Elevated polysaccharide production by mutants of the fungus Aureobasidium pullulans

Abstract Two mutants of the fungus Aureobasidium pullulans ATCC 42023 were isolated that exhibited elevated polysaccharide production. Both mutants were isolated using a combination of chemical mutagenesis and resistance to growth inhibitors. It was found that both mutants elaborated higher polysaccharide levels after 7 days of growth on corn syrup or sucrose, respectively, compared to ATCC 42023. The dry weights of the mutant cells were found not to differ greatly from those of the parent cells whether corn syrup or sucrose served as the carbon source. The pullulan content of the polysaccharide synthesized by the mutants or parent cells on sucrose was consistently lower than polysaccharide synthesized on corn syrup. Using corn syrup as a carbon source, the pullulan content of the polysaccharide elaborated by the parent was higher than either mutant. The inverse was found to occur with respect to pullulan content when the strains were grown on sucrose as a carbon source.     

Short Communication: Effect of manganese on polysaccharide production and cellular pigmentation in the fungus Aureobasidium pullulans

Manganese supplementation resulted in higher polysaccharide levels and reduced cellular pigmentation by more than 8- or 17-fold after growth for 7d of the fungus Aureobasidium pullulans ATCC 42023 on sucrose or corn syrup, respectively, as a carbon source. The melanin content of the polysaccharide elaborated by ATCC 42023 cells also decreased if MnCl2 was added to the medium. The pullulan content of the polysaccharide synthesized by ATCC 42023 on sucrose was found to increase with increasing levels of manganese, whereas it was lower during growth on corn syrup if manganese was present.     

Polysaccharide production by sponge-immobilized cells of the fungus Aureobasidium pullulans

T.P. WEST AND B.R.-H. STROHFUS. 1996. Cells of the fungus Aureobasidium pullulans ATCC 42023 were immobilized in sponge cubes and examined for their ability to elaborate the polysaccharide pullulan in relation to carbon source. It was found that fungal cells grown on corn syrup, sucrose or glucose as a carbon source could be immobilized in sponge cubes and that comparable cell weights and viable cell concentrations were immobilized. Independent of the carbon source tested, the immobilized fungal cells could be used at least three times for the production of polysaccharide. The immobilized A. pullulans cells elaborated the highest polysaccharide levels in the culture medium after 5–7 d of growth at 30°C.     

Polysaccharide production by a reduced pigmentation mutant of Aureobasidium pullulans NYS-1

AIMS: To isolate a reduced pigmentation mutant of Aureobasidium pullulans NYS-1 and characterize its cellular pigmentation plus its polysaccharide and biomass production relative to carbon source. METHODS AND RESULTS: Cellular pigmentation, polysaccharide levels and biomass production by the isolated mutant NYSRP-1 were analysed relative to carbon source. Cellular pigmentation of the mutant was lower than its parent strain using either carbon source. The mutant elaborated higher polysaccharide levels on sucrose than on corn syrup. The pullulan content of the polysaccharide synthesized and biomass production by the mutant rose as the carbon source concentration was increased. CONCLUSION: It is feasible to isolate a reduced pigmentation mutant from strain NYS-1 that exhibits elevated polysaccharide production using corn syrup as a carbon source. SIGNIFICANCE AND IMPACT OF THE STUDY: The mutant provides an advantage for commercial pullulan production because of its reduced pigmentation and enhanced polysaccharide synthesis.     

Effect of nitrogen source on pullulan production by Aureobasidium pullulans grown in a batch bioreactor

Pullulan production by Aureobasidium pullulans ATCC 201253 using selected nitrogen sources was studied in a medium using corn syrup as a carbon source. Independent of the corn syrup concentration present, the use of corn steep liquor or hydrolysed soy protein as a nitrogen source instead of ammonium sulphate did not elevate polysaccharide production by ATCC 201253 cells grown in an aerated, batch bioreactor containing 4 litres of medium. Pullulan production on corn steep liquor or hydrolysed soy protein as a nitrogen source became more comparable as the concentration of corn syrup was increased. Cell weights after 7 days of growth on any of the nitrogen sources were similar. The viscosity of the polysaccharide on day 7 was highest for cells grown on ammonium sulphate and 12.5% corn syrup. The pullulan content of the polysaccharide elaborated by ammonium sulphate-grown cells on day 7 decreased as the corn syrup level rose in the medium while the pullulan content of polysaccharide produced by cells grown on corn steep liquor or soytone generally increased.     

