Continuous simultaneous saccharification and fermentation of starch using Zymomonas mobilis

A continuous process involving simultaneous saccharification and fermentation of liquefied starch has been developed using Zymomonas mobilis. Amyloglucosidase retention and cell recycle have been effected by using an Amicon hollow-fiber membrane system with a MW cutoff of 5000. Relatively high productivities of up to 60 g L(-1) h(-1) have been achieved at ethanol concentrations of 60-65 g/L. The system also offers the potential for reduced enzyme requirements for saccharification.     

Simultaneous saccharification and ethanol fermentation of sago starch using immobilized Zymomonas mobilis

Simultaneous saccharification and ethanol fermentation (SSF) of sago starch using amyloglucosidase (AMG) and immobilized Zymomonas mobilis ZM4 on sodium alginate was studied. The immobilized Zymomonas cells were more thermo-stable than free Zymomonas cells in this system. The optimum temperature in the SSF system was 40°C, and 0.5% (v/w) AMG concentration was adopted for the economical operation of the system. The final ethanol concentration obtained was 68.3 g/l and the ethanol yield, Y_p/s, was 0.49 g/g (96% of the theoretical yield). After 6 cycles of reuse at 40°C with 15% sago starch hydrolysate, the immobilized Z. mobilis retained about 50% of its ethanol fermenting ability.     

Continuous ethanol production from sago starch using immobilized amyloglucosidase and Zymomonas mobilis

To produce ethanol more economically than in a conventional process, it is necessary to attain high productivity and low production cost. To this end, a continuous ethanol production from sago starch using immobilized amylogucosidase (AMG) and Zymomonas mobilis cells was studied. Chitin was used for immobilization of AMG and Z. mobilis cells were immobilized in the form of sodium alginate beads. Ethanol was produced continuously in an simultaneous saccharification and ethanol fermentation (SSF) mode in a pacekd bed reactor. The maximum ethanol productivity based on the void volume, V_v, was 37 g/l/h with ethanol yield, Y_p/s, 0.43 g/g (84% of the theoretical ethanol yield) in this system. The steady-state concentration of ethanol (46 g/l could be maintained in a stable manner over two weeks at the dilution rate of 0.46 h⁻.     

Optimization of fermentation conditions for the production of ethanol from sago starch by co-immobilized amyloglucosidase and cells of Zymomonas mobilis using response surface methodology

Statistical experimental design was used to optimize the conditions of simultaneous saccharification and fermentation (SSF), viz. temperature, pH and time of fermentation of ethanol from sago starch with co-immobilized amyloglucosidase (AMG) and Zymomonas mobilis MTCC 92 by submerged fermentation. Maximum ethanol concentration of 55.3 g/l was obtained using a starch concentration of 150 g/l. The optimum conditions were found to be a temperature of 32.4 °C, pH of 4.93 and time of fermentation of 17.24 h. Thus, by using SSF process with co-immobilized AMG and Z. mobilis cells MTCC 92, the central composite design (CCD) was found to be the most favourable strategy investigated with respect to ethanol production and enzyme recovery.     

Development of new unstructured model for simultaneous saccharification and fermentation of starch to ethanol by recombinant strain

