Improved operational stability of cell-free glucose-fructose oxidoreductase from Zymomonas mobilis for the efficient synthesis of sorbitol and gluconic acid in a continuous ultrafiltration membrane reactor

For the continuous, enzymatic synthesis of sorbitol and gluconic acid by cell-free glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis, the principal determinants of productivity have been identified. Most important, the rapid inactivation of the soluble enzyme during substrate conversion can be avoided almost completely when weak bases such as tris(hydroxymethyl)aminomethan or imidazol are used for the titration of the produced gluconic acid and when 5-10 mM dithiothreitol are added to prevent thiol oxidations. With regard to a long-term operational stability of the enzyme for continuous syntheses, thermal deactivation becomes significant at reaction temperatures above 30 degrees C. Without any additional purification being required, the crude cell extract of Z. mobilis can be employed in a continuous ultrafiltration membrane reactor over a time period of more than 250 h without significant decrease in substrate conversion or enzyme activity. The use of soluble GFOR thus appears to be an interesting alternative to employing permeabilized cells of Zymomonas for the production of sorbitol and gluconic acid and may be superior with regard to reactor productivities, at comparable operational stabilities.     

Elucidation of the cytoplasmic and vacuolar components in the inorganic phosphate region in the 31P NMR spectrum of yeast

Subcellular compartments, such as the vacuole in yeast, play important roles in cell metabolism and in cell response to external conditions. Concentrations of inorganic phosphate and pH values of the vacuole and cytoplasm were determined for anaerobic Saccharomyces cerevisiae cells based upon (31)P NMR spectroscopy. A new approach allows the determination of these values for the vacuole in cases when the resonance for inorganic phosphate in the cytoplasm overlaps with the resonance for inorganic phosphate in the vacuole. The intracellular inorganic phosphate resonance was first decomposed into two components by computer analysis. The assignments of the components were determined from in vivo correlations of P(i) chemical shift and the chemical shifts of the cytoplasmic sugar phosphates, and the pH dependency of the resonance of pyrophosphate and the terminal phosphate of poly-phosphate (PP(1)) which reside in the vacuole. An in vivo correlation relating PP(1) and P(i) (vac) chemical shifts was established from numerous evaluations of intracellular compositions for several strains of S. cerevisiae. This correlation will aid future analysis of (31)P NMR spectra of yeast and will extend NMR studies of compartmentation to cellular suspensions in phosphate-containing medium. Application of this method shows that both vacuolar and extracellular P(i) were phosphate reserves during glycolysis in anaerobic S. cerevisiae. Net transport of inorganic phosphate across the vacuolar membrane was not correlated with the pH gradient across the membrane.     

Filament formation and ethanol production by Zymomonas mobilis in adsorbed cell bioreactors

Zymomonas mobilis immobilized on microporous ion exchange resins has previously been shown to allow the attainment of high ethanol productivities in packed-bed bioreactors. The formation of bacterial filaments after several days of continuous operation, however, had resulted in excessive pressure increases across the reactor bed. The present work examines techniques for controlling filament formation by Z. mobilis in two reactor sizes (161 mL and 7.85 L) and a feed glucose concentration of 100 g/L. By controlling the fermentation temperature at 20-25 degrees C it has been possible to eliminate filament formation by Z. mobilis and to operate the larger bioreactor for 232 h with an ethanol productivity of 50 g/L h (based on total reactor volume). The rate of ethanol production has been shown to be very sensitive to temperature in the range 20-30 degrees C, and it is likely that slightly higher temperatures than those used in this study will improve ethanol productivity while still permitting long-term operation.     

Continuous simultaneous saccharification and fermentation of starch using Zymomonas mobilis

A continuous process involving simultaneous saccharification and fermentation of liquefied starch has been developed using Zymomonas mobilis. Amyloglucosidase retention and cell recycle have been effected by using an Amicon hollow-fiber membrane system with a MW cutoff of 5000. Relatively high productivities of up to 60 g L(-1) h(-1) have been achieved at ethanol concentrations of 60-65 g/L. The system also offers the potential for reduced enzyme requirements for saccharification.     

