Continuous ethanol production from sugar beet molasses using an osmotolerant mutant strain of Zymomonas mobilis

A laboratory process was established for ethanol production by fermentation of sugar beet molasses with the bacterium Zymomonas mobilis. Sucrose in the molasses was hydrolyzed enzymatically to prevent levan formation. A continuous system was adopted to reduce sorbitol formation and a two-stage fermentor was used to enhance sugar conversion and the final ethanol concentration. This two-stage fermentor operated stably for as long as 18 d. An ethanol concentration of 59.9 g/l was obtained at 97% sugar conversion and at high ethanol yield (0.48 g/g, 94% of theoretical). The volumetric ethanol productivity (3.0 g/l·h) was superior to that of batch fermentation but inferior to that of a single-stage continuous system with the same medium. However, the thanol concentration was increased to a level acceptable for economical recovery. The process proposed in this paper is the first report of successful fermentation of sugar beet molasses in the continuous mode using the bacterium Z. mobilis.     

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.     

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     

Effects of ethanol concentration on immobilized Zymomonas mobilis for continuous production of ethanol

The effects of ethanol concentration on the ethanol productivity and activity of immobilized Zymomonas mobilis cells during continuous fermentation of glucose has been studied at various ethanol concentrations. On changing the inlet ethanol concentration, Po, from 0.0 kg/m³ to any other level, 8 h were required to fully experience the effects of a change in Po, whereas 8 h to 2 days, depending on Po, were required to reach the steady state on switching back to the ethanol free medium. The volumetric ethanol productivity decreased from 92.5 to 0.0 kg/m³·h as the ethanol concentration in the bioreactor was changed from 46.3 to 126 kg/m³. The activity of the immobilized cells recovered up to 63% in 2 days even after exposing the cells to 126 kg/m³ of ethanol.     

Continuous ethanol production by immobilized cells of Zymomonas mobilis

Summary Studies have been carried out with a highly productive strain of Zymomonas mobilis in an immobilized cell reactor using both Ca alginate and -carrageenan as supporting matrices. Productivities above 50 g/l/h have been found at ethanol concentrations in excess of 60 g/l. With immobilized cells of Z. mobilis, there was a decline of approximately 30s% in activity after 800 h operation.     

Effect of calcium chloride on the growth and ethanol production by free cells of Zymomonas mobilis

Summary The effect of calcium chloride concentration on the growth rate and ethanol production using free cells of Zymomonas mobilis was studied. There was no appreciable change in rates of cell mass production and ethanol formation in the medium containing upto 2g/L CaCl₂. On further increase in CaCl₂ concentration, the rates started decreasing. However, the ethanol yield decreased and biomass yield increased with increase in CaCl₂ concentration.     

Continuous ethanol production by a flocculent strain of Zymomonas mobilis

Summary Cell retention and ethanol production using the flocculent bacterium Zymomonas mobilis NRRL B-12526 were studied in three bioreactor configurations. The flocculent growth characteristic of this strain and a special reactor design were combined to achieve relatively high cell concentrations in a continuous bioreactor for the conversion of glucose to ethanol.Research sponsored by the Office of Energy Research, U.S. Department of Energy, under contract W-7405-eng-26 with the Union Carbide Corporation.     

Kinetic analysis of ethanol production by an acetate-resistant strain of recombinant Zymomonas mobilis

Zymomonas mobilis ZM4/Ac^R (pZB5), a mutant recombinant strain with increased acetate resistance, has been isolated following electroporation of Z. mobilis ZM4/Ac^R. This mutant strain showed enhanced kinetic characteristics in the presence of 12 g sodium acetate l^–1 at pH 5 in batch culture on 40 g glucose, 40 g xylose l^–1 medium when compared to ZM4 (pZB5). In continuous culture, there was evidence of increased maintenance energy requirements/uncoupling of metabolism for ZM4/Ac^R (pZB5) in the presence of sodium acetate; a result confirmed by analysis of the effect of acetate on other strains of Z. mobilis. Nomenclature m Cell maintenance energy coefficient (g g^–1 h^–1)Maximum overall specific growth rate (1 h^–1)Maximum specific ethanol production rate (g g^–1 h^–1)Maximum specific total sugar utilization rate (g g^–1 h^–1)Biomass yield per mole of ATP (g mole^–1 Ethanol yield on total sugars (g g^–1)Biomass yield on total sugars (g g^–1)True biomass yield on total sugars (g g^–1)     

Cellulosic ethanol production by Zymomonas mobilis harboring an endoglucanase gene from Enterobacter cloacae

Enterobactercloacae was isolated from the gut of the wood feeding termite, Heterotermesindicola, and a 2.25-kb fragment conferring cellulase activity was cloned in Escherichiacoli. The cloned fragment contained a 1083-bp ORF which could encode a protein belonging to glycosyl hydrolase family 8. The cellulase gene was introduced into Zymomonasmobilis strain Microbial Type Culture Collection centre (MTCC) on a plasmid and 0.134 filter paper activity unit (FPU)/ml units of cellulase activity was observed with the recombinant bacterium. Using carboxymethyl cellulose and 4% NaOH pretreated bagasse as substrates, the recombinant strain produced 5.5% and 4% (V/V) ethanol respectively, which was threefold higher than the amount obtained with the original E.cloacae isolate. The recombinant Z. mobilis strain could be improved further by simultaneous expression of cellulase cocktails before utilizing it for industrial level ethanol production.     

