Levan Formation Research Articles

Enhancement of ethanol production from sucrose by Zymomonas mobilis by the addition of immobilized invertase

In the production of ethanol by Zymomonas mobilis ATCC 10988, using sucrose as the substrate, we added immobilized invertase to the fermentation broth to promote sucrose hydrolysis. The rapid accumulation of glucose in the medium caused the inhibition of levan formation and an enhancement of the growth rate at the initial stage of fermentation. The formation of sorbitol could also be reduced by addition of immobilized invertase, because the accumulation of glucose and fructose was observed to be even lower than without immobilized invertase. After the addition of 0.2 g immobilized invertase (activity, 2, 100 U g −1, using 0.8 m sucrose at pH 5 and 30°C) to a 1.2-l batch fermenter containing 200 g l −1 sucrose, the formation of byproducts was significantly reduced and the ethanol yield was increased from 0.29 to 0.40 g g −1. High ethanol yields were also obtained from continuous cultures. When 0.2 g immobilized invertase was added to the fermenter, the dilution rate for maximal ethanol productivity was determined to be 0.2 h −1. The coupled microbe-immobilized-invertase system proved to be better than the two-stage (separate hydrolysis and fermentation) system in the yield of ethanol produced.

Identification and characterisation of the extracellular sucrases of Zymomonas mobilis UQM 2716 (ATCC 39676)

An investigation was conducted to isolate, and characterise the extracellular sucrases of Zymomonas mobilis UQM 2716. Levansucrase (EC 2.4.1.10) was the only extracellular sucrase produced by this organism. This enzyme was responsible for sucrose hydrolysis, levan formation, and oligosaccharide production. It had a molecular mass of 98 kDa, a Michaelis constant (Km) of 64 mm, and a pH optimum of 5.5. It was inhibited by glucose, but not by fructose, ethanol, sorbitol, NaCl, TRIS or ethylenediaminetetraacetic acid (EDTA). The formation of levan was the principal reaction catalysed by this enzyme at low temperatures. However, levan formation was thermolabile, being irreversibly lost when levansucrase was heated to 35°C. S This did not effect sucrose hydrolysis or oligosaccharide formation, which were optimal at 45°C. Sucrose concentration greatly influenced the type of acceptor molecule used in the transfructosylation reactions catalysed by levansucrase. At low sucrose concentration, the predominant reaction catalysed was the hydrolysis of sucrose to free glucose and fructose. At high sucrose concentrations, oligosaccharide production was the major reaction catalysed.

Bioreaction-separation on continuous chromatographic systems

Combined bioreaction and separation has been carried out successfully on a simulated moving column continuous countercurrent chromatographic reactor-separator (SCCR-S) system. The continuous inversion of sucrose to glucose and fructose using invertase and the biosynthesis of dextran from sucrose in the presence of the enzyme dextransucrase have been studied. 100% sucrose conversions were achieved and product purities of up to 95% w/v were obtained in the inversion studies. The operation was carried out on a preparative scale with throughputs in excess of 16 kg of dry sugar solids per cubic metre of resin per hour used. The simultaneous inversion and product separation was found to overcome problems associated with substrate inhibition. Complete reaction and separation was obtained at feed concentrations as high as 55% w/v. Previous studies on the biosynthesis of dextran on batch chromatographic systems showed significant improvements in product yields. The simultaneous removal of the acceptor by product fructose from the reaction mixture led to the formation of greater amounts of high molecular weight dextran (over 80% more than the conventional process at 20% w/v sucrose concentration). To carry out dextran biosynthesis on the SCCR-S system, it was necessary to repack it with larger-size resin to allow for the high pressures caused by the more viscous dextran. Complete conversions have been achieved at sucrose concentrations of 5% w/v with dextran and fructose product purities of up to 100% and 97% respectively obtained under certain conditions. Some levan formation occurred after about 50 h of operation, which is readily removed from the dextran in the normal dextran processing procedures.

Continuous ethanol production from sugar beet molasses using an osmotolerant mutant strain of Zymomonas mobilis

Abstract A laboratory process was established for ethanol production by fermentation of sugar beet molasses with the bacterium Zymomonas mobilis . Sucrose in the molasses was hydrolyzed enzymatically to prevent levan formation. A continuous system was adopted to reduce sorbitol formation and a two-stage fermentor was used to enhance sugar conversion and the final ethanol concentration. This two-stage fermentor operated stably for as long as 18 d. An ethanol concentration of 59.9 g/ l was obtained at 97% sugar conversion and at high ethanol yield (0.48 g/g, 94% of theoretical). The volumetric ethanol productivity (3.0 g/ l ·h) was superior to that of batch fermentation but inferior to that of a single-stage continuous system with the same medium. However, the thanol concentration was increased to a level acceptable for economical recovery. The process proposed in this paper is the first report of successful fermentation of sugar beet molasses in the continuous mode using the bacterium Z. mobilis .