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.     

Continuous production of gluconic acid and sorbitol from Jerusalem artichoke and glucose using an oxidoreductase of Zymomonas mobilis and inulinase

Gluconic acid and sorbitol were simultaneously produced from glucose and Jerusalem artichoke using a glucose-fructose oxidoreductase of Zymomonas mobilis and inulinase. Inulinase was immobilized on chitin by cross-linking with glutaraldehyde. Cells of Z. mobilis permeabilized with toluene were coimmobilized with chitin-immobilized inulinase in alginate beads. The optimum amounts of both chitin-immobilized inulinase and permeabilized cells for coimmobilization were determined, and operational conditions were optimized. In a continuous stirred tank reactor operation, the maximum productivities for gluconic acid and sorbitol were about 19.2 and 21.3 g/L/h, respectively, at the dilution rate of 0.23 h(-1) and the substrate concentration of 20%, but operational stability was low because of the abrasion of the beads. As an approach to increase the operational stability, a recycle packed-bed reactor (RPBR) was employed. In RPBR operation, the maximum productivities for gluconic acid and sorbitol were found to be 23.4 and 26.0 g/L/h, respectively, at the dilution rate of 0.35 h(-1) and the substrate concentration of 20% when the recirculation rate was fixed at 900 mL/h. Coimmobilized enzymes were stable for 250 h in a recycle packed-bed reactor without any loss of activity, while half-life in a continuous stirred tank reactor (CSTR) was observed to be about 150 h.     

Continuous enzymatic production of lactobionic acid using glucose-fructose oxidoreductase in an ultrafiltration membrane reactor

Glucose-fructose oxidoreductase from Zymomonas mobilis catalyzed the oxidation of various aldose sugars to the corresponding aldonic acids. The enzyme was used for the selective and high-yield conversion of lactose to lactobionic acid in batch, fed-batch and continous reaction mode. A productivity of 110 g L d was obtained in an ultrafiltration membrane reactor, operated for 70 h.     

Separation of membrane fractions of Zymomonas mobilis

Abstract Membranes of Zymomonas mobilis ZM4 were separated by centrifugation on discontinuous density sorbitol gradient using a 2-step purification procedure. Four bands of respective densities 1.17 (L1), 1.20 (L2), 1.22 (H1), and 1.32 (H2) were obtained. NADH oxidase activity was detected in L1 and L2 fractions, indicating that they were derived from cytoplasmic membrane. H1 and H2 fractions gave a positive Limulus polyphemus lysate test of outer membrane endotoxin. Proteins of 2 cytoplasmic membrane bands and of 2 outer membrane bands showed respectively similar patterns when separated by electrophoresis.     

Differences in response of Zymomonas mobilis and Saccharomyces cerevisiae to change in extracellular ethanol concentration

In high cell density batch fermentations, Zymomonas mobilis produced 91 g L(-1) ethanol in 90 min but culture viability fell significantly. Similar viability losses in rapid fermentations by yeast have recently been shown to be attributable in part to the high rate of change of the extracellular ethanol concentration. However, in simulated rapid fermentations in which ethanol was pumped continuously to low cell density Z. mobilis suspensions, increases in the rate of change of ethanol concentration in the range 21-83 g L(-1) h(-1) did not lead to accelerated viability losses. The lag phase of Zymomonas cultures exposed to a 30-g L(-1) step change in ethanol concentration was much shorter than that of Saccharomyces cerevisiae, providing evidence that the comparative insensitivity of Zymomonas to high rates of change of ethanol concentration is due to its ability to adapt to changes in ethanol concentration more rapidly than yeast. (c) 1994 John Wiley & Sons, Inc.     