Isolation of a mutant strain of Pseudomonas sp ATCC 31461 exhibiting elevated polysaccharide production

A mutant strain of the bacterium Pseudomonas sp. ATCC 31461 that exhibited elevated production of the polysaccharide gellan on glucose or corn syrup as a carbon source was isolated. Gellan production by the mutant strain was about twofold higher than its parent strain on glucose or corn syrup after 48 h of growth, and about 1.4-fold higher after 72 h. An increase in biomass production was not correlated with enhanced gellan synthesis by the mutant strain. The increased gellan production by the mutant strain on either carbon source resulted in an increase in its culture medium viscosity and the viscosity of the isolated polysaccharide produced by glucose-grown cells. No differences in the glucuronic acid content of the polysaccharides produced by the mutant and parent strains were observed. Journal of Industrial Microbiology & Biotechnology (2002) 29, 185–188 doi:10.1038/sj.jim.7000278 Received 13 February 2002/ Accepted in revised form 20 May 2002     

Polysaccharide production by immobilized Aureobasidium pullulans cells in batch bioreactors.

Cells of the fungus Aureobasidium pullulans ATCC 201253 were entrapped within 4% agar cubes or 5% calcium alginate beads and were examined for their production of the polysaccharide pullulan in batch bioreactors. The batch bioreactors were utilized twice for 168 hours of polysaccharide production in medium containing corn syrup as a carbon source. The agar-entrapped cells produced nearly equivalent pullulan concentrations during both production cycles. The alginate-entrapped cells produced higher polysaccharide levels during the second cycle compared to the levels observed during the initial cycle. The agar-entrapped cells elaborated a polysaccharide with a higher pullulan content than did the alginate-entrapped cells during both production cycles.     

A comparison of polyssacharides from strains of Aureobasidium pullulans

Abstract Production of pullulan by five strains of Aureobasidium pullulans was compared in three media with three carbohydrate sources. Our goal was to screen strains and media to obtain pullulan in maximal yield, purity, and stability. Pullulan yields and properties were strongly affected by strain specificity, but a single medium performed best with most strains. Sucrose was the preferred carbohydrate for all five strains. A "color variant" strain of Aureobasidium , NRRL Y-12974, possibly representing a distinct species, produced a polysaccharide of intermediate molecular weight which was stable during storage at 4°C, heating to 100°C, and high shearing action. This polysaccharide was 70% pullulanase sensitive and contained the least contaminating melanin pigment.     

Role of pH and Nitrogen Limitation in the Elaboration of the Extracellular Polysaccharide Pullulan by Pullularia pullulans

During the growth of the yeastlike fungus Pullularia pullulans on glucose as sole carbon source, the cell-mass does not increase concomitantly with the elaboration of the extracellular polysaccharide pullulan, but precedes it. The conditions generated in the culture medium which activate the secretion of polysaccharide have been sought for, and, in particular, the roles of extracellular pH and nitrogen limitatation are examined.     

Production of the fungal exopolysaccharide pullulan by batch-wise and continuous fermentation

A mutant strain of the deuteromycete Aureobasidium pullulans deficient in melanin synthesis was used to investigate the production of the exopolysaccharide pullulan and biomass, respectively. Shake-flask experiments with different carbon sources showed significant differences in pullulan elaboration. Sucrose was most suitable for pullulan synthesis among the carbon sources examined. Fermentations were carried out both batch-wise and continuously in a stirred vessel fermentator. In batch fermentations about 45% of the glucose offered was converted into pullulan at maximum formation rates of 0.16 g/l per hour using standard medium. The yield of polysaccharide could be maintained at 45% in continuous fermentations. At a dilution rate of 0.05 l/h, the formation rate of polysaccharide increased up to 0.35 g/l per hour. Alterations in the nitrogen content of the feed significantly affected the consumption rate of glucose and the production rate of polysaccharide, but final concentrations of biomass were hardly affected. Correspondence to: R. Schuster     

Polysaccharide production by immobilized Aureobasidium pullulans cells in batch bioreactors

Cells of the fungus Aureobasidium pullulans ATCC 201253 were entrapped within 4% agar cubes or 5% calcium alginate beads and were examined for their production of the polysaccharide pullulan in batch bioreactors. The batch bioreactors were utilized twice for 168 hours of polysaccharide production in medium containing corn syrup as a carbon source. The agar-entrapped cells produced nearly equivalent pullulan concentrations during both production cycles. The alginate-entrapped cells produced higher polysaccharide levels during the second cycle compared to the levels observed during the initial cycle. The agar-entrapped cells elaborated a polysaccharide with a higher pullulan content than did the alginate-entrapped cells during both production cycles.     

