Zymomonas mobilis mutants blocked in fructose utilization

Summary A mutant ofZymomonas mobilis deficient in the utilization of fructose for growth and ethanol formation was shown to lack fructokinase activity. When grown in media which contained glucose+fructose or sucrose, both the mutant and wild type produced sorbitol in amounts up to 60 g·l^-1, depending on the initial concentrations of sugars. Sorbitol formation was accompanied by an accumulation of acetaldehyde, gluconate, and acetoin. A ferricyanide-dependent sorbitol dehydrogenase could be localized in the cell membrane; it thus resembles the sorbitol dehydrogenase ofGluconobacter suboxydans. Neither a NAD(P)H dependent reduction of fructose nor a NAD(P) dependent dehydrogenation of sorbitol could be detected in cell-free extracts. The use of fructose-negative mutants ofZ. mobilis for the enrichment of fructose in glucose+fructose mixtures is discussed.     

Production of sorbitol and gluconic acid by permeabilized cells of Zymomonas mobilis

Summary Zymomonas mobilis is able to convert glucose and fructose to gluconic acid and sorbitol. The enzyme, glucose-fructose oxidoreductase, catalysing the intermolecular oxidation-reduction of glucose and fructose to gluconolactone and sorbitol, was formed in high amounts [1.4 units (U)·mg^-1] when Z. mobilis was grown in chemostats with glucose as the only carbon source under non-carbon-limiting conditions. The activity of a gluconolactone-hydrolysing lactonase was constant at 0.2 U·mg^-1. Using glucose-grown cells for the conversion of equimolar fructose and glucose mixtures up to 60% (w/v), a maximum product concentration of only 240 g·1^-1 of sorbitol was found. The gluconic acid accumulated was further metabolized to ethanol. After permeabilizing the cells using cationic detergents, maximum sorbitol and gluconic acid concentrations of 295 g·1^-1 each were reached; no ethanol production occurred. In a continuous process with -carrageenan-immobilized and polyethylenimin-hardened, permeabilized cells no significant decrease in the conversion yield was observed after 75 days. The specific production rates for a high yield conversion ( > 98%) in a continuous two-stage process were 0.19 g·g^-1·h^-1 for sorbitol and 0.21 g·g^-1·h^-1 for gluconic acid, respectively. For the sugar conversion of cetyltrimethylammonium bromide-treated -carrageenan-immobilized cells a V _max of 1.7 g·g^-1·h^-1 for sorbitol production and a K _m of 77.2 g·1^-1 were determinedOffprint requests to: B. Rehr     

Metabolic state of Zymomonas mobilis in glucose-, fructose-, and xylose-fed continuous cultures as analysed by 13C- and 31P-NMR spectroscopy

The reasons for the well-known significantly different behaviour of the anaerobic, gram-negative, ethanologenic bacterium Zymomonas mobilis during growth on fructose (i.e. decreased growth and ethanol yields, increased by-product formation) as compared to that on its second natural substrate, glucose, have remained unexplained. A xylose-fermenting recombinant strain of Z. mobilis that was recently constructed in our laboratory also unexpectedly displayed an increased formation of by-products and a strongly reduced growth rate as compared to the parent strain. Therefore, a comprehensive study employing recently developed NMR-based methods for the in vivo analysis of intracellular phosphorylated pool sizes and metabolic fluxes was undertaken to enable a global characterization of the intracellular metabolic state of Z. mobilis during growth on ¹³C-labelled glucose, fructose and xylose in defined continuous cultures. The ¹³C-NMR flux analysis indicated that ribose 5-phosphate is synthesized via the nonoxidative pentose phosphate pathway in Z. mobilis, and it identified a metabolic bottleneck in the recombinant xylose-fermenting Z. mobilis strain at the level of heterologous xylulokinase. The ³¹P-NMR analyses revealed a global alteration of the levels of intracellular phosphorylated metabolites during growth on fructose as compared to that on glucose. The results suggest that this is primarily caused by an elevated concentration of intracellular fructose 6-phosphate. Received: 7 January 1999 / Accepted: 22 March 1999     

Continuous ethanol production by Zymomonas mobilis in a fluidized bed reactor. Part II: Process development for the fermentation of hydrolysed B-starch without sterilization

