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.     

Inactivation of fructose diphosphatase by sucrose in yeast.

In Saccharomyces carlsbergensis, the addition of sucrose to the medium led to the inactivation of fructose diphosphatase. Sucrose itself probably triggered this process.     

Study of the production of fructose and ethanol from sucrose media by Saccharomyces cerevisiae

The production of ethanol and enriched fructose syrups from a synthetic medium with various sucrose concentrations using the mutant Saccharomyces cerevisiae ATCC 36858 was investigated. In batch tests, fructose yields were above 90% of theoretical values for the sucrose concentrations between 35 g/l and 257 g/l. The specific growth rates and biomass yields were from 0.218 to 0.128 h(-1) and from 0.160 to 0.075 g biomass/g of glucose and fructose consumed, respectively. Ethanol yields were in the range of 72 to 85% of theoretical value when sucrose concentrations were above 81 g/l. The volumetric ethanol productivity was 2.23 g ethanol/(l h) in a medium containing 216 g/l sucrose. Fructo-oligosaccharides and glycerol were also produced in the process. A maximum fructo-oligosaccharides concentration (up to 9 g/l) was attained in the 257 g/l sucrose medium in the first 7 h of the fermentation. These sugars started to be consumed when the concentrations of sucrose in the media were less than 30% of its initial values. The fructo-oligosaccharides mixture was composed of 6-kestose (61.5%), neokestose (29.7%) and 1-kestose (8.8%). The concentration of glycerol produced in the process was less than 9 g/l. These results will be useful in the production of enriched fructose syrups and ethanol using sucrose-based raw materials.     

Continuous culture study of transient behaviour of Saccharomyces cerevisiae growing on sucrose and fructose

During fully aerobic continuous growth of Saccharomyces cerevisiae on fructose- and sucrose-limited cultures, there exists the possibility of a number of distinct steady states at higher dilution rates. It is thus postulated that a number of discrete “states” exist for yeast cells under constant environmental conditions and that the particular “state” obtained is a function of the time spent under these conditions (adaptation) and the manner in which the steady state was approached. These observations are significant since they provide an insight into the large range of responses possible by yeasts in continuous cultures and may help to explain why unified views of sugar utilisation by yeasts have been so elusive.     

Radiorespirometric study of the utilization of exogenous sucrose,glucose and fructose by germinating apple pollen

Po?adí intensity, s jakou prodýchávají pylové lá?ky testované cukry z 0,3 M roztok?, je sacharosa> glukosa> invertní cukr> fruktosa. Stejné po?adí je zaehováno na cukr-agarových mediích s výjimkou prvých dvou hodin inkubace, během kterých jsou sacharosa, glukosa a fruktosa prodýchávány témě? stejnou rychlostí. Během této doby se v prost?edí fruktosy, stejně jako v kontrole bez cukru, nevytvo?ily pylové lá?ky, zatím za p?itomnosti sacharosy dosahovaly délky a? 450 µ. Jestli?e byla pou?ita pro radioaktivní cukry jako nosi? sacharosa, byla fruktosa-¹⁴C prodýchávána a? 12krát, glukosa-¹⁴C a? 6krât intensivnëji neá sacharosa-¹⁴C. Za pou?ití nosi?e sacharosa+glukosa ?i sacharosa+fruktosa (molárni poměry 1:1), prodýchávaji pylové lácky sacharosu-¹⁴C pomaleji ne? p?íslu?ny monosaeharid a rovně? pomaleji ne? z prost?edí samotnéé sacharosy. Jestli?e byla nosi?em sacharosy-¹⁴C glukosa nebo fruktosa, byla (v některých ?asových úsecíeh pokusu) produkce¹⁴CO₂ pylovými láckami několik desítek procent mohutněj?í ne? za pou?ití nosi?e sacharosového. Z prost?edí invertního cukru je p?ednostně prodýchäväna fruktosa. Je tedy kapacita pylových enzymových systém? za?leňujících sledované cukry do jejich dýchacích cest pro fruktosu>glukosu> sacharosu, co? je opa?né po?adí ne? platí pro intensitu r?stového ú?inku těchto cukr? a ne? jaké bylo zji?těno pro rychlost jejich prodýchávání, jestli?e nebyly navzájem kombinovány. Ve specifickém r?stovém efektu sacharosy nem??e tedy být primárním faktorem ani rychlost její absorpce, ani intensita jejího prodýcháváni. Rychlá utilisace samotné sacharosy je následkem intensivněj?ího r?stu v jejím prost?edí. Získané výsledky dále ukazují, ?e sacharosa je vyu?ívána p?edev?ím cestou její inverse, p?i ?em? je p?ednostně prodýchávána fruktosová slo?ka.     