Production of high molecular weight pullulan by Aureobasidium pullulans HP-2001 with soybean pomace as a nitrogen source

The production of pullulan by Aureobasidium pullulans HP-2001 was enhanced by yeast extract as a nitrogen source as well as soybean pomace. The highest production of pullulan by A. pullulans HP-2001 with yeast extract was 5.5 g/l whereas that of pullulan with soybean pomace was 7.5 g/l. The gas chromatogram of pullulan produced by A. pullulans HP-2001 with soybean pomace as a nitrogen source showed that the major and minor components were glucose and mannose. The FTIR spectra of pullulans produced with yeast extract, a mixture of yeast extract and soybean pomace, and soybean pomace alone exhibited similar features. The increase in content of reducing sugars after pullulanase treatment of pullulans produced with different nitrogen sources indicated that all the pullulans had alpha-(1,6) glucosidic linkages of alpha-(1,4) linked maltotriose units. The average molecular weights of pullulans produced with various concentrations of yeast extract and soybean pomace ranged from 0.17 to 1.32x10(6) and from 1.32 to 5.66x10(6), respectively. All pullulans produced by A. pullulans HP-2001 in this study had the same basic structures, but their ratios of monomeric components were a little different, which might result in the production of pullulans with different molecular weights.     

Pullulan-hyperproducing color variant strain of Aureobasidium pullulans FB-1 newly isolated from phylloplane of Ficus sp

The studies were carried out for the isolation of efficient pullulan producing strains of Aureobasidium pullulans. Five strains were isolated from phylloplane of different plants. Amongst these, three were producing black pigment melanin, while the remaining two produced pink pigment. These two color variant isolates of A. pullulans were designated as FB-1 and FG-1, and obtained from phylloplane of Ficus benjamina and Ficus glometa, respectively. The parameters employed for the identification of the isolates included morphology, nutritional assimilation patterns and exopolysaccharide (EPS) production. Isolates were compared with standard cultures for EPS production. A. pullulans FB-1 was the best producer of pullulan giving up to 1.9, 1.4 and 1.7 times more pullulan than the control of A. pullulans NCIM 976, NCIM 1048 and NCIM 1049, respectively. The IR spectra of the isolates and standard strains revealed that the polysaccharide was pullulan, but not aubasidan. The study also supported the fact that A. pullulans is a ubiquitous organism and phylloplane being the important niche of the organism.     

Synthesis of Pullulan by Acetone-dried Cells and Cell-free Enzyme from Pullularia pullulans,and the Participation of Lipid Intermediate

It was demonstrated that the polysaccharide, pullulan, was synthesized from sucrose by acetone-dried cells of Pullularia pullulans or from UDPG by cell-free enzyme preparations prepared from the organism. The pullulan formed was estimated by precipitation with ethanol, and determining maltotriose produced after treating the precipitate with Aerobacter isoamylase (pullulanase). Acetone cells (5 g) shaken with 200 ml of 10% sucrose produced over 250 mg of pullulan per 100 ml after 90 hr at 30°C and pH 6.0. Cell-free enzyme produced pullulan from UDPG in the presence of ATP. ATP was essential for the biosynthesis, and ADPG could not replace for UDPG.

In addition, it was observed that a lipid containing glucose residue was formed during, the reaction. The nature of this glucolipid was examined, and possible participation of a lipid intermediate was assumed in the pullulan biosynthesis.     

出芽短梗霉发酵产生普罗蓝糖的研究

对一株野生型的出芽短梗霉(Aureobasidium pullulans)Ft1和从Ft1出发经原生质体再生筛选出的菌株R45进行了摇瓶发酵产普罗蓝糖的比较研究,结果表明R45无论从形态,菌体生长情况,还是从普罗蓝糖的产量,黑色素的产生等方面都与亲株Ft1有明显的区别,说明R45是一株具有一定生产价值的变异菌株.     

Optimization of high molecular weight pullulan production by Aureobasidium pullulans in batch fermentations

Of five strains of Aureobasidium pullulans studied, NRRL Y-2311-1 yielded the highest titer (26.2 g/L) of pullulan and formed the lowest amount of melanin-like pigment. Sucrose was superior to glucose as the carbon and energy source on the basis of yield and titer of pullulan produced. Pullulan titer was higher (26.2 vs 5.1 g/L), biomass concentration was lower (6.9 vs 12.7 g/L), and DO was lower (0 vs 60% of saturation) when the fermenter was agitated by a marine propeller compared to Rushton impellers. Pullulan produced by strain NRRL Y-2311-1 ranged in weight-average molar mass (M(w)) from 486 KDa and number-average molar mass (M(n)) from 220 Da on day 1 of growth to 390 KDa and 690 Da on day 6; M(w) declined by about 35% from day 1 to day 3, the day of maximum pullulan titer. For the other strains, the ranges of molar mass on the day of maximum pullulan titer were 338-614 KDa (M(w)) and 100-6820 Da (M(n)).     