A continuous fluidized bed reactor operation system has been developed for ethanol production by Zymomonas mobilis using hydrolysed B-starch without sterilization. The operation system consists of two phases. In the first phase macroporous glass carriers in a totally mixed fluidized bed reactor were filled up totally with a monoculture of Z. mobilis by fast computer-controlled colonization, so that in the subsequent production phase no contaminants, especially lactic-acid bacteria, could penetrate into the carrier beads. In the production phase the high concentration of immobilized Z. mobilis cells in the fluidized bed reactor permits unsterile fermentation of hydrolysed B-starch to ethanol at short residence times. This results in wash-out conditions for contaminants from the substrate. Long-term experimental studies (more than 120 days) of unsterile fermentation of hydrolysed B-starch in the laboratory fluidized bed reactor (2.2 l) demonstrated stable operation up to residence times of 5 h. A semi-technical fluidized bed reactor plant (cascade of two fluidized bed reactors, each 55 l) was operated stably at a mean residence time of 4.25 h. Glucose conversion of 99% of the unsterile hydrolysed B-starch was achieved at 120 g glucose/l^–1 in the substrate, resulting in an ethanol concentration of 50 g·l^–1 and an ethanol space-time yield of 13 g·l^–1·h^–1. This is a factor of three compared to ethanol fermentation of hydrolysed B-starch with Z. mobilis in a continuous stirred tank reactor, which can only be operated stably under sterile conditions. Correspondence to: D. Weuster-Botz     

By-products in the fermentation of sucrose by different Zymomonas-strains

Summary Eight Zymomonas strains were compared with respect to their sucrose hydrolysing activity and subsequent ethanol, levan and sorbitol formation. The ethanol yields obtained were within narrow limits, 0.40–0.43 g·g^-1 of sucrose. The distribution of by-products differed significantly between the strains tested. A low sucrose hydrolysis rate seemed to be associated with the formation of levan and a high sucrose hydrolysis rate with the formation of sorbitol through accumulation of monomeric sugars. Fructo-oligomers consisting of two fructose and one glucose unit represented the greatest loss of sucrose in the fermentation conditions used.     

The biotechnological production of sorbitol

Sorbitol, a polyol found in many fruits, is of increasing industrial interest as a sweetener, humectant, texturizer and softener. At present, it is produced chemically. The bacterium Zymomonas mobilis is able to produce sorbitol and gluconic acid from fructose and glucose, respectively. This is possible in a one-step reaction via a glucose-fructose oxidoreductase so far only known from Z. mobilis. The possibilities for the industrial production of sorbitol by Z. mobilis are discussed, and compared with the current chemical production method as well as other microbiological processes. Electronic Publication     

Alcoholic fermentation of glucose and xylose by Pichia stipitis,Candida shehatae,Saccharomyces cerevisiae and Zymomonas mobilis: oxygen requirement as a key factor

Summary To investigate simultaneous alcoholic fermentation of glucose and xylose derived from lignocellulosic material by separate or co-culture processes, the effect of oxygen transfer rate (OTR) on the fermentation of 50 g/l xylose by Pichia stipitis NRRL Y 7124 and Candida shehatae ATCC 22984, and the fermentation of 50 g/l glucose by Saccharomyces cerevisiae CBS 1200 and Zymomonas mobilis ATCC 10988 was carried out in batch cultures. The kinetic parameters of the xylose-fermenting yeasts were greatly dependent on the OTR. The optimum OTR values were found to be 3.9 and 1.75 mmol·1^–1·h^–1 for C. shehatae and P. stipitis, respectively. By contrast the fermentative parameters of S. cerevisiae were poorly affected by the OTR range tested (0.0–3.5 mmol·l^–1·h^–1) Under these conditions the ethanol yields ranged from 0.41 g·g^–1 to 0.45 g·g^–1 and the specific ethanol productivity was around 0.70 g·g^–1·h^–1. Z. mobilis gave the highest fermentative performance under strictly anaerobic conditions (medium continually flushed with nitrogen): under these conditions, the ethanol yield was 0.43 g·g^–1 and the average specific ethanol productivity was 2.3 g·g^–1·h^–1. Process considerations in relation to the effect of OTR on the fermentative performance of the tested strains are discussed. Offprint requests to: J. P. Delgenes     

