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.     

Pullulan production by <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> 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.     

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     

Effect of carbon source and dissolved oxygen level on cell growth and pullulanase production byBacillus stearothermophilus G-82

Cell growth and extracellular pullulanase production ofBacillus stearothermophilus G-82 were investigated in batch culture using a defined medium with glucose, maltose, pullulan or amylopectin as carbon source. Maximum enzyme activity was with pullulan or amylopectin. Cell growth in batch culture was better under oxygen unlimited conditions, while higher total and specific enzyme activities, using pullulan or amylopectin, were obtained in oxygen-limited conditions. Enzyme accumulation took place in the late growth phase. The highest enzyme production of 300 U/I was reached when pullulan was used as carbon source in conditions of oxygen limitation.     

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.     

Pullulan production by a non-pigmented strain of Aureobasidium pullulans using batch and fed-batch culture

The production of pigment-free pullulan by Aureobasidium pullulans in batch and fed-batch culture was investigated. Batch culture proved to be a better fermentation system for the production of pullulan than the fed-batch culture system. A maximum polysaccharide concentration (31.3 g l⁻¹), polysaccharide productivity (4.5 g l⁻¹ per day), and sugar utilization (100%) were obtained in batch culture. In fed-batch culture, feed medium composition influenced the kinetics of fermentation. For fed-batch culture, the highest values of pullulan concentration (24.5 g l⁻¹) and pullulan productivity (3.5 g l⁻¹ per day) were obtained in culture grown with feeding substrate containing 50 g l⁻¹ sucrose and all nutrients. The molecular size of pullulan showed a decline as fermentation progressed for both fermentation systems. At the end of fermentation, the polysaccharide isolated from the fed-batch culture had a slightly higher molecular weight than that of batch culture. Structural characterization of pullulan samples (methylation and enzymic hydrolysis with pullulanase) revealed the presence of mainly α-(1→4) (∼66%) and α-(1→6) (∼31%) glucosidic linkages; however, a small amount (<3%) of triply linked (1,3,4-, 1,3,6-, 1,2,4- and 1,4,6-Glc p) residues were detected. The molecular homogeneity of the alcohol-precipitated polysaccharides from the fermentation broths as well as the structural features of pullulan were confirmed by ¹³C-NMR and pullulanase treatments followed by gel filtration chromatography of the debranched digests.     

Optimization and characterization of pullulan production by a newly isolated high-yielding strain Aureobasidium melanogenum

Abstract

Pullulan is an extracellular water-soluble polysaccharide with wide applications. In this study, we screened strains that could selectively produce high molecular weight pullulan for application in industrial pullulan production. A new fungus strain A4 was isolated from soil and identified as Aureobasidium melanogenum based on colony characteristics, morphology, and internally transcribed spacer analysis. Thin-layer chromatography, Fourier-transform infrared spectroscopy, and nuclear magnetic resonance analysis suggested that the dominant exopolysaccharide produced by this strain, which presented a molecular weight of 1.384?×?10⁶ Dalton in in-gel permeation chromatography, was pullulan. The culture conditions for A. melanogenum A4 were optimized at 30?°C and 180?rpm: carbon source, 50?g/L maltose; initial pH 7; and 8?g/L Tween 80. Subsequently, batch fermentation was performed under the optimized conditions in a 5-L stirred-tank fermentor with a working volume of 3?L. The fermentation broth contained 303?g/L maltose, which produced 122.34?g/L pullulan with an average productivity of 1.0195?g/L/h and 82.32?g/L dry biomass within 120?h. The conversion efficiency of maltose to pullulan (Y%) and specific production rate (g/h/g dry cells) (Qs) reached 40.3% and 0.0251?g/L/g dry cells, respectively. The results showed strain A4 could be a good candidate for industrial production.     

Exopolysaccharide production on low-grade maple sap by Enterobacter agglomerans grown in small scale bioreactors

Enterobacter agglomerans isolated from a soil sample collected in the vicinity of a sugar house was grown on low-grade maple sap: When cultivated in shake flask, Ent. agglomerans produced exopolysaccharide and α-ketoglutarate. No polysaccharide and no α-ketoglutarate were produced during growth at dissolved oxygen concentrations lower than 1% saturation in a 3.5 1 bioreactor, but succinate, lactate and formate were accumulated. In order to increase polysaccharide production, various agitation systems were adapted to 7 1 bioreactors with working volumes of 41. Highest polysaccharide yield (Y_p/s= 0.70) and biomass yield (Y_x/s= 0.17) were obtained with bioreactors agitated with four-and six-bladed Rushton turbines. Accumulation of mixed organic acids during polysaccharide production indicated that both aerobic and anaerobic conditions prevailed inside the bioreactors. Maple sap concentration higher than 60 g 1⁻¹ (as sucrose equivalent) decreased both growth and exopolysaccharide 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.     

