Effect of two-stage temperature on pullulan production by Aureobasidium pullulans

The effect of a two-stage cultivation temperature on the production of pullulan synthesized by Aureobasidium pullulans CGMCC1234 was investigated. Pullulan production was affected by temperature; although the optimum temperature for pullulan production was 26°C, the optimal temperature for cell growth was 32°C. Maximum pullulan production was achieved by growing A. pullulans in a first stage of 32°C for 2 days, and then in a second stage of 26°C for 2 days. Pullulan production using these two-stage temperatures significantly increased: about 27.80% (w/w) compared to constant-temperature fermentation (26°C for 4 days). The morphology of the A. pullulans (CGMCC 1234) was also affected by temperature; the lower temperature (26°C) supported unicellular biomass growth. Results of this study indicate that fermentation using two temperature stages is a promising method for pullulan production.     

Sodium chloride improves pullulan production by Aureobasidium pullulans but reduces the molecular weight of pullulan

Applied Microbiology and Biotechnology - The effect of sodium chloride (NaCl) on pullulan production by batch culture of Aureobasidium pullulans CCTCC M 2012259 was investigated. NaCl at...     

Optimization of conditions for the production of pullulan and high molecular weight pullulan by Aureobasidium pullulans

Aureobasidium pullulans had a maximum yield coefficient of pullulan (Y _p/s=0.24) with an initial pH of the culture broth of 6.5 in a shake-flask culture. In a batch culture, the maximum pullulan yield coefficient of 0.30 was obtained at the aeration rate of 0.5 vvm. A yeast-like form and mycelial form of cells were found at the culture broth with pH controlled at 4.5 with a maximum yield coefficient of pullulan of 0.27. However, a high portion (35%) of high molecular weight pullulan (M _w>2 000 000) was produced at pH 6.5 with a yeast-like morphology of the cells.     

Biosynthesis of pullulan using immobilized Aureobasidium pullulans cells

Immobilization of Aureobasidium pullulans by adsorption on solid supports and entrapment in open pore polyurethane foam were attempted. By adsorption, the highest cell loading of 0.012-0.018 g dry wt/cm(2) support was obtained in pH 2.0 medium. Under this acidic condition, the net surface charges (zeta potentials) of both the cells and supports were close to zero and no pullulan was synthesized. Cationic coatings of Cytodex and polyethylenimine were not efficient in enhancing the binding strength between the cells and the supports. Surface immobilized cells and polyurethane foam entrapped cells exhibited a similar fermentation characteristics resulting in ca. 18 g/L pullulan and ca. 5 g/L leaked cells. However, cells entrapped in the polyurethane foam were more shear resistant. The immobilized cells thus could be repeatedly used for pullulan biosynthesis.     

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)).     

Isolation of Aureobasidium pullulans and the effect of different conditions for pullulanase and pullulan production

Isolation and production of pullulahase by a new Aureobasidium pullulans isolate from the Fayoum Governorate (AUMC 2997) which was identified by the Assiut University Mycological Center was investigated. Another isolate from the Aswan Governorate (AUMC 1695) was kindly provided by the Assiut University Mycological Center. Acetone 2× gave better results for the precipitation of protein than 80% ammonium sulfate in the case of the media containing yeast extract. Very low protein production occurred in media without yeast extract. No enzyme production occurred in the first two days and the production of the enzyme started on the third day. Statistical analysis determined that the optimum conditions for the production of pullulanase were: incubation at 25°C for 5 days, pH 5.5, with sucrose as carbon source at 100 g/L and sodium nitrate as nitrogen source at 2 g/L. Addition of manganese chloride to the medium (1, 2 and 3 g/L) caused inhibition of pullulanase. Also, while the lowest pullulan + pigment concentrations were attained at the fifth day, pH 5.5, at 15°C, 100 g/L sucrose, 2 g/L nitrogen sources, the pullulan + pigment production increased with increasing the concentrations of manganese chloride.     

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.     

