Kinetic Study of the Alcoholic Fermentation Process, in the Presence of Free and Immobilized Saccharomyces Cerevisiae Cells, at Different Initial Glucose Concentrations by Reversed Flow GC

Reversed flow gas chromatography (RFGC) was applied for the kinetic study of the alcoholic fermentation processes conducted with cells of the alcohol-resistant and psychrophilic Saccharomyces cerevisiae AXAZ-1 yeast strain, either free or immobilized on wheat, barley and corn grains as well as on potato pieces. Repeated alcoholic fermentations with must of varying initial glucose concentrations were performed in order to estimate the catalytic efficiency of the biocatalysts used in the present study. With the RFGC method, the distinction of the duration of alcoholic fermentation phases was achieved, which may be correlated to the phases of AXAZ-1 cells growth cycle. The rate constants of ethanol production for each phase of the alcoholic fermentations, conducted with free and immobilized cells, were also determined with the aid of RFGC, confirming the predominance of the immobilized against free cells in the fermentation process. Comparing the supports used for immobilization, wheat and barley grains seemed to be more efficient than corn grains and potato pieces, as they provided a higher number of immobilized cells and rate constant values.     

Development of continuous fermentation using immobilized yeast cells for wine cooler process development

The continuous wine fermentation process, which employs a newly designed tapered column type bioreactor and immobilized yeast cells (Montrachet 522), was studied and its fermentation performance was compared with batch and suspended cell continuous wine fermentation systems. It was found that a stable continuous culture fermentation process could be maintained for a period of 2–3 mo when the new bioreactor system packed with immobilized yeast cells was employed. The new bioreactor containing immobilized yeast cells performed significantly better than the suspended cell culture system or batch culture. The effluent wine from the continuous fermentor system contained 7.1% (v/v) ethanol and 0.18% (w/v) residual sugar at 0.01 h^-1 dilution rate. The new continuous bioreactor system also gave 17–34 times higher maximum ethanol productivity compared to the conventional batch wine fermentation. At a low dilution rate, 0.01^-1, as high as 92% sugar to ethanol yield was achieved. Based on the results obtained from this study, the possibility of developing a continuous wine cooler fermentation process was demonstrated. A two-stage continuous wine fermentation system may be designed and operated. The grape juice can be fed into the first-stage that is operated at about 0.2 h^-1 dilution rate and the effluent from the first-stage is fed into the second-stage continuous fermentor operated at about 0.01 h^-1 dilution rate. By doing so, a wine cooler can be produced continuously and efficiently, by employing the newly designed tapered column type bioreactor charged with the immobilized yeast cells.     

Ethanol Production by Fermentation Using Immobilized Cells of Saccharomyces cerevisiae in Cashew Apple Bagasse

In this work, cashew apple bagasse (CAB) was used for Saccharomyces cerevisiae immobilization. The support was prepared through a treatment with a solution of 3% HCl, and delignification with 2% NaOH was also conducted. Optical micrographs showed that high populations of yeast cells adhered to pre-treated CAB surface. Ten consecutive fermentations of cashew apple juice for ethanol production were carried out using immobilized yeasts. High ethanol productivity was observed from the third fermentation assay until the tenth fermentation. Ethanol concentrations (about 19.82–37.83 g L^?1 in average value) and ethanol productivities (about 3.30–6.31 g L^?1 h^?1) were high and stable, and residual sugar concentrations were low in almost all fermentations (around 3.00 g L^?1) with conversions ranging from 44.80% to 96.50%, showing efficiency (85.30–98.52%) and operational stability of the biocatalyst for ethanol fermentation. Results showed that cashew apple bagasse is an efficient support for cell immobilization aiming at ethanol production.     

Production of ethanol from waste paper using immobilized yeasts

This work is aimed at a selection of yeast strains suitable for simultaneous saccharification and fermentation of waste paper. The waste paper, as a lignocellulosic material, represents an unconventional source for the production of ethanol which is a promising alternative fuel. The yeast strains Saccharomyces cerevisiae and Pichia kudriavzevii produced the highest amounts of ethanol at 30 °C and were also resistant at 40 °C during the first 92 h of fermentation. These two strains were immobilized by entrapment into poly(vinyl alcohol) hydrogel lens-shaped particles LentiKats^®. The immobilized S. cerevisiae was a better ethanol producer and retained higher metabolic activity in repeated batch fermentations than P. kudriavzevii. The immobilized S. cerevisiae was also suitable for a long-term storage, with 23% decrease in the ethanol production ability after 1-year storage of yeast cells.     

