Ethanol production by Zymomonas mobilis in fed-batch fermentations

The production of ethanol by Zymomonas mobilis NRRL B-4286 was studied in fed-batch cultures. Initial percent (w/v) glucose, rate of feed, and quantity of 50%; (w/v) glucose feed were varied. Glucose inhibition of growth rate occurred at concentrations greater than 8% (w/) Feed was begun after 4 h incubation. Feed volume was ca. 36%; of starting batch volume to get ca. 10%; (w/v) ethanol at harvest. The range of feed rates studied varied from 16%; batch volume/h (glucose concentration increased to an inhibitory level) to 4%; batch volume/h (glucose concentration dropped rapidly to zero and was limiting). Increasing feed volume to 46%; of starting volume at the best feed rate (ca. 10%; feed volume/h) increased final ethanol concentration to 11.3%; (w/v). However, the resultant increase in fermentation time from ca. 21 to 29 h decreased ethanol volumetric productivity from 5.2 to 4.6 g/L h.     

Extractive fermentation by Zymomonas mobilis and the control of oscillatory behavior

Summary Extractive fermentation is shown to greatly improve the performance ofZymomonas mobilis in continuous culture during the conversion of concentrated substrates to ethanol, and it is also used to eliminate the oscillatory behavior often exhibited byZ. mobilis in conventional fermentations. An ethanol productivity of 15.6 g/Lh is achieved with the near-conversion of a 295 g/L glucose feed at a medium dilution rate of 0.11 h^–1 and solvent dilution rate of 1.5 h^–1. This is more than triple the productivity obtained during conventional fermentation of a 135 g/L glucose feed at the same medium dilution rate.     

Refolding and purification of Zymomonas mobilis levansucrase produced as inclusion bodies in fed-batch culture of recombinant Escherichia coli

Zymomonas mobilis levansucrase was overproduced by the fed-batch culture of recombinant Escherichia coli harboring a novel expression system that is constitutively expressed by the promoter from the Rahnella aquatilis levansucrase gene. Most of the levansucrase was produced as inclusion bodies in the bacterial cytoplasm, accounting for approximately 20% of the total cellular protein. Refolding after complete denaturation by high concentrations of urea or guanidine hydrochloride was not successful, resulting in large amounts of insoluble aggregates. During the development of the refolding method, it was found that direct solubilization of the inclusion bodies with Triton X-100 reactivated the enzyme, with a considerable refolding efficiency. About 65% of inclusion body levansucrase was refolded into active levansucrase in the renaturation buffer containing 4% (v/v) Triton X-100. The in vitro refolded enzyme was purified to 95% purity by single-step DEAE-Sepharose ion exchange chromatography. Triton X-100 was removed by this ion exchange chromatography.     

Alcoholic fermentation of cellulose hydrolysate by Zymomonas mobilis

Summary Growth and ethanol production by three strains of the bacterium Zymomonas mobilis were tested, at 40°C, in a medium containing cellulose hydrolysate (hexose fraction) as carbon source. The thermotolerant mutant C107 exhibited the best growth compared to wild type ZM4 and to the osmotolerant mutant SBE15. When cultivated in media supplemented with various nutrients, growth was only observed in presence of yeast extract (10 g/l) which acted both as a vitamin supplier and pH stabilizer. Using calcium pantothenate instead of yeast extract and sodium acetate to control pH resulted in growth inhibition by the high medium osmolality. Batch fermentation with pH control (KOH addition) showed good growth and ethanol production with the mineral medium.     

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.     

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.     