The development of simultaneous saccharification and fermentation of starch to ethanol (SSFSE) by genetically modified microbial strains has been studied intensively [M.M. Altintas, B. Kirdar, Z.Ï. Önsan, K.Ö. Ülgen, Cybernetic modelling of growth and ethanol production in a recombinant Saccharomyces cerevisiae strain secreting a bifunctional fusion protein, Process Biochem. 37 (2002) 1439–1445; G. Birol, Z.Ï. Önsan, B. Kirdar, S.G. Oliver, Ethanol production and fermentation characteristics of recombinant Saccharomyces cerevisiae strains grown on starch, Enzyme Microb. Technol. 22 (1998) 672–677; F. Kobayashi, Y. Nakamura, Effect of repressor gene on stability of bioprocess with continuous conversion of starch into ethanol using recombinant yeast, Biochem. Eng. J. 18 (2004) 133–141; F. Kobayashi, Y. Nakamura, Mathematical model of direct ethanol production from starch in immobilized recombinant yeast culture, Biochem. Eng. J. 21 (2004) 93–101; M.M. Altintas, K.Ö. Ülgen, B. Kirdar, Z.Ï. Önsan, S.G. Oliver, Improvement of ethanol production from starch by recombinant yeast through manipulation of environmental factors, Enzyme Microb. Technol. 31 (2002) 640–647; K.Ö. Ülgen, B. Saygili, Z.Ï. Önsan, B. Kirdar, Bioconversion of starch into ethanol by a recombinant Saccharomyces cerevisiae strain YPG-AB, Process Biochem. 37 (2002) 1157–1168]. Saccharomyces cerevisiae YPB-G strain secretes a bifunctional fusion protein containing enzymatic activity of the B. subtilis alpha-amylase and of the Aspergillus awamori glucoamylase [M.M. Altintas, B. Kirdar, Z.Ï. Önsan, K.Ö. Ülgen, Cybernetic modelling of growth and ethanol production in a recombinant Saccharomyces cerevisiae strain secreting a bifunctional fusion protein, Process Biochem. 37 (2002) 1439–1445], and therefore is distinguished in relation to SSFSE step. In this work we have used the experimental data, presented in the paper [M.M. Altintas, B. Kirdar, Z.Ï. Önsan, K.Ö. Ülgen, Cybernetic modelling of growth and ethanol production in a recombinant Saccharomyces cerevisiae strain secreting a bifunctional fusion protein, Process Biochem. 37 (2002) 1439–1445] to develop two-hierarchic-level unstructured mathematical model describing kinetics of direct bioconversion of starch to ethanol. The first level has modeled enzymatic hydrolysis of starch to glucose by bifunctional protein and the second level includes utilization and bioconversion of glucose to ethanol by yeasts. The second level has unified the enzymatic degradation of starch, and glucose metabolization to ethanol by microorganisms. The response surface analysis was used to develop the rates models. A hybrid genetic algorithm and a decomposition approach were used in the nonlinear parameters identification procedure. The proposed model demonstrated excellent flexibility for different operational conditions of SSFSE process, and can be used successfully to describe microbial physiology of genetically modified strains.     

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.     

Ethanol fermentation of cassava starch by Zymomonas mobilis NRRL B-4286

Four strains of Zymomonas mobilis were compared for ethanol production from enzymatically hydrolysed cassava starch. Strain NRRL B-4286 performed efficiently, producing 80 g/litre ethanol from 171 g/litre initial sugar concentration. Addition of yeast extract, calcium pantothenate, ammonium sulphate or magnesium sulphate did not significantly increase ethanol production by this strain.     

High efficiency ethanol fermentation by entrapment of Zymomonas mobilis into LentiKats

AIMS: To examine the potential of Zymomonas mobilis entrapped into polyvinylalcohol (PVA) lens-shaped immobilizates in batch and continuous ethanol production. METHODS AND RESULTS: Cells, free or immobilized in PVA hydrogel-based lens-shaped immobilizates - LentiKats, were cultivated on glucose medium in a 1 l bioreactor. In comparison with free cell cultivation, volumetric productivity of immobilized batch culture was nine times higher (43.6 g l(-1) h(-1)). The continuously operated system did not improve the efficiency (volumetric productivity of the immobilized cells 30.7 g l(-1) h(-1)). CONCLUSIONS: We demonstrated Z. mobilis capability, entrapped into LentiKats, in the cost-efficient batch system of ethanol production. SIGNIFICANCE AND IMPACT OF THE STUDY: The results reported here emphasize the potential of bacteria in combination with suitable fermentation technology in industrial scale. The innovation compared with traditional systems is characterized by excellent long-term stability, high volumetric productivity and other technological advantages.     

Characteristics of Zymomonas mobilis immobilized by photo-crosslinkable resin in ethanol fermentation

Zymomonas mobilis, an ethanol-producing bacterium, was immobilized in hydrophilic photo-crosslinked resin gels to form a biocatalyst. The molecular structure of the photo-crosslinkable resin could be modulated so as to minimize a disadvantage of this bacterium—poor-tolerance to salts in molasses. Characteristics of Z. mobilis immobilized by photo-crosslinkable resin gel, such as fermentability, cell growth in gel, the potential of gel materials, diffusion of materials, and salt distribution are discussed. ENTG-3800 photo-crosslinkable resin was selected as the most suitable entrapping material for Z. mobilis, especially in using molasses.     

