Pullulans Cells Research Articles

Applicability of non-invasive and live-cell holotomographic imaging on fungi

The ability to acquire three-dimensional (3D) information of cellular structures without the need for fluorescent tags or staining makes holotomographic imaging a powerful tool in cellular biology. It provides valuable insights by measuring the refractive index (RI), an optical parameter describing the phase delay of light that passes through the living cell.Here, we demonstrate holotomographic imaging on industrial relevant ascomycete fungi and study their development and morphogenesis. This includes conidial germination, subcellular dynamics, and cytoplasmic flow during hyphal growth in Aspergillus niger. In addition, growth and budding of Aureobasidium pullulans cells are captured using holotomographic microscopy. Coupled to fluorescence imaging, lipid droplets, vacuoles, the mitochondrial network, and nuclei are targeted and analyzed in the 3D RI reconstructed images. While lipid droplets and vacuoles can be assigned to a specific RI pattern, mitochondria and nuclei were not pronounced. We show, that the lower sensitivity of RI measurements derives from the fungal cell wall that acts as an additional barrier for the illumination light of the microscope. After cell wall digest of hyphae and protoplast formation of A. niger expressing GFP-tagged histone H2A, location of nuclei could be determined by non-invasive RI measurements. Furthermore, we used coupled fluorescence microscopy to observe migration of nuclei in unperturbed hyphal segments and duplication during growth on a single-cell level. Detailed micromorphological studies in Saccharomyces cerevisiae and Trichoderma reesei are challenging due to cell size restrictions.Overall, holotomography opens up new avenues for exploring dynamic cellular processes in real time and enables the visualization of fungi from a new perspective.

Production of the Polysaccharide Pullulan by Aureobasidium pullulans Cell Immobilization

This review examines the immobilization of A. pullulans cells for production of the fungal polysaccharide pullulan. Pullulan is a water-soluble gum that exists structurally as a glucan consisting primarily of maltotriose units, which has a variety of food, non-food and biomedical applications. Cells can be immobilized by carrier-binding or entrapment techniques. The number of studies utilizing carrier-binding as a method to immobilize A. pullulans cells appears to outnumber the investigations using cell entrapment. A variety of solid supports, including polyurethane foam, sponge, diatomaceous earth, ion-exchanger, zeolite and plastic composite, have been employed to immobilize pullulan-producing A. pullulans cells. The most effective solid support that was used to adsorb the fungal cells was polyurethane foam which produced polysaccharide after 18 cycles of use. To entrap pullulan-producing fungal cells, agents such as polyurethane foam, polyvinyl alcohol, calcium alginate, agar, agarose, carrageenan and chitosan were investigated. Polysaccharide production by cells entrapped in polyurethane foam, polyvinyl alcohol or calcium alginate was highest and the immobilized cells could be reutilized for several cycles. It was shown that the pullulan content of the polysaccharide synthesized by cells entrapped in calcium alginate beads was low, which limits the method’s usefulness for pullulan production. Further, many of the entrapped fungal cells synthesized polysaccharide with a low pullulan content. It was concluded that carrier-binding techniques may be more effective than entrapment techniques for A. pullulans cell immobilization, since carrier-binding is less likely to affect the pullulan content of the polysaccharide being synthesized.

Effect of chemical structure of benzofuran derivatives and reaction conditions on enantioselective properties of Aureobasidium pullulans microorganism contained in Boni Protect antifungal agent.

