Cells Of Aureobasidium Pullulans 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.

Enantioselective Bioreduction of Prochiral Pyrimidine Base Derivatives by Boni Protect Fungicide Containing Live Cells of Aureobasidium pullulans

The enzymatic enantioselective bioreduction of prochiral 1-substituted-5-methyl-3-(2-oxo-2-phenylethyl)pyrimidine-2,4(1H,3H)-diones to corresponding chiral alcohols by Boni Protect fungicide containing live cells of Aureobasidium pullulans was studied. The microbe-catalyzed reduction of bulky-bulky ketones provides enantiomerically pure products (96–99% ee). In the presence of A. pullulans (Aureobasidium pullulans), one of the enantiotopic hydrides of the dihydropyridine ring coenzyme is selectively transferred to the si sides of the prochiral carbonyl group to give secondary alcohols with R configuration. The reactions were performed under various conditions in order to optimize the procedure with respect to time, solvent, and temperature. The present methodology demonstrates an alternative green way for the synthesis of chiral alcohols in a simple, economical, and eco-friendly biotransformation.

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.

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.

One step separation of Aureobasidium pullulans from β‐poly(L‐malic acid) fermentation broth by membranes technology

AbstractBACKGROUNDβ‐Poly (L‐malic acid) (PMLA) can be used as a pro‐drug or for a drug‐delivery system, while the separation of PMLA producer cells of Aureobasidium pullulans from its fermentation broth is a challenge. This paper reports the separation of A. pullulans from PMLA fermentation broth by membranes technology.RESULTSThe best separation efficiency was obtained by the membrane with a molecular weight cut off of 300 kDa, an initial feed volume of 1300 mL, a volume reduction ratio (VRR) of 7.2, a cross‐flow velocity of 6 m s−1, a trans‐membrane pressure (TMP) of 0.2 MPa, and a temperature of 40°C. The average flux reached 55.97 L m−2 h−1 with a VRR of 7.2. The main fouling mechanism during the filtration of cell suspension solution was cake layer formation, while it was pore blocking for fermentation broth and cell free broth. Since the cake layer induced by cells would reject some macromolecular substances (i.e. reduce the pore blocking and enhance the fluxes), one step separation of A. pullulans with a 300 kDa membrane was better than that with centrifugation followed by membrane filtration.CONCLUSIONOne step separation of A. pullulans by membranes technology would be beneficial for PMLA bio‐refinery. © 2016 Society of Chemical Industry

Copper sulfate improves pullulan production by bioconversion using whole cells of Aureobasidium pullulans as the catalyst

The effects of mineral salts on pullulan production by bioconversion using whole cells of Aureobasidium pullulans CCTCC M 2012259 as the catalyst were investigated. Copper sulfate (CuSO4) improved pullulan production by 36.2% and 42.3% when added at the optimum concentration of 0.2mg/L to the bioconversion broth or seed medium, respectively, as compared with controls without CuSO4 addition. Pullulan production was further enhanced when CuSO4 was added to both seed medium and bioconversion broth simultaneously. In order to probe the mechanism of CuSO4 improvement, cell viability, membrane integrity, intracellular adenosine triphosphate (ATP) levels and the activities of key enzymes involved in pullulan biosynthesis were determined. As a result, CuSO4 increased the activities of key biosynthetic enzymes, maintained intracellular ATP at a higher level, and accelerated the rate of pullulan secretion, all of which contributed to improved pullulan production by bioconversion.

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.

Industrial production of fructooligosaccharides by immobilized cells of Aureobasidium pullulans in a packed bed reactor

Continuous production of fructooligosaccharides (FOS) by Aureobasidium pullulans immobilized on calcium alginate beads with a packed bed was investigated at a plant scale reactor. Optimum conditions were with 770g sucrose/l, being fed at 200l/h at 50°C which gave a productivity of 180g FOS/lh. Initial activity was maintained for more than 100days. The reactor was successfully scaled up to a production scale of 1.2m(3).

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.

