Effects of plastic composite support and pH profiles on pullulan production in a biofilm reactor

Pullulan is a linear homopolysaccharide which is composed of glucose units and often described as α-1, 6-linked maltotriose. The applications of pullulan range from usage as blood plasma substitutes to environmental pollution control agents. In this study, a biofilm reactor with plastic composite support (PCS) was evaluated for pullulan production using Aureobasidium pullulans. In test tube fermentations, PCS with soybean hulls, defatted soy bean flour, yeast extract, dried bovine red blood cells, and mineral salts was selected for biofilm reactor fermentation (due to its high nitrogen content, moderate nitrogen leaching rate, and high biomass attachment). Three pH profiles were later applied to evaluate their effects on pullulan production in a PCS biofilm reactor. The results demonstrated that when a constant pH at 5.0 was applied, the time course of pullulan production was advanced and the concentration of pullulan reached 32.9 g/L after 7-day cultivation, which is 1.8-fold higher than its respective suspension culture. The quality analysis demonstrated that the purity of produced pullulan was 95.8% and its viscosity was 2.4 centipoise. Fourier transform infrared spectroscopy spectra also supported the supposition that the produced exopolysaccharide was mostly pullulan. Overall, this study demonstrated that a biofilm reactor can be successfully implemented to enhance pullulan production and maintain its high purity.     

Pullulan: biosynthesis,production, and applications

Pullulan is a linear glucosic polysaccharide produced by the polymorphic fungus Aureobasidium pullulans, which has long been applied for various applications from food additives to environmental remediation agents. This review article presents an overview of pullulan’s chemistry, biosynthesis, applications, state-of-the-art advances in the enhancement of pullulan production through the investigations of enzyme regulations, molecular properties, cultivation parameters, and bioreactor design. The enzyme regulations are intended to illustrate the influences of metabolic pathway on pullulan production and its structural composition. Molecular properties, such as molecular weight distribution and pure pullulan content, of pullulan are crucial for pullulan applications and vary with different fermentation parameters. Studies on the effects of environmental parameters and new bioreactor design for enhancing pullulan production are getting attention. Finally, the potential applications of pullulan through chemical modification as a novel biologically active derivative are also discussed.     

Production of pure β-glucan by <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> after pullulan synthetase gene disruption

By disruption of the pullulan synthetase gene (pul) of Aureobasidium pullulans IMS822 KCTC11179BP, we constructed a mutant strain, A. pullulans NP1221, which produced a pure β-glucan exopolysaccharide. The mutant NP1221 was white, whereas the wild-type strain produced a black dye. When we compared fermentation kinetics between wide-type and mutant strains, the mutant NP1221 did not produce pullulan. Substrate uptake rate and β-glucan production were similar in both strains. However, the biomass yield of mutant NP1221 was 2.3-fold (9.2 g l⁻¹) greater than that of wild-type.     

Continuous pullulan fermentation in a biofilm reactor

Biofilm is a natural form of cell immobilization in which microorganisms attach onto solid support. In this study, a pigment-reduced pullulan-producing strain, Aureobasidium pullulans (ATCC 201253), was used for continuous pullulan fermentation in a plastic composite support (PCS) biofilm reactor. Optimal conditions for the continuous pullulan production were determined by evaluating the effects of the feeding medium with various concentrations of ammonium sulfate and sucrose and dilution rate. Pullulan concentration and production rate reached maximum (8.3 g/l and 1.33 g/l/h) when 15 g/l of sucrose, 0.9 g/l of ammonium sulfate, and 0.4 g/l of yeast extract were applied in the medium, and the dilution rate was at 0.16 h⁻¹. The purity of produced pullulan was 93.0%. The ratio of hyphal cells of A. pullulans increased when it was grown on the PCS shaft. Overall, the increased pullulan productivity can be achieved through biomass retention by using PCS biofilm reactor.     

Pullulan content of the ethanol precipitate from fermented agro-industrial wastes