Semiconductor gas sensors in bioreactor control

A simple semiconductor gas sensor (TGS 812) is used for the on-line measurement and control of indole during the production of l-tryptophan from indole and l-serine with immobilized E. coli cells. Indole is estimated in the reactor gas space. In combination with an automatic indole supply system, a feed-batch process became possible. The indole concentration was monitored and kept within the optimal range (300–600 mg l^?1). A simple gas-sensing electrode dipped in the reaction medium provides direct measurement of organic solvents and gases in the liquid. Such a system is suitable for on-line determination of ethanol (10–70 g l^?1) during continuous production of ethanol with immobilized yeast cells.     

The Use of Microporous Divinyl Benzene Copolymer for Yeast Cell Immobilization and Ethanol Production in Packed-Bed Reactor

Microporous divinyl benzene copolymer (MDBP) was used for the first time as immobilization material for Saccharomyces cerevisiae ATCC 26602 cells in a bed reactor and ethanol production from glucose was studied as a model system. A very homogenous thick layer of yeast cells were seen from the scanning electron micrographs on the outer walls of biopolymer. The dried weight of the cells was found to be approximately 2 g per gram of cell supporting material. Hydrophobic nature of polymer is an important factor increasing cell adhesion on polymer pieces. The dynamic flow conditions through the biomaterial due to its microporous architecture prevented exopolysaccharide matrix formation around cells and continuous washing out of toxic metabolites and dead and degraded cells from the reactor provided less diffusional limitations and dynamic living environment to the cells. In order to see the ethanol production performance of immobilized yeast cells, a large initial concentration range of glucose between 6.7 and 300 g/l was studied at 1 ml/min in continuous packed-bed reactor. The inhibition effect of glucose with increasing initial concentration was observed at above 150 g/l, a relatively high substrate concentration. The continuous fluid flow around the microenvironment of the attached cells and mass transferring ability of cell immobilized on MDBP can help in decreasing the inhibition effect of ethanol accumulation and high substrate concentration in the vicinity of the cells.     

Butanol production by Clostridium beijerinckii BA101 in an immobilized cell biofilm reactor

Acetone butanol ethanol was produced in a continuous immobilized cell (biofilm) plug-flow reactor inoculated with Clostridium beijerinckii BA101. To achieve high reactor productivity, C. beijerinckii BA101 cells were immobilized by adsorption onto clay brick. The continuous plug-flow reactor offers high productivities owing to reduced butanol inhibition and increased cell concentration. Although high productivity was achieved, it was at the expense of low sugar utilization (30.3%). To increase sugar utilization, the reactor effluent was recycled. However, this approach is complicated by butanol toxicity. The effluent was recycled after removal of butanol by pervaporation to reduce butanol toxicity in the reactor. Recycling of butanolfree effluent resulted in a sugar utilization of 100.7% in addition to high productivity of 10.2g/(L·h) at a dilution rate of 1.5 h⁻¹. A dilution rate of 2.0h⁻¹ resulted in a reactor productivity of 16.2g/(L·h) and sugar utilization of 101.4%. It is anticipated that this reactor-recovery system would be economical for butanol production when using C. beijerinckii BA101.     

Immobilization of growing cells by polyethyleneimine-modified alginate

A unique polymer matrix that is suitable for immobilizing growing cells has been developed. Alginate was chemically modified with polyethyleneimine (PEI), and the resultant polymer aggregate was evaluated as a cell carrier. Our method of immobilization depends on reversible gelation of the PEI-modified alginate. Our hypothesis is that immobilized cells grow by dissolving the surrounding gel matrix; the dissolved polymer adduct is displaced peripherally and gelled again by the influx of calcium ion from the surrounding fermentation broth, retaining both cells and carrier polymer in the gel beads. Thus, the immobilized cells gain space for growth by expanding the carrier matrix. The PEI modification offers the following advantages: (1) improved mechanical strength; (2) improved cell retention; (3) increased catalyst life; (4) ease of pelletization; and (5) an apparent bacteriostatic capability. When immobilized yeast cells were applied to a continuous ethanol fermentation, 94% theoretical conversion of glucose to ethanol was observed, with a reactor productivity of 15–30 g/L/h in a nonsterile reactor. A 3-mo catalyst life and minimal cell washout were observed.     