A hollow-fiber reactor design for NMR studies of microbial cells

A hollow-fiber membrane reactor was designed and constructed to allow perfusion of entrapped, dense Escherichia coli cells with nutrient medium during examination of cell metabolism using nuclear magnetic resonance (NMR) spectroscopy. Phosphorus-31 NMR spectra of the perfused cells included peaks for nucleoside di- and triphosphates, sugar phosphates, and pH-sensitive peaks for inorganic phosphate. The observed intensity of the lumenal inorganic phosphate peak was found to depend on flow rate, ruling out the use of this peak as a concentration reference. Absolute intracellular pH values obtained from NMR measurements were found to be accurate to 0.2 pH units due to uncertainties in intracellular ionic concentrations. Relative pH values, however, were found to be sensitive to cell energetic status. The response of E. coli intracellular pH following a shift to carbon starvation medium was monitored with a resolution of 3 min. Use of a hollow-fiber reactor for cell containment and perfusion during NMR spectroscopy enables metabolic experiments of longer duration and of greater variety than is possible using standard, nonperfused sample tubes.     

Experimental investigation and modeling of oscillatory behavior in the continuous culture of Zymomonas mobilis

The mechanism causing oscillation in continuous ethanol fermentation by Zymomonas mobilis under certain operating conditions has been examined. A new term, "dynamic specific growth rate," which considers inhibitory culture conditions in the recent past affecting subsequent cell behavior, is proposed in this article. Based on this concept, a model was formulated to simulate the oscillatory behavior in continuous fermentation of Zymomonas mobilis. Forced oscillation fermentation experiments, in which exogenous ethanol was added at a controlled rate to generate oscillatory behavior, were performed in order to obtain estimates for the model parameters and to validate the proposed model. In addition, data from a literature example of a sustained oscillation were analyzed by means of the model, and excellent agreement between the model simulation and experimental results was obtained. The lag in the cells' response to a changing environment, i.e., ethanol concentration change rate experienced by the cells, was shown to be the major factor contributing to the oscillatory behavior in continuous fermentation of Zymomonas mobilis under certain operating conditions. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 99-105, 1997.     

Fermentation kinetics of Zymomonas mobilis near zero growth rate

The fermentation kinetics Zymomonas mobilis were studied near zero growth rate in fed-batch cultures and continuous cultures with complete cell recycle. The results show the ethanol enhances that specific substrate conversion rate under these conditions. The maximum achievable ethanol concentration in continuous cultures with cell recycle (66 g/L) was significantly lower than in fed-batch cultures (100 g/L). The results indicate that growth-rate-independent metabolism is not instantaneous and can lag behind steadily increasing ethanol concentrations in fed-batch fermentations. A model is proposed to account for this slow adaptation.     

Continuous ethanol production and cell growth in an immobilized-cell bioreactor employing Zymomonas mobilis

Ethanol-producing bioreactors employing cells of Zymomonas mobilis attached to glass-fiber pads were operated continuously for as long as 28 days. Ethanol production, which is related to bed-associated biomass levels, was found to occur in three distinct phases: an exponential phase, a linear phase, and a "steady-state" phase. After prolonged operation, a bacterial floc developed in the reactor. The maximum effluent ethanol concentration and the maximum volumetric productivity were 6.4% and 152 g L(-1) h(-1), respectively, and both were attained at a liquid residence time of from 10-15 min. Both maxima occurred after the development of the bacterial floc. The flocculant bacterium has been isolated and tentatively identified as a flocculant strain of Z. mobilis.     

Ethanol transport in Zymomonas mobilis measured by using in vivo nuclear magnetic resonance spin transfer.

