A study on the ethanol production by immobilized cells ofZymomonas mobilis

Cells ofZymomonas mobilis immobilized in Ca-alginate matrix were used for ethanol production under various conditions. Immobilized cells showed broad optimum pH profile and their operational optimum temperature shifted from 30‡C to 40‡C upon immobilization. As reportedlyZ. mobilis did get the substrate inhibition by glucose, but at high concentration level of glucose the reduction of activity for ethanol production was less severe than that for yeast. The used beads of the immobilizedZ. mobilis were reactivated by incubating them in the activation medium. The increase in cell number and the enhancement of the specific activity per each cell are considered the two major factors responsible for the overall activation. A packed-bed reactor with the feed glucose concentration of 20% (W/V) gave an ethanol productivity as high as 33.0 g/l.hr at the flow rate of 58.5 ml/hr. A comparison between the experimental results from the real packed-bed reactor and the simulation results of an ideal case showed a two-fold inferior performance by the real reactor and this is at least partly attributed to the CO₂ gas effect.     

Zymomonas mobilis: an alternative ethanol producer

Zymomonas mobilis is a unique bacterium in the microbial world, and offers a number of advantages over the existing ethanol‐producing microorganisms. Being a prokaryote, it is more amenable to genetic manipulations. Thus, it has attracted great attention in the ethanol production world and efforts have been made to commercialize its application for the purpose. Despite the various efforts made worldwide, none of the processes using this microbe has been commercialized owing to certain bottlenecks. To circumvent the hindrances currently associated with a Zymomonas process, researchers have made various attempts to improve the technology using different techniques. This paper reviews the different substrates and the genetic improvement techniques with special emphasis on mutagenesis and recombinant DNA technology used for ethanol production by Zymomonas strains. Copyright © 2006 Society of Chemical Industry     

Direct ethanol production from dextran industrial waste water by Zymomonas mobilis

The direct production of ethanol from dextran industrial waste water was investigated by using Zymomonas mobilis via batch and semi-continuous fermentation mode. In batch fermentation, pretreated waste water (unsterilized and sterilized), pH value (3.8 and 6.0), and Mg²⁺ (with and without) was compared with OD₆₀₀, sugar and ethanol concentration. After 24 h fermentation, sugar in the dextran waste water was almost exhausted, and the amount of ethanol accumulated reached 24.33–29.92 g/l, which is nearly 99% of the theoretical yield of ethanol. Kinetic parameters of Z. mobilis in batch fermentation were also investigated. The raw dextran waste water was also used in semi-continuous fermentation. After 48 h fermentation, the production of ethanol was 28.65 g/l. These results indicated that dextran waste water may be used as a candidate substrate and Z. mobilis could convert the raw material into ethanol directly.     

Stabilized production of highly concentrated bioethanol from fermentation broths by Zymomonas mobilis by pervaporation using silicone rubber‐coated silicalite membranes

Since pervaporation performance of ethanol‐permselective silicalite membrane, which is an aluminum‐free hydrophobic zeolite, in the separation of fermentation broths by yeast are negatively affected by succinic acid, the potential of pervaporation using silicone rubber‐coated silicalite membranes of ethanol fermentation broths, not containing succinic acid, by Zymomonas mobilis was investigated for the reliable production of concentrated bioethanol. In the separation of fermentation broths, the pervaporation performance was influenced by nutrients used for the preparation of fermentation broths. In the separation of a broth prepared with yeast extract, pervaporation performance was greatly compromised by accumulation of a substance(s) having an ultraviolet absorption maximum at approximately 260 nm not only in total flux, but also in permeate ethanol concentration compared to the separation of binary ethanol/water mixtures. When supplying a prepared broth with corn steep liquor without the accumulation of a substance(s) having an ultraviolet absorption maximum at approximately 260 nm, the permeate ethanol concentration did not decrease. Treating the prepared broth with activated carbon was effective in restraining the decrease in total flux. Pervaporation performance is also deteriorated by the adsorption of lactic acid contained in corn steep liquor onto the silicalite crystals. In the separation of ternary mixtures of ethanol/water/lactic acid, accomplished by adjusting the ternary mixtures to pH > 5, more than 90% of the permeation flux in the separation of binary ethanol/water mixtures was obtained, and the permeate ethanol concentration was comparable to that obtained in the separation of binary mixtures. For stably performing pervaporation, it is important to prepare ethanol fermentation broths by Zymomonas mobilis in which lactic acid concentration is as low as possible. Copyright © 2007 Society of Chemical Industry     

