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)     

Continuous ethanol fermentation ofZymomonas mobilis using soy flour as a protective agent

Biotechnology Letters - From the analysis of steady state data, an increase of 81.6% in ethanol concentration and fermentor productivity was obtained due to the addition of soy flour to a...     

High productivity ethanol fermentation on a mineral medium using a flocculent strain ofZymomonas mobilis

Summary A flocculent strain ofZymomonas mobilis (ZM4F JM1) was isolated in continuous culture. The parent strain, ZM4F, had lost its flocculating properties. The isolation was done in a conical fermentor at high dilution rate. Ethanol production by the new strain was then compared on a rich and mineral medium. The mineral medium showed high performance and could be used for industrial production of ethanol since it reduced one hundred fold the vitamin cost of the fermentation.     

Characterization of a superior thermotolerant mutant ofZymomonas mobilis ZM4 for ethanol production in glucose medium

Summary Nitrosoguanidine-induced, stable theromotolerant mutant (ZMI₂) ofZymomonas mobilis ZM4 was found to possess almost normal cell morphology, and a better ethanol tolerance at 42°C than the parent strain (ZM4). Its kinetic parameters, in converting different concentrations of glucose to ethanol, were comparable to ZM4 at 30°C, and significantly superior at 42°C. In a 200 g/L glucose medium in a pH-stat (5.0) at 42°C, the mutant yielded more ethanol (71.0 g/L) (improved to 73.7 g/L at pH 5.5) and alcohol dehydrogenase (ADH) than the parent strain. The ADH levels in both the strains were repressed, depending upon the increased level of sugar and degree of temperature.     

Ethanol production from cellulose by a coculture ofZymomonas mobilis and a clostridium

Zymomonas mobilis and a mesophilic cellulolytic clostridium (strain C7) were grown in coculture in a medium containing cellulose as fermentable substrate. The coculture was stable through at least ten serial transfers and produced markedly higher amounts of ethanol than monocultures of the cellulolytic clostridium. Glucose and cellobiose, derived from the breakdown of cellulose, accumulated in strain C7 monocultures, but not in cocultures. The molar ratio of ethanol to acetate was higher in cocultures than in monocultures of strain C7. The cellulolytic clostridium was relatively ethanol-tolerant, inasmuch as it grew and fermented cellulose in media containing up to 7 g of ethanol/100 ml. Cellulase (Avicelase) activity of strain C7 was inhibited by cellobiose, but not by glucose.     

Intracellular ethanol and cell viability ofZymomonas mobilis during fed-batch alcoholic fermentation

Summary The effect of intracellular as well extracellular ethanol concentration on the viability ofZymomonas mobilis during a fed-batch fermentation was examined. The cells retained their viability until ethanol attained 69.5 ± 1.55 and 69 ± 1.6g/l for respectively, extracellular and intracellular values.Z. mobilis does not therefore accumulate ethanol in the cells. The number of dead cells increased after exposure to ethanol. The maximal efficiency of the fermentation was 95.5% (3.82 mol of ethanol-mol sucrose).

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Contenu intra-cellulaire en éthanol et viabilité des cellules de Zymomonas mobilis pendant une fermentation éthanolique en milieu non renouvelé à alimentation étagée

Résumé On a examiné l'effet de la concentration en éthanol tant inta- que extra-cellulaire sur la viabilité deZymomonas mobilis pendant une fermentation en milieu non renouvelé à alimentation étagée. Les cellules ont gardé leur viabilité jusqu'à ce que la concentration atteigne 69.5 ± 1.55 et 69 ±a 1.6 g/l respectivement pour les valeurs extra- et intra-cellulaires. Il en résulte queZymomonas mobilis n'accumule pas l'éthanol dans les cellules. Le nombre de cellules mortes augmentait après l'exposition à l'éthanol. L'efficience maximum de la fermentation était de 95.5% (3.82 mol d'éthanol/mol de sucrose).

     

Caracterization of flocculation in a strain ofZymomonas mobilis

Summary Populations ofZymomonas mobilis flocs, cultivated in a continuous tower reactor, can be completly described with a single parameter: the settling velocity. The floc size distribution is directly linked to the average settling rate. This property will simplify the design of continuous settlers.     