The unique role of siderophore in marine-derived Aureobasidium pullulans HN6.2

The l-ornithine-N ⁵-monooxygenase structural gene (SidA gene, accession number: FJ769160) was isolated from both the genomic DNA and cDNA of the marine yeast Aureobasidium pullulans HN6.2 by inverse PCR and RT-PCR. An open reading frame of 1,461 bp encoding a 486 amino acid protein (isoelectric point: 7.79) with calculated molecular weight of 55.4 kDa was characterized. The promoter of the gene (intronless) was located from −1 to −824 and had three HGATAR boxes which were putative binding motifs for the respective DNA-binding motifs and one CATA box. The SidA gene in A. pullulans HN6.2 was disrupted by integrating the hygromycin B phosphotransferase (HPT) gene into Open Reading Frame of the SidA gene using homologous recombination. Of all the disruptants obtained, one strain S6 (∆sidA) did not synthesize both intracellular and extracellular fusigen so that it could not inhibit growth of the pathogenic bacteria Vibrio anguillarum and Vibrio parahaemolyticus. The disruptant S6 did not grow in the iron-deplete medium and seawater medium because cell budding was stopped, but could grow in the iron-replete medium with 10 μM Fe³⁺ and Fe²⁺. H₂O₂ in the medium was more toxic to the disruptant S6 than to its wild type HN6.2. Thus, we infer that the fusigen produced by the marine-derived A. pullulans HN6.2 can play a unique role in chelating, uptake and concentration of iron to maintain certain proper physiological functions within the cells and secretion of siderophore may represent an efficient tool to eliminate competitors to compete for limiting nutritional resources in marine environments.     

Production of high molecular weight pullulan by Aureobasidium pullulans using glucosamine

Pullulan productivity was optimized in Aureobasidium pullulans ATCC 42023 with 54 g glucose l^–1. Pullulan with its higher molecular weight (>1000000) was produced using 2% (w/v) glucose and 3% (w/v) glucosamine together. The maximum concentration of pullulan was 8 g l^–1 at 140 h with shake-flask culture.     

Simultaneous production of pullulan and biosorption of metals by Aureobasidium pullulans strain CH-1 on peat hydrolysate

It was demonstrated that during the growth of Aureobasidium pullulans strain CH-1 on the acid hydrolysate of peat from the Vlasina Lake, the content of metals (Cu, Fe, Zn, Mn, Pb, Cd, Ni and Cr) decreased due to biosorption. The reduction in the metal content was found to be in the range (%): 38.2-62.2, 67.7-97.3, 0.02-62.05, 0.05-23.97, 0.16-4.24, 3.45-51.72, 1.18-35.82, 0.86-44.44, for Cu, Fe, Zn, Mn, Pb, Cd, Ni and Cr, respectively. During this process, the metals were accumulated in the biomass, while pullulan, an extracellular polysaccharide produced by Aureobasidium pullulans strain CH-1, was found not to bind the above-mentioned metals.     

Adhesion of Aureobasidium pullulans is controlled by uronic acid based polymers and pullulan

Aureobasidium pullulans is a potentially pathogenic microfungus that produces and secretes the polysaccharide pullulan and other biomacromolecules, depending on the microbe's physiological state. The role of these macromolecules in mediating adhesion and attachment were examined. Interfacial forces and adhesion affinities of A. pullulans were probed for early-exponential phase (EEP) and late-exponential phase (LEP) cells, using atomic force microscopy (AFM). Biochemical assays showed that A. pullulans produces both pullulan and a uronic acid based polymer. The pullulan is not produced until the LEP, and it can be removed by treatment with pullulanase. Both adhesion forces between the microbe and the AFM tip (silicon nitride) and attachment of the cells to quartz sand grains were controlled by the density of the uronic acid polymer. Uronic acid polymers doubled in density between the EEP and the LEP and were unaffected by the enzyme pullulanase. Retention to quartz in a packed column was quantified using the collision efficiency (alpha), the fraction of collisions between the microbes, and the sand grains, that result in attachment. Adhesion forces and retention on glass were well correlated, with these values being higher for EEP cells (F(adh) = 7.65 +/-4.67 nN; alpha = 1.15) than LEP (F(adh) = 2.94 +/- 0.75; alpha = 0.49) and LEP + pullulanase cells (F(adh) = 2.33 +/-2.01 nN; alpha = 0.43). Steric interactions alone do not describe the adhesion behavior of this fungus, but they do provide information regarding the length and density of the macromolecules studied.     