Ethanol fermentation by Z. mobilis ATCC10988 in repeated batch cultures

Repeated-batch fermentations of glucose to ethanol by Z. mobilis (ATCC 10988) were examined in two semi-defined nutrient media. The measurement of acid produced by the microorganism was used to study its transient fermentation characteristics. An examination of the inhibitory effect of ethanol in repeated-batch fermentations supports an earlier finding on the presence of a ‘slow-acting inhibitory mechanism’ at high ethanol concentration in Z. mobilis (ATCC 10988) cultures. The experimental results also suggest that both microbially produced and exogeneously added ethanol have similar inhibitory effects on the fermentation behavior of this bacterium.     

Two‐Parameter Continuation and Bifurcation Strategies for Oscillatory Behavior Elimination from a Zymomonas mobilis Fermentation System

Two‐parameter continuation and bifurcation analysis strategies were applied to deal with the oscillatory phenomena of a Zymomonas mobilis ethanol fermentation system. A structured verified non‐linear mathematical model considering the physiological limitations of microorganisms for a single continuous fermenter for ethanol production using Z. mobilis was built to identify the Hopf bifurcation (HB) points, which indicate the oscillatory behavior, using the inlet substrate concentration and the dilution rate as bifurcation parameters. The path of the HB points can be determined with different controlling operating parameters. It was found that with the addition of a small amount of cells or ethanol to the feed stream or by increasing the dilution rate, the oscillations could be eliminated and steady‐state behavior was attained. Using a two‐parameter continuation strategy, the Z. mobilis fermentation system could operate at steady state without oscillatory behavior.     

Elimination of oscillations in fermentation processes

Fermentation processes that are used to produce ethanol exhibit oscillatory behavior and for periods of time during these oscillations, ethanol production decreases substantially and there is considerable loss of residual substrates. There has been a considerable amount of work demonstrating oscillatory behavior in fermentation processes. The aim of this work is to demonstrate simple strategies to eliminate the oscillatory behavior in fermentation processes involving both Zymomonas mobilis and Saccharomyces cerevisiae. The oscillatory behavior is caused by the existence of Hopf bifurcations and it is demonstrated that very minor changes in the input conditions can eliminate the Hopf bifurcation points. In the case of Zymomonas mobilis, it is shown that the addition of a small amount of substrate and/or the key component of the biomass and/or product in the input stream causes the disappearance of the Hopf bifurcation points while in Saccharomyces cerevisiae fermentation process, a very minor increase in the input oxygen supply produces the same result. The aim of this work is not only to demonstrate the existence of the Hopf bifurcations in the fermentation problems, but also to provide strategies to eliminate them. © 2010 American Institute of Chemical Engineers AIChE J, 2011     

The Quorum Sensing Auto-Inducer 2 (AI-2) Stimulates Nitrogen Fixation and Favors Ethanol Production over Biomass Accumulation in Zymomonas mobilis

Autoinducer 2 (or AI-2) is one of the molecules used by bacteria to trigger the Quorum Sensing (QS) response, which activates expression of genes involved in a series of alternative mechanisms, when cells reach high population densities (including bioluminescence, motility, biofilm formation, stress resistance, and production of public goods, or pathogenicity factors, among others). Contrary to most autoinducers, AI-2 can induce QS responses in both Gram-negative and Gram-positive bacteria, and has been suggested to constitute a trans-specific system of bacterial communication, capable of affecting even bacteria that cannot produce this autoinducer. In this work, we demonstrate that the ethanologenic Gram-negative bacterium Zymomonas mobilis (a non-AI-2 producer) responds to exogenous AI-2 by modulating expression of genes involved in mechanisms typically associated with QS in other bacteria, such as motility, DNA repair, and nitrogen fixation. Interestingly, the metabolism of AI-2-induced Z. mobilis cells seems to favor ethanol production over biomass accumulation, probably as an adaptation to the high-energy demand of N₂ fixation. This opens the possibility of employing AI-2 during the industrial production of second-generation ethanol, as a way to boost N₂ fixation by these bacteria, which could reduce costs associated with the use of nitrogen-based fertilizers, without compromising ethanol production in industrial plants.     