Bioorganic asymmetric reduction of carbonyl compounds is one of the most important fundamental and practical reactions for producing chiral alcohols. The stereoselective bioreduction of prochiral ketones of benzofuran derivatives in the presence of yeast-like fungus Aureobasidium pullulans contained in the antifungal Boni Protect agent was studied. Biotransformations were carried out under moderate conditions in an aqueous and two-phase system and without multiplication of the bioreagent. Despite similar chemical structure, each of the used ketone has been reduced with varying efficiency and selectivity. One of the reasons for these results is the presence of a whole set of oxidoreductases in A. pullulans cells that are sensitive to the smallest changes in the structure of prochiral substrate. The unsymmetrical methyl ketones were biotransformed with the highest selectivity. Aureobasidium pullulans microorganism is less effective in the reduction of unsymmetrical halomethyl ketones. The presence of a heteroatom in the alkyl group significantly decreases the selectivity of the process. Finally, as a result of the preferred hydride ion transfer from the dihydropyridine ring of the cofactor to the carbonyl double bond on the re side, secondary alcohols of the S and R configuration were obtained with moderate to high enantioselectivity (55-99%).

Ferrous chloride and ferrous sulfate improve the fungicidal efficacy of cold atmospheric argon plasma on melanized Aureobasidium pullulans

Since cold atmospheric pressure plasma (CAP) has not only bactericidal activity but also fungicidal activity without toxic residues and thermal damage, it is considered as an alternative method for sterilization of fungi on the surfaces of perishable foodstuffs and human bodies. Aureobasidium pullulans is a ubiquitous yeast-like fungus and called black yeast because it produces melanin, a dark biological pigment. It is well known that various melanized fungi show hyper-resistance to extreme stress conditions including high levels of radioactivity. Curiously, however, there is very little information about the fungicidal effects of CAP on melanized fungi. Therefore, we herein investigated the effects of CAP on A. pullulans, using cold atmospheric argon plasma (Ar plasma). We found that ammonium sulfate repressed the synthesis of melanin in A. pullulans as well as Aureobasidium melanogenum. Although the non-melanized A. pullulans cells were efficiently killed by the exposure of Ar plasma, the melanized cells showed the significant resistance to Ar plasma as well as to hydrogen peroxide and thermal stress. In order to improve the fungicidal efficacy of Ar plasma, we examined the combination of Ar plasma and Fenton reaction. We realized that FeCl2 and FeSO4 significantly improved the sterilization efficacy of Ar plasma on the melanized A. pullulans.

Highly concentrated populations of Aureobasidium pullulans cells in biocatalytic pullulan production processes

Results are presented from studies and a comparative analysis of highly concentrated populations of free and immobilized Aureobasidium pullulans Y-4137 cells in the biocatalytic processes of pullulan production in glucose-containing media. The possibility of effectively using the developed biocatalyst in the form of immobilized cells is demonstrated. The process characteristics are determined for pullulan production from hydrolysates of various sources of renewable feedstocks (Jerusalem artichoke tubers, aspen wood, Chlorella vulgaris microalgal biomass, and potato pulp) under the action of the catalyst. It is established that A. pullulans cells immobilized in a polyvinyl alcohol (PVA) cryogel consume glucose 1.5 times faster and accumulate a 1.7 times higher concentration of the target polysaccharide in the medium than free cells. The immobilized cells can function for at least 15 operating cycles with a slight (no more than 10%) reduction in their metabolic activity. Analysis of the obtained data confirms that cell immobilization in a PVA gel for the production of pullulan allows us to shorten the duration of operating cycles in similar processes by a factor of 1.4 while reaching a comparable yield of the target product.

The Biocatalytic Process of Pullulan Production by Auerobasidium pullulans Cells Being in a Form of High Density Populations

The results of investigation and comparative analysis of biocatalytic process of pullulan production by a high cell density populations of Auerobasidium pullulans cells taken in suspended and immobilized into poly(vinyl alcohol) cryogel forms are performed in the work. The possible effective using of the immobilized biocatalyst was demonstrated and the characteristics of the process of pullulan production from different hydrolisates of renewable raw materials such as tubers of Jerusalem artichoke, aspen, Chlorella vulgaris biomass, potato pulp were determined. It was revealed that A. pullulans cells entrapped into poly(vinyl alcohol) cryogel can 1.5-times faster convert glucose to pullulan and accumulate the polysaccharide in cultural broth in 1.7-times higher concentration as compared to suspended cells. Immobilized cells appeared to retain a high functional metabolic action during at least 15 working cycles with minimum (not more than 10 %) loss of activity. The analysis of experimental data confirmed that use of cells immobilized into poly(vinyl alcohol) cryogel enables decrease in 1.5–3 times the duration of the working cycle in the same process while achieving comparable level of product yield.