Effect of Acetic Acid on Saccharomyces Carlsbergensis ATCC 6269 Batch Ethanol Production Monitored by Flow Cytometry

Bioethanol produced from lignocellulosic materials has been considered a sustainable alternative fuel. Such type of raw materials have a huge potential, but their hydrolysis into mono-sugars releases toxic compounds such as weak acids, which affect the microorganisms' physiology, inhibiting the growth and ethanol production. Acetic acid (HAc) is the most abundant weak acid in the lignocellulosic materials hydrolysates. In order to understand the physiological changes of Saccharomyces carlsbergensis when fermenting in the presence of different acetic acid (HAc) concentrations, the yeast growth was monitored by multi-parameter flow cytometry at same time that the ethanol production was assessed. The membrane potential stain DiOC₆(3) fluorescence intensity decreased as the HAc concentration increased, which was attributed to the plasmic membrane potential reduction as a result of the toxic effect of the HAc undissociated form. Nevertheless, the proportion of cells with permeabilized membrane did not increase with the HAc concentration increase. Fermentations ending at lower external pH and higher ethanol concentrations depicted the highest proportions of permeabilized cells and cells with increased reactive oxygen species levels. Flow cytometry allowed monitoring, near real time (at-line), the physiological states of the yeast during the fermentations. The information obtained can be used to optimize culture conditions to improve bioethanol production.     

Characteristics of an Immobilized Yeast Cell System Using Very High Gravity for the Fermentation of Ethanol

The characteristics of ethanol production by immobilized yeast cells were investigated for both repeated batch fermentation and continuous fermentation. With an initial sugar concentration of 280?g/L during the repeated batch fermentation, more than 98% of total sugar was consumed in 65?h with an average ethanol concentration and ethanol yield of 130.12?g/L and 0.477?g ethanol/g consumed sugar, respectively. The immobilized yeast cell system was reliable for at least 10 batches and for a period of 28?days without accompanying the regeneration of Saccharomyces cerevisiae inside the carriers. The multistage continuous fermentation was carried out in a five-stage column bioreactor with a total working volume of 3.75?L. The bioreactor was operated for 26?days at a dilution rate of 0.015?h^?1. The ethanol concentration of the effluent reached 130.77?g/L ethanol while an average 8.18?g/L residual sugar remained. Due to the high osmotic pressure and toxic ethanol, considerable yeast cells died without regeneration, especially in the last two stages, which led to the breakdown of the whole system of multistage continuous fermentation.     

Comparison of Alcoholic Fermentation Performance of the Free and Immobilized Yeast on Water Hyacinth Stem Pieces in Medium with Different Glucose Contents

Ethanol fermentation with Saccharomyces cerevisiae cells was performed in medium with different glucose concentrations. As the glucose content augmented from 200 to 250 g/L, the growth of the immobilized cells did not change while that of the free cells was reduced. At higher glucose concentration (300, 350, and 400 g/L), the cell proliferation significantly decreased and the residual sugar level sharply augmented for both the immobilized and free yeast. The specific growth rate of the immobilized cells was 27–65 % higher than that of the free cells, and the final ethanol concentration in the immobilized yeast cultures was 9.7–18.5 % higher than that in the free yeast cultures. However, the immobilized yeast demonstrated similar or slightly lower ethanol yield in comparison with the free yeast. High fermentation rate of the immobilized yeast was associated with low unsaturation degree of fatty acids in cellular membrane. Adsorption of S. cerevisiae cells on water hyacinth stem pieces in the nutritional medium decreased the unsaturation degree of membrane lipid and the immobilized yeast always exhibited lower unsaturation degree of membrane lipid than the free yeast in ethanol fermentation.     

Lipase-catalyzed acylation in a continuous down-flow fixed-bed reactor

The kinetic resolution of racemic 1-phenylethanol with ethyl acetate was investigated in a down-flow fixed-bed reactor operated in a continuous mode mainly at the molar ratio of 1: 3 in 400 mL toluene at 70°C. The catalytic activity of the immobilized lipase was studied by: (i) changing the flow rates, (ii) utilizing different substrate concentrations, (iii) applying step changes using ethyl acetate, ethyl benzene, acetic acid, acetophenone etc., (iv) investigating the inhibitory effect of either the desired or the stoichiometric products (R)-1-phenylethyl acetate and ethanol, respectively), (v) elucidating the effect of water on the activity and stability of the immobilized lipase. The residence time distribution and the reactor hydrodynamics were also discussed along with kinetic modelling. The results were linked to the one-pot reactions.     