Production of ethanol by continuous fermentation and liquid–liquid extraction

A continuous pilot plant was constructed for fermentation production of ethanol, using liquid–liquid extraction to remove the product and with recycle of the fermented broth raffinate. The plant was operated for up to 18 days with feed glucose concentrations in the range 10·0–45·8% (w/w). The solvent was n-dodecanol and immobilised yeast was used to overcome the problem of emulsification. The concentration of by-products in the fermented broth had no adverse effect on the rate of ethanol production. A mathematical model to predict the time required for achievement of 99% of the steady-state by-products concentrations was shown to be in good agreement with the experimentally determined concentration of the main by-product, glycerol. At a feed glucose concentration of 45·8% (w/w), the aqueous purge was equivalent to 2·8 m³ of effluent per m³ of ethanol produced and represented a 78% reduction in the volume of the aqueous purge compared with using a feed containing 10% (w/w) glucose.     

Platelet‐activating factor detection,metabolism, and inhibitors in the ethanologenic bacterium Zymomonas mobilis

Platelet‐activating factor (PAF) is a signaling phospholipid with a significant physiological role in multicellular and unicellular organisms, including fermentative organisms such as yeast. Zymomonas mobilis is an ethanologenic α‐proteobacterium currently studied for bioethanol production. In order to examine the presence of PAF and/or PAF inhibitors in Z. mobilis, a new one‐step high performance liquid chromatography (HPLC) separation procedure of total lipids was performed, using a C8 reversed‐phase semi‐preparative column. According to this method and to bioassays based on washed rabbit platelet aggregation, two lipid molecules with PAF‐like activity and same retention times as those of standard PAF were detected; electron‐spray ionization MS and MS/MS analysis revealed that they share similar structure with 16:0 and 18:0 PAF. Furthermore, other lipids extracted from Z. mobilis were found to exhibit a potent anti‐PAF activity. Enzyme activities indicative of key PAF biosynthetic enzymes, such as dithiothreitol‐insensitive cholinephosphotransferase (PAF‐CPT) and lyso‐PAF acetyltransferase were detected in Z. mobilis homogenates. As for PAF degradation, activity similar to that of PAF acetylhydrolase was also discovered. Overall, the presence of PAF, PAF‐specific inhibitors, and enzyme activities relating to PAF metabolism, suggests that PAF may play an intrinsic role in this biotechnological organism. Practical applications: Z. mobilis is a platform microorganism for bioethanol production and a potential source of high‐value chemicals of interest to the food and healthcare industries. Further investigation of PAF's role is bound to affect applications involving this and other biotechnological organisms. The finding that Z. mobilis lipids exhibit potent anti‐PAF activity opens up prospects for their identification, overproduction and pharmaceutical use. The presented HPLC method for lipid fractionation accomplishes a one‐step separation of lipids from dense samples, which may be successfully employed to other lipid‐rich sources such as blood.     

Ethanol and chitosan production from wheat hydrolysate by Mucor hiemalis

BACKGROUND: High glucose and ethanol tolerance is among the most important requirements of ethanol‐producing microorganisms. The purpose of this study was evaluation of filamentous fungus Mucor hiemalis for ethanol production from wheat and starch hydrolysates with high glucose concentration. RESULTS: The results showed high tolerance of the fungus in fermentation of the hydrolyzates with high glucose concentrations (as high as 190 g L^?1). Interestingly, increasing the glucose concentration from 15 to 190 g L^?1 was accompanied by enhancement of initial sugar uptake rate. Ethanol was the most important metabolite obtained during all fermentations and its concentration reached over 50 g L^?1. Beside ethanol, chitosan was another valuable product of the process. Glucosamine, a precursor of chitosan, made up 37.3–46.7% of the cell wall of this fungus. CONCLUSIONS: M. hiemalis is a promising microorganism for simultaneous production of ethanol and chitosan from substrates with high sugar concentrations. © 2012 Society of Chemical Industry     