Ethanol-precipitated substances after fermentation of various agro-industrial wastes by Aureobasidium pullulans were examined for their pullulan content. Grape skin pulp extract, starch waste, olive oil waste effluents and molasses served as substrates for the fermentation. A glucose-based defined medium was used for comparison purposes. Samples were analysed by an enzyme-coupled assay method and by high-performance anion-exchange chromatography with pulsed amperometric detection after enzymic hydrolysis with pullulanase. Fermentation of grape skin pulp extract gave 22.3 g l⁻¹ ethanol precipitate, which was relatively pure pullulan (97.4% w/w) as assessed by the coupled-enzyme assay. Hydrolysed starch gave only 12.9 g l⁻¹ ethanol precipitate, which increased to 30.8 g l⁻¹ when the medium was supplemented with NH₄NO₃ and K₂HPO₄; this again was relatively pure pullulan (88.6% w/w). Molasses and olive oil wastes produced heterogeneous ethanol-precipitated substances containing small amounts of pullulan, even when supplemented with nitrogen and phosphate. Overall, grape skin pulp should be considered as the best substrate for pullulan production. Starch waste requires several hydrolyis steps to provide a usable carbon source, which reduces its economic attraction as an industrial process. Received: 24 October 1997 / Received revision: 10 February 1998 / Accepted: 15 February 1998     

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.     

Enhanced production of poly (γ-glutamic acid) from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> NCIM 2324 in solid state fermentation

This work reports on the optimization of PGA production by Bacillus licheniformis NCIM 2324 in solid state fermentation (SSF). In the first step, the one factor-at-a-time method was used to investigate the effect of solid substrates, initial moisture content, pH, and additional carbon and nitrogen source on PGA production; subsequently, response surface methodology (RSM) was used to establish the optimum concentrations of the key nutrients for higher PGA production. In the second step, the effects of amino acids and TCA cycle intermediates on the production of PGA were studied. The final optimized medium gave a maximum yield of 98.64 ± 1.61 mg gds⁻¹ of PGA, which is significantly higher than that reported in the literature.     

Pullulan production and physiological characteristics of Aureobasidium pullulans under acid stress

In this study, batch processes of pullulan production by Aureobasidium pullulans CCTCC M 2012259 under different pH environments were evaluated. The pH of the medium decreased quickly to an acid stress condition under batch fermentation without pH control. A higher pullulan production was always obtained with a lower biomass under a given glucose concentration with constant pH control, and vice versa. Based on the nonlinear regression analysis of the results obtained from diverse pH control modes, a constant controlled pH of 3.8 was predicted as an optimum pH for efficient pullulan production using a one-element cubic equation. A maximum pullulan concentration of 26.8 g/L and a minimum biomass of 8.1 g/L were achieved under the optimal pH of 3.8, which were in good agreement with the results predicted by the mathematical model. Further information on the physiological characteristics of A. pullulans CCTCC M 2012259 such as intracellular pH, NADH/NAD⁺, ATP/ADP, and glutathione generation under moderate or severe acidic conditions were investigated, and the results presented more evidence on why pullulan biosynthesized with high efficiency under moderate acid stress (e.g., pH 3.8), which would also help us to better understand the response of the cells to acid stress.     

Optimization of pH for high molecular weight pullulan

Summary Pullulan is a polysaccharide produced by Aureobasidium pullulans. In this study, the effect of pH on the molecular weight of pullulan was investigated. High concentration of pullulan was obtained when initial pH was 6. Pullulan having molecular weight of 500,000–600,000 was produced at initial pH of 3.0, while pullulan with molecular weight of 200,000–300,000 was produced at pH above 4.5. To obtain high molecular weight pullulan with high concentration, pH was initially controlled at pH 6, followed by pH shift from pH 6 to pH 3. Transition of pH at 2 days of fermentation was observed to be optimum. Higher molecular weight pullulan was also obtained when sucrose concentration was 50 g/l compared to the result obtained at initial sucrose concentration of 20 g/l. Sucrose concentration and pH of the fermentation broth seem to be important parameters in obtaining high molecular weight of pullulan.     

Optimization of thermal-dilute sulfuric acid pretreatment for enhancement of methane production from cassava residues

In this study, the pretreatment of cassava residues by thermal-dilute sulfuric acid (TDSA) hydrolysis was investigated by means of a statistically designed set of experiments. A three-factor central composite design (CCD) was employed to identify the optimum pretreatment condition of cassava residues for methane production. The individual and interactive effects of temperature, H₂SO₄ concentration and reaction time on increase of methane yield (IMY) were evaluated by applying response surface methodology (RSM). After optimization, the resulting optimum pretreatment condition was 157.84 °C, utilizing 2.99% (w/w TS) H₂SO₄ for 20.15 min, where the maximum methane yield (248 mL/g VS) was 56.96% higher than the control (158 mL/g VS), which was very close to the predict value 56.53%. These results indicate the model obtained through RSM analysis is suit to predict the optimum pretreatment condition and there is great potential of using TDSA pretreatment of cassava residues to enhance methane yield.     

