Continuous ethanol production at high glucose concentrations by a passively immobilized Zymomonas mobilis system

Summary The effect of high glucose concentrations on continuous ethanol production by passively immobilized Zymomonas mobilis cells has been studied. High effluent ethanol concentrations always led to low productivities. The maximum ethanol concentration attained was 92.8 g/l (98% glucose conversion) at a dilution rate of 0.14 h^-1 with 200 g/l glucose medium. The observed enhancement of cell immobilization in the fibrous support at high glucose concentrations in the feed input seems to be related to the formation of bacterial filaments.Preliminary results from this work were previously presented at the Second Spanish Conference on Biotechnology (Barcelona, 1988)     

Electron microscopic investigations of zymomonas mobilis cells grown in low and high glucose concentrations

Summary Zymomonas mobilis cells grown in the presence of low and high glucose concentrations were examined by electron microscopy. Using ultrathin sectioning and agar diffusion method, significant changes in morphology were observed. Although the fine structure resembles that of a typical gram-negative bacterium, changes in glucose concentration and phases of growth lead to large cell wall vesicle or blebs formation. The possible implications of this morphological change to glucose uptake and ethanol formation are discussed.     

Formation of ethanol and higher alcohols by immobilized zymomonas mobilis in continuous culture

Cells of Zymomonas mobilis ATCC 10988 were immobilized in 1.5% calcium alginate and packed in a column bioreactor for a series of fermentations utilizing 10.0% glucose media with the addition of one of the following amino acids or keto acids: L-leucine, L-isoleucine, L-valine, α-ketoisocaproic acid, α-ketobutyric acid, or α-ketoisovaleric acid. This was done in order to study the rates of production of higher alcohols during ethanolic fermentations at varying dilution rates while under the influence of amino acids or keto acids. Results indicate that the EHRLICH mechanism is operative in Zymomonas sp. α-Ketobutyrate enhanced the production of n-propanol and act-amyl alcohol. α-Ketoisocaproic acid stimulated the production of isoamyl alcohol. α-Ketoisovaleric acid increased the levels of isobutanol. The amino acids also gave rise to their corresponding alcohols but to a far lesser degree than did the keto acids. During high glucose utilization, ethanol yields ranged from 87% to 94% of theoretical with productivity ranging from 60.08 g/l/h in one fermentation (at a dilution rate of 1.35 h^?1) to 70.42 g/l/h in another (at a dilution rate of 1.58 h^?1). At dilution rates of 1.58 h^?1, higher alcohol productivity rose to as high as 4,313 mg/l/h in the presence of α-ketoisocaproic acid, 1,734.49 mg/l/h using α-ketoisovaleric acid, and 1,618.05 mg/l/h in α-ketobutyric acid. The concomitant production of ethanol and higher alcohols in all of the fermentations indicates that glucose is required for the production of the higher alcohols from their corresponding amino acids or keto acids.     

Improvement of productivity and efficiency in ethanol production with ca-alginate immobilized zymomonas mobilis

Summary A three-stage reactor system was used to obtain high ethanol concentration (74 g/1) with immobilized Zymomonas mobilis DSM 424. The catalyst efficiency was improved by formation of small diameter beads and arrangement of the biocatalyst in a reactor configuration adapted to the kinetics of ethanol production. A higher productivity than reported before was obtained with 56,5 g/lh based on total working volume at a sugar conversion rate of 98%. The bioreactor was run continuously for 65d.     

Kinetics of alcohol production by zymomonas mobilis at high sugar concentrations

Summary Studies on the growth ofZ.mobilis revealed that high concentrations of glucose (10-25%) can be efficiently and rapidly converted to ethanol in batch culture. By comparison withS. carlsbergensis,Z.mobilis had specific glucose uptake rates and specific ethanol productivies several times greater than the yeast.Z.mobilis also had ethanol yields of up to 97% of a theoretical value.     

Rapid ethanol fermentation with immobilized Zymomonas mobilis in a three stage reactor system

Summary Using the Zymomonas mobilis NRRL B 14023 strain for ethanol fermentation with immobilized cells the combination of two external loop reactors followed by a plug flow reactor was the most effective reactor configuration. A maximal productivity of 92 and 108 g/l·h at practically complete sugar consumption was obtained with Carrageenan and Alginate catalysts respectively. Due to the high dilution rate nonsterile operation for extended periods of weeks was possible.     

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⁻.     

Examination of immobilized cells in a rotating packed drum reactor for the production of ethanol from d-glucose

A rotating packed drum reactor has been proposed as an immobilized whole cell reactor and its performance for ethanol production has been studied with yeast cells immobilized in calcium alginate gel. In a continuous operation with synthetic d-glucose medium containing 125 g d-glucose l^?1, ethanol productivity was 20 g l^?1 h^?1 at a space velocity of 0.38 l (l gel)^?1 h^?1. With intermittent aeration the viability of yeast cells after 270 h of operation remained above 65%. CO₂ removal was easy, but d-glucose conversion was low at a high space velocity.     

A mutant of Zymomonas mobilis ZM4 capable of ethanol production from glucose in the presence of high acetate concentrations

Growth of Zymomonas mobilis ZM4 in media containing sodium acetate was inhibited above 12 g sodium acetate/l at pH 5.0. Following mutagenesis of ZM4, an acetate-tolerant strain was isolated in continuous culture that grew in the presence of 20 g sodium acetate/l at pH 5.0. In continuous culture with complete cell recycle at 30 deg C and pH 5.4 using media containing 110 g glucose/l, the maintenance energy coefficient (m) for the mutant was found to increase from 1.9 g glucose/g cell dry wt.h at 12 g sodium acetate/l up to 3.9 g/g.h at 20 g/l.     