Production of bioethanol by immobilized Saccharomyces Cerevisiae onto modified sodium alginate gel

BACKGROUND: Microbial bioethanol production is an important option in view of the finite global oil reserves. Bioethanol fermentation was carried out using immobilized microorganisms (Saccharomyces cerevisiae, Zymomonas mobilis, Pichia stipitis, etc.), which has many advantages compared with the use of free cells. Various support materials have been used for bioethanol fermentation, and alginate gels have been one of the most widely used matrices for cell entrapment. The aim of this study was increased bioethanol production by Saccharomyces cerevisiae immobilized on alginate gels. First, N‐vinyl‐2‐pyrrolidone was grafted onto sodium alginate. Then, the properties of ethanol production were investigated using the matrix obtained. RESULTS: The performance of ethanol fermentation was affected by calcium chloride concentration, N‐vinyl‐2‐pyrrolidone grafted onto the sodium alginate, sugar concentration and the percentage of immobilized cell beads. These effects were optimized to give maximum ethanol production. Ethanol production was accelerated when sodium alginate polymer was modified with N‐vinyl‐2‐pyrrolidone. The maximum concentration, productivity and yield of ethanol were 69.68 g L^?1, 8.71 g L^?1 h^?1 and 0.697 g g^?1, respectively. CONCLUSION: The new polymeric matrix, when compared with sodium alginate, showed better ethanol production due to the hydrophilic property of N‐vinyl‐2‐pyrrolidone. The results suggest that the proposed method for immobilization of Saccharomyces cerevisiae has potential in industrial applications of the ethanol production process. Copyright © 2011 Society of Chemical Industry     

Kinetic study on ethanol production using Saccharomyces cerevisiae ITV‐01 yeast isolated from sugar cane molasses

BACKGROUND: Bio‐ethanol production from renewable sources, such as sugar cane, makes it a biofuel that is both renewable and environmentally friendly. One of the strategies to reduce production costs and to make ethanol fuel economically competitive with fossil fuels could be the use of wild yeast with osmotolerance, ethanol resistance and low nutritional requirements. The aim of this work was to investigate the kinetics of ethanol fermentation using Saccharomyces cerevisiae ITV‐01 yeast strain in a batch system at different glucose and ethanol concentrations, pH values and temperature in order to determine the optimum fermentation conditions. RESULTS: This strain showed osmotolerance (its specific growth rate (µ_max) remained unchanged at glucose concentrations between 100 and 200 g L^?1) as well as ethanol resistance (it was able to grow at 10% v/v ethanol). Activation energy (Ea) and Q₁₀ values calculated at temperatures between 27 and 39 °C, pH 3.5, was 15.6 kcal mol^?1 (with a pre‐exponential factor of 3.8 × 10¹² h^?1 (R² = 0.94)) and 3.93 respectively, indicating that this system is biologically limited. CONCLUSIONS: The optimal conditions for ethanol production were pH 3.5, 30 °C and initial glucose concentration 150 g L^?1. In this case, a maximum ethanol concentration of 58.4 g L^?1, ethanol productivity of 1.8 g L^?1 h^?1 and ethanol yield of 0.41 g g^?1 were obtained. Copyright © 2010 Society of Chemical Industry     

Fuel ethanol production from concentrated food waste hydrolysates in immobilized cell reactors by Saccharomyces cerevisiae H058

BACKGROUND: Continuous ethanol fermentation of concentrated food waste hydrolysates has been studied. The process was carried out in an immobilized cell reactor with beads of calcium‐alginate containing immobilized Saccharomyces cerevisiae H058 at temperature 30 °C and pH 5.0. RESULTS: The total residual sugar decreased with increase of hydraulic retention time (HRT) under various reducing sugar concentrations. Ethanol production by immobilized cells increased with increase in HRT, regardless of the substrate concentrations employed. The highest ethanol concentration of 89.28 g L^?1 was achieved at an HRT of 5.87 h and reducing sugar concentration of 200 g L^?1. At an HRT of 1.47 h, the maximum volumetric ethanol productivity of 49.88 g L^?1 h^?1 and the highest ethanol yield of 0.48 g g^?1 were achieved at reducing sugar concentration of 160 and 200 g L^?1, respectively. The difference between the fresh and the 30‐day Ca–alginate immobilized cell was also shown by scanning electronic micrographs of beads taken from their outer and inner surfaces. CONCLUSIONS: Continuous ethanol production from concentrated food waste hydrolysates using immobilized yeast cells is promising in view of the high ethanol productivity obtained at relatively high conversion and excellent reactor stability. Copyright © 2011 Society of Chemical Industry     