Fermentation capabilities ofZymomonas mobilis glycolytic enzymes

Summary Cell-free extracts ofZymomonas mobilis were capable of fermenting glucose to ethanol and CO₂ when stimulated by arsenate to act as an ATP uncoupler. 2M glucose was completely converted resulting in a final concentration of 16.5 % w/v ethanol. 1 M glucose was completely converted at temperatures up to 50°C. The results demonstrate that the glycolytic enzymes are more resistant to temperature and ethanol than are the living cells.     

Genetics and genetic engineering ofZymomonas mobilis

Present knowledge on the genetics of the ethanologenic anaerobeZymomonas mobilis includes background information on: size, restriction, and to some extent hybridization, analysis of indigenous plasmids; mutagenesis and isolation of a wide variety of auxotrophic, drug resistant and conditional mutants; construction of shuttle cloning vectors able to replicate and express inZ. mobilis; development of gene transfer systems based on conjugal mobilization of plasmids fromEscherichia coli donors toZ. mobilis; expression of heterologous genes inZ. mobilis; cloning and analysis of genes encoding enzymes of the Entner-Doudoroff pathway. Moreover, preliminary data on recombinational repair mechanisms and plasmid stability, which are now available, makeZ. mobilis an attractive model system for molecular genetic research and, furthermore, they contribute towards expansion of the substrate and product range of this industrial microorganism.G.A. Sprenger is with the Institut für Biotechnologie I, Forschungszentrum, KFA Julich, Postfach 1913, D-5170 Julich, Germany. M.A. Typas is with the Department of Biochemistry, Molecular & Cellular Biology and Genetics. University of Athens, Kouponia 105 71 Athens, Greece. C. Drainas is with the Sector of Organic Chemistry and Biochemistry, Department of Chemistry, University of loannina, 451 10 loannina, Greece.     

Growth ofZymomonas mobilis CP4 on mannitol

Summary The ethanologenZymomonas mobilis has a restricted substrate range, namely glucose, fructose and sucrose. It would be useful to expand its substrate range to include other carbohydrates.Z. mobilis was screened for growth on 30 different carbohydrates and organic acids. A single spontaneous mutant,Z mobilis CP4.60, was isolated which illustrated feeble growth on mannitol as the sole carbohydrate source after three months of incubation. Growth ofZ. mobilis CP4.60 for several months in continuous culture with excess mannitol, and including a round of NTG (N-methyl-N'-nitro-N-nitrosoguanidine) mutagenesis in the chemostat, led to the isolation a sequential series of mutants (CP4.62, CP4.64 and CP4.66), each with improved growth rates on mannitol. Metabolism of mannitol byZ. mobilis is oxygen-dependent, resulting in limited production of ethanol and incresed production of lactic acid. This is an initial example of extension of the substrate range ofZymomonas. The conversion of mannitol to fructose could be via an altered alcohol dehydrogenase.     

Determination of the energy maintenance coefficient ofZymomonas mobilis

Summary Continuous culture experiments withZymomonas mobilis Z-1-81, indicated the existence of linearity between specific substrate uptake rates and dilution rates. The value of the energy maintenance coefficient was estimated at 2,521 g/g.h. The percentage of carbon used for maintenance energy was shown to increase with the decrease in the dilution rate.     

Kinetic studies on alac-containing strain ofZymomonas mobilis

Summary Batch and continuous culture studies have been carried out on a strain ofZ.mobilis (ZM6306) which can convert lactose directly to ethanol. Previous strain development has established that thelac operon encoded on the transposon Tn951 can be expressed inZ.mobilis. Using a medium containing 80 g/l glucose and 40 g/l lactose, it was found that strain ZM6306 could convert about 13 g/l lactose to 4 g/l ethanol and 6 g/l galactose in continuous culture. Further lactose conversion is likely with increased cell concentration using a cell recycle system.     