Screening and selection of potential carriers to immobilize Aureobasidium pullulans cells for fructo-oligosaccharides production

Fructo-oligosaccharides (FOS) are non-digestible sweeteners with improved functional and technological properties for food and pharmaceutical industry applications. The immobilization of FOS-producing microorganism’s whole cells can be used to improve the production of FOS and so, an accurate choice of the carrier is of crucial importance. Here we present a screening and selection of carriers for immobilization of Aureobasidium pullulans cells with potential for yielding high FOS production. Synthetic, agro-industrial by-products and mineral materials were tested and selected regarding their ability to immobilize cells, as well as their potential to produce FOS, through shaken-flask fermentations. Increased amounts of immobilized cells were found for carriers with higher porosity observed at the microscope, higher water absorption index (WAI) and lower critical humidity point (CHP). Reticulated polyurethane foam was one of the most efficient synthetic carriers immobilizing cells (over 75% (w/w) of the total cells were immobilized). Also, high FOS production was obtained: FOS concentration, purity and yield increased in 15, 8 and 12% (w/w), respectively, as compared to free cells conditions. Although walnut shell had a much lower immobilization efficiency, the high amount of cells grown while using this carrier led to a highest increment of FOS concentration, purity and yield, namely, 27, 10 and 25% (w/w). Cells immobilization with the selected carriers holds great promise for FOS production on a larger scale.

Why sucrose is the most suitable substrate for pullulan fermentation by Aureobasidium pullulans CGMCC1234?

This paper studies the metabolic pathway of sucrose in pullulan fermentation by Aureobasidium pullulans. Because of its high pullulan production, sucrose proved to be the best carbon source for pullulan synthesis by A. pullulans CGMCC1234 (36.3g/L). Compared to other carbon sources, A. pullulans cells reached the stationary phase more quickly in the presence of sucrose. The specific sugar types and concentrations occurring during pullulan fermentation were detected using High Performance Liquid Chromatography (HPLC). HPLC results revealed that sucrose did not simply break down into glucose and fructose in the medium employed. Kestose (22.69g/L) also accumulated during early stages of fermentation (24h), which reduced the osmotic pressure of the extracellular fluid and diminished the inhibition of cell growth and pullulan production. β-Fructofuranosidase activity strongly depended on the carbon source. Sucrose was the best inducer of β-fructofuranosidase production. However, β-fructofuranosidase production did not directly and/or proportionally correlate with the growth of A. pullulans cells.

Antagonistic interactions between Aureobasidium pullulans and Fusarium culmorum, a fungal pathogen of winter wheat

The effect of the biological control agent Aureobasidium pullulans (de Bary) G. Arnaud on the development of Fusarium head blight (FHB) on winter wheat and kernel contamination with fungi of the genera Fusarium, Acremonium, Cladosporium and Penicillium was analyzed in a greenhouse experiment. Scanning electron microscopy was used to evaluate the distribution of A. pullulans cells and aggregates on wheat kernels, infection structures of Fusarium culmorum (W.G. Smith) Sacc and the antagonist-pathogen interactions. Biological control with A. pullulans reduced FHB severity by 21.67 % and improved grain filling by 5.02 %, compared with the control treatment. The survival of A. pullulans was good (to 31 cells per kernel), in particular on the surface and in the crease of kernels, including in pathogen-inoculated wheat plants. A. pullulans cells firmly adhered to F. culmorum hyphae, and damaged them. In most cases, autochthonous communities of filamentous fungi of the genera Acremonium and Penicillium developed at a slower rate after kernel inoculation with the pathogen.