Comparison of ethanol production by Zymomonas mobilis from sugar beet substrates

The potential of four sugar beet substrates from the sugar industry [syrup (S), crystallizer effluent 1 (CE1), crystallizer effluent 2 (CE2) and molasses (M)] were compared for ethanol production using an osmotolerant mutant strain of the bacterium Zymomonas mobilis. Sucrose of the substrates was enzymatically hydrolysed to avoid levan formation during fermentation. Nutrient supplementation experiments have shown that reproducible growth and ethanol production could be obtained on the four substrates supplemented only with magnesium sulphate (CE2 and M) or additionally with ammonium sulphate (S and CE1). Thus, addition of costly yeast extract could be avoided. All 20% (w/v) substrates showed nearly complete sugar conversion (>94.9%), good growth (0.16 h(-1)) and ethanol production (>40 g 1(-1)). However, sorbitol formation reduced the ethanol yield (73-79% of the theoretical value) significantly. Batch kinetic parameters and studies of instantaneous parameters showed that enhanced osmolality of substrates (S<CE1<CE2<M) inhibited biomass production more strongly than ethanol production. In conclusion, all four sugar beet substrates could be utilized for ethanol production using this mutant strain of Z. mobilis with appropriate supplementation.

Cloning and sequencing of the sacA gene: characterization of a sucrase from Zymomonas mobilis

The Zymomonas mobilis gene (sacA) encoding a protein with sucrase activity has been cloned in Escherichia coli and its nucleotide sequence has been determined. Potential ribosome-binding site and promoter sequences were identified in the region upstream of the gene which were homologous to E. coli and Z. mobilis consensus sequences. Extracts from E. coli cells, containing the sacA gene, displayed a sucrose-hydrolyzing activity. However, no transfructosylation activity (exchange reaction or levan formation) could be detected. This sucrase activity was different from that observed with the purified extracellular protein B46 from Z. mobilis. These two proteins showed different electrophoretic mobilities and molecular masses and shared no immunological similarity. Thus, the product of sacA (a polypeptide of 58.4-kDa molecular mass) is a new sucrase from Z. mobilis. The amino acid sequence, deduced from the nucleotide sequence of sacA, showed strong homologies with the sucrases from Bacillus subtilis, Salmonella typhimurium, and Vibrio alginolyticus.

Levan production with a flocculent strain ofzymomonas mobilis

Abstract Levan is a fructose polymer with potential importance as fructose source or thickening agent in food technology in case that it could be produced in a simple and cheap process. Levan concentrations of up to 42.5 g/L and a yield from fructose of 032 g/g were obtained with a new flocculent mutant of Zymomonas mobilis. A sucrose concentration of 250 g/L, pH‐values around 5.0 and temperatures of 27 to 30 °C were optimal. Higher pH‐values and high starting concentrations of levan caused hydrolysis of the polymer by the bacterium itself. However, adding levan up to 1.5 g/L led to nearly 40 % increase of the levan synthesis. Sorbitol also increased levan formation due to a decrease of sucrose and levan hydrolysis. Presumably by induction of the levansucrase, fructose addition caused a higher levan yield too. In continuous operation, a levan productivity of 16 g/L.h and an ethanol productivity of 26.3 g/L.h resulted. That was 5 to 12 times, respectively the productivity of batch processes. These results ...

The effect of pH, temperature and sucrose concentration on high productivity continuous ethanol fermentation using Zymomonas mobilis

A flocculent strain of Zymomonas mobilis was used for ethanol production from sucrose. Using a fermentor with cell recycle (internal and external settler) high sugar conversion and ethanol productivity were obtained. At a dilution rate of 0.5 h-1 (giving 96% sugar conversion) the ethanol productivity, yield and concentrations respectively were 20 g/l/h, 0.45 g/g and 40 g/l using a medium containing 100 g/l sucrose. At a sucrose concentration of 150 g/l, the ethanol concentration reached 60 g/l. The ethanol yield was 80% theoretical due to levan and fructo-oligomer formation. No sorbitol was detected. This fermentation was conducted at a range of conditions from 30 to 36°C and from pH 4.0 to 5.5.

Studies on the Extracellular Polysaccharides (EPS) Produced in vitro by Pseudomonas phaseolicola III. Kinetics of Levan and Alginate Formation in Batch Culture and Demonstration of Levansucrase Activity in Crude EPS

AbstractIn batch culture Pseudomonas phaseolicola converted ca. 8–11% of the substrate (sucrose) into extracellular polysaccharides (EPS). The percentage of conversion was independent of the concentration of sucrose in the medium. However, the increase of total EPS observed with enhanced substrate concentration was largely due to a drastic stimulation of the levan produced. Production of alginate, in contrast, was stimulated to a much lower extent. EPS production was highest during the middle phaseof exponential growth and gradually terminated as the cultures grew older. With sucrose as substrate, the formation of alginate followed the levan synthesis. With glucose or gluconate, however, there was no production of levan, and the alginate synthesis, started immediately after the lag‐phase of the culture.Levansucrase activity was detected in the protein fraction of crude EPS produced by a culture grown in sucrose containing medium. Enzyme activity was demonstrated both by the release of glucose and the formation of levan from sucrose. Upon ion exchange chromatography on DEAE‐fractogel levansucrase behaved as a rather acidic protein. Native polyacrylamide gel electrophoresis of the enzyme fraction revealed a major band with an apparent molecular weight of ca. 68,000 d. Several grams of levan were synthesized with a partly purified enzyme preparation in the absence of bacteria.