Molecular basis of fructose utilization by the wine yeast Saccharomyces cerevisiae: a mutated HXT3 allele enhances fructose fermentation

Fructose utilization by wine yeasts is critically important for the maintenance of a high fermentation rate at the end of alcoholic fermentation. A Saccharomyces cerevisiae wine yeast able to ferment grape must sugars to dryness was found to have a high fructose utilization capacity. We investigated the molecular basis of this enhanced fructose utilization capacity by studying the properties of several hexose transporter (HXT) genes. We found that this wine yeast harbored a mutated HXT3 allele. A functional analysis of this mutated allele was performed by examining expression in an hxt1-7Delta strain. Expression of the mutated allele alone was found to be sufficient for producing an increase in fructose utilization during fermentation similar to that observed in the commercial wine yeast. This work provides the first demonstration that the pattern of fructose utilization during wine fermentation can be altered by expression of a mutated hexose transporter in a wine yeast. We also found that the glycolytic flux could be increased by overexpression of the mutant transporter gene, with no effect on fructose utilization. Our data demonstrate that the Hxt3 hexose transporter plays a key role in determining the glucose/fructose utilization ratio during fermentation.     

Regulation of glucose, fructose and sucrose catabolism in Rhodopseudomonas capsulata.

Regulation of glucose, fructose and sucrose catabolism was studied in Rhodopseudomonas capsulata grown under phototrophic conditions. The sequence of preference for the utilization of the sugar substrates was fructose, glucose, sucrose. The presence of a preferred substrate did not completely suppress the utilization of the less preferred. Glucose-6-phosphate dehydrogenase, the key enzyme of glucose and sucrose catabolism, exhibited sigmoidal substrate saturation curves and was inhibited by phosphoenolpyruvate, whereas 1-phosphofructokinase, the key enzyme of fructose catabolism, exhibited hyperbolic substrate saturation curves and was not inhibited by phosphoenolpyruvate. Since phosphoenolpyruvate is a common intermediate of glucose, fructose and sucrose catabolism, the control of glucose-6-phosphate dehydrogenase may be responsible for the preferential utilization of fructose.     

The utilization of fructose by Escherichia coli. Properties of a mutant defective in fructose 1-phosphate kinase activity

1. The isolation and properties of a mutant of Escherichia coli devoid of fructose 1-phosphate kinase activity are described. 2. This mutant grew in media containing any one of a variety of substances, including hexoses, hexose 6-phosphates, sugar acids and glucogenic substrates, at rates not significantly different from those at which the parent organism grew on these substrates. However, only the parent grew on fructose or fructose 1-phosphate. 3. Fructose and fructose 1-phosphate inhibit the growth of the mutant, but not of its parent, on other carbon sources. 4. Even though not previously exposed to fructose, the mutant took up [(14)C]fructose rapidly but to only a small extent: [(14)C]fructose 1-phosphate was identified as the predominant labelled product. In contrast, the equally rapid but more extensive uptake of [(14)C]fructose by the parent organism required prior growth in the presence of fructose.     

The effect of pH on the kinetics of beef-liver fructose bisphosphatase.