Conversion efficiency of glucose/xylose mixtures for ethanol production using Saccharomyces cerevisiae ITV01 and Pichia stipitis NRRL Y‐7124

BACKGROUND: Efficient conversion of glucose/xylose mixtures from lignocellulose is necessary for commercially viable ethanol production. Oxygen and carbon sources are of paramount importance for ethanol yield. The aim of this work was to evaluate different glucose/xylose mixtures for ethanol production using S. cerevisiae ITV‐01 (wild type yeast) and P. stipitis NRRL Y‐7124 and the effect of supplying oxygen in separate and co‐culture processes. RESULTS: The complete conversion of a glucose/xylose mixture (75/30 g L^?1) was obtained using P. stipitis NRRL Y‐7124 under aerobic conditions (0.6 vvm), the highest yield production being Y_p/s = 0.46 g g^?1, volumetric ethanol productivity Q_pmax = 0.24 g L^?1 h^?1 and maximum ethanol concentration P_max = 34.5 g L^?1. In the co‐culture process and under aerobic conditions, incomplete conversion of glucose/xylose mixture was observed (20.4% residual xylose), with a maximum ethanol production of 30.3 g L^?1, ethanol yield of 0.4 g g^?1 and Q_pmax = 1.26 g L^?1 h^?1. CONCLUSIONS: The oxygen present in the glucose/xylose mixture promotes complete sugar consumption by P. stipitis NRRL Y‐7124 resulting in ethanol production. However, in co‐culture with S. cerevisiae ITV‐01 under aerobic conditions, incomplete fermentation occurs that could be caused by oxygen limitation and ethanol inhibition by P. stipitis NRRL Y‐7124; nevertheless the volumetric ethanol productivity increases fivefold compared with separate culture. Copyright © 2011 Society of Chemical Industry     

Ethanol production from sweet sorghum residual

In this work, the ethanol production from sweet sorghum residue was studied. Sweet sorghum residue was hydrolyzed with phosphoric acid under mild conditions. The liquid hydrolysate was fermented by Pachysolen tannophilus, and the hydrolysis residue was fermented by the simultaneous saccharification and fermentation (SSF) using Saccharomyces cerevisiae with cellulase (60 FPU/g dry materials). Orthogonal experiments were carried out to investigate the effects of main reaction condition factors, such as temperature, acid concentration, time and dry-matter content, on the reducing sugar yield. The results show that the optimal reaction conditions should be 120°C, 80 g/L, 80 min and 10%, respectively. Under these conditions, 0.3024 g reducing sugar/g dry material was obtained. The liquid hydrolysate was then fermented by P.tannophilus with the highest ethanol concentration of 14.5 g/L. At a water-insoluble solid concentration of 5%, 5.4 g/L ethanol was obtained after 12 h of SSF. The total ethanol yield was 0.147 g/g dry material, which would be beneficial for the application of ethanol production from sweet sorghum residue. __________ Translated from Journal of Beijing University of Chemical Technology, 2007, 34(6): 637-639, 652 [译自: 北京化工大学学报]     

Kinetics of Bioethanol Production Employing Mono‐ and Co‐Cultures of Saccharomyces cerevisiae and Pichia stipitis

The kinetics of bioethanol production using mono‐ and co‐cultures of Saccharomyces cerevisiae and Pichia stipitis with glucose, xylose, and glucose‐xylose sugar mixtures were investigated. A MATLAB® program was formulated for simulation of experimental results in order to get predicted values of ethanol production and sugar consumption and for kinetic parameter estimation. Kinetic parameters implied less extent of substrate and/or product inhibition when the co‐culture scheme of immobilized S. cerevisiae and free P. stipitis was employed for fermentation of mixed sugars. In addition, a high ethanol yield was achieved by applying this co‐culture strategy to wheat straw hydrolysates.     