A structured kinetic model for Zymomonas mobilis ATCC10988

The inhibitory effects of glucose and ethanol on Zymomonas mobilis ATCC10988 were isolated through kinetic analysis of transient batch fermentation data. Growth of Z. mobilis was inhibited above a glucose concentration of 80 g/L. Growth was mildly inhibited by ethanol to 50 g/L, and severely inhibited above this concentration. Specific rates of ethanol production and glucose uptake were essentially invariant during batch fermentation. A structured kinetic model was developed, by way of augmentation of the Extended Bottleneck model, to quantify the kinetics of the growth and product formation processes. The model successfully describes the transient batch fermentation of Z. mobilis over a wide range of initial glucose concentration in a semidefined medium.     

A kinetic model for simultaneous saccharification and fermentation of Avicel with Saccharomyces cerevisiae

This work describes a numerical model for predicting simultaneous saccharification and fermentation of Avicel, an insoluble crystalline cellulose polymer. Separate anoxic cultivations of 40 g/L glucose and 100 g/L Avicel were conducted to verify model predictions and obtain parameters to describe the reaction kinetics. Saccharification of Avicel was achieved with Trichoderma reesei cellulases from the enzyme preparation Spezyme CP with an enzyme loading of 10 FPU/g cellulose. Cultivations were supplemented with 50 IU/g cellulose of β‐glucosidase from Novozym 188 to prevent product inhibition by cellobiose. Saccharomyces cerevisiae MH‐1000 is a robust industrial strain and was used to ferment glucose to ethanol, glycerol, and carbon dioxide. The numerical model presented in this paper differs from previous models by separating the endoglucanase and exoglucanase enzyme kinetics and allowing for inhibitive site competition. Assuming all enzymes remain active and that each enzyme complex has a corresponding constant specific activity, the model is capable of predicting adsorbed enzyme concentrations with reasonable accuracy. Comparison of predicted values to experimental measurements indicated that the numerical model was capable of capturing the significant elements involved with cellulose conversion to ethanol. Biotechnol. Bioeng. 2011; 108:924–933. © 2010 Wiley Periodicals, Inc.     

Batch fermentation kinetics of ethanol production by Zymomonas mobilis on cellulose hydrolysate

Summary Growth and ethanol production by three strains (MSN77, thermotolerant, SBE15, osmotolerant and wild type ZM4) of the bacterium Zymomonas mobilis were tested in a rich medium containing the hexose fraction from a cellulose hydrolysate (Aspen wood). The variations of yield and kinetic parameters with fermentation time revealed an inhibition of growth by the ethanol produced. This inhibition may result from the increase in medium osmolality due to ethanol formation from glucose.Nomenclature S glucose concentration (g/L) - C conversion of glucose (%) - t fermentation time (h) - q_S specific glucose uptake rate (g/g.h) - q_p specific ethanol productivity (g/g.h) - Q_p volumetric ethanol productivity (g/L.h) - Q_X volumetric biomass productivity (g/L.h) - Y_X/S biomass yield (g/g) - Y_p/S ethanol yield (g/g) - specific growth rate (h^-1)     

Vacuum fermentation for ethanol production using strains of Zymomonas mobilis

Summary Using strains of Z.mobilis, a vacuum fermentation system has been evaluated. The system was designed with the fermentor at atmospheric pressure and an external vacuum vessel (50 mm Hg). Sequential operation of the vacuum vessel was under microprocessor control. The use of Z.mobilis together with the two-stage design of the vacuum system has been found to overcome the problems of oxygen addition and the possibility of contamination reported previously for vacuum fermentations with yeasts. The productivity of 85 g/1/h found in the continuous cell recycle experiments was similar to that reported previously for a strain of S.cerevisiae.     