1. The kinetics of the reaction catalysed by fructose bisphosphatase have been studied at pH 7.2 and at pH 9.5. The activity of the enzyme was shown to respond sigmoidally to increasing concentrations of free Mg2+ or Mn2+ ions at pH 7.2, whereas the dependence was hyperbolic at pH 9.5. At both pH values the enzyme responded hyperbolically to increasing concentrations of fructose 1,6-bisphosphate, although inhibition was observed at higher concentrations of this substrate. This high substrate inhibition was shown to be partial in nature and the enzyme was found to be more sensitive at pH 7.2 than at pH 9.5. 2. The properties of the enzyme, are consistent with the enzyme obeying either a random-order equilibrium mechanism or a compulsory-order steady-state mechanism in which fructose bisphosphate binds to the enzyme before the cation. 3. Reaction of the enzyme with a four-fold molar excess of p-chloromercuribenzoate caused activation of the enzyme when its activity was assayed in the presence of MN2+ ions but inhibition when Mg2+ ions were used. Higher concentrations of p-chloromercuribenzoate caused inhibition. This activation at low p-chloromercuribenzoate concentrations, and the reaction of 5,5'-dithio-bis(2-nitrobenzoate) with the four thiol groups in the enzyme that reacted rapidly with this reagent, were prevented or slowed by the presence of inhibitory, but not non-inhibitory, concentrations of fructose bisphosphate. After reaction with a four-fold molar excess of p-chloromercuribenzoate the enzyme was no longer sensitive to high substrate inhibition by fructose bisphosphate.     

Dynamics and regulation of sucrose phosphorylase formation in Leuconostoc mesenteroides fermentations

The production of Leuconostoc mesenteroides sucrose phosphorylase has been studied in 10- and 20-L batch fermentations. A fermentation medium was devised combining rapid growth, high cell yield, and high enzyme levels. Overall fermentation dynamics and enzyme fermentation patterns are elucidated here in detail. Sucrose is phosphorolyzed into fructose and glucose-1-phosphate (G-1-P) with G-1-P preferentially utilized (thus saving ATP). Subsequently, fructose is gradually metabolized and is also converted to mannitol. Invertase activity is absent. Sucrose phosphorylase is formed transitorily with peak levels toward the end of active growth; a sharp decline in enzyme activity occurs upon further fermentation. The moment of cell (enzyme) harvest is thus critical in view of obtaining active cell or enzyme preparations for sucrose phosphorolysis. Microaerophilic and strictly anaerobic fermentations displayed no appreciable difference in sucrose phosphorylase formation profile. The enzyme is intracellularly located. It is constitutively formed in the absence of sucrose, contrary to that of Pseudomonas species; other disaccharide phosphorylases are not formed.     

Effect of feed zone in fed-batch fermentations of Saccharomyces cerevisiae

In production-scale, fed-batch fermentations, feed is often added to a single point at the top of the fermentor, which, combined with poor mixing, results in formation of a "feed zone" rich in nutrients. Frequent exposure of the culture to high concentrations of nutrients in the feed zone for sufficient duration can produce unexpected effects on its performance. The effect of the feed zone was evaluated by conducting aerobic fed-batch fermentations of Saccharomyces cerevisiae with both complex and defined media. The broth was recirculated between a recycle loop and a bench-scale fermentor, and feed was intermittently added into the recycle loop to simulate the circulation of cells through the feed zone. Experiments were carried out for a range of residence times in the recycle loop from 0.5 to 12 min. Biomass yields from the complex-media fermentations were not affected by exposure to high nutrient levels in the recycle loop for residence times up to 12 min. Ethanol consumption was reduced by as much as 50% for residence time in the loop up to 3 min. Very long exposure of yeast cells to excess nutrient levels (12 min) gave acetic acid formation. In a defined medium, the simulated feed zone effect increased biomass yield by up to 10%, but had no effect on ethanol levels. This study indicates that the feed zone effect on biomass yield in yeast fermentation, using complex substrates, will be negligible under fully aerobic conditions.     

The production of ethanol plus fructose sweetener using fructose utilization negative mutants of Zymomonas mobilis

Summary Two mutants, unable to utilize fructose (Fru^–) as a sole source of carbon and energy, were isolated fromZymomonas mobilis following ethyl methane sulfonate (EMS) mutagenesis. The frequency of stable Fru^– mutants among survivors of mutagenesis was 1 in 10⁴. The two Fru^– mutants were able to cleave sucrose to glucose and fructose, and then ferment only the glucose to ethanol while accumulating fructose close to the theoretical value. Under controlled fermentation conditions, sucrose was converted to ethanol plus 80% or higher purity fructose syrup in a single-stage batch fermentation process, improving the Sucrotech Process significantly.     