Ethanol fermentation using a fructose-negative mutant of Zymomonas mobilis

Summary The fermentation of an equimolar mixture of glucose and fructose into ethanol and sorbitol by a fructose negative mutant of Zymomonas mobilis is analysed using a recently described methodology (Ait-Abdelkader and Baratti, Biotechnol. Tech. 1993,329–334) based on polynomial fitting and calculation of instantaneous and overall parameters. These parameters are utilized to describe this mixed-substrate mixed-product fermentation.Nomenclature X biomass concentration, g/l - S total sugar concentration, g/l - Glu glucose concentration, g/l - Fru fructose concentration, g/l - Sor sorbitol concentration, g/l - P ethanol concentration, g/l - t fermentation time, h - specific growth rate, h^-1 - q_s specific sugar uptake rate, g/g.h - q_g specific glucose uptake rate, g/g.h - q_F specific fructose uptake rate, g/g.h - q_P specific ethanol productivity, g/g.h - q_Sor specific sorbitol productivity, g/g.h - Y_X/S biomass yield on total sugar, g/g - Y_P/S ethanol yield on total sugar, g/g - Y_Sor/S sorbitol yield on total sugar, g/g - Y_Sor/F sorbitol yield on fructose, (g/g) - Y_P/G ethanol yield on glucose, (g/g)     

Zymomonas mobilis CP4 fed-batch fermentations of glucose-fructose mixtures to ethanol and sorbitol

Zymomonas mobilis CP4 fed-batch fermentations of glucose-fructose mixtures were carried out at different operational conditions (aeration, feed rate and substrate concentration) to test their effects on the system productivity. In these fermentations, the main products were ethanol and sorbitol. Kinetic parameters were calculated using the experimental data. However, parameters in the sorbitol synthesis rate were estimated from data recorded in different experiments in order to avoid the effect of the simultaneous cell growth and ethanol synthesis. In this case, the crude cell extract was used as source of the enzyme responsible for the sorbitol synthesis. The highest degree of conversion of fructose into sorbitol obtained with the extract was equal to 71% in a sugar mixture with an initial concentration of 200 g/l. Results obtained in the fed-batch fermentations showed that aeration of the culture has a positive effect on the final biomass concentration. However, final ethanol concentration is lower under aerated conditions. The best sugar yields to biomass and ethanol were 0.032 and 0.411 g/g, respectively. On the other hand, the highest sorbitol yield in the fed-batch fermentations was 0.148 g/g.     

Pentose metabolism in Zymomonas mobilis wild-type and recombinant strains

The enzyme activities of the pentose phosphate pathway in the ethanologenic, Gram-negative bacterium Zymomonas mobilis were studied in order to construct a xylose catabolic pathway. In cell-free extracts of wild-type Z. mobilis CP4, activities of the enzymes transketolase (TKT) [2 munits (U)/mg], phosphoribose epimerase (640 mU/mg), phosphoribose isomerase (1600 mU/mg) and 6-phosphogluconate dehydrogenase (2 mU/mg) were determined. However, no transaldolase activity could be detected. Recombinant strains of Z. mobilis were constructed that carried the xylAB genes of the xylose catabolic pathway from Klebsiella pneumoniae. Expression of xylose isomerase (XI, 150 mU/mg) and xylulokinase (XK) (1300 mU/mg) were found in recombinant strains but no growth on pentose as sole carbon source occurred. The xyl-recombinant cells were moreover growth-inhibited in the presence of xylose and were found to accumulate xylitol phosphate due to the subsequent action of a novel enzyme, an NADPH-dependent aldose reductase, and a side reaction of XK on xylitol. From the xylAB recombinant strains, mutants were isolated that were less inhibited and formed less xylitol phosphate when grown in the presence of xylose. The tkt gene of E. coli was cloned on the xylAB plasmid and introduced into Z. mobilis strains. This led to higher TKT activities (150 mU/mg) and, in cooperation with the enzymes XI and XK, mediated a conversion of small amounts of xylose to CO₂ and ethanol. However, no growth on xylose as sole carbon source was detected, instead sedoheptulose 7-P accumulated intracellularly. Correspondence to: G. Sprenger     

Fructose production by Zymomonas mobilis in fed-batch culture with minimal sorbitol formation

Summary Fed-batch cultures of Zymomonas mobilis (UQM 2864), a mutant unable to metabolise fructose, grown on diluted sugar cane syrup (200 g/l sucrose) achieved yields of 90.5 g/l fructose and 48.3 g/l ethanol with minimal sorbitol formation and complete utilization of the substrate. The effect of inoculum size on sorbitol formation in the batch stage of fed-batch fermentation are reported. Fermentation of sucrose (350 g/l) supplemented with nutrients yielded 142 g/l fructose and 76.5 g/l ethanol. Some fructose product loss at high fructose concentrations was observed. The fed-batch fermentation process offers a method for obtaining high concentrations of fructose and ethanol from sucrose materials.     

