w) of the 1, 3, and 5% glucose to alcohol continuously for one week. However, both the yeast and bacterium were inhibited in the highest glucose (20% w/w) solution. All of the immobilized cultures produced some alcohol for at least 14 days. Immobilized S. cerevisiae was the best alcohol producer of all of the glucose concentrations; the yeast yielded 4.7 g ethanol/100 g solution within 72 h in the 10% glucose solution. After 7-8 days in the 10% solution, S. cerevisiae produced ethanol at 100% of theoretical yield (5.0 g ethanol/100 g solution), with a gradual decrease in alcohol production by 14 days. Immobillized S. uvarum produced a maximum of 4.0 g ethanol/100 g solution within 2 days and then declined to ca. 1.0 g ethanol/100 g solution after 7 days continuous fermentation in the 10% glucose solution. Zymomonas mobilis reached its maximum ethanol production at 4 days (4.7 g/100 g solution), and then diminished similarly to S. uvarum. The development of a multiple disk shaft eliminated the problem both of uneven distribution of alginate-encapsulated cells and of glucose channeling within the continuous-flow fermentor column. This invention improved alcohol production about threefold for the yeast cells.