For the first time, unidirectional rate constants of ethanol diffusion through the lipid membrane of a microorganism, the bacterium Zymomonas mobilis, were determined, thus replacing indirect inferences with direct kinetic data. The rate constants k1 (in to out) were 6.8 +/- 0.4s(-1) at 29 degrees C and 2.7 +/- 0.3s(-1) at 20 degrees C. They were determined by using 1H selective nuclear magnetic resonance spin magnetization transfer. The measurements were done on l-ml cell suspensions. No addition of radiotracers, withdrawing of aliquots, physical separation methods, or chemical manipulations were required. Until now, the rate constants of ethanol transport in microorganisms have been unknown because ethanol diffuses through the cytoplasmic membrane too quickly for radiolabel approaches. Net velocities of ethanol exchange were calculated from unidirectional rate constants and cytoplasmic volume, which was also determined with the same nuclear magnetic resonance experiments. The results (i) confirmed that ethanol would not be rate limiting during the conversion of glucose by Z. mobilis and (ii) indicated that ethanol can serve as an in vivo marker of cytoplasmic volume changes. This was verified by monitoring for the first time the changes of both cytoplasmic volume and extracytoplasmic and cytoplasmic concentrations of alpha and beta anomers of D-glucose in cell suspensions of a microorganism. These findings may open up new possibilities for kinetic studies of ethanol and sugar transport in Z. mobilis and other organisms.     

Cell immobilization in kappa-carrageenan for ethanol production

A combination of extended Monod kinetics and the diffusional equation was used for evaluating the effectiveness factor of entrapped immobilized cells. Based on the kinetics of Zymomonas mobilis reported in the literature, the numerical results have revealed that the problem of mass transfer diffusional restrictions can be neglected by using small beads (1 mm in diameter) with a corresponding cell loading up to 276 g/L gel. On the basis of the numerical results obtained, the application of immobilized cells for continuous ethanol production was investigated. The kappa-carrageenan method was utilized to entrap Z. mobilis CP4, a potential ethanol producer. A two stage fermentation process has also been developed for ethanol production by the Z. mobilis carrageenan-bound cells. About 90 g/L ethanol was produced by immobilized cells at a total residence time of 1.56 h. The ethanol yield was estimated to be 93% of theoretical. The results obtained in this study also indicated that the control of optimum pH in an immobilized cell column is necessary to enhance the rate of ethanol production.     

Pulsed high-field gradient in vivo NMR spectroscopy to measure diffusional water permeability in Corynebacterium glutamicum

Pulsed high-field gradient in vivo NMR spectroscopy was used to measure diffusional water permeability in cell suspensions of the Gram-positive bacterium Corynebacterium glutamicum. Two different regions of H2O mobility were detected. One was characterized by the apparent coefficient of self-diffusion, D(1 app) = (4.6-12.7)x10(-8) cm(2) s(-1), depending on the observation time t. The other region was characterized by D(2) = 1.4x10(-5) cm(2) s(-1). The value of D(2) was similar to the diffusion coefficient of H2O in free water and in extracellular biological fluids. Restricted diffusion could be demonstrated for the slower process (D(1)). It was attributed to the cytoplasm of the cells. The membrane permeability, P(d H2O), for C. glutamicum was (4.8+/-0.4)x10(-3) cm s(-1). It compared favorably with values reported for human erythrocytes and was higher by a factor of about 100 compared to the diffusional permeability for ethanol, P(d ethanol), in Zymomonas mobilis. Addition of HgCl2, a water channel inhibitor in eukaryotes, decreased P(d H2O) in C. glutamicum by a factor of approximately 8. To our knowledge, these are the first functional studies of water transport in prokaryotes that yielded quantitative data, viz., transmembrane water permeability expressed through D(H2O) and P(d H2O).     

Detection and characterization of acidic compartments (vacuoles) in Chlorella vulgaris 11h cells by 31P-in vivo NMR spectroscopy and cytochemical techniques

