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.     

Effects of ethanol concentration and stripping temperature on continuous fermentation rate

The operation of a pilot plant consisting of a 14-l fermentor, 10-cm packed column and condenser for continuous fermentation and stripping of ethanol was stable for more than 100 days. The feed consisted of a non-sterile solution of 560 g/l glucose with 100 g/l corn steep water. Fouling of the packing in the column with attached growth of yeast cells was controlled by in situ washing at intervals of 3–6 days. A computer simulation of the pilot plant was developed and used to analyze the data. The productivity of the continuous fermentor varied from 14 g ethanol to 17 g ethanol l⁻¹ h⁻¹. The yield was equal to the maximum theoretically possible: 0.51 g ethanol/g glucose consumed. Results are fit to linear models for the effects of ethanol concentration on specific growth rate and cell yield, and for the effect of stripping temperature on specific growth rate. Received: 16 October 1996 / Received revision: 3 January 1997 / Accepted: 24 January 1997     

Fermentation of Detoxified Acid-Hydrolyzed Pyrolytic Anhydrosugars into Bioethanol with <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> 2.399

Pyrolysate obtained from the pyrolysis of waste cotton is a source of fermentable sugars that could be fermented into bioethanol fuel and other chemicals via microbial fermentation. However, pyrolysate is a complex mixture of fermentable and non-fermentable substrates causing inhibition of the microbial growth. The aim of this study was to detoxify the hydrolysate and then ferment it into bio-ethanol fuel in shake flasks and fermenter applying yeast strain Saccharomyces cerevisiae 2.399. Pyrolysate was hydrolyzed to glucose with 0.2 M sulfuric acid, neutralized with Ba(OH)₂ followed by treatment with ethyl acetate and activated carbon to remove fermentation inhibitors. The effect of various fermentation parameters such as inoculum concentration, pH and hydrolysate glucose was evaluated in shake flasks for optimum ethanol fermentation. With respect to inoculum concentration, 20% v/v inoculum i.e. 8.0 × 10⁸–1.2 × 10⁹ cells/mL was the optimum level for producing 8.62 ± 0.33 g/L ethanol at 9 h of fermentation with a maximum yield of 0.46 g ethanol/g glucose. The optimum pH for hydrolysate glucose fermentation was found to be 6.0 that produced 8.57 ± 0.66 g/L ethanol. Maximum ethanol concentration, 14.78 g/L was obtained for 4% hydrolysate glucose concentration after 16 h of fermentation. Scale-up studies in stirred fermenter produced much higher productivity (1.32 g/L/h^–1) compared to shake flask fermentation (0.92 g/L/h^–1). The yield of ethanol reached a maximum of 91% and 89% of the theoretical yield of ethanol in shake flasks and fermenter, respectively. The complex of integrated models of development was applied, that has been successfully tested previously for the mathematical analysis of the fermentation processes.     

Continuous ethanol production using induced yeast aggregates

Summary The induction of yeast cell aggregates in a column reactor was initiated by packing yeast cell paste of Saccharomyces uvarum into the column, and then YMP broth was fed into the column from the bottom at a linear flow rate of 2.5 cm/h. Thereafter, yeast cells aggregated in the column within 48 h without a supply of oxygen. When this yeast aggregate column reactor was used for continuous ethanol production, a final ethanol concentration of 10.8% (w/v) was obtained from 23% (w/v) of glucose in a YMP broth with a dilution rate of 0.05 h^-1, and 4.9% (w/v) was obtained from 10% (w/v) of glucose with a dilution rate of 0.6 h^-1. The theoretical yield was above 97% in both cases. The ethanol production rates were 13 g¹ h^-1 l^-1 and 90 g¹ h^-1 l^-1 for producing 10.8% (w/v) and 4.9% (w/v) of ethanol respectively. This column reactor was maintained at a steady state for more than one month.     