Kinetics of ethanol production from carob pods extract by immobilizedSaccharomyces cerevisiae cells

Kinetics of ethanol production from carob pods extract by immobilizedS. cerevisiae cells in static and shake flask fermentation have been investigated. Shake flask fermentation proved to be a better fermentation system for the production of ethanol than static fermentation. The optimum values of ethanol concentration, ethanol productivity, ethanol yield, and fermentation efficiency were obtained at pH range 3.5–6.5 and temperature between 30–35°C. A maximum ethanol concentration (65 g/L), ethanol productivity (8.3 g/Lh), ethanol yield (0.44 g/g), and fermentation efficiency (95%) was achieved at an initial sugar concentration of 200, 150, 100, and 200 g/L, respectively. The highest values of specific ethanol production rate and specific sugar uptake rate were obtained at pH 6.5, temperature 40°C, and initial sugar concentration of 100 g/L. Other kinetic parameters, biomass concentration, biomass yield, and specific biomass production rate were maximum at pH 5.5, temperature 30°C, and initial sugar concentration 150 g/L. Under the same fermentation conditions non-sterilized carob pod extract gave higher ethanol concentration than sterilized medium. In repeated batch fermentations, the immobilizedS. cerevisiae cells in Ca-alginate beads retained their ability to produce ethanol for 5 d.     

Yeasts and Lactic Acid Bacteria Mixed-Specie Biofilm Formation is a Promising Cell Immobilization Technology for Ethanol Fermentation

We previously found that some Saccharomyces cerevisiae and Lactobacillus plantarum remarkably formed mixed-specie biofilm in a static co-culture and deduced that this biofilm had potential as immobilized cells. We investigated the application of mixed-specie biofilm formed by S. cerevisiae BY4741 and L. plantarum HM23 for ethanol fermentation in repeated batch cultures. This mixed-specie biofilm was far abundantly formed and far resistant to washing compared with S. cerevisiae single biofilm. Adopting mixed-specie biofilm formed on cellulose beads as immobilized cells, we could produce enough ethanol from 10 or 20 % glucose during ten times repeated batch cultures for a duration of 10 days. Cell numbers of S. cerevisiae and L. plantarum during this period were stable. In mixed-specie biofilm system, though ethanol production was slightly lower compared to S. cerevisiae single-culture system due to by-production of lactate, pH was stably maintained under pH 4 without artificial control suggesting high resistance to contamination. Inoculated model contaminants, Escherichia coli and Bacillus subtilis, were excluded from the system in a short time. From the above results, it was indicated that the mixed-specie biofilm of S. cerevisiae and L. plantarum was a promising immobilized cell for ethanol fermentation for its ethanol productivity and robustness due to high resistance to contamination.     

Biotransformation from geraniol to nerol by immobilized grapevine cells (V. vinifera)

The abilities of two grapevine cell suspensions (Vitis vinifera L. cv. Gamay Fréaux andVitis vinifera L. cv. Monastrell) to biotransform geraniol into nerol in a biphasic system based on the culture medium and Miglyol 812 were compared. The Gamay grape cell suspension was able to transform higher concentrations of geraniol into nerol than the Monastrell one. Gamay grape cells were immobilized in both calcium alginate beads and polyurethane foams. The cytotoxic effect of increasing concentrations of geraniol, as well as the ability of the immobilized cells to biotransform geraniol into nerol, was checked. Immobilization proved to be advantageous in protecting cells against the toxicity of the substrate. Furthermore, immobilization also seemed to have an effect on the secondary metabolism, the cells immobilized in polyurethane foams being more efficient at performing the isomerization process (40% conversion of geraniol into nerol) than both the freely suspended and calcium alginate immobilized cells (20% conversion).     

Dynamic modeling of an immobilized cell reactor

A mathematical model has been developed to describe the dynamic aerobic reaction occurring in a semibatch type of mixed flow reactor, containing cells immobilized in gel beads. This modeling is an extension of that developed in our previous study, for an immobilized cell reactor involving ethanol fermentation. In contrast to anaerobic reactions such as ethanol fermentation, (wherein the influent substrate concentration can be set at any desired level), aeration becomes necessary to provide additional substrate (oxygen) for most aerobic reactions occurring in immobilized cell reactors. Tobacco cell cultivation was chosen as a representative aerobic reaction, and the effect of aeration was assessed in terms of the volumetric coefficient of oxygen from gas to liquid phases.     

