Effective diffusivity of galactose in calcium alginate gels containing immobilized Zymomonas mobilis.

The effective diffusivity of galactose was measured for calcium alginate gel membranes containing immobilized live Zymomonas mobilis cells at concentrations ranging from 0 to 150 g dry wt/L of gel. Since galactose is not taken up by living Z. mobilis organisms, the diffusion of this representative six-carbon sugar could be studied independently of sugar consumption. Various immobilized biomass loadings were achieved by two different techniques: addition of biomass at known concentrations to the sodium alginate solution before membrane formation and growth of cells in the gel to various biomass concentrations. The highest immobilized cell concentration, attained by in situ growth, corresponds to the maximum of this system, as growth beyond this maximum concentration led to disintegration of the gel membrane. The galactose effective diffusivity measurements for both methods of immobilized cell loading overlap within experimental error and follow the same general monotonic decline with entrapped biomass concentration. Most of the data fall below the upper bound predicted by Hashin and Shtrikman (1962) and show good agreement with the random pore model of Wakao and Smith (1962, 1964). Available effective diffusivity data from the literature provide evidence that the random pore model is an excellent predictor of sugar effective diffusivity in gel immobilized cell systems in general.     

Ethanol production by immobilized cells of Zymomonas mobilis

Summary Columnar reactors containing immobilized cells of Zymomonas mobilis were utilized for the continuous production of ethanol from glucose. Two different immobilization strategies were investigated. In one case, cells were entrapped in borosilicate glass fiber pads, while in the other, cells were immobilized via flocculation. The reactors were operated in both the fixed-bed and expanded-bed manner. Ethanol productivities as high as 132 g/l·h were achieved. Data obtained from studies employing 5.0 and 10.0% glucose concentrations are presented. Problems encountered during the operation of the continuous, immobilized cell reactors are discussed.Operated by Union Carbide Corporation under contract W-7405-eng-26 with the U.S. Department of Energy.     

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.     

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.     

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.     

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⁻.     

Preparation and characterization of immobilized growing cells of Zymomonas mobilis for ethanol production

Zymomonas mobilis cells were entrapped in K. carrageenan. Growth was observed with the immobilized cell preparation. The kinetic and yield parameters for the conversion of fructose to ethanol were nearly identical to free cells. The same preparation of immobilized cells was used in six repeated batch runs and at the end sixthbatch fructose was converted to ethanol more rapidly and efficiently with ethanol productivity of 14 g/L h and 96% conversion of fructose. The effect of high fructose and ethanol levels on specific fructose uptake rate and ethanol productivity was studied and quantitatively analyzed.     

Ethanol production by immobilized Saccharomyces cerevisiae, Saccharomyces uvarum, and Zymomonas mobilis

Saccharomyces cerevisiae NRRL Y-2034, S, uvarum NRRL Y-1347, and Zymomonas mobilis NRRL B-806 each were separately immobilized in a Ca-alginate matrix and incubated in the presence of a free-flowing and continuous 1, 3, 5, 10, or 20% (w/w) glucose solution. In general, the yeast cells, converted 100percnt; of the 1, 3, and 5% glucose to alcohol within 48 h and maintained such a conversion rate for at least two weeks. The bacterium converted ca. 90% (w/w) of the 1, 3, and 5% glucose to alcohol continuously for one week. However, both the yeast and bacterium were inhibited in the highest glucose (20% w/w) solution. All of the immobilized cultures produced some alcohol for at least 14 days. Immobilized S. cerevisiae was the best alcohol producer of all of the glucose concentrations; the yeast yielded 4.7 g ethanol/100 g solution within 72 h in the 10% glucose solution. After 7-8 days in the 10% solution, S. cerevisiae produced ethanol at 100% of theoretical yield (5.0 g ethanol/100 g solution), with a gradual decrease in alcohol production by 14 days. Immobillized S. uvarum produced a maximum of 4.0 g ethanol/100 g solution within 2 days and then declined to ca. 1.0 g ethanol/100 g solution after 7 days continuous fermentation in the 10% glucose solution. Zymomonas mobilis reached its maximum ethanol production at 4 days (4.7 g/100 g solution), and then diminished similarly to S. uvarum. The development of a multiple disk shaft eliminated the problem both of uneven distribution of alginate-encapsulated cells and of glucose channeling within the continuous-flow fermentor column. This invention improved alcohol production about threefold for the yeast cells.     