Continuous ethanol fermentation using immobilized yeast cells

Growing cells of Saccharomyces cerevisiae immobilized in calcium alginate gel beads were employed in fluidizedbed reactors for continuous ethanol fermentation from cane molasses and other sugar sources. Some improvements were made in order to avoid microbial contamination and keep cell viability for stable long run operations. Notably, entrapment of sterol and unsaturated fatty acid into immobilized gel beads enhanced ethanol productivity more than 50 g ethanol/L gel h and prolonged life stability for more than one-half year. Cell concentration in the carrier was estimated over 250 g dry cell/L gel. A pilot plant with a total column volume of 4 kL was constructed and has been operated since 1982. As a result, it was confirmed that 8-10%(v/v)ethanol-containing broth was continuously produced from nonsterilized diluted cane molasses for over one-half year. The productivity of ethanol was calculated as 0.6 kL ethanol/kL reactor volume day with a 95% conversion yield versus the maximum theoretical yield for the case of 8.5% (v/v) ethanol broth.     

Fermentation of enzymatically saccharified sunflower stalks for ethanol production and its scale up

Pretreated sunflower stalks saccharified with a Trichoderma reesei Rut-C 30 cellulase showed 57.8% saccharification. Enzyme hydrolysate concentrated to 40 g/l reducing sugars was fermented under optimum conditions of fermentation time (24 h), pH (5.0), temperature (30 degrees C) and inoculum size (3% v/v) and, showed a maximum ethanol yield of 0.444 g/g ethanol. Ethanol production scaled up in a 1 l and a 15 l fermenter under optimum conditions revealed maximum ethanol yields of 0.439 and 0.437 g/g respectively.     

Tower fermentation using Zymomonas mobilis for ethanol production

Summary Flocculation was induced in a pure strain of the bacteria Zymomonas mobilis. When fermenting glucose to ethanol, cell densities of up to 40g/l were achieved and sustained in a 0.92 litre tower fermenter with dilution rates of up to 2.3 hr^-1. A maximum productivity of 100g EtOH/l/hr with 98% conversion of the 105g/l glucose feed was achieved. The limitation to performance with increase in throughput arose from incomplete fermentation of the feed glucose, rather than washout of the flocculated bacteria.     

The physiology of lactate production by Lactobacillus delbreuckii in a chemostat with cell recycle

The physiology of lactate production by Lactobacillus delbreuckii NRRL B-445 in a continuous fermenter with partial cell recycle has been studied and compared with that observed in a conventional chemostat. Partial cell recycle was achieved using a hollow-fiber ultrafiltration cartridge. The biomass growth yield was reduced in the recycle fermenter while culture viability and the cellular content of polysaccharide, protein, carbon, and nitrogen remained constant, suggesting an enlarged specific rate of glucose consumption for nonanabolic (e.g., maintenance) functions. The volumetric productivity of lactate was enhanced in the recycle fermenter due to the complete utilization of glucose. The yield of lactate from biomass and the molar product ratio, lactate: ethanol plus acetate, decreased with increasing recycle ratio. Enhanced formation of ethanol and acetate occurred in the recycle fermenter although lactate remained the major product. The change in product profile was due to glucose limitation. The specific activity of lactate dehydrogenase remained constant during recycle fermentation. These physiological observations have implications for the future application of cell recycle to production processes.     

Control of packed column fouling in the continuous fermentation and stripping of ethanol

By recycling the contents of a 14 L fermentor through a stripping column to continuously remove ethanol and reduce product inhibition, continuous complete conversion of nutrient feed containing 600 g/L glucose was achieved in a small pilot plant. Ethanol was recovered from the carbon dioxide stripping gas in a refrigerated condenser, and the gas was reheated with steam and recycled by a blower. Productivity of ethanol in the fermentor as high as 15.8 g/L/h and condensate production of up to 10 L/day of almost 50% by volume ethanol were maintained for up to 60 days of continuous operation. Weekly washing of the column packing in situ was required to prevent loss of performance caused by attached growth of yeast cells, which restricts the gas flow rate through the stripping column. (c) 1996 John Wiley & Sons, Inc.     