Kinetic modeling to optimize pentose fermentation in Zymomonas mobilis

Zymomonas mobilis engineered to express four heterologous enzymes required for xylose utilization ferments xylose along with glucose. A network of pentose phosphate (PP) pathway enzymatic reactions interacting with the native glycolytic Entner Doudoroff (ED) pathway has been hypothesized. We have investigated this putative reaction network by developing a kinetic model incorporating all of the enzymatic reactions of the PP and ED pathways, including those catalyzed by the heterologous enzymes. Starting with the experimental literature on in vitro characterization of each enzymatic reaction, we have developed a kinetic model to enable dynamic simulation of intracellular metabolite concentrations along the network of interacting PP and ED metabolic pathways. This kinetic model is useful for performing in silico simulations to predict how varying the different enzyme concentrations will affect intracellular metabolite concentrations and ethanol production rate during continuous fermentation of glucose and xylose mixtures. Among the five enzymes whose concentrations were varied as inputs to the model, ethanol production in the continuous fermentor was optimized when xylose isomerase (XI) was present at the highest level, followed by transaldolase (TAL). Predictions of the model that the interconnecting enzyme phosphoglucose isomerase (PGI) does not need to be overexpressed were recently confirmed through experimental investigations. Through such systematic analysis, we can develop efficient strategies for maximizing the fermentation of both glucose and xylose, while minimizing the expression of heterologous enzymes.     

Batch fermentation kinetics of sugar beet molasses by Zymomonas mobilis

A new osmotolerant mutant strain of Zymomonas mobilis was successfully used for ethanol production from beet molasses. Addition of magnesium sulfate to hydrolyzed molasses allowed repeated growth without the need of yeast extract addition. The kinetics and yields parameters of fermentation on media with different molasses concentrations were calculated. The anabolic parameters (specific growth rate, mu, and biomass yield, Y(X/S)) were inhibited at elevated molasses concentrations while the catabolic parameters (specific ethanol productivity, q(p), and ethanol yield, Y(p/s)) were not significantly affected. In addition to ethanol and substrate inhibition, osmotic pressure effects can explain the observed results.     

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.     

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     

Extractive fermentation by Zymomonas mobilis and the use of solvent mixtures

Summary A systematic and comprehensive screening of 1330 solvents, individually and as mixtures, was undertaken with the aim of improving on present extractive fermentation technology. The screening resulted in the selection of a solvent mixture of 5% by volume 4-heptanone and 95% Adol 85 NF (primarily oleyl alcohol). The performance of this solvent mixture in an extractive fed-batch fermentation configuration was compared with that of pure Adol 85 NF. The solvent mixture had a distribution coefficient 12% higher than that of pure Adol 85 NF and had no significant inhibitory effect on the fermentation under typical operating conditions.     

Modeling of ethanol fermentation using Zymomonas mobilis ATCC 10988 grown on the media containing glucose and fructose

A model of ethanol fermentation by Zymomonas mobilis ATCC 10988 on the medium containing glucose and fructose is proposed. This model was developed on the basis of metabolic analysis and many experimental findings. When glucose was used as the substrate, the dependence of the carbon fraction (α) assimilating to biomass on the specific growth rate (μ) could be well correlated to α = 0.25μ + 0.012. This correlation resulted in a novel equation for specific glucose uptake rate, which could describe the Z. mobilis fermentation in both batch and continuous modes. When fructose and glucose were both presented in the liquid medium, the model could predict the uptake of glucose and fructose as well as the formation of biomass, ethanol and sorbitol by Z. mobilis. All parameters used in the model were independently evaluated on the basis of various experimental findings. Good agreement was found between the model predictions and data of Z. mobilis fermentation on media containing both glucose and fructose. The proposed model could also describe the behavior of ethanol fermentation on sucrose medium supplemented with immobilized invertase.     

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.     

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.