Biochemical study of the relationship of extracellular glucan to adherence and cariogenicity in Streptococcus mutans and an extracellular polysaccharide mutant.

A mutant of Streptococcus mutans, GS-5, which differed in extracellular polysaccharide (EPS) produced from sucrose, was used to study the role of EPS in the production of dental caries. The mutant proved to be identical to the parent strain in sugar fermentation, growth rate, and serotype. Strain GS-5 synthesized an EPS, which in electron micrographs appeared to be of fibrillar structure, whereas the mutant produced no fibrillar material but only a globular EPS. Analysis of the EPS revealed that about 30% of the glucose units in the GS-5 polymer carried (1-3)-like bonds either as branch points or as part of the linear backbone and that the mutant material contained only about 3% of these linkages. When grown in sucrose broth, the proportion of the mutant culture adherent to the glass vessel was dramatically less than that of the parent strain. Caries scores produced in conventional rats by the mutant were significantly lower than those obtained with the parent strain. Since the only difference discovered between strain GS-5 and the mutant was the inability of the mutant to synthesize either a fibrillar EPS or an EPS with more than about 3% (1-3)-like linkages, it was concluded that the fibrillar EPS of strain GS-5 contained about 30% (1-3)-like linkages and was necessary for adherence of the bacteria to surfaces and for production of dental caries in test animals.     

Constitutive mutants for dextransucrase from Leuconostoc mesenteroides NRRL B-512F

Constitutive mutants for dextransucrase were isolated from cells of Leuconostoc mesenteroides NRRL B-512F by treatment with N-methyl-N′-nitro-N-nitrosoguanidine, growing on an agar plate containing glucose as a carbon source and overlaying a soft agar with sucrose and tetracycline. These mutants were able to produce the enzyme in a liquid media containing sugars other than sucrose, such as glucose, fructose and maltose, without simultaneous synthesis of dextran. The enzyme activity of one mutant strain, SH 3002, was 2- to 3-fold higher than that of the wild strain grown on sucrose. When the concentration of glucose in the medium was increased from 2 to 4%, a 1.7-fold increase of enzyme activity was obtained for the mutant, whereas only a slight increase of the activity was observed on sucrose for both the wild strain and the mutant.     

Lactobacilli isolated from sugary kefir grains capable of polysaccharide production and minicell formation

Homo- and heterofermentative species of Lactobacillus have been isolated from sugary kefir grains. Most of the homofermentative strains fermented tagatose and aldonitol and presented 48–54% of homology with Lactobacillus paracasei ssp. paracasei NCDO 151 (ex Lactobacillus casei ). The two variants of a heterofermentative species, although fermenting arabinose, were related to Lactobacillus hilgardii NCDO 264 (type strain) with 88% of homology. One of them produced polysaccharide from sucrose at pH 4–8 and 30°C; the best glucose conversion into polysaccharide was obtained from 3% of sucrose (81–8%), and the maximum production occurred about 35 hours after the end of the log phase of growth, in MRS sucrose broth. Polysaccharide formation did not occur above 40°C, a temperature at which no growth was observed. The two variants were forming minicells by abnormal divisions.     

Pullulan production by tropical isolates of <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis>

Tropical isolates of Aureobasidium pullulans previously isolated from distinct habitats in Thailand were characterized for their capacities to produce the valuable polysaccharide, pullulan. A. pullulans strain NRM2, the so-called “color variant” strain, was the best producer, yielding 25.1 g pullulan l⁻¹ after 7 days in sucrose medium with peptone as the nitrogen source. Pullulan from strain NRM2 was less pigmented than those from the other strains and was remarkably pure after a simple ethanol precipitation. The molecular weight of pullulan from all cultures dramatically decreased after 3 days growth, as analyzed by high performance size exclusion chromatography. Alpha-amylase with apparent activity against pullulan was expressed constitutively in sucrose-grown cultures and induced in starch-grown cultures. When the alpha-amylase inhibitor acarbose was added to the culture medium, pullulan of slightly higher molecular weight was obtained from late cultures, supporting the notion that alpha-amylase plays a role in the reduction of the molecular weight of pullulan during the production phase.Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may also be suitable.