Role of antimicrobial agents in simultaneous saccharification and fermentation of paddy malt mash to ethanol by mixed cultures of Saccharomyces cerevisiae PH03 and Zymomonas mobilis ZM4

Summary Among various antimicrobial plant extracts, chemicals and antibiotics used for simultaneous saccharification and fermentation, penicillin G prevented contamination and did not inhibit amylase activity and growth of the synergistic co-cultures Saccharomyces cerevisiae PH03 and Zymomonas mobilis ZM4 during a 7-day fermentation of paddy malt (25.0%) mash (18.0% dextrose equivalent) to ethanol at 30°C and pH 5.5. The treatment yielded 10.1% (v/v) ethanol from the mash which was significantly more than that of the boiled and fermented mash (9.3% v/v) and equal to that of the mash boiled and fermented (10 2% v/v) after added amylases treatment. Most of the other compounds (kanamycin, streptomycin, polymyxin, tetracycline) had growth inhibitory effect especially on Z.mobilis.     

Direct fermentation of cassava starch to ethanol by mixed cultures of Endomycopsis fibuligera and Zymomonas mobilis: Synergism and limitations

Summary A mixed culture of Endomycopsis fibuligera NRRL 76 and Zymomonas mobilis ZM4 could directly and more efficiently ferment cassava starch (22.5% w/v) to ethanol (10.5% v/v) than the monocultures. The combination of culture filtrate of E.fibuligera containing amylases and Z.mobilis simultaneously saccharified and fermented the cassava starch to ethanol equally well. Glucoamylase (0.01%) added to the fermenting medium improved ethanol (13.2% v/v) production by the above mixed culture to almost the theoretical level (98%) indicating that this enzyme is a rate-limiting factor in E.fibuligera. Z. mobilis alone converted the enzymehydrolyzed starch only to almost theoretical level (98%).     

Estimation of kinetic and yield parameters: ethanol fermentation of a glucose and fructose mixture using Zymomonas mobilis

Summary A new method is presented to calculate the kinetic and yield parameters of a fermentation. This method is based on polynomial fitting of the variations of substrates and biomass concentrations with time and calculation of instantaneous and overall parameters. Application to a mixed substrate and mixed product fermentation by the bacterium Zymomonas mobilis is presented.     

Mechanism of ethanol inhibition of fermentation in Zymomonas mobilis CP4.

Accumulation of alcohol during fermentation is accompanied by a progressive decrease in the rate of sugar conversion to ethanol. In this study, we provided evidence that inhibition of fermentation by ethanol can be attributed to an indirect effect of ethanol on the enzymes of glycolysis involving the plasma membrane. Ethanol decreased the effectiveness of the plasma membrane as a semipermeable barrier, allowing leakage of essential cofactors and coenzymes. This leakage of cofactors and coenzymes, coupled with possible additional leakage of intermediary metabolites en route to ethanol formation, is sufficient to explain the inhibitory effects of ethanol on fermentation in Zymomonas mobilis.     

Kinetic control of ethanol production by Zymomonas mobilis

Summary Zymomonas mobilis UQM 2716 was grown anaerobically in continuous culture (D = 0.1/h; 30° C) 3nder glucose or nitrogen limitation at pH 6.5 or 4.0. The rates of glucose consumption and ethanol production were lowest during glucose-limited growth at pH 6.5, but increased during growth at pH 4.0 or under nitrogen limitation, and were highest during nitrogen-limited growth at pH 4.0. The uncoupling agent CCCP substantially increased the rate of glucose consumption by glucose-limited cultures at pH 6.5, but had much less effect at pH 4.0. Washed cells also metabolised glucose rapidly, irrespective of the conditions under which the original cultures were grown, and the rates were variably increased by low pH and CCCP. Broken cells exhibited substantial ATPase activity, which was increased by growth at low pH. It was concluded that the fermentation rates of cultures growing under glucose or nitrogen limitation at pH 6.5, or under glucose limitation at pH 4.0, are determined by the rate at which energy is dissipated by various cellular activities (including growth, ATP-dependent proton extrusion for maintenance of the protonmotive force and the intracellular pH, and an essentially constitutive ATP-wasting reaction that only operates in the presence of excess glucose). During growth under nitrogen limitation at pH 4.0 the rate of energy dissipation is sufficiently high for the fermentation rate to be determined by the inherent catalytic activity of the catabolic pathway.Abbreviations CCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - qG rate of glucose consumption (g glucose/g dry wt cells/h) - qE rate of ethanol production (g ethanol/g dry wt cells/h) - Y growth yield (g dry wt cells/g glucose) - D dilution rate Offprint requests to: C. W. Jones