Effect of different fermentation parameters on bioethanol production from corn meal hydrolyzates by free and immobilized cells of Saccharomyces cerevisiae var. ellipsoideus

BACKGROUND: Bioethanol produced from renewable biomass, such as corn meal, is a biofuel that is both renewable and environmentally friendly. Significant scientific and technological investments will be needed to achieve substitution of conventional fossil fuels with alternative fuels. The ethanol fermentation of enzymatically obtained corn meal hydrolyzates by free and immobilized cells of Saccharomyces cerevisiae var. ellipsoideus yeast in a batch system was studied. The initial glucose and inoculum concentration and the time required for the efficient ethanol production were optimized taking into account parameters such as ethanol concentration, ethanol yield, percentage of the theoretical yield of ethanol and volumetric productivity in both immobilized and free cell systems. RESULTS: The yeast cells were immobilized in Ca–alginate by an electrostatic droplet generation method. An optimal initial inoculum concentration of 2% (v/v) and optimal fermentation time of 38 h for both immobilized and free yeasts were determined. An optimal initial glucose concentration of 150 g L^?1 for free system was achieved. At the initial glucose concentration of 176 g L no substrate or product inhibition were achieved with immobilized yeast. CONCLUSION: By immobilization of the yeast into Ca–alginate using the method of electrostatic droplet generation a superior system was realized, which exhibited lower substrate inhibition and higher tolerance to ethanol. The cells of S. cerevisiae var. ellipsoideus yeast entrapped in Ca–alginate showed good physical and chemical stability, and no substrate and product diffusion restrictions were noticed. Copyright © 2008 Society of Chemical Industry     

Continuous ethanol fermentation using immobilized yeast in a fluidized bed reactor

Continuous ethanol fermentation of glucose using fluidized bed technology was studied. Saccharomyces cerevisiae were immobilized and retained on porous microcarriers. Over two-thirds of the total reactor yeast cell mass was immobilized. Ethanol productivity was examined as dilution rate was varied, keeping all other experimental parameters constant. Ethanol yield remained high at an average of 0.36 g ethanol g^?1 glucose (71% of theoretical yield) as the dilution rate was increased stepwise from 0.04 h^?1 to 0.14 h^?1. At a dilution rate of 0.15 h^?1, the ethanol yield steeply declined to 0.22 g ethanol g^?1 glucose (44% of theoretical yield). The low maximum percentage of theoretical yield is primarily due to an extended mean cell residence time, and possibly due to the inhibitory effect of a high dissolved carbon dioxide concentration, enhanced by the probable intermittent levels of low pH in the reactor. Constant ethanol production was possible at a high glucose loading rate of 840 g dm^?3 day^?1 (attained at a dilution rate of 0.14 h^?1). Although the highest average ethanol concentration (97.14 g dm^?3) occurred at the initial dilution rate of 0.04 h^?1, the peak average ethanol production rate (2.87 g (g yeast)^?1 day^?1) was reached at a greater dilution rate of 0.11 ^h-1. Thus, the optimal dilution rate was determined to be between 0.11 h^?1 and 0.14 h^?1. Ethanol inhibition on yeast cells was absent in the reactor at average bulk-liquid ethanol concentrations as high as 97.14 g dm^?3. In addition, zero-order kinetics on ethanol production and glucose utilization was evident.     

Mathematical modelling of the alcoholic fermentation of glucose by Zymomonas mobilis mobilis

The kinetics of alcoholic fermentation of a strain of Zymomonas mobilis, isolated from sugarcane juice, has been studied with the objective of determining the constansts of a non-structured mathematical model that represents the fermentation process. Assays in batch and in continuous culture have been carried out with different initial concentrations of glucose. The final concentrations of glucose, ethanol and biomass were determined. The following kinetic parameters were obtained: μ_max, 0·5 h^?1; K_s, 4·64 g dm^?3; P_max, 106 g dm^?3; Y_x/s, 0·0265 g g^?1; m, 1·4 g g^?1 h^?1; α, 17·38 g g^?1; β, 0·69 g g^?1 h^?1.