Polysaccharide production by a reduced pigmentation mutant of the fungus Aureobasidium pullulans

Abstract A reduced pigmentation mutant was isolated from Aureobasidium pullulans ATCC 42023 by chemical mutagenesis and was subsequently characterized. The pigment melanin was present not only in A. pullulans cells but also contaminated the elaborated polysaccharide and thus, was measured in both fractions. Cellular and polysaccharide melanin levels of the mutant strain were at least 11-fold and 18-fold reduced, respectivelu, compared toits parent strain after 7 days of growth at 30°C whether sucrose or glucose served as the carbon source in the culture medium. Polysaccharide and cell dry weight levels of the mutant were very similar to those observed for the parent after growth on sucrose or glucose as the source of carbon over a period of 7 days at 30°C. The pullulan content of the polysaccharide produced by the parent or mutant strain was lower for sucrose-grown cells than for glucose-grown cells. It was also noted that the pullulan content of the polysaccharide elaborated by the mutant strain was slightly higher than that of the polysaccharide produced by the parent strain after growth on either sucrose or glucose.     

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.     

Feasibility study on the use of calcium alginate-entrapped hybridoma cells for IgM production

In order to test the feasibility of using calcium alginate-entrapped hybridoma cells for IgM production, HO-22-1 hybridoma cells entrapped into calcium alginate beads with varying alginate concentrations were cultivated in spinner flasks. It was observed that the IgM produced by the entrapped cells could diffuse out of the calcium alginate beads regardless of alginate concentrations tested (0.8–2.5%). Since the increase in alginate concentrations showed an adverse effect on cell growth and maximum cell concentration, the use of lower alginate concentration was desirable for higher volumetric monoclonal antibody (MAb) productivity. When the entrapped cells in 0.8% alginate beads were cultivated in repeated-fed batch mode, the reduction of serum concentration in the medium from 10% to 1% did not decrease the volumetric IgM production. Taken together, the data obtained here showed the feasibility of using calcium alginate-entrapped hybridoma cells for IgM production.Alginate was generously provided by the Kelco company. This work was supported by the Ministry of Science and Technology, Korea.     

β-Fructofuranosidase production by repeated batch fermentation with immobilized <Emphasis Type="Italic">Aspergillus japonicus</Emphasis>

The fungus Aspergillus japonicus ATCC 20236 was immobilized in vegetal fiber and used in repeated batch fermentations of sucrose (200 g/l) for the production of β-fructofuranosidases (FFase). The assays were performed during eight consecutive cycles that were completed in a total period of 216 h. After each 24-h cycle of fermentation (except for the first cycle, which lasted 48 h), the fermented broth was replaced by fresh medium, and the FFase activity was determined in the replaced medium. The average value of FFase activity was a constant 40.6 U/ml at the end of the initial seven cycles, but had decreased by 22% at the end of the eighth cycle. Concurrent with these high and constant FFase values, the hydrolyzing activity of this enzyme increased during the cycles, while the transfructosylating activity decreased. As a consequence, the maximum production of fructooligosaccharides of 134.60 g/l observed in the initial 30 h of fermentation (first cycle) had gradually decreased by the end of the subsequent cycles, reaching approximately 23% of this value during cycles 4–8. Based on these results, we conclude that the present immobilization system has a great potential for application in a semi-continuous process for the production of FFase, but further studies are necessary to maintain the FFase transfructosylation activity at high levels during the overall process.     

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.     

Downstream processing of pullulan from fermentation broth

pullulan, a water soluble extracellular polysaccharide, was produced by downstream fermentation employing the strain Aureobasidium pullulans. To obtain pure biopolymer from the fermentation broth, it is necessary to harvest cells, heat the broth, remove the melanin pigments co-produced during fermentation, concentration, precipitate and dry. Centrifugation of the fermentation broth at 10,000 rpm for 15 min gave cell pellets that were discarded and a green–black supernatant containing melanin pigment was subjected to the heat treatment at 80 °C for 20 min in order to remove the protein in the fermentation broth. The supernatant was demelanized by oxidation with hydrogen peroxide, concentrated under vacuum, precipitated with ethanol and dried at 60 °C for 30 min. This procedure produced high purity pullulan that was comparable in color and texture to the commercial samples.     

Biosensor online control of citric acid production from glucose by Yarrowia lipolytica using semicontinuous fermentation

Our study aimed at the development of an effective method for citric acid production from glucose by use of the yeast Yarrowia lipolytica. The new method included an automated bioprocess control using a glucose biosensor. Several fermentation methodologies including batch, fed‐batch, repeated batch and repeated fed‐batch cultivation were tested. The best results were achieved during repeated fed‐batch cultivation: Within 3 days of cycle duration, approximately 100 g/L citric acid were produced. The yields reached values between 0.51 and 0.65 g/g and the selectivity of the bioprocess for citric acid was as high as 94%. Due to the elongation of the production phase of the bioprocess with growth‐decoupled citric acid production, and by operating the fermentation in cycles, an increase in citric acid production of 32% was achieved compared with simple batch fermentation.     