Rate and yield limiting factors in continuous fermentation of sucrose byZymomonas mobilis

The initial transfer of fructosyl units in the utilization of sucrose led to the formation of fructose, oligomers or levan and was apparently controlled by the concentrations of sugars in the medium. In continuous fermentation, the rather low levels of monomeric sugars in the broth prevented the formation of sorbitol and oligomers, whereas the production of levan was increased compared to that in batch fermentation. The fructooligomers contained approximately one mole of glucose per two, three or four moles of fructose. The overall ethanol production rate was limited by the uptake rates of glucose and especially of fructose, which was decreased due to transfructosylation reactions.

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

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.

Formation of levan and sorbitol from sucrose by Zymomonas mobilis

Ethanol yields produced by Zymomonas strains from sucrose are significantly lower than from glucose or fructose. The low yield is a consequence of the formation of both levan and sorbitol as by-products. Most of the levan is in a non-precipitable form, indicating low molecular weight. Formation of sorbitol was observed with both the Zymomonas strains studied. The measured amounts of levan and sorbitol were 8% and 11% of the original sucrose content, respectively.

Study on Levan formation during fermentation ofzymomonas mohilis on sucrose

Levan was formed from sucrose by a non-growingZ.mobilis cell suspension most markedly at pH 5 and 30°C. The formation of levan was substantially reduced at 35°C. Levan was also formed significantly during sucrose fermentation at 30°C. Fermentation at 35°C formed levan in small amount, whereas production of ethanol and C02 increased slightly. Higher concentration of sucrose produced more levan.

The kinetics of ethanol production by Zymomonas mobilis on fructose and sucrose media

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.

Marginal Blight of Cucumber Caused by a Strain of Pseudomonas marginalis (Brown) Stevens

A bacterial disease was found on matured lower leaves of cucumber grown under vinyl house conditions in winter of 1974. The disease rarely occurred on inmature leaves soon after unfolding. The disease usually started from leaf edges near hydathodes and quickly enlarged to form V-shaped lesions frequently surrounded by chlorotic areas. These lesions finally coalesced to form the severe blight along the whole margin of the leaf. From these symptoms, “marginal blight of cucumber” was proposed as the disease name. The cultural and biochemical characters of the pathogen were compared with those of Pseudomonas lachrymans, the pathogen of angular leaf spot of cucumber. These two bacteria differed in the important characteristics such as production of fluorecin, degree of gelatin liquefaction, levan formation, margarine hydrolysis, arginine dihydrolase reaction, potato rot, and utilization of sucrose, sorbitol and tartarate. The marginal blight pathogen was rather close to soft-rotting pseudomonads in bacteriological properties. Although the similarity was not complete, therefore, we are inclined to identify the present bacterium as a strain of Pseudomonas marginalis (Brown) Stevens based on the detailed survey of pathological, cultural and physiological characteristics. The isolates of P. lachrymans used in the study showed also some conflicts in physiological properties with the description of the type culture suggesting the presence of strains in this bacterium.

Levan formation by whole cells of Actinomyces viscosus ATCC 15987.

Levan formation by whole cells of Actinomyces viscosus ATCC 15987.

Levan formation by Odontomyces viscosus.

The authors present some physiological parameters involved in levan formation by Odontomyces viscosus, a filamentous organism in gingival plaque of the hamster.

Mechanisms of biopolymer growth: the formation of dextran and levan.

Mechanisms of biopolymer growth: the formation of dextran and levan.

LEVAN FORMATION BY ACETOMONAS

The mucilage formed by growth of Acetomonas on a medium containing sucrose has been isolated and purified. Partial hydrolysis, methylation followed by hydrolysis, periodate oxidation and optical rotation of the product and its methylated derivative showed it to be a 2β,6′-linked levan having branch points with 2β,1′ linkage and an average unit chain of 12 anhydrofructofuranose residues; the molecular weight was of the order of several hundred thousand. In this property of levan formation, Acetomonas resembles the related genus Pseudomonas and differs from the dextran-forming genus Acetobacter.

Sucrose utilization by Zymomonas mobilis: formation of a levan.

1. Molar growth-yield coefficients of Zymomonas mobilis for glucose, fructose, glucose plus fructose, and sucrose are reported. Yield coefficients for sucrose are appreciably lower than those for the equivalent concentrations of glucose plus fructose. 2. Only 2.6% of [U-(14)C]glucose supplied in the growth medium is incorporated into cell substance by Z. mobilis utilizing glucose as the energy source. 3. During growth on sucrose a levan is formed. It has been characterized and shown to resemble other bacterial levans. 4. Levan formation from sucrose could be demonstrated with both washed cell suspensions and cell extracts of Z. mobilis. 5. Sucrose phosphorylase could not be demonstrated in extracts of the organism.