Glucose and fructose utilization in human platelets. Effects of diamide

In this study we have reported that platelets metabolize fructose more slowly than glucose and probably by a different mechanism. While formed lactate is correlated with glucose utilized, in the presence of fructose an overproduction of lactate was demonstrated. The different behaviour of glucose and fructose was also shown by utilizing diamide at various concentrations. Low diamide concentrations increase glucose consumption, whereas higher concentrations inhibit. Fructose is gradually inhibited by increasing oxidant quantities. Data obtained suggests that diamide interferes with the transport process across platelet membrane. It is likely that glucose and fructose do not share the same transport mechanism. On the other hand only high diamide concentrations inhibit sugar metabolism by acting on the glycolytic flux at the level of some key enzymes.     

Mitochondrial control of sugar utilization in Saccharomyces cerevisiae.

When a number of wild-type strains of Saccharomyces cerevisiae—all capable of utilizing the three sugars galactose, maltose, and α-methyl-d-glucoside for growth—were converted by ethidium bromide (EtdBr) mutagenesis to stable cytoplasmic petite (rho^?) mutants, the latter lost the ability to grow on one or more of these sugars. The actual pattern of retention (or loss) or sugar utilization by these mutants depended on the wild-type strain, but was independent of the length of exposure to EtdBr during mutagenesis. This treatment varied from 0.5 to 24 h, by which time the majority of the mutants must have been of the mitochondrial (mt) DNA-deficient rho⁰ type. Furthermore, with one exception—involving the ability of one set of mutants to utilize α-methyl-glucoside—all rho^? mutants derived from the same wild type exhibited the same, discrete pattern of sugar utilization. Respiration-deficient mutants with defined lesions in their mtDNA (mit^? mutants) exhibited the same pattern of sugar utilization as did the petite mutants of the same strain. Diploid petite strains also exhibited discrete, but less stringent, patterns of sugar utilization. For any one genotype this pattern was identical whether the mutant was generated by crossing two haploid rho^? strains, themselves derived by EtdBr mutagenesis, or by EtdBr mutagenesis of the diploid obtained from a haploid wild-type × wild-type cross. In such mutant diploids the sugar-positive phenotype was usually dominant, but there were indications in some instances of modulation of this effect by virtue of nuclear gene interactions. Various respiration-deficient mutants incapable of utilizing α-methylglucoside also were unable to form α-glucosidase, but were able to do so after being rendered permeable by exposure to dimethyl sulfoxide. Arguments are advanced that respiring mitochondria generate an entity—probably not directly related to ATP production—required for the expression of nuclear genes or their products, some of which may be necessary for plasma membrane function.     

Adenosine utilization in cordycepin-sensitive mutants of Saccharomyces cerevisiae.

A purine-requiring, wild-type yeast strain was cordycepin resistant and failed to grow in medium containing adenosine; in contrast, a cordycepin-sensitive mutant (also purine requiring) grew well in medium containing adenosine. The cordycepin-sensitive mutant incorporated [8-14C]adenosine at nine times the wild-type rate, and adenosine completely fulfilled the purine requirement of the cells. Exogenous adenosine rapidly entered the mutant cells, apparently as free nucleoside, and was phosphorylated; uptake displayed concentration-dependent saturation kinetics (Km, 6 mM). Within 10 min 14C radioactivity was being incorporated into nucleic acids.     

Regulation of sugar utilization by Saccharomyces cerevisiae.

There are several kinds of regulation that enable microbes to cope with rapidly changing supplies of nutrients. This is exemplified by sugar metabolism in Saccharomyces cerevisiae. Some readily reversible controls affect the activity of enzymes, either by allosteric activation and deactivation, which often occur within seconds, or by covalent modification, within minutes. Other controls regulate the amount of enzyme present in the cells, either by irreversible proteolytic inactivation of the enzyme, or by influencing enzymic synthesis. The nomenclature of these processes is often confused.