Oxidative phosphorylation in Zymomonas mobilis

The obligately fermentative aerotolerant bacterium Zymomonas mobilis was shown to possess oxidative phosphorylation activity. Increased intracellular ATP levels were observed in aerated starved cell suspension in the presence of ethanol or acetaldehyde. Ethanolconsuming Z. mobilis generated a transmembrane pH gradient. ATP synthesis in starved Z. mobilis cells could be induced by external medium acidification of 3.5–4.0 pH units. Membrane vesicles of Z. mobilis coupled ATP synthesis to NADH oxidation. ATP synthesis was sensitive to the protonophoric uncoupler CCCP both in starved cells and in membrane vesicles. The H⁺-ATPase inhibitor DCCD was shown to inhibit the NADH-coupled ATP synthesis in membrane vesicles. The physiological role of oxidative phosphorylation in this obligately fermentative bacterium is discussed.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - CCCP carbonyl cyanide m-chlorophenylhydrazone     

R-Plasmid Transfer in Zymomonas mobilis

Conjugal transfer of three IncP1 plasmids and one IncFII plasmid into strains of the ethanol-producing bacterium Zymomonas mobilis was obtained. These plasmids were transferred at high frequencies from Escherichia coli and Pseudomonas aeruginosa into Z. mobilis and also between different Z. mobilis strains, using the membrane filter mating technique. Most of the plasmids were stably maintained in Z. mobilis, although there was some evidence of delayed marker expression. A low level of chromosomal gene transfer, mediated by plasmid R68.45, was detected between Z. mobilis strains. Genetic evidence suggesting that Z. mobilis may be more closely related to E. coli than to Pseudomonas or Rhizobium is discussed.     

Fructose metabolism in Zymomonas mobilis

Summary In the metabolism of fructose by Zymomonas, the ethanol yield is decreased due to the formation of dihydroxyacetone, mannitol and glycerol. The reduction of fructose to mannitol by an NADPH-dependent mannitol dehydrogenase is apparently coupled to the oxidation of glucose-6-phosphate by glucose-6-phosphate dehydrogenase, which exhibits higher activity with NADP than with NAD as cofactor. The relatively low cell yield on fructose can partly be explained by the loss of ATP in the formation of dihydroxyacetone and glycerol and partly by the toxic effect of dihydroxyacetone and acetaldehyde on the growth of the organism.     

High ethanol productivity from lactose by immobilized cells of Kluyveromyces fragilis and Zymomonas mobilis

Ethanol production from 200 g lactose/l by Kluyveromyces fragilis immobilized in calcium alginate was 63 g/l whereas with co-immobilized K. fragilis and Zymomonas mobilis 72 g ethanol/l was attained. With free cells of K. fragilis, only 52 g ethanol/l was obtained. The beads were relatively stable without significant reduction in activity for about six batches of fermentation.The authors are with the Department of Microbiology and Microbial Technology, School of Biological Sciences, Madurai Kamaraj University, Madurai 625 021, India.This paper is dedicated to Professor M. Lakshmanan, Vice-Chancellor, Madurai Kamaraj University, in commemoration of his 60th birthday.     

Isolation and Properties of Mutants of Zymomonas mobilis Deficient in Sugar Assimilation

An enrichment method using d-cycloserine was designed for the isolation of spontaneous mutants of Zymomonas mobilis deficient in glucose or fructose utilization. The mutants could easily be isolated since they represented 80 to 90% of the population after two and three enrichment cycles. Glucokinase and fructokinase activities in the mutants were affected.     

Effect of temperature and pH on immobilized Zymomonas mobilis for continuous production of ethanol

The effects of temperature and inlet pH of the medium on the ethanol productivity and activity of the immobilized Z. mobilis cells during continuous fermentation of glucose have been studied at various temperatures and pH. On changing the temperature from one steady state level to a new one, 6-8 h were required in order to fully experience the effect of a change in temperature; whereas 8-20 h were required on changing the pH. The optimum temperature of 37 degrees C and a broad pH range of 4.4-6.0 were observed for maximum ethanol productivity and ethanol yield.     

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 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.