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⁻.     

Ethanol production from cassava and sago starch using Zymomonas mobilis

Summary Cassava and sago starch were evaluated for their feasibilities as substrates for ethanol production using Zymomonas mobilis ZM4 strain. Before fermentation, the starch materials were pretreated employing two commercial enzymes, Termamyl (thermostable -amylase) and AMG (amyloglucosidase). Using 2 l/g of Termamyl and 4 l/g of AMG, effective conversion of both cassava and sago starch into glucose was found with substrate concentration up to 30%(w/v) dry substances. Fermentation study performed using these starch hydrolysates as substrates resulted in ethanol yield at an average of 0.48g/g by Z. Mobilis ZM4.     

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.     

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.     

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     

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     

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.     

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.     

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.     

Cloning and expression in Escherichia coli of the dnaK gene of Zymomonas mobilis.

The DnaK protein of Zymomonas mobilis (DnaKz) was identified and found to be 80% identical to the DnaK protein of Escherichia coli on the basis of the sequence of the N-terminal 21 amino acids. The dnaKz gene was cloned and found to be expressed in a thermosensitive dnaK mutant of Escherichia coli. Expression of the foreign gene restored a thermoresistant phenotype but failed to modulate the heat shock response in E. coli.     

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.     

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.     

Continuous production of ethanol from fructose by immobilized growing cells of Zymomonas mobilis

Immobilized growing cells of Zymomonas mobilis were found to ferment rapidly and efficiently media containing 100 g/L fructose in a continuous reactor. A volumetric ethanol productivity of 94.8 g/L h was achieved at a substrate conversion of 75.5%. With 97% conversion of substrate the productivity was 28.4 g/L h. At fructose concentrations of 150 and 200 g/L substrate and product inhibitions limited the performance of the reactor. Ethanol production was constant over a period of 55 days.     

枯草芽孢杆菌Mn-SOD基因的克隆及原核表达

为了实现Mn-SOD基因在大肠杆菌(E.coli)中的可溶性表达,根据枯草芽孢杆菌(Bacillus subtilis)168sodA核酸序列设计引物,以枯草芽孢杆菌ATCC 9372基因组为模板,PCR扩增获得了Mn-SOD基因.将此基因重组至原核表达载体pET-28a,构建含Mn-SOD基因的重组表达质粒,并转化至大肠杆菌BL21(DE3).异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达获得Mn-SOD,蛋白分子量约为26kD,占全菌蛋白的5.6%.改良的连苯三酚自氧化法测定SOD活力,菌体可溶性总蛋白SOD比活为51.09U·mg^-1,是对照组的.8倍.枯草芽孢杆菌ATCC 9372 Mn-SOD基因在大肠杆菌BL21(DE3)中首次成功表达,产物具有较高的可溶性和活性,为大量制备Mn-SOD奠定了基础.     

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.     

Cloning of fibrinolytic enzyme gene from Bacillus subtilis isolated from Cheonggukjang and its expression in protease-deficient Bacillus subtilis strains

Bacillus subtilis CH3-5 was isolated from cheonggukjang prepared according to traditional methods. CH3-5 secreted at least four different fibrinolytic proteases (63, 47, 29, and 20 kDa) into the culture medium. A fibrinolytic enzyme gene, aprE2, encoding a 29 kDa enzyme was cloned from the genomic DNA of CH3-5, and the DNA sequence determined. aprE2 was overexpressed in heterologous B. subtilis strains deficient in extracellular proteases using a E. coli-Bacillus shuttle vector. A 29 kDa AprE2 band was observed and AprE2 seemed to exhibit higher activities towards fibrin rather than casein.     

Xanthan gum and ethanol production by Xanthomonas campestris and Zymomonas mobilis from peach pulp

The wild type of Xanthomonas campestris and a mutant strain of Zymomonas mobilis CP4, tolerant to sucrose up to 40% (w/v), were used to produce either xanthan gum or ethanol, respectively, from peach pulp supplemented with different salts. Both bacteria grew well (2.7 mg/ml for X. campestris and 1.45 mg/ml for Z. mobilis) in fine peach pulp and the production of xanthan gum or ethanol was 0.1–0.2 g/l or 110 g/l, respectively.