Performance of rotating packed disk reactor with immobilized glucose oxidase

Summary Reactor performance was studied to investigate whether a rotating packed disk reactor (RPDR) can be used for the enzymatic oxidation of biochemicals. The disks were packed with calcium alginate beads with immobilized glucose oxidase and catalase, which catalyze the reaction of glucose and oxygen. The production rate of gluconic acid increased with the speed of rotation and the bulk flow rate. An optimum submergence for maximum productivity existed.     

Ethanol production by zymomonas mobilis: Effect of temperature on cell growth,ethanol production and intracellular ethanol accumulation

Summary The kinetic behaviour of Zymomonas mobilis is studied at temperatures from 27 °C to 34.5 °C. Increases of temperature (above a critical value) have a negative effect on the yield of ethanol, mainly by raising the maintenance coefficient. Cell growth is less inhibited by the ethanol accumulation inside the cell than is the fermentative pathway. The specific rate of ethanol production is depressed when the intracellular ethanol concentration is maximal. An increase of temperature enhances this phenomenon.     

Kinetics of ethanol production by immobilized cells of Zymomonas mobilis at varying d-glucose concentrations

Ethanol fermentation by cells of Zymomonas mobilis immobilized in calcium alginate gel has been studied using 5 to 30 wt% initial d-glucose in the medium. Up to 27% d-glucose was completely fermented and the maximum ethanol concentration of 12.6% (w/v) was obtained using an immobilized cell concentration of 58 g dry wt l^?1 of bead volume. The ethanol yield coefficient was almost unaffected by initial d-glucose concentration and its value was >95% of theoretical. The rates of ethanol production and d-glucose utilization first increased, with an increase in initial d-glucose concentration up to 13.6%, and then started to decrease upon a further increase in initial d-glucose concentration. Cell leakage from the calcium alginate beads was very low.     

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.     

Production of ethanol by a stirred catalytic basket reactor with immobilized yeast cells

A stirred catalytic basket reactor with immobilized yeast cells was used for the batchwise production of ethanol. Fractional conversions up to 0.99 in 10 h were attained, depending on the agitation rates, initial glucose, and cell densities. The volumetric productivity of the reactor was considerably better than that of conventional stirred tank reactors. Productivities were strongly dependent on the stirred speed.     

High ethanol productivities using small Ca-alginate beads of immobilized cells of Zymomonas mobilis

Summary Zymomonas mobilis cells were immobilized into small 1 mm diameter beads of Ca-alginate in order to minimize mass transfer limitations and maximize immobilized cell activity. A combination of small bead size with a high cell concentration of 58 g dry wt. cell per lit. bead volume resulted in high ethanol productivities using a newly designed packed bed bioreactor system. Steady-state dilution rates ranging from 0.4 h^-1 to 3.9 h^-1 were run resulting in a maximum productivity of 102 g ethanol/l/h for an inlet substrate concentration of 100 g glu/l and 87% conversion. The bioreactor was run continuously at a fixed dilution rate for 384 h and short intermittent treatment of the beads with CaCl₂ temporarily increased ethanol productivity to a maximum of 116 g ethanol/l/h.     

The long-term effects of ethanol on immobilized cell reactor performance using K. fragilis

The effects of ethanol on reactor performance were studied in a small, 5-cm packed height, "differential" type immobilized cell reactor. Lactose utilizing yeast cells, Kluyveromyces fragilis, were absorbed to a porous adsorbant sponge matrix in a gas continuous reactor. Step changes in the feed ethanol concentration to the column (10-130 g/L) were used to test the reactor response over extended periods of time (about 30-50 h per dosage level) followed by a return to basal zero inlet ethanol feed. Effluent cell density and effluent cell viability were measured at intervals. An inhibitory response in ethanol productivity to feed dosage ethanol levels above 20 g/L was detected almost immediately, with a near steady state response noted within 2.5 h of initiating the dosage. Feed ethanol levels above 50 g/L resulted in a subsequent gradual decrease in reactor productivity over time, which was associated with a decrease in the fraction of viable shed cells in the reactor effluent. The reactor response to a step removal of the ethanol inhibition was also monitored. Quick and complete rebounding of the fermentation rate to the original basal rate was noted following dosage concentrations of under 50 g/L ethanol. Recovery rates slowed following ethanol dosage levels above 50 g/L. Viable shed cell density improved overtime during the slow recovery periods. Growth rates (as determined by shed cell density) were more strongly inhibited than productivity. Growth responded more slowly to changes in ethanol environment as growth rates at 30 h fell to about 40% of the rates measured 7.5 h after initiation of a dosage level. It is concluded that ethanol contributions to cell injury and death (and consequent ICR performance degradation) may be more important than ethanol inhibition of productivity rates in the long-term operation of immobilized cell reactors at ethanol concentrations over 50 g/L.     

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.     

Production of ethanol from sugar cane molasses by Zymomonas mobilis

Summary Zymomonas mobilis strains were compared with each other and with a Saacharomyces cerevisiae strain for the production of ethanol from sugar cane molasses in batch fermentations. The effect of pH and temperature on ethanol production by Zymomonas was studied. The ability of Z. mobilis to produce ethanol from molasses varied from one strain to another. At low sugar concentrations Zymomonas compared favourably with S. cerevisiae. However, at higher sugar concentrations the yeast produced considerably more ethanol than Zymomonas.