Identification of saccharolytic enzymes ofZymomonas mobilis CP4

Studies on sucrose hydrolysis withZymomonas mobilis CP4 have revealed, via electrophoresis and zymogram staining, three enzymes that hydrolyze sucrose. One electrophoretic band corresponds to the well characterized levansucrase, while the two remaining bands appear to be invertases (Inv A and Inv B). Inv A occurs intracellularly in CP4, while Inv B occurs in the supernatant as well as intracellularly.     

Characterization ofZymomonas mobilis alkaline phosphatase activity inEscherichia coli

Zymomonas mobilis phoA gene encoding alkaline phosphatase was expressed inEscherichia coli CC118 carrying the recombinant plasmid pZAP1. The pH optimum for this enzyme was 9.0 and showed a peak activity at 42°C. This enzyme required Zn²⁺ for its catalytic activity; however, Mg²⁺ or Ca²⁺ significantly affected the activity. This enzyme was found to be ethanolabile, and ethanol inhibition was reversed by addition of Zn²⁺. Kinetics ofZ. mobilis alkaline phosphatase production inE. coli CC118 (pZAP1) showed that the enzyme activity was growth associated and localized in the cellular fraction, and the maximum activity was found in the stationary phase.     

Buffering capacity and membrane H+ conductance ofZymomonas mobilis

Buffering power and membrane conductance to H⁺ were measured inZymomonas mobilis subspmobilis ATCC 29191 by a pulse technique. Over the pH range studied, from 4.02 to 7.44,Z. mobilis presented very high values of cytoplasmic buffering capacity; it was a significant proportion of the total buffering capacity. These results support the idea that the cytoplasmic buffering power might be part of the pH homeostatic mechanism.     

Ethanol production by immobilized whole cells ofZymomonas mobilis in a continuous flow expanded bed bioreactor and a continuous flow stirred tank bioreactor

Continuous ethanol fermentation by immobilized whole cells ofZymomonas mobilis was investigated in an expanded bed bioreactor and in a continuous stirred tank reactor at glucose concentrations of 100, 150 and 200 g L^–1. The effect of different dilution rates on ethanol production by immobilized whole cells ofZymomonas mobilis was studied in both reactors. The maximum ethanol productivity attained was 21 g L^–1 h^–1 at a dilution rate of 0.36 h^–1 with 150 g glucose L^–1 in the continuous expanded bed bioreactor. The conversion of glucose to ethanol was independent of the glucose concentration in both reactors.     

Ethanol fermentation of cassava starch by Zymomonas mobilis NRRL B-4286

Four strains of Zymomonas mobilis were compared for ethanol production from enzymatically hydrolysed cassava starch. Strain NRRL B-4286 performed efficiently, producing 80 g/litre ethanol from 171 g/litre initial sugar concentration. Addition of yeast extract, calcium pantothenate, ammonium sulphate or magnesium sulphate did not significantly increase ethanol production by this strain.     

Improved technique for the isolation of stable mutants ofZymomonas mobilis

Addition of caffeine to the recovering medium after mutagenesis ofZymomonas mobilis by N-methyl-N-nitrosoguanidine increased 4-fold the number of auxotrophic mutants obtained. Moreover, while the mutants isolated without caffeine survived only a few repeated serial transfers on minimal medium supplemented with the required growth factor, 40 % of those obtained in the presence of caffeine were stable.     

High productivity continuous ethanol fermentation with a flocculating mutant strain of Zymomonas mobilis

High fermenter (volumetric) ethanol productivities (80 g/lh^–1) were attained in a simple single-stage continuous-stirred-tank-reactor (CSTR) employing a flocculent mutant of Zymomonas mobilis with a feed containing 100g/l glucose. Under these conditions a final ethanol concentration of 47.6 g/l was obtained, representing a maximum conversion efficiency of 97% of theoretical.Nomenclature SR = Medium glucose concentration (g/l)X Biomass concentration (g/l) - P Ethanol concentration (g/l) - VP Volumetric productivity (g ethanol/l/h) - Yp/s Product yield coefficient (g ethanol/g glucose consumed) - Qp Specific rate of ethanol formation (g ethanol/g cells/h) - D Dilution rate (h^–1) - Dmax Maximum dilution rate: ie., highest dilution rate at which the effluent glucose concentration 4g/l (h^–1)