The effects of pressure on the growth of Aureobasidium pullulans and the synthesis of pullulan

Summary The feasibility of pressurized culture was explored for the growth of Aureobasidium pullulans and the synthesis of pullulan. For all volumetric flow rates of air, the production of biomass increased with pressure up to a critical value ranging from 0.50 to 0.75 MPa, at which point a drastic decrease in biomass production and a change in cellular morphology was observed. For pullulan synthesis, the same dramatic decrease was observed at approximately the same critical pressure. In the pressure range 0.1–0.65 MPa, the synthesis of pullulan was subject to what is believed to be the competing effects of oxygen availability and pressure inhibition.Offprint requests to: J. Thibault     

A self-tuning vision system for monitoring biotechnological processes: I. Application to production of pullulan by Aureobasidium pullulans

A prototype of a self-tuning vision system (STVS) has been developed to monitor cell population in fermentations. The STVS combines classical image processing techniques, neural networks and fuzzy logic technologies. By combining these technologies the STVS is able to analyze sampled images of the culture. The proposed system can be "tailored" with minimum effort by an expert who can "teach" the system to recognize cells by showing examples of different morphologies. After adaptation, the STVS is able to capture images, isolate the different cells, classify them according to the expert's criteria, and provide the profile of the cell's population. The system was applied to the classification and analysis of Aureobasidium pullulans. The importance of understanding the changes of population distribution during the fermentation and its effect in the production of pullulan are emphasized. The STVS can be used for monitoring and control of the cell population in small research fermentors or in large-scale production.     

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.     

Optimization of tannase production by Aureobasidium pullulans DBS66

Tannase production by Aureobasidium pullulans DBS66 was optimized. The organism produced maximum tannase in the presence of 1% tannic acid after 36 h. Maximum gallic acid accumulation was observed within 36 h and tannic acid in the fermented broth was completely degraded after 42 h of growth. Glucose had a stimulatory effect on tannase synthesis at 0.1% (w/v) concentration. The organism showed maximum tannase production with (NH4)2HPO4 as nitrogen source. Shaking speed of 120 rpm and 50-ml broth volume have been found to be suitable for maximum tannase production.     

Extracellular cellulase production by tropical isolates of Aureobasidium pullulans

Cellulase production by Aureobasidium pullulans from the temperate regions has remained speculative, with most studies reporting no activity at all. In the current study, tropical isolates from diverse sources were screened for cellulase production. Isolates were grown on a synthetic medium containing cell walls of Msasa tree (Brachystegia sp.) as the sole carbon source, and their cellulolytic activities were measured using carboxymethyl cellulose and alpha-cellulose as substrates. All isolates studied produced carboxymethyl cellulase (endoglucanase) and alpha-cellulase (exoglucanase) activity. Endoglucanase-specific activities of ten selected isolates ranged from 2.375 to 12.884 micromol glucose.(mg protein)-1.h-1, while activities on alpha-cellulose (exoglucanase activity) ranged from 0.293 to 22.442 micromol glucose.(mg protein)-1.day-1. Carboxymethyl cellulose induced the highest cellulase activity in the selected isolates, while the isolates showed variable responses to nitrogen sources. The current study indicates that some isolates of A. pullulans of tropical origin produce significant extracellular cellulolytic activity and that crude cell walls may be good inducers of cellulolytic activity in A. pullulans.     

Lipase production by diverse phylogenetic clades of Aureobasidium pullulans

Thirty-nine strains representing 12 diverse phylogenetic clades of Aureobasidium pullulans were surveyed for lipase production using a quantitative assay. Strains in clades 4 and 10 produced 0.2–0.3 U lipase/ml, while color variant strain NRRL Y-2311-1 in clade 8 produced 0.54 U lipase/ml. Strains in clade 9, which exhibit a dark olivaceous pigment, produced the highest levels of lipase, with strain NRRL 62034 yielding 0.57 U lipase/ml. By comparison, Candida cylindracea strain NRRL Y-17506 produced 0.05 U lipase/ml under identical conditions. A. pullulans strain NRRL 62034 reached maximal lipase levels in 5 days on lipase induction medium, while A. pullulans strain NRRL Y-2311-1 and strains in clades 4 and 10 were highest after 6 days. A. pullulans strain NRRL Y-2311-1 and strains in clade 9 produced two extracellular proteins in common, at >50 and <37 kDa.