Fermentation and costs of fuel ethanol from corn with quick-germ process

The Quick-Germ process developed at the University of Illinois at Urbana-Champaign is a way to obtain corn oil, but with lower capital costs than the traditional wet-milling process. Quick-Germ has the potential to increase the coproduct credits and profitability of the existing dry-grind fuel ethanol process, but the fermentability of the corn remaining after oil recovery has not been tested. Therefore, a series of pilot scale (50 L) fermentations was carefully controlled and monitored with unique methods for standard inoculation and automatic sampling. It was found that the concentration of suspended solids was significantly reduced in the Quick-Germ fermentations. When compared at the same concentration of fermentable sugars, the fermentation rate and yield were not statistically different from controls. When Quick-Germ was integrated into a state-of-the-art dry-grind fuel ethanol process, computer simulation and cost models indicated savings of approx $0.01/L of ethanol ($0.04/gal) with the Quick-Germ process. Additional savings associated with the lower suspended solids could not be quantified and were not included. However, the savings are sensitive to the price of corn oil. Mention of brand or firm name does not constitute an endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned.     

Fluorometric determination of ethanol in liquor samples by flow-injection analysis using an immobilized enzyme-reactor column with packing prepared by coupling alcohol oxidase and peroxidase onto chitosan beads

A flow-injection system was developed for the determination of ethanol with an immobilized enzyme-reactor column. This system, which consisted of hand-made reactor columns packed with alcohol oxidase and horseradish peroxidase immobilized onto chitosan beads, and a fluorometric detector, was applied to the determination of ethanol in liquor samples. Under the recommended conditions, the ethanol, which was present in the pretreated samples, was converted to hydrogen peroxide when it was passed through the immobilized alcohol oxidase (AOD) column with 0.1 mol/dm3 phosphate buffer (pH 7.0). A sample can be analyzed with this system in <10 min. The calibration curve for ethanol was linear from 2.0 to 0.1 mg/dm3. The determination limit, which was defined by the difference between the sample peak and blank peak, was estimated to be 50 microg/dm3 for ethanol. Interferences from some substances present in actual liquor samples decreased the analytical response and activity of the immobilized AOD-reactor column, but they were removed by dilution and pretreatment with an octyldecylsilane cartridge.     

Impact of High Temperature on Ethanol Fermentation by Kluyveromyces marxianus Immobilized on Banana Leaf Sheath Pieces

Ethanol fermentation was carried out with Kluyveromyces marxianus cells at various temperatures (30, 35, 40, and 45 °C). Fermentation performance of the immobilized yeast on banana leaf sheath pieces and the free yeast were evaluated and compared. Generally, ethanol production of the immobilized and free yeast was stable in a temperature range of 30–40 °C. Temperature of 45 °C restricted yeast growth and lengthened the fermentation. The immobilized yeast demonstrated faster sugar assimilation and higher ethanol level in the fermentation broth in comparison with the free yeast at all fermentation temperatures. Change in fatty acid level in cellular membrane was determined to clarify the response of the free and immobilized yeast to thermal stress. The free cells of K. marxianus responded to temperature increase by increasing saturated fatty acid (C16:0 and C18:0) level and by decreasing unsaturated fatty acid (C18:1 and C18:2) level in cellular membrane. For fermentation at 40 °C with immobilized cells of K. marxianus, however, the changes were not observed in both saturated fatty acid (C16:0) and unsaturated fatty acid (C18:1 and C18:2) level.     

A Study of the Hydrolysis of Waste Paper Cellulose with a Vertically Hanging Immobilized Cellulase Reactor and the Reuse of the Immobilized Cellulase

A commercialized cellulase from Trichoderma reesei has been successfully immobilized by using calcium alginate gel in our laboratory. The waste paper cellulose was hydrolyzed with a special design of the reactor to form a vertically hanging immobilized cellulase under the optimum conditions of pH 4.0 and 45 °C. Glucose, cellobiose and xylose are the major hydrolysis products. The glucose production from the hydrolysis with the vertically hanging immobilized cellulase was about 1.73‐fold better than the freely suspended immobilized cellulase. The average diameter of the immobilized cellulase pellets was 4.190 ± 0.291 mm. UV light irradiation deactivates the activity of the immobilized cellulase. The advantage of the vertically hanging immobilized cellulase reactor is an easy recycle and reuse of the immobilized cellulase. Washing and soaking the recycled immobilized cellulase with distilled water for one day can restore its activity to a small extent. Overall, the application of the hanging immobilized cellulase reactor for waste paper cellulose hydrolysis is successful.