Growth of Trichoderma reesei RUT C-30 in stirred tank and reciprocating plate bioreactors

A series of fed-batch experiments at different agitation speeds were performed using the industrially important strain Trichoderma reesei RUT C-30 in two different bioreactors to understand the close relationship that exists between the shear field within a bioreactor, the morphology of the microorganism, the rheology of cultivation broth, and the process performance. The two bioreactors, stirred tank bioreactor (STB) and reciprocating plate bioreactor (RPB), are characterized by a significantly different shear field to which microorganisms are exposed. Highest biomass concentration (ca. 15 g l⁻¹) was obtained at higher agitation rates in both bioreactors due to better oxygen supply. However, better filter paper activities per mg of protein were obtained at lower agitation in both bioreactors. In both bioreactors, young and healthier fungi in the batch phase were not affected by shear even at higher agitation rates. However, during the fed-batch phase, higher degree of fragmentation of clump morphology at high agitation intensity was confirmed by image analysis. Also, the rheological analysis showed an increase in apparent viscosity during the batch phase and early fed-batch phase due to the increase in the biomass concentration. During the late stages of cultivation, the apparent viscosity decreased due to cell lysis and spore formation.     

Evaluation of mycelial inocula of edible<Emphasis Type="Italic"> Lactarius</Emphasis> species for the production of<Emphasis Type="Italic"> Pinus pinaster</Emphasis> and<Emphasis Type="Italic"> P. sylvestris</Emphasis> mycorrhizal seedlings under greenhouse conditions

Different methods to inoculate seedlings of Pinus pinaster and P. sylvestris with edible Lactarius species under standard greenhouse conditions were evaluated. Fungal inoculations were performed both under pure culture synthesis in vitro, followed by transplantation of acclimatized seedlings, and directly in the greenhouse using different techniques for inocula production (mycelial slurries, vegetative inoculum grown in peat-vermiculite and alginate-entrapped mycelium). In vitro inoculations with L. deliciosus produced thoroughly colonized seedlings. However, a sharp decrease in mycorrhizal colonization was detected on transplanted seedlings after 4 month's growth in the greenhouse. On the other hand, all the inocula applied directly in the greenhouse, except the alginate-entrapped mycelium, produced a variable number of mycorrhizal seedlings and colonization rates after the first growing season, depending on the plant-fungal combination and the inoculation method. Inoculations with vegetative inocula of the strain 178 of L. deliciosus were the most effective in producing mycorrhizal seedlings. All the seedlings inoculated with this strain were colonized although the colonization rates were relatively low. The commercial feasibility of the different inoculation methods for the production of seedlings colonized with edible Lactarius species is discussed.     

Production of cellulase in a rotating disc fermenter using immobilized Trichoderma reesei cells

The production of cellulase was investigated in repeated batch experiments using immobilized cells of two Trichoderma reesei mutants in a rotating disc fermenter under very low shear stress. The enzyme production with one of the mutants was maintained for three successive batch cycles (ca. 30 days), while with the other mutant the cellulase formation lasted only one batch cycle (14 days) because of a genetic instability. The enzymatic hydrolysis of microcrystalline cellulose by the cellulase complex formed in the rotating disc fermenter is distinctly higher than that of cellulase produced in a stirred tank reactor, in which the higher shear stress partially damages the enzyme molecules, mainly those of cellobiohydrolase. The higher specific activity of the cellulase produced in the disc fermenter correlates with its higher capacity of adsorption onto microcrystalline cellulose.     

Influence of different sugars on pullulan production and activities of α-phosphoglucose mutase, UDPG-pyrophosphorylase and glucosyltransferase involved in pullulan synthesis in Aureobasidium pullulans Y68

Effects of different sugars on pullulan production, UDP-glucose level, and activities of α-phosphoglucose mutase, UDPG-pyrophosphorylase and glucosyltransferase in Aureobasidium pullulans Y68 were examined. It was found that more pullulan was produced when the yeast strain was grown in the medium containing glucose than when it was cultivated in the medium supplementing other sugars. Our results demonstrate that when more pullulan was synthesized, less UDP-glucose was left in the cells of A. pullulans Y68. However, it was observed that more pullulan was synthesized, the cells had higher activities of α-phosphoglucose mutase, UDPG-pyrophosphorylase and glycosyltransferase. Therefore, high pullulan yield is related to high activities of α-phosphoglucose mutase, UDPG-pyrophosphorylase and glucosyltransferase in A. pullulans Y68 grown on different sugars. A pathway of pullulan biosynthesis in A. pullulan Y68 was proposed based on the results of this study and those from other researchers. This study will be helpful to metabolism-engineer the yeast strain to further enhance pullulan yield.