Fructan Biosynthesis by Intra- and Extracellular Zymomonas mobilis Levansucrase after Simultaneous Production of Ethanol and Levan

Summary The chemical composition of the Zymomonas mobilis biomass and the culture liquid after ethanol and levan synthesis were studied. The activities of intra- and extracellular levansucrase produced by the Z. mobilis strain 113 ”S” under optimum conditions both for levan and fructooligosaccharide (FOS) synthesis were also determined. It was shown that levan production relates to the reduction of the carbohydrate and lipid content in the biomass by increasing the nucleic acid and protein content. The levan producing activity of cellular levansucrase after ethanol and levan synthesis was approximately 30–40% of the total activity in the second fermentation stage. It was established that th…

Stillage as a Source of Growth Promoting Biofactors and a Stimulator of Levan and Extracellular Levansucrase Synthesis for Zymomonas mobilis

In the present work, the fermentation of simultaneous production of ethanol and levan by Zymomonas mobilis grown on different growth media has been studied. Yeast extract, rye stillage or sugar beet molasses stillage were used as additives to the basic sucrose media and the chemical composition, including vitamins, of the cultivation liquids have been determined. It has been shown that 0.5 % of yeast extract dry weight additive could be substituted by 10.0 % of native stillage additive. It was established that molasses stillage stimulates the ethanol synthesis, but rye stillage additive is more preferable for levan production. The extracellular levansucrase obtained from the culture liquid …

A novel and simple method for the purification of extracellular levansucrase from Zymomonas mobilis.

A new and simple method for the purification of extracellular levansucrase from Zymomonas mobilis from highly viscous fermentation broth was developed. After incubation of the fermentation broth with a fructose-polymer cleaving enzyme preparation (Fructozyme, Novozymes, DK) for 48 h, levansucrase precipitated as aggregates and was redissolved in a 3 M urea solution. By ongoing size-exclusion chromatography on Sephacryl S-300 the final levansucrase preparation was purified 100-fold and exhibited a specific activity of 25-35 U/mg(protein). The levansucrase was stable in 3 M urea solution for at least four months without inactivation. To maximize the enzyme yield the dynamic changes of extrace…

Formation of Levan from Raffinose by Levansucrase ofZymomonas mobilis

Levansucrase (EC 2.4.1.10.) of Zymomonas mobilis 113S can perform the polymerisation of fructose moiety from raffinose to levan concomitantly with a release of non-catabolised melibiose into the medium. The kinetic parameters of the levansucrase-catalysed reaction provide even higher reaction velocities on raffinose as compared to sucrose, particularly at low substrate concentrations. A decreased value in the number of the average molecular mass (Mn = 1693 kDa), an increased intrinsic viscosity (η = 49.47 cm3/g), and a diminished Huggin's constant (K' = 0.67) are intrinsic to the levan synthesis from raffinose, indicating certain structural peculiarities compared to a polysaccharide obtaine…

Stillage as a Source of Growth Promoting Biofactors and a Stimulator of Levan and Extracellular Levansucrase Synthesis for Zymomonas mobilis

In the present work, the fermentation of simultaneous production of ethanol and levan by Zymomonas mobilis grown on different growth media has been studied. Yeast extract, rye stillage or sugar beet molasses stillage were used as additives to the basic sucrose media and the chemical composition, including vitamins, of the cultivation liquids have been determined. It has been shown that 0.5 % of yeast extract dry weight additive could be substituted by 10.0 % of native stillage additive. It was established that molasses stillage stimulates the ethanol synthesis, but rye stillage additive is more preferable for levan production. The extracellular levansucrase obtained from the culture liquid …

OATS AND FAT-FREE MILK BASED FUNCTIONAL FOOD PRODUCT

The present study addresses problems and solutions related to new functional bio-active food product creation. Commercial rolled oats and fat-free milk were used as raw materials. Limited enzymatic hydrolysis of oat starch was carried out by α-amylase. The lactic acid bacteria strains Lactobacillus acidophilus, Bifidobacterium sp., and commercial starter cultures ABT-1 were cultivated in enzymatically hydrolysed oat mash. Lactobacillus acidophilus was a more active producer of lactic acid, while Bifidobacterium sp. improved the taste properties. A fat-free milk additive was applied to stabilise the texture of the oat mash, and increase the protein content of the final product. All procedure…

The effect of osmo-induced stress on product formation by Zymomonas mobilis on sucrose

The intensification of biosynthesis of fructooligosaccharides in the presence of high salt concentrations was observed during sucrose (10%) fermentation by Zymomonas mobilis 113S. A 0.6 M NaCl concentration led to an increase of oligosaccharide productivity by 3.5-fold. Sorbitol formation was increased in the presence of 0.16 M NaCl and was inhibited at highest salt concentrations. In a medium with high (65%, w/w) sucrose content the salts gave inhibitory effects on fructooligosaccharide production by lyophilised Z. mobilis cells. Influence of salts on gluconic acid and sorbitol formation under these conditions was studied. The ratio of oligosaccharides and gluconic acid productivity (Qolig…

The effect of mixing on glucose fermentation by Zymomonas mobilis continuous culture

Abstract The influence of mixing (in the range from 300 to 1100 rpm) on the performance of Zymomonas mobilis anaerobic continuous culture was studied. Biomass yield and ethanol productivity were improved at higher stirring intensities, in parallel with a decrease in byproduct synthesis. A mixing-dependent formation of minor amounts of fructose was first demonstrated in Z. mobilis anaerobic culture on glucose. The rate of fructose synthesis and the specific activity of the key enzyme in the pathway to fructose, phosphoglucose isomerase (EC 5.3.1.9), had a maximum at 700 rpm.