Production of multiple extracellular enzyme activities by novel submerged culture of <Emphasis Type="Italic">Aspergillus kawachii</Emphasis> for ethanol production from raw cassava flour

Cassava is a starch-containing root crop that is widely used as a raw material in a variety of industrial applications, most recently in the production of fuel ethanol. In the present study, ethanol production from raw (uncooked) cassava flour by simultaneous saccharification and fermentation (SSF) using a preparation consisting of multiple enzyme activities from Aspergillus kawachii FS005 was investigated. The multi-activity preparation was obtained from a novel submerged fermentation broth of A. kawachii FS005 grown on unmilled crude barley as a carbon source. The preparation was found to consist of glucoamylase, acid-stable α-amylase, acid carboxypeptidase, acid protease, cellulase and xylanase activities, and exhibited glucose and free amino nitrogen (FAN) production rates of 37.7 and 118.7 mg/l/h, respectively, during A. kawachii FS005-mediated saccharification of uncooked raw cassava flour. Ethanol production from 18.2% (w/v) dry uncooked solids of raw cassava flour by SSF with the multi-activity enzyme preparation yielded 9.0% (v/v) of ethanol and 92.3% fermentation efficiency. A feasibility study for ethanol production by SSF with a two-step mash using raw cassava flour and the multi-activity enzyme preparation manufactured on-site was verified on a pilot plant scale. The enzyme preparation obtained from the A. kawachii FS005 culture broth exhibited glucose and FAN production rates of 41.1 and 135.5 mg/l/h, respectively. SSF performed in a mash volume of about 1,612 l containing 20.6% (w/v) dry raw cassava solids and 106 l of on-site manufactured A. kawachii FS005 culture broth yielded 10.3% (v/v) ethanol and a fermentation efficiency of 92.7%.     

Application of multistage continuous fermentation for production of fuel alcohol by very-high-gravity fermentation technology

A fermentation system to test the merging of very-high-gravity (VHG) and multistage continuous culture fermentation (MCCF) technologies was constructed and evaluated for fuel ethanol production. Simulated mashes ranging from 15% to 32% w/v glucose were fermented by Saccharomyces cerevisiae and the dilution rates were adjusted for each glucose concentration to provide an effluent containing less than 0.3% w/v glucose (greater than 99% consumption of glucose). The MCCF can be operated with glucose concentrations up to 32% w/v, which indicates that the system can successfully operate under VHG conditions. With 32% w/v glucose in the medium reservoir, a maximum of 16.73% v/v ethanol was produced in the MCCF. The introduction of VHG fermentation into continuous culture technology allows an improvement in ethanol productivity while producing ethanol continuously. In comparing the viability of yeast by methylene blue and plate count procedures, the results in this work indicate that the methylene blue procedure may overestimate the proportion of dead cells in the population. Ethanol productivity (Yps) increased from the first to the last fermentor in the sequence at all glucose concentrations used. This indicated that ethanol is more effectively produced in later fermentors in the MCCF, and that the notion of a constant Yps is not a valid assumption for use in mathematical modeling of MCCFs. Journal of Industrial Microbiology & Biotechnology (2001) 27, 87–93. Received 20 January 2001/ Accepted in revised form 28 April 2001     

The inhibitory effect of ethanol on ethanol production by Zymomonas mobilis

Summary In an effort to establish the reasons for the limitations in the final ethanol concentration of Zymomonas mobilis fermentation, the effects of CO₂ and ethanol on the fermentation were investigated using continuous and fed-batch cultivation systems. The nucleation and stripping out of CO₂ from the fermenter using diatomaceous earth or nitrogen gas or both exhibited a profound effect on the glucose uptake rate during the early stages of fed-batch fermentation, but did not improve final ethanol yields. The addition of ethanol together with above mentioned experiments confirmed conclusively that ethanol inhibition is responsible for the final ethanol concentration obtainable during Zymomonas mobilis fermentation. The final concentration lies between 90 and 110 gl⁻¹ or approximately 12–15% (v/v) ethanol.     

Growth of Streptococcus faecalus in dense culture

A fermentation system was designed and constructed to study the growth characteristics of microorganisms at low and high cell concentrations. The technique used to develop high cell densities utilized a rotating microfiltration unit to permit the removal of cell-free product from the fermenter. The fermenter volume and the filter were contained in a single unit composed of a series of concentric cylinders. Annuli contained the fermenter volume while the second outermost cylinder supported a microfiltration membrane. Feed to the system was pumped at constant rates, and the internal pressure built up to a value, which would effect the required filtration rate. The system was operated batchwise and continuously with and without filtration. The anaerobie growth characteristics of Streptococcus faccalus were determined at 37°C and pH 7.0 for batch, continuous, and continuous with filtration modes of operation. The growth characteristics were unchanged when the cell density was increased. Changes in cell yield peer model of glucose consumed were clearly illustrated during thee course of single run by operating the fermenter in the unsteady state with filtration. No consumption of glucose for developed was 40% packed cell volume, a value 45 times larger than could be grown in simple batch culture.     