Modeling of ethanol fermentation using Zymomonas mobilis ATCC 10988 grown on the media containing glucose and fructose

A model of ethanol fermentation by Zymomonas mobilis ATCC 10988 on the medium containing glucose and fructose is proposed. This model was developed on the basis of metabolic analysis and many experimental findings. When glucose was used as the substrate, the dependence of the carbon fraction (α) assimilating to biomass on the specific growth rate (μ) could be well correlated to α = 0.25μ + 0.012. This correlation resulted in a novel equation for specific glucose uptake rate, which could describe the Z. mobilis fermentation in both batch and continuous modes. When fructose and glucose were both presented in the liquid medium, the model could predict the uptake of glucose and fructose as well as the formation of biomass, ethanol and sorbitol by Z. mobilis. All parameters used in the model were independently evaluated on the basis of various experimental findings. Good agreement was found between the model predictions and data of Z. mobilis fermentation on media containing both glucose and fructose. The proposed model could also describe the behavior of ethanol fermentation on sucrose medium supplemented with immobilized invertase.     

31P Nuclear magnetic resonance studies of ethanol inhibition in Zymomonas mobilis

Ethanol inhibition of glucose catabolism in Zymomonas mobilis was investigated using ³¹P NMR spectroscopy in vivo and of perchloric acid extracts from cell suspensions incubated with 0, 5 and 10% (w/v) ethanol. In vivo ³¹P NMR experiments revealed slower glucose utilization and decreased levels of nucleoside triphosphates in the presence of 10% ethanol as compared to controls. Using ³¹P NMR spectroscopy of perchloric acid extracts, intracellular accumulation of 3.4 mM 3-phosphoglycerate was found when 10% ethanol was present in the medium. No accumulation of this metabolite occurred in cells incubated with 0 and 5% ethanol. Enzyme assays confirmed that phosphoglycerate-mutase and enolase were inhibited 31 and 40%, respectively, in the presence of 10% ethanol in the test system. Therefore, under the conditions used the decrease in the fermentative activity of Z. mobilis at high ethanol concentrations is due to inhibition of phosphoglycerate-mutase and enolase.Abbreviation KDPG 2-keto-3-deoxy-6-phosphogluconate     

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.     

The relationship between sucrose hydrolysis,sorbitol formation and mineral ion concentration during bioethanol formation using Zymomonas mobilis 2716

Summary Investigations into the relationship between sucrose hydrolysis, sorbitol formation and mineral ion concentration during bioethanol formation by Zymomonas mobilis 2716 revealed two distinct phenomena responsible for carbon flow diversion, a sucrose effect and a salt effect. Neither of the two phenomena affects sucrose hydrolysis, but they divert carbon flow of the fructose monomer leading to its own accumulation, sorbitol or oligosaccharide formation. Sucrose concentrations in excess of 15% (w/v) led to sorbitol formation, the level of which may exceed 2% (w/v) depending upon glucose accumulation during sucrose hydrolysis. Increasing mineral ion concentrations led initially to carbon losses and finally to fructose accumulation instead of sorbitol formation. This carbon loss can be corrected by the addition of invertase, which in turn leads to an increase in sorbitol, fructose and ethanol. Potassium and chloride are the dominant ions responsible for suppression of sorbitol formation and fructose uptake, encouraging oligosaccharide formation. These fructooligosaccharides must be of a type which can be converted to fructose, sorbitol and ethanol through the action of invertase. The requirement of invertase addition to prevent fructooligosaccharide formation is indirect evidence that Z. mobilis 2716 does not produce invertase.Offprint requests to: H. W. Doelle     

Fructose production by Zymomonas mobilis in fed-batch culture with minimal sorbitol formation

Summary Fed-batch cultures of Zymomonas mobilis (UQM 2864), a mutant unable to metabolise fructose, grown on diluted sugar cane syrup (200 g/l sucrose) achieved yields of 90.5 g/l fructose and 48.3 g/l ethanol with minimal sorbitol formation and complete utilization of the substrate. The effect of inoculum size on sorbitol formation in the batch stage of fed-batch fermentation are reported. Fermentation of sucrose (350 g/l) supplemented with nutrients yielded 142 g/l fructose and 76.5 g/l ethanol. Some fructose product loss at high fructose concentrations was observed. The fed-batch fermentation process offers a method for obtaining high concentrations of fructose and ethanol from sucrose materials.     