Sucrose medium osmolality as a regulator of anabolic and catabolic parameters inZymomonas culture

This study focuses on the growth of Zymomonas mobilis strain 113 S and its ethanol and levan production under the conditions of increasing sucrose medium osmolality caused by NaCl, KCl, sorbitol or maltose. The increase in medium osmolality (700-1,500 mosml/kg) was accompanied by the inhibition of growth (growth rate, biomass yield) and ethanol production (specific productivity and yield). In contrast, levan synthesis was less affected or even stimulated and, as a consequence, levan specific productivity was increased significantly. A decrease in the anabolic growth parameters correlated with a parallel inhibition of glucose-6-P dehydrogenase and alcohol dehydrogenase (isoenzyme ADH II) act…

2-Phenylethanol production by Kluyveromyces marxianus on glucose and lactose substrates

Levansucrases from Pseudomonas syringae pv. tomato and P. chlororaphis subsp. aurantiaca: Substrate specificity, polymerizing properties and usage of different acceptors for fructosylation

Levansucrases of Pseudomonas syringae pv. tomato DC3000 (Lsc3) and Pseudomonas chlororaphis subsp. aurantiaca (also Pseudomonas aurantiaca) (LscA) have 73% identity of protein sequences, similar substrate specificity and kinetic properties. Both enzymes produce levan and fructooligosaccharides (FOS) of varied length from sucrose, raffinose and sugar beet molasses. A novel high-throughput chip-based nanoelectrospray mass spectrometric method was applied to screen alternative fructosyl acceptors for levansucrases. Lsc3 and LscA could both transfructosylate D-xylose, D-fucose, L- and D-arabinose, D-ribose, D-sorbitol, xylitol, xylobiose, D-mannitol, D-galacturonic acid and methyl-α-D-glucopyra…

Levan production byZymomonas mobilis cells. Attached to plaited spheres

In this work, an immobilization method for polymer-levan production by a non-flocculating Z mobilis culture was developed. The extent of cell attachment to the stainless steel wire surface, culture growth and product synthesis were described. It was established that during short-term passive immobilization of non-flocculating Z mobilis cells on a stainless steel wire surface, sufficient amounts of biomass for proper levan and ethanol fermentation could not be obtained. Adherence of cells was improved by pressing the paste-like biomass within stainless steel spheres knitted from wire with subsequent dehydration. Biomass fixed in metal spheres was used for repeated batch fermentation of levan…

Fructan-Biosynthese durch in Membranen immobilisierte Levansucrase

Stability of levan produced by Zymomonas mobilis

Abstract The stability of levan produced by Zymomonas mobilis 113 “S” was investigated during storage of culture liquid at different pH and temperatures. Levan was not degraded if stored in a culture liquid for 120 h at 30 or 25 °C, but at 55 or 60 °C the degree of degradation was inversely proportional to pH. Cell biomass and levan sediment components did not cause the degradation of levan. The influence of organic acids (lactic-, acetic-, and gluconic) and HCl as levan hydrolyzing agents at pH 3.7, temperature 30 °C or 55 °C was investigated in a model solution. The hydrolytic effect was similar in the case of organic acids—ensuring more fructose formation, while HCl ensured higher amount…

The influence of fermentation conditions on biomass composition during ethanol biosynthesis from cheese whey lactose concentrate by Kluyveromyces marxianus

Ethanol production improvement in Zymomonas mobilis by supplementation of fructan-cleaving enzymes on sucrose containing substrates

Buckwheat based substrate fermentation - possibilities for non-traditional alcoholic beverage production

Sugar beet diffusion juice and syrup as media for ethanol and levan production byZymomonas mobilis

Abstract Sugar beet is an important crop cultivated widely in Europe. Roots contain up to 6–9 t/ha of fermentable sugar. Our laboratory is targeting to investigate the production possibilities of ethanol and levan by Zymomonas mobilis bacteria, using sugar beet juice or their syrup as raw material. The Z.mobilis strain 113 “S”;, selected in our institute, produces simultaneously with ethanol up to 40–70 g/1 fructose polymer levan in sucrose medium. It was established that Z.mobilis can convert sucrose into ethanol and levan in batch fermentation using natural sugar beet diffusion juice, without additives of mineral salts and growth factors. Syrup obtained in sugar industrial production by c…

Whey lactose bioconversion to valuable products by non-conventional yeasts Kluyveromyces marxianus

Fructooligosaccharide and levan producing activity of Zymomonas mobilis extracellular levansucrase

Abstract The present work was devoted to investigations of the fructooligosaccharide (FOS) and levan forming activity of ethanol producing bacteria Zymomonas mobilis and their extracellular levansucrase. After cell separation the culture liquid was treated with ethanol to precipitate levan together with extracellular levansucrase. Levan–levansucrase sediment can be used as biocatalyst for fructooligosaccharide (FOS) production in sucrose syrup and levan sediment as soluble fibre source. The dynamics of sucrose conversion and glucose, fructose, and FOS formation by extracellular levansucrase showed that the fructose content increased only during the first 6 h, while the glucose content conti…

Model-based biotechnological potential analysis of Kluyveromyces marxianus central metabolism

Abstract The non-conventional yeast Kluyveromyces marxianus is an emerging industrial producer for many biotechnological processes. Here, we show the application of a biomass-linked stoichiometric model of central metabolism that is experimentally validated, and mass and charge balanced for assessing the carbon conversion efficiency of wild type and modified K. marxianus. Pairs of substrates (lactose, glucose, inulin, xylose) and products (ethanol, acetate, lactate, glycerol, ethyl acetate, succinate, glutamate, phenylethanol and phenylalanine) are examined by various modelling and optimisation methods. Our model reveals the organism’s potential for industrial application and metabolic engi…

Utilization of different carbohydrates by various Kluyveromyces marxianus strains