Production of fructosyltransferase in mechanically stirred and air-lift bioreactors

AbstractAureobasidium pullulans cells were cultivated in a 3 dm3 stirred bioreactor to optimize the production of fructosyltransferase (FTase). Batch experiments were focused on the influence of the initial sucrose concentration (150–350 g dm−3), sodium nitrate concentration (10–25 g dm−3), and stirring rate (180–540 min−1) on the process. It was found that the FTase specific activity per cell mass was positively influenced by the conditions that impeded the cell growth such as higher sucrose concentrations or lower oxygen transfer rates. Higher content of inorganic nitrogen substrate slightly increased the overall FTase production. The presence of fructose moiety-containing saccharides in the cultivation medium was observed to be indispensable for FTase production. An addition of a concentrated sucrose solution in the 40th cultivation hour therefore essentially resumed the FTase production. Finally, scale-up experiments were performed in 12 dm3 and 100 dm3 mechanically stirred bioreactors and in 20 dm3 and 60 dm3 air-lift bioreactors.

English

A novel fermentor system containing Aureobasidium pullulans in polyurethane foam was designed to achieve continuous production of pullulan. A. pullulans cells were immobilized in 10 to 20 g polyurethane foam with pore size of 1000 Ǻ. The system has specialized aeration provision with 80 perforations of 4 mm. Pullulan production achieved was approximately 37g/l in 18 cycles at 42°C, pH 5.5 and at aeration rate of 0.5 vvm. The concentration of sucrose and ammonium sulphate was 3 and 0.5%, respectively. This novel design could serve as an excellent fermentor system in industries for large scale continuous production of pullulan.   Key words: Aureobasidium pullulans, polyurethane foam, pullulan, immobilized cells.

Efficient production of polymalic acid from raw sweet potato hydrolysate with immobilized cells of Aureobasidium pullulans CCTCC M2012223 in aerobic fibrous bed bioreactor

AbstractBACKGROUNDPolymalic acid (PMA) is a water‐soluble polyester with many attractive properties for biomedical application. Its monomer L‐malic acid is widely used in the food industry and is also a potential C4 platform chemical. Hydrolysate of raw sweet potato as a low‐cost renewable feedstock was applied for the production of PMA by immobilized Aureobasidium pullulans cells in an aerobic fibrous bed bioreactor system (AFBB).RESULTSUnder the initial hydrolysate sugar concentration 120 g L‐1, yeast extract 5 g L‐1, and citrate 7.5 g L‐1, PMA production in a shake flask was improved. Comparison of the kinetics of fed‐batch immobilized‐cell fermentation using the AFBB with those of batch or fed‐batch free‐cell fermentation in a 5 L fermentor showed that immobilized‐cell fermentation gave much higher PMA production and productivity. The maximum PMA production and productivity in AFBB were 57.5 g L‐1 and 0.34 g L‐1 h, which was 30.6% and 21.4% higher than that in free‐cell mode.CONCLUSIONRaw sweet potato is an efficient renewable feedstock for PMA fermentation. This present work demonstrated the great potential of AFBB and renewable feedstocks for the economical production of PMA and malic acid at industrial scale. © 2013 Society of Chemical Industry

Pullulan production by Aureobasidium pullulans cells immobilized in chitosan beads

Fungal cells of Aureobasidium pullulans ATCC 201253 were immobilized by entrapment in chitosan beads, and the immobilized cells were investigated for their ability to produce the polysaccharide pullulan using batch fermentation. The 1% chitosan-entrapped fungal cells were capable of producing pullulan for two cycles of 168 h using corn syrup as a carbon source. Pullulan production by the immobilized cells increased by 1.6-fold during the second production cycle (5.0 g/l) relative to the first production cycle (3.1 g/l) with the difference in production being statistically significant after 168 h. The productivity of the immobilized cells increased during the second production cycle while its pullulan content decreased. The level of cell leakage from the support remained unchanged for both production cycles.