Continuous gluconic acid production by the yeast-like Aureobasidium pullulans in a cascading operation of two bioreactors

The application of a new developed process for the continuous production of gluconic acid using a cascade of two bioreactors in a continuous process is shown reaching the highest concentration of gluconic acid described in the literature for continuous culture fermentation. Very high gluconic acid concentrations of 272-308 g/l have been achieved under continuous cultivation of free-growing cells of Aureobasidium pullulans in the first bioreactor at residence times (RT) between 19.5 and 24 h with formation rates for the generic product between 12.7 and 13.9 g/(l h). Gluconic acid, 350-370 g/l, was continuously reached in the second bioreactor at a total RT of 30.8-37 h with R (j) of 9.2-12 g/(l h). The highest specific gluconic acid production (m (p)) of 3.6 g/(g h) was found in the first bioreactor at the lowest RT of 19.5 h. The highest selectivity of 93.6% was determined in the first bioreactor as well. Complete glucose consumption was obtained at 37 h total residence time in the second bioreactor. Gluconic acid, 433 g/l, was continuously produced in the second bioreactor at a total RT of 37 h.

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.

Specific Cd2 + uptake of encapsulated Aureobasidium pullulans biosorbents

Calcium alginate capsules containing Aureobasidium pullulans cells were prepared by an in-situ one-step method. The Cd2+uptakes of “free biosorbent” and “capsule biosorbent” were described well by the Freundlich isotherms. The Cd2+ uptake of “capsule biosorbent” was lower than that of “free biosorbent” with Cd2 +at 100 mg l−1. The total cadmium uptake of “capsule biosorbent” increased with the increase in encapsulated cell density and plateaued at a value of 23 μg Cd2+/capsule with 150 g dry biosorbent l−1 core volume of a capsule. The specific cadmium uptake of encapsulated biosorbent was 85% to that of “free biosorbent” at 35 g biosorbent dry weight l−1 core volume of a capsule, decreased to 35% at 176 g biosorbent dry weight l−1.

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.

Ascorbic acid stimulation of production of a highly branched ,beta-1,3-glucan by Aureobasidium pullulans K-1--oxalic acid, a metabolite of ascorbic acid as the stimulating substance.

The production of a highly branched beta-1,3-glucan by Aureobasidium pullulans K-1 in Czapek's medium has been found to be stimulated by ascorbic acid. When the culture supernatant, after removal of polysaccharide from the culture filtrate by ethanol precipitation, was concentrated, then added to a new medium and this strain was cultured in the medium, the polysaccharide production was stimulated the same as when L-ascorbic acid was added to the medium. The stimulating substance was partially purified from the supernatant, and was found to be oxalic acid; 0.03% oxalic acid was the most effective concentration for the stimulation of polysaccharide production. The stimulating substance, oxalic acid, was proved to be derived from ascorbic acid added to a medium in an experiment using L-[1-14C]ascorbic acid. We suggest that oxalic acid generated from the metabolism of ascorbic acid in cells of Aureobasidium pullulans K-1 participated in the stimulation of the polysaccharide production by ascorbic acid.

Polysaccharide production by Aureobasidium pullulans cells immobilized by entrapment

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. The immobilized fungal cells were utilized twice in batch cultures for 7 days of polysaccharide production in medium containing corn syrup as a carbon source. The entrapped cells produced higher pullulan levels during the second cycle when compared to the first cycle polysaccharide levels. The agar-entrapped cells exhibited a higher level of productivity and pullulan content than did the alginate-entrapped cells during both production cycles.

Continuous production of isomalto-oligosaccharides from maltose syrup by immobilized cells of permeabilizedAureobasidium pullulans

Continuous production of isomalto-oligosaccharides from maltose syrup by the permeabilized cells ofAureobasidium pullulans immobilized into calcium alginate gel was studied using a column reactor. The immobilized cell column maintained its full activity over 45 days when the reactor was operated at a velocity of 0.1 h−1 at 50°C using 60%(w/v) maltose syrup as a substrate, and the maximum productivity achieved was around 60 g/1h.

Production of panose from maltose by intact cells of Aureobasidium pullulans

Panose, a major component of isomalto-oligosaccharides, was selectively produced from maltose using transglucosylation reaction catalyzed by intact cells of Aureobasidium pullulans. When 50 %(w/v) maltose was used as a substrate, the maximum concentration of panose accumulated in the final reaction mixture was about 50 %(w/w) after 120 hr reaction at 55 °C.

The existence of a dispensable fibrillar layer on the wall surface of mycelial but not yeast cells of Aureobasidium pullulans

The existence of a dispensable fibrillar layer on the wall surface of mycelial but not yeast cells of Aureobasidium pullulans