Selection and characterization of a newly isolated thermotolerant Pichia kudriavzevii strain for ethanol production at high temperature from cassava starch hydrolysate

Pichia kudriavzevii DMKU 3-ET15 was isolated from traditional fermented pork sausage by an enrichment technique in a yeast extract peptone dextrose (YPD) broth, supplemented with 4 % (v/v) ethanol at 40 °C and selected based on its ethanol fermentation ability at 40 °C in YPD broth composed of 16 % glucose, and in a cassava starch hydrolysate medium composed of cassava starch hydrolysate adjusted to 16 % glucose. The strain produced ethanol from cassava starch hydrolysate at a high temperature up to 45 °C, but the optimal temperature for ethanol production was at 40 °C. Ethanol production by this strain using shaking flask cultivation was the highest in a medium containing cassava starch hydrolysate adjusted to 18 % glucose, 0.05 % (NH₄)₂SO₄, 0.09 % yeast extract, 0.05 % KH₂PO₄, and 0.05 % MgSO₄·7H₂O, with a pH of 5.0 at 40 °C. The highest ethanol concentration reached 7.86 % (w/v) after 24 h, with productivity of 3.28 g/l/h and yield of 85.4 % of the theoretical yield. At 42 °C, ethanol production by this strain became slightly lower, while at 45 °C only 3.82 % (w/v) of ethanol, 1.27 g/l/h productivity and 41.5 % of the theoretical yield were attained. In a study on ethanol production in a 2.5-l jar fermenter with an agitation speed of 300 rpm and an aeration rate of 0.1 vvm throughout the fermentation, P. kudriavzevii DMKU 3-ET15 yielded a final ethanol concentration of 7.35 % (w/v) after 33 h, a productivity of 2.23 g/l/h and a yield of 79.9 % of the theoretical yield.     

Mathematical modeling of ethanol production by immobilized Zymomonas mobilis in a packed bed fermenter

A mathematical model which describes ethanol production in a packed bed fermenter containing. Zymomonas mobilis entrapped in small spheres of calcium alginate within a packed bed fermenter has been developed. The equations combine simultaneous diffusion and reaction as well as a complex flow pattern to calculate glucose and ethanol profiles in the column type reactor. As part of the study, diffusivity values for glucose and ethanol in cell-loaded calcium alginate were determined. Also a freecell kinetic expression for Z. mobilis at 33 degrees C and ph 6.0 was developed. Comparison of the model with actual experimental results were made showing average deviations of ca. 30-40%.     

Ethanol fermentation coupled with complete cell recycle pervaporation system: Dependence of glucose concentration

Summary A membrane bioreactor system comprised of a fermenter and a flat pervaporation module was developed for continuous ethanol fermentation by Saccharomyces cerevisiae. In order to obtain the guidelines for high sugar concentration fermentation, the dependence of glucose concentration on the coupled system was investigated. Fed by 158 and 290g glucose/l, the improvement in productivity was obtained with 1. 58 and 1. 86 times, and the ethanol yield was 0. 45 and 0. 395, respectively. With the fermentation proceeding, the permeate flux decreased but the selectivity kept unaltered.     

Expression of a heterologous xylose transporter in a <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain engineered to utilize xylose improves aerobic xylose consumption

The goal of this investigation was to determine the effect of a xylose transport system on glucose and xylose co-consumption as well as total xylose consumption in Saccharomyces cerevisiae. We expressed two heterologous transporters from Arabidopsis thaliana in recombinant xylose-utilizing S. cerevisiae cells. Strains expressing the heterologous transporters were grown on glucose and xylose mixtures. Sugar consumption rates and ethanol concentrations were determined and compared to an isogenic control strain lacking the A. thaliana transporters. Expression of the transporters increased xylose uptake and xylose consumption up to 46% and 40%, respectively. Xylose co-consumption rates (prior to glucose depletion) were also increased by up to 2.5-fold compared to the control strain. Increased xylose consumption correlated with increased ethanol concentration and productivity. During the xylose/glucose co-consumption phase, strains expressing the transporters had up to a 70% increase in ethanol production rate. It was concluded that in these strains, xylose transport was a limiting factor for xylose utilization and that increasing xylose/glucose co-consumption is a viable strategy for improving xylose fermentation.     