Pullulan production by Aureobasidium pullulans cells immobilized on ECTEOLA-cellulose

Cells of the fungus Aureobasidium pullulans ATCC 42023 were immobilized by adsorption on the ion-exchange resin ECTEOLA (epichlorohydrin triethanolamine)-cellulose and the immobilized cells were examined for their ability to produce the polysaccharide pullulan using batch fermentation. It was found that the cells immobilized on the ECTEOLA-cellulose at pH 2.0 produced higher pullulan levels than those cells immobilized at pH 3.0, 4.0, 5.0, 6.0 and 7.0 after 72 h at 30°C. The pH 2.0-immobilized cells were capable of producing pullulan for 2 cycles of 168 h. Pullulan production by the immobilized cells decreased slightly during the second production cycle but the difference in production was not statistically significant after 168 h.

Optimization of pullulan production using Ca‐alginate‐immobilized Aureobasidium pullulans by response surface methodology

AbstractBACKGROUND: The production of pullulan from synthetic medium by Aureobasidium pullulans P56 immobilized in Ca‐alginate beads was investigated using batch and repeated batch fermentation systems.RESULTS: The highest pullulan concentration (19.52 ± 0.37 g dm−3) was obtained with 2.0‐2.4 mm beads prepared from 2% sodium alginate solution. Pullulan production was mainly accomplished by immobilized fungal cells since leaked cells in the fermentation medium comprised 17.4% of the total fungal population at the end of fermentation. The pullulan proportion was 84.5% of the total polysaccharide in the fermentation medium. Response surface methodology was used to investigate the effects of three fermentation parameters (initial pH, agitation speed and incubation time) on the concentration of pullulan. Results of the statistical analysis showed that the fit of the model was good in all cases. The maximum pullulan concentration of 21.07 ± 0.48 g dm−3 was obtained at the optimum concentrations of process variables (pH 7.31, agitation speed 191.5 rpm, incubation time 101.2 h). The gel beads produced pullulan under the optimized conditions for six consecutive batch fermentations without marked activity loss and deformation.CONCLUSION: The results of this study suggest that the immobilization of A. pullulans cells in Ca‐alginate gel beads is suitable for batch and repeated batch production of pullulan. Copyright © 2007 Society of Chemical Industry

Novel method for cell immobilization and its application for production of oligosaccharides from sucrose.

The purpose of the present investigation was to develop a novel method for cell immobilization. Aureobasidium pullulans cells were mixed with an alginate solution, and the mixture was extruded to form small gel beads as hydrated-immobilized cells. The beads were then placed at -15 degrees C for 6-24 h to induce freeze-dehydration. The freeze-dehydration resulted in shrinkage of beads as a result of water removal reducing bead volume by 82% and bead weight by 85%. The dehydrated beads were successfully used for the production of fructo-oligosaccharides in a model reactor system. Dehydrated beads may provide some commercial advantages over conventional immobilized cells. This study shows that bioreactor performance can be improved up to two times by the use of the dehydrated beads.

Production of fructo-oligosaccharides from molasses by Aureobasidium pullulans cells

Three different strains of Aureobasidium pullulans were grown in batch cultures to compare their abilities for the production of fructo-oligosaccharides. Specific intracellular enzyme activity was the highest with strain KCCM 12017 and enzyme production was closely coupled to growth. Using A. pullulans cells, 166 g/l fructo-oligosaccharides was produced from 360 g/l molasses sugar as sucrose equivalent at 55 °C and pH 5.5 after 24 h incubation.