Growth ofZymomonas on lactose: Gene cloning in combination with mutagenesis

Summary Wild-type strains ofZymomonas mobilis have a limited substrate range of glucose, fructose and sucrose. In order to expand this substrate range, transconjugants ofZ. mobilis containing Lac⁺ plasmids have been constructed. Although -galactosidase is expressed in such strains, they lack the ability to grow on lactose. We now report the development ofZ. mobilis strains capable of growth on lactose. This was achieved in two stages. First, a broad host range plasmid was constructed (pRUT102) which contained the lactose operon under the control of aZ. mobilis promoter plus genes for galactose utilization.Z. mobilis CP4.45 containing pRUT102 was then subjected to mutagenesis combined with continued selection pressure for growth on lactose. One strain,Z. mobilis SB6, produced a turbid culture that yielded 0.25% ethanol from 5% lactose (plus 2% yeast extract) in 15 days.     

Purification and some properties of extracellular invertase B from Zymomonas mobilis

Zymomonas mobilis is a Gram-negative ethanologen that can ferment glucose, fructose, and sucrose. Three enzymes that hydrolyze sucrose were found in a zymogram of electrophoretically separated proteins of Z. mobilis CP4. Two were invertase,, Inv A and Inv B; the latter was studied. Inv B is extracellular and accounts for at least 60% of the saccharolytic activity found in the culture broth of Z. mobilis CP4. The enzyme was purified 51-fold in 17% yield from culture broth of Z. mobilis grown on sucrose. It is a -fructosidase, monomeric with a molecular mass of 47 kDa and pI of 4.3. Its K _m for sucrose is 86 mm, and it has high catalytic activity (V _max = 1800 mol product/min per milligram protein). The purification and some properties of Inv B are presented. Correspondence to: D. E. Eveleigh     

Hexose metabolism in discs excised from developing potato (Solanum tuberosum L.) tubers

Metabolism of radiolabelled hexoses by discs excised from developing potato (Solanum tuberosum L.) tubers was been investigated in the presence of acid invertase to prevent accumulation of labelled sucrose in the bathing medium (Viola, 1996, Planta 198: 179–185). When the discs were incubated with either [U-¹⁴C]glucose or [U-¹⁴C]fructose without unlabelled hexoses, the unidirectional rate of sucrose synthesis was insignificant compared with that of sucrose breakdown. The inclusion of unlabelled fructose in the medium induced a dramatic increase in the unidirectional rate of sucroses synthesis in the tuber discs. Indeed, the decline in the sucrose content observed when discs were incubated without exogenous sugars could be completely prevented by including 300 mM fructose in the bathing medium. On the other hand, the inclusion of unlabelled glucose in the medium did not significantly affect the relative incorporation of [U-¹⁴C]glucose to starch, sucrose or glycolytic products. Substantial differences in the intramolecular distribution of ¹³C enrichment in the hexosyl moieties of sucrose were observed when the discs were incubated with either [2-¹³C]fructose or [2-¹³C]glucose. The pattern of ¹³C enrichment distribution in sucrose suggested that incoming glucose was converted into sucrose via the sucrose-phosphate synthase pathway whilst fructose was incorporated directly into sucrose via sucrose synthase. Quantitative estimations of metabolic fluxes in vivo in the discs were also provided. The apparent maximal rate of glucose phosphorylation was close to the extractable maximum catalytic activity of glucokinase. On the other hand, the apparent maximal rate of fructose phosphorylation was much lower than the maximum catalytic activity of fructokinase, suggesting that the activity of the enzyme (unlike that of glucokinase) was regulated in vivo. Although in the discs incubated with or without fructose the rates of starch synthesis or glycolysis were similar, the relative partitioning of metabolic intermediates into sucrose was much higher in discs incubated with fructose (0.6% and 32.6%, respectively). It is hypothesised that the equilibrium of the reaction catalysed by sucrose synthase in vivo is affected in discs incubated with fructose as a result of the accumulation of the sugar in the tissue. This results in the onset of sucrose cycling. Incubation with glucose enhanced all metabolic fluxes. In particular, the net rate of starch synthesis increased from 2.0 mol · hexose · g FW^–1 · h^–1 in the absence of exogenous glucose to 3.7 mol · hexose · g FW^–1 · h^–1 in the presence of 300 mM glucose. These data are taken as an indication that the regulation of fructokinase in vivo may represent a limiting factor in the utilisation of sucrose for biosynthetic processes in developing potato tubers.Abbreviations ADPGlc adenosine 5-diphosphoglucose - Glc6P glucose-6-phosphate - hexose-P hexose phosphate - NMR nuclear magnetic resonance - UDPGlc uridine 5-diphosphoglucose Many thanks to L. Sommerville for skillfull assistance and to J. Crawford and J. Liu for useful discussions on flux analysis. The research was funded by the Scottish Office Agriculture and Fisheries Department.     