Production of pigment-free pullulan by swollen cell in <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> NG which cell differentiation was affected by pH and nutrition

A black yeast strain “NG” was isolated from strawberry fruit and identified as Aureobasidium pullulans. Strain NG displayed yeast-like cell (YL), swollen cell (SC), septate swollen cell (SSC), meristematic structure (MS), and chlamydospore (CH) morphologies. pH was the key factor regulating cell morphogenesis of strain NG. Differentiation of YL controlled by extracellular pH had no relationship with nutrition level. YL was maintained at pH >6.0, but was transformed into SC at pH ∼4.5. SC, a stable cell type of A. pullulans, could bud, septate, or transform into MS or CH, in response to nutrition level and low pH. SC produced swollen cell blastospores (SCB) at pH 2.1 with abundant nutrition, and could transform into MS at lower pH (1.5). SC was induced to form CH by low level nutrition and pH <3, and this transition was suppressed by adjusting pH to ∼4.5. Crude polysaccharides without pigment (melanin) were produced by SC of strain NG. Pullulan content of the polysaccharides was very high (98.37%). Fourier-transform infrared spectroscopy confirmed that chemical structures of the polysaccharides and standard pullulan were identical. Swollen cells produced 2.08 mg/ml non-pigmented polysaccharides at 96 h in YPD medium. Controlling pH of fermentation is an effective and convenient method to harvest SC for melanin-free pullulan production.     

Sweet potato: A novel substrate for pullulan production by Aureobasidium pullulans

A strain Aureobasidium pullulans AP329, was used for the production of pullulan by employing hydrolysed sweet potato as cultivation media. Hydrolysis with α-amylase alone resulted in the lowest yields of pullulan. In contrast continuous hydrolysis with pullulanase and the β-amylase in sweet potato itself gave higher yields, but prolonged hydrolysis with amyloglucosidase decreased the yield. The maximum pullulan yield (29.43 g/l) was achieved at the dextrose equivalent value of 45 and pH of 5.5 for 96 h. As a substitute of sucrose, hydrolysed sweet potato was found to be hopeful and the yield of pullulan was higher than that of glucose and sucrose. The molecular weight of pullulan obtained from hydrolysed sweet potato media was much higher than that of sucrose and glucose media. Results of this work indicated that sweet potato was a promising substrate for the economical production of pullulan.     

Poly(β-L-malic acid) production by diverse phylogenetic clades of Aureobasidium pullulans

Poly(β-L-malic acid) (PMA) is a natural biopolyester that has pharmaceutical applications and other potential uses. In this study, we examined PMA production by 56 strains of the fungus Aureobasidium pullulans representing genetically diverse phylogenetic clades. Thirty-six strains were isolated from various locations in Iceland and Thailand. All strains from Iceland belonged to a newly recognized clade 13, while strains from Thailand were distributed among 8 other clades, including a novel clade 14. Thirty of these isolates, along with 26 previously described strains, were examined for PMA production in medium containing 5% glucose. Most strains produced at least 4 g PMA/L, and several strains in clades 9, 11, and 13 made 9–11 g PMA/L. Strains also produced both pullulan and heavy oil, but PMA isolated by differential precipitation in ethanol exhibited up to 72% purity with no more than 12% contamination by pullulan. The molecular weight of PMA from A. pullulans ranged from 5.1 to 7.9 kDa. Results indicate that certain genetic groups of A. pullulans are promising for the production of PMA.     

Production of pullulan from xylose and hemicellulose hydrolysate by Aureobasidium pullulans AY82 with pH control and DL-dithiothreitol addition

Xylose, the second most abundant sugar in lignocellulosic materials, is not efficiently utilized in current lignocellulose biotransformation processes, such as cellulosic ethanol production. The bioconversion of xylose to value-added products, such as pullulan, is an alternative strategy for efficient lignocellulose biotransformation. This paper reports the production of pullulan from xylose and hemicellulose hydrolysate by Aureobasidium pullulans AY82. The effects of DL-dithiothreitol (DTT) and pH on pullulan production from xylose were also intensively investigated. A maximal increase of 17.55% of pullulan production was observed in flasks added with 1.0 mM DTT. Batch fermentations with controlled pH were also conducted, and the optimal pH for cell growth and pullulan synthesis was 3.0 and 5.0, respectively. Based on these findings, two-stage pH control fermentations were performed, in which the pH of the medium was first adjusted to 3.0 for cell growth, and then changed to 5.0 for pullulan synthesis. However, the earlier the pH was changed to 5.0, the more pullulan was produced. Fermentation with controlled pH of 5.0 acquired the highest pullulan production. Under the optimized conditions (with the addition of 1.0 mM DTT and controlled pH of 5.0), the maximal pullulan production obtained from xylose was 17.63 g/L. A. pullulans AY82 also readily fermented hemicellulose hydrolysate under these optimized conditions, but with lower pullulan production (12.65 g/L). Fourier transform infrared spectroscopy and high-performance liquid chromatography showed that the structure of the pullulan obtained in this study was identical to that of the pullulan standard.     