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)     

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.     

Effect of substrate concentration on ethanol production by Zymomonas mobilis on cellulose hydrolysate

Summary A cellulose hydrolysate from Aspen wood, containing mainly glucose, was fermented into ethanol by a thermotolerant strain MSN77 of Zymomonas mobilis. The effect of the hydrolysate concentration on fermentation parameters was investigated. Growth parameters (specific growth rate and biomass yield) were inhibited at high hydrolysate concentrations. Catabolic parameters (specific glucose uptake rate, specific ethanol productivity and ethanol yield) were not affected. These effects could be explained by the increase in medium osmolality. The results are similar to those described for molasses based media. Strain MSN77 could efficiently ferment glucose from Aspen wood up to a concentration of 60 g/l. At higher concentration, growth was inhibited.Nomenclature S glucose concentration (g/l) - X biomass concentration (g/l) - P ethanol 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) - YINX/S biomass yield (g/g) - Y_p/S ethanol yield (g/g) - specific growth rate (h^-1)     

Comparison of ethanol production by Zymomonas mobilis from sugar beet substrates

Summary The potential of four sugar beet substrates from the sugar industry [syrup (S), crystallizer effluent 1 (CE1), crystallizer effluent 2 (CE2) and molasses (M)] were compared for ethanol production using an osmotolerant mutant strain of the bacterium Zymomonas mobilis. Sucrose of the substrates was enzymatically hydrolysed to avoid levan formation during fermentation. Nutrient supplementation experiments have shown that reproducible growth and ethanol production could be obtained on the four substrates supplemented only with magnesium sulphate (CE2 and M) or additionally with ammonium sulphate (S and CE1). Thus, addition of costly yeast extract could be avoided. All 20% (w/v) substrates showed nearly complete sugar conversion (>94.9%), good growth (0.16 h^–1) and ethanol production (>40 g 1^–1). However, sorbitol formation reduced the ethanol yield (73–79% of the theoretical value) significantly. Batch kinetic parameters and studies of instantaneous parameters showed that enhanced osmolality of substrates (SZ. mobilis with appropriate supplementation. Offprint requests to: J. Baratti     

Sorbitol production by Zymomonas mobilis

Summary High resolution ¹³C Nuclear Magnetic Resonance (NMR) spectroscopy has been employed to determine the chemical composition of the unknown major products in a sucrose or fructose plus glucose fermentation to ethanol by the bacterium Zymmonas mobilis. When grown on these sugars Z.mobilis was found to produce significant amounts of sorbitol, up to 43 g·l^-1 for strain ZM31 when grown on 250 g·l^-1 sucrose.The production of sorbitol and decrease of glucose, fructose, or sucrose was followed throughout batch fermentations by NMR and HPLC. Sorbitol was shown to be derived only from fructose by [¹⁴C]-feeding experiments. Additionally ³¹P NMR spectroscopy was utilized to determine the concentrations of both glucose 6-phosphate and fructose 6-phosphate relative to their respective concentrations in Z.mobilis cells fermenting glucose or fructose alone.It is suggested that free glucose inside the cell inhibits fructokinase. Free intracellular fructose may then be reduced to sorbitol via a dehydrogenase type enzyme. Attempts to grow Z.mobilis on sorbitol were unsuccessful, as were experiments to induce growth via mutagenesis.This work was supported in part by the National Energy Research, Development and Demonstration Council of Australia     

The production of ethanol from sucrose using Zymomonas mobilis

Summary A new single-batch fermentation process for the commercial production of ethanol from refined sucrose, raw sugar, sugar cane juice and sugar cane syrup has been developed using a highly adapted and efficient strain of Zymomonas mobilis. The process gives a 94–98% sucrose hydrolysis efficiency and a 95–98% ethanol conversion efficiency. Within 24–30 h, 200 g/l sucrose is converted to produce 95.5 g/l ethanol. Reinoculation is carried out from the fermented broth without the need for centrifugation or membrane filtration.     

The kinetics of ethanol production by Zymomonas mobilis on fructose and sucrose media

Summary Z.mobilis is strain ZM4 was grown on 250 g/l fructose and sucrose media in batch culture and on 100 and 150 g/l sucrose media in continuous culture. With fructose, a significant reduction in the growth rate and the cell yield was apparent although the other kinetic parameters were similar to those previously reported for fermentation of glucose. With sucrose the major differences were a reduction in ethanol yield, (due to levan formation) and a lower final ethanol concentration. Ethanol inhibition of sucrose metabolism occurred at relatively low ethanol concentrations compared to those inhibiting glucose metabolism.