Continuous ethanol production by Zymomonas mobilis in a fluidized bed reactor. Part I. Kinetic studies of immobilization in macroporous glass beads

A model has been developed to calculate the ethanol production in a well-mixed fluidized bed reactor. This model takes into account diffusion and the reaction inside porous glass beads and the activity of suspended cells in the fluidized bed reactor. The associated model parameters have been determined from the literature and by kinetic studies with Zymomonas mobilis in a continuous stirred tank reactor. The model permits good predictions of steady-state data in a fluidized bed reactor at residence times longer than 1–1.5 h. The immobilization of Z. mobilis in a fluidized bed reactor results in high ethanol space-time yields of more than 50 g·^–1·h^–1 at a glucose conversion of 80% (glucose in substrate: 120 gl^–1). At 99% conversion a space-time yield of 30 g·^–1·^–1 can be achieved when two fluidized bed reactors operate as cascade.     

Fermentation pattern of sucrose to ethanol conversions by Zymomonas mobilis

General patterns of sucrose fermentation by two strains of Zymomonas mobilis, designated Z7 and Z10, were established using sucrose concentrations from 50 to 200 g/liter. Strain Z7 showed a higher invertase activity than Z10. Strain Z10 showed a reduced specific growth rate at high sucrose concentration while Z7 was unaffected. High sucrose hydrolyzing activity in strain Z7 lead to glucose accumulation in the medium at high sucrose concentrations. Ethanol production and fermentation time depend on the rate of catabolism of the products of sucrose hydrolysis, glucose and fructose. The metabolic quotients for sucrose utilization, q_s, and ethanol production, q_p (g/g·hr), are unsuitable for describing sucrose utilization by Zymomonas mobilis, as the logarithmic phase of growth precedes the phase of highest substrate utilization (g/liter·hr) and ethanol production (g/liter·hr) in batch culture.     

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.     

Fermentation pattern of Zymomonas mobilis at high sucrose concentrations

Summary Zymomonas mobilis was grown in batch concentrations between 200 and 400 g/l sucrose. The fermentation pattern revealed that the efficiency of sucrose hydrolysis dropped only from 94 to 78.6% whereas the efficiency with which the hydrolyzed products were converted to ethanol decreased from 94 to 43%. The ethanol yields were relatively constant for final concentrations which lay between 80 and 132 g/l. Fermentation times increased to 72 hours at the higher sucrose concentrations. Sorbitol and fructose were identified as the major by-products. Preliminary evidence suggests that the ratio between the two by-products depends on the pH of the culture medium. Results suggest the possibility of processes producing ethanol plus fructose, ethanol plus fructose and sorbitol, or ethanol plus sorbitol in a single-stage batch fermentation.     

Hydrogen sulphide production by Zymomonas and its elimination in a mutant strain

Strains of Zymomonas mobilis grown in media containing either glucose or sucrose were assessed for the production of hydrogen sulphide (H₂S). In a liquid medium with low glucose concentration (20 g l^?1) only a proportion of the strains tested formed H₂S, but in medium containing a higher glucose concentration (100 g l^?1) all the strains tested produced H₂S. Four Z. mobilis strains were assayed quantitatively for H₂S production and strain ZM4 was found to produce the most H₂S in glucose medium. The amount of yeast extract and glucose, and the type of sugar used in the medium affected the amount of H₂S formed by strain ZM4. A mutant, designated ZM4701, of strain ZM4 was isolated which did not produce any detectable H₂S in liquid medium containing yeast extract plus either glucose or sucrose. The nutritional requirements of ZM4701 were investigated.