Population biology ofAureobasidium pullulanson apple leaf surfaces

Colonization of apple leaves by the yeast-like fungus Aureobasidium pullulans was studied in the field on eight dates over 2 years. Population densities from adaxial leaf surfaces were approximately log10 0.5-2.6 U higher when enumerated directly along line transects as microscopic counts of A. pullulans cells specifically identified by fluorescence in situ hybridization (FISH) than indirectly as CFU obtained by plating leaf washings from comparable surfaces onto nutrient media. Site-specific mapping of the leaf landscape colonized by A. pullulans was facilitated by use of geographic information system (GIS) software. Colonization was plotted and analyzed both qualitatively (as occupancy) and quantitatively (as density). Overall, when expressed as mean occupancy per date, 22-42% of the microscope fields (each 0.196 mm2) contained > or = 1 A. pullulans cell. Occupancy on a microscope field basis was greater over the midvein (47-89%) or smaller veins (49-65%) than over interveinal regions (11-21%). Intensity of colonization, whether expressed as percentage of total A. pullulans cells associated with a particular leaf feature or as cell density per unit area, was also significantly greater (P < 0.05) over the veinal areas compared with the interveinal areas. The primary fungal morphotypes involved in colonization were blastospores, swollen cells, and chlamydospores; only infrequently were hyphae or pseudohyphae seen. Numbers of microcolonies (> or = 10 clustered cells) and percentage of total A. pullulans cells that occurred as microcolonies increased over the growing season and were significantly greater (P < 0.05) over veinal regions compared with interveinal regions. Locally high concentrations of A. pullulans were associated with naturally occurring micro-wounds in interveinal areas. We conclude that A. pullulans colonizes the phylloplane predominantly as single cells and groups thereof in a highly heterogeneous fashion and that sites exist that are relatively conducive (veins; wounds) or nonconducive (unwounded interveinal areas) for epiphytic fungal growth.

Monitoring and control of pullulan production using vision sensor

The production of the polysaccharide pullulan by the yeast-like fungi, Aureobasidium pullulans, is accompained by cellular morphogenetic changes. High productivity and yield of the process have been found to correlate with high concentration of yeast-like cells in the culture. The morphogenetic changes of A. pullulans cells depend on the culture conditions, e.g., dissolved oxygen, shear rate and medium composition. In order to improve the productivity of the process, a novel control law was formulated. A feeding strategy dependent on the culture cellular composition was designed and aimed to keep the yeast-like cell concentration high. The culture morphogenetic composition during the process was monitored by a recently developed vision sensor. Feeding was actuated when the yeast-like cell concentration decreased below a threshold. The proposed control strategy improved pullulan production by increasing both productivity and yield of the cells by 67% and 80%, correspondingly. The results point to the advantage and the potential of using the monitoring and control system and algorithm to increase productivity and yield in cellular bioprocesses.

Biocontrol activity and induced resistance as a possible mode of action for Aureobasidium pullulans against grey mould of strawberry fruit

Efficacy and mode of action were investigated fox Aureobasidium pullulans, a potential biocontrol agent for grey mould on strawberry fruit. Wound inoculation of detached green strawberry cv. Elsanta fruit with Aureobasidium pullulans prevented grey mould rot on fruit inoculated 2 days later with Botrytis cinerea. Treatment of white, pink and red-ripe fruit did not, however, control grey mould. Treatment of wound sites on green fruit with both live and heat-killed A. pullulans cells reduced B. cinerea infection compared to controls. Dip-inoculation of unwounded green strawberry fruit with A pullulans when still attached to the plant delayed the development of grey mould after harvest at the fully ripe stage. The (i) efficacy of A. pullulans on green but not on ripening fruit, (ii) partial inhibitory effect of both live and heat-killed A. pullulans cells and (iii) absence of evidence for antibiotic production from in vitro competition tests suggest that control of grey mould on green fruit is at least partly due to a mechanism other than antagonism and / or competition. Bioassays showed that skin tissue from green fruit treated with A. pullulans had greater antifungal activity than control tissue. Thus, enhanced natural disease resistance in green strawberry fruit contributed to grey mould rot suppression by A. pullulans.