Kinetics and modelling of kojic acid production by Aspergillus flavus Link in batch fermentation and resuspended mycelial system

An unstructured model based on logistic and Luedeking-Piret equations was proposed to describe growth, substrate consumption and kojic acid production by Aspergillus flavus Link strain 44-1 in batch fermentation and also in a resuspended cell system. The model showed that kojic acid production was non-growth associated. The maximum kojic acid and cell concentrations obtained in batch fermentations using the fermenter with optimized dissolved oxygen control (32.5 g/l and 11.8 g/l, respectively) and using a shake-flask (36.5 and 12.3 g/l, respectively) were not significantly different. However, the maximum specific growth rate and a non-growth-associated rate constant for kojic acid formation (n) for batch fermentation using the fermenter (0.085/h and 0.0125 g kojic acid/g cell.h, respectively) were approximately three and two times higher than the values obtained for fermentation using a shake-flask, respectively. Efficient conversion of glucose to kojic acid was achieved in a resuspended pellet or mycelial system, in a solution containing only glucose with citrate buffer at pH 3.5 and at a temperature of 30 °C. The resuspended cell material in the glucose solution was still active in synthesizing kojic acid after prolonged incubation (up to about 600 h). The rate constant of kojic acid production (n) in a resuspended cell system using 100 g glucose/l was almost constant at an average value of 0.011 g kojic acid/g cell.h up to a cell concentration of 19.2 g/l, above which it decreased. A drastic reduction of n was observed at a cell concentration of 26.1 g/l. However, the yield based on glucose consumed (0.45 g/g) was similar for all cell concentrations investigated.     

Effect of oleic acid on the production of ethanol and fructose from glucose/fructose mixtures in an immobilized cell reactor

Saccharomyces cerevisiae ATCC 39859 was immobilized onto small cubes of wood to produce ethanol and very enriched fructose syrup from glucose/fructose mixtures through the selective fermentation of glucose. A maximum ethanol productivity of 21.9 g/l-h was attained from a feed containing 9.7% (w/v) glucose and 9.9% (w/v) fructose. An ethanol concentration, glucose conversion and fructose yield of 29.6 g/l, 62% and 99% were obtained, respectively. This resulted in a final fructose/glucose ratio of 2.7. At lower ethanol productivity levels the fructose/glucose ratio increases, as does the ethanol concentration in the effluent. The addition of 30 mg/l oleic acid to the medium increased the ethanol productivity and its concentration by 13% at a dilution rate of 0.74 h^?1.     

Isolation of a novel high erythritol-producing <Emphasis Type="Italic">Pseudozyma tsukubaensis</Emphasis> and scale-up of erythritol fermentation to industrial level

This study isolated a novel erythritol-producing yeast strain, which is capable of growth at high osmolarity. Characteristics of the strain include asexual reproduction by multilateral budding, absence of extracellular starch-like compounds, and a negative Diazonium blue B color reaction. Phylogenetic analysis based on the 26S rDNA sequence and physiological analysis indicated that the strain belongs to the species Pseudozyma tsukubaensis and has been named P. tsukubaensis KN75. When P. tsukubaensis KN75 was cultured aerobically in a fed-batch culture with glucose as a carbon source, it produced 245 g/L of erythritol, corresponding to 2.86 g/L/h productivity and 61% yield, the highest erythritol yield ever reported by an erythritol-producing microorganism. Erythritol production was scaled up from a laboratory scale (7 L fermenter) to pilot (300 L) and plant (50,000 L) scales using the dissolved oxygen as a scale-up parameter. Erythritol production at the pilot and plant scales was similar to that at the laboratory scale, indicating that the production of erythritol by P. tsukubaensis KN75 holds commercial potential.