Acidic inorganic phosphate (Pi) pool (pH around 6) was detected besides the cytoplasmic pool in intact cells of Chlorella vulgaris 11h by ³¹P-in vivo nuclear magnetic resonance (NMR) spectroscopy. It was characterized as acidic compartments (vacuoles) in combination with the cytochemical technique; staining the cells with neutral red and chloroquine which are known as basic reagents specifically accumulated in acidic compartments. Under various conditions, the results obtained with the cytochemical methods were well correlated with those obtained from in vivo NMR spectra; the vacuoles were well developed in the cells at the stationary growth phase where the acidic Pi signal was detected. In contrast, cells at the logarithmic phase in which no acidic Pi signal was detected contained only smaller vesicles that accumulated these basic reagents. No acidic compartment was detected by both cytochemical technique and ³¹P-NMR spectroscopy when the cells were treated with NH₄OH. The vacuolar pH was lowered by the anaerobic treatment of the cells in the presence of glucose, while it was not affected by the external pH during the preincubation ranging from 3 to 10. Possible vacuolar functions in unicellular algae especially with respect to intracellular pH regulation are discussed.Non-standard abbreviations EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MDP methylene diphosphonic acid - NMR nuelear magnetic resonance - PCA perchloric acid - PCV packed cell volume - Pi inorganic phosphate - Pic sytoplasmic inorganic phosphate - Piv vacuolar inorganic phosphate - ppm parts per million - SP sugar phosphates - TCA trichloroacetic acid     

The effect of 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide on the rat liver microsomal glucose-6-phosphatase system

The effect of 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMC) on the reactions catalyzed by the glucose-6-phosphatase system of rat liver microsomes was studied. Modification of the intact microsomes by CMC leads to the inhibition of the glucose-6-phosphatase, pyrophosphate:glucose and carbamoyl-phosphate : glucose phosphotransferase activities of the system. The activities are restored by the disruption of the microsomal permeability barrier. The mannose-6-phosphate, pyrophosphate, and carbamoyl-phosphate phosphohydrolase activities of the intact as well as the disrupted microsomes were not affected by CMC. It follows from the results obtained that CMC inactivates the microsomal glucose-6-phosphate translocase, the inactivation is a result of the modification of a single sulfhydryl or amino group of the translocase; pyrophosphate, carbamoyl phosphate and inorganic phosphate are transported across the microsomal membrane without participation of the glucose-6-phosphate translocase; pyrophosphate and carbamoyl phosphate may act as the phosphate donors in the glucose phosphorylation reactions in vivo.     

NMR studies of [1-2H]glucose metabolism in Zymomonas mobilis

In complementary experiments the metabolism of [1-2H]glucose in H2O and of unlabelled glucose in 2H2O by Zymomonas mobilis was examined. The utilization of [1-2H]glucose by Z. mobilis was monitored by high-resolution 2H NMR. The deuterium-labelling pattern and stereochemistry of the ethanols produced from the metabolism of [1-2H]glucose and unlabelled glucose in 2H2O were determined by a combination of 13C and 1H NMR and selective enzyme action. The labelling patterns were explained in terms of enzyme mechanisms and stereospecificity, and metabolite enolization.     

Kinetic and nuclear magnetic resonance studies of xylose metabolism by recombinant Zymomonas mobilis ZM4(pZB5)

The specific rates of growth, substrate utilization, and ethanol production as well as yields of biomass and ethanol production on xylose for the recombinant Zymomonas mobilis ZM4(pZB5) were shown to be much less than those on glucose or glucose-xylose mixtures. Typical fermentations with ZM4(pZB5) growing on glucose-xylose mixtures followed two-phase growth kinetics with the initial uptakes of glucose and xylose being followed by slower growth and metabolic uncoupling on xylose after glucose depletion. The reductions in rates and yields from xylose metabolism were considered in the present investigation and may be due to a number of factors, including the following: (i) the increased metabolic burden from maintenance of plasmid-related functions, (ii) the production of by-products identified as xylitol, acetate, lactate, acetoin, and dihydroxyacetone by (13)C-nuclear magnetic resonance (NMR) spectroscopy and high-performance liquid chromatography, (iii) growth inhibition due to xylitol by the putative inhibitory compound xylitol phosphate, and (iv) the less energized state of ZM4(pZB5). In vivo (31)P-NMR studies have established that the levels of NTP and UDP sugars on xylose were less than those on glucose, and this energy limitation is likely to restrict the growth of the recombinant strain on xylose media.