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.     

Development of aqueous two‐phase systems comprising poly ethylene glycol and dextran for purification of pullulan: Phase diagrams and fiscal analysis

Pullulan is a commercially important Exopolysaccharide (EPS) with wide‐spread applications which is produced by Aureobasidium pullulans. The alternative α (1 4) & α (1 6) configuration in pullulan provides it the specific structural and conformational properties. Pullulan is currently being exploited in food, health care, pharmacy, lithography, cosmetics. The fermented broth is processed by organic solvent precipitation for isolation and purification of pullulan. In this study, we have tried to analyze the potential of aqueous two phase system as an alternate technique to extract pullulan from fermented broth. Including this viability of ATPS was also compared with conventional organic solvent precipitation system in terms of cost and time. It was found that ATPS process produced a higher yield of pullulan (80.56%) than organic solvent precipitation method (71.6%). ATPS was also found more economical and less time consuming method.     

Physiological and enzymatic characterization of a novel pullulan-degrading thermophilic Bacillus strain 3183

Summary A new thermophilic Bacillus strain 3183 (ATCC 49341) was isolated from hot-spring sediments. The organism grew on pullulan as a carbon source and showed optimum pH and temperature at pH 5.5 and 62° C, respectively, for growth. The strain reduced nitrate to nitrite both aerobically and anaerobically. It produced extracellular thermostable pullulanase and saccharidase activities which degraded pullulan and starch into maltotriose, maltose, and glucose. Medium growth conditions for pullulanase production were optimized. The optimum pH and temperature for pullulanase activity were at pH 6.0 and 75° C, respectively. The enzyme was stable at pH 5.5-7.0 and temperature up to 70° C in the absence of substrate. The K _m for pullulan at pH 6.0 and 75° C was 0.4 mg/ml. The pullulanase activity was stimulated and stabilized by Ca²⁺. It was inhibited by ethylenediaminetetraacetate (EDTA), beta and gamma-cyclodextrins but not by alpha-cyclodextrin and reagents that inhibit essential enzyme SH-groups. Offprint requests to: B. C. Saha     

The amylopullulanase of Bacillus sp. DSM 405

The amylopullulanse produced by Bacillus sp. DSM 405 was purified to homogeneity. It exhibited dual activity, cleaving the α1-4 bonds in starch, releasing a range of malto-oligosaccharides, and also cleaving the α1-6 bonds in pullulan, releasing maltotriose as the sole end-product. The enzyme was a glycoprotein and had a relative molecular mass of 126 000 and an isoelectric point of 4.3. While the enzyme was optimally active on starch at pH 6.5 and at pH 6.0 on pullulan, activity on both substrates was maximal at 70 °C. Kinetic analyses of the enzyme in a system that contained both starch and pullulan as two competing substrates demonstrated the dual specificity of the enzyme. Chemical modification of the carboxyl groups within the active centre of the protein showed that one active site was responsible for hydrolysis of the α1-4 and α1-6 bonds in starch and pullulan respectively. This is the first comprehensive investigation of an amylopullulanse produced by an aerobic bacterium, showing a single active site responsible for both activities. Received: 3 August 1998 / Received revision: 13 October 1998 / Accepted: 16 October 1998     

High pullulan yield is related to low UDP-glucose level and high pullulan-related synthases activity inAureobasidium pullulans Y68

Effects of different pH and carbon sources on pullulan production, UDP-glucose level and pullulan-related synthases activity inAureobasidium pullulans Y68 were examined. It was found that more pullulan was produced when the yeast strain was grown in the medium with initial pH 7.0 than when it was grown in the same medium with constant pH 6.0. The results also show that higher pullulan yield was obtained when the cells were grown in the medium containing glucose than when they were cultivated in the medium supplementing other carbon sources. Our results demonstrate that the more pullulan was synthesized, the less UDP-glucose was left in the cells ofA. pullulans Y68. However, it was observed that more pullulan was synthesized; the cells had higher pullulan-related synthase activity. Therefore, high pullulan yield was related to low UDP-glucose level and high pullulan-related synthases activity inAureobasidium pullulans Y68.