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.     

Optimization of mineral salts in medium for enhanced production of pullulan by <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> HP-2001 using an orthogonal array method

Based on intuitive analyses and statistical calculations using data from orthogonal array experiments, the optimal concentrations of K₂HPO₄, NaCl, MgSO₄·7H₂O, and (NH₄)₂SO₄ in cell growth medium of Aureobasidium pullulans HP-2001 were measured as 7.5, 1.0, 0.1, and 2.4 g/L, respectively, whereas those for the production of pullulan were 2.5, 0.25, 0.8, and 0.3 g/L, respectively. The most important factor for cell growth and production of pullulan by A. pullulans HP-2001 was identified as K₂HPO₄. Optimal concentrations of glucose and yeast extract, along with the initial pH of the cell growth medium of A. pullulans HP-2001 containing optimized salt concentrations, were found to be 100.0, 10.0, and 6.0 g/L, respectively, whereas those for the production of pullulan were 100.0, 2.5, and 6.0 g/L, respectively. Conversion rates of pullulan from 10.0, 25.0, 50.0, 75.0, and 100.0 g/L of glucose in the presence of optimized salt concentrations were 26.0, 25.2, 22.4, 17.9, and 14.1%, respectively, whereas those in the presence of previously reported salt concentrations were 26.6, 25.2, 19.9, 14.3, and 11.7%, respectively. Optimal salt concentrations for the production of pullulan by A. pullulans HP-2001 varied according to the concentrations of the carbon and nitrogen sources, especially at higher concentrations.     

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.     

Optimization of physico‐chemical and nutritional parameters for a novel pullulan‐producing fungus,Eurotium chevalieri

Aims: To isolate the novel nonmelanin pullulan‐producing fungi from soil and to optimize the physico‐chemical and nutritional parameters for pullulan production. Methods and Results: A selective enrichment method was followed for the isolation, along with development of a suitable medium for pullulan production, using shake flask experiments. Pullulan content was confirmed using pure pullulan and pullulanase hydrolysate. Eurotium chevalieri was able to produce maximum pullulan (38 ± 1·0 g l^?1) at 35°C, pH 5·5, 2·5% sucrose, 0·3% ammonium sulfate and 0·2% yeast extract in a shake flash culture medium with an agitation rate of 30 rev min^?1 for 65 h. Conclusions: The novel pullulan‐producing fungus was identified as E. chevalieri (MTCC no. 9614), which was able to produce nonmelanin pullulan at from poorer carbon and nitrogen sources than Aureobasidium pullulans and may therefore be useful for the commercial production of pullulan. Significance and Impact of the Study: Eurotium chevalieri could produce pullulan in similar amounts to A. pullulans. Therefore, in future, this fungus could also be used for commercial pullulan production, because it is neither polymorphic nor melanin producing, hence its handling during pullulan fermentation will be easier and more economical.     

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.     

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.     

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.     

Improved β-glucan yield using an Aureobasidium pullulans M-2 mutant strain in a 200-L pilot scale fermentor targeting industrial mass production

β-(1→3)-D-glucans with β-(1→6)-glycosidic linked branches are known to be immune activation agents and are incorporated in anti-cancer drugs and health-promoting supplements. β-Glucan concentration was 9.2 g/L in a 200-L pilot scale fermentor using mutant strain Aureobasidium pullulans M-2 from an imperfect fungal strain belonging to A. pullulans M-1. The culture broth of A. pullulans M-2 had a faint yellow color, whereas that of the wild-type had an intense dark green color caused by the accumulation of melanin-like pigments. β-Glucan produced by A. pullulans M-2 was identified as a polysaccharide of D-glucose monomers linked by β-(1→3, 1→6)-glycosidic bonds through GC/MS and NMR analysis. When a conventional medium was used in the culture of A. pullulans M-2 in a 3-L jar fermentor, β-glucan concentration was 1.4-fold that produced by the wild-type. However, when a medium optimized by statistical experimental design was used with dissolved oxygen at 10%, the β-glucan concentration was 9.9 g/L with a yield of 0.52 (g β-glucan/g consumed sucrose), 2.9-fold that of the wild-type. This level of productivity was reproduced when the fermentation was scaled up 200-L. The industrial production of high β-glucan without melanin-like pigments is highly expected, as a health-promoting supplement or functional food.     

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.     

Improving the production of S-adenosyl-L-methionine in Escherichia coli by overexpressing metk

S-adenosyl-L-methionine (SAM) has important applications in many fields including chemical therapy and pharmaceutical industry. In this study, the recombinant Escherichia coli strain was constructed for effective production of SAM by introducing the SAM synthase gene (metK). This strain produced 34.5?mg/L of SAM in basic medium in shake flask. Yeast extract, pH, and loaded volume had a significant positive effect on the yield of SAM. Their optimal values were 35?g/L, 7.5, and 30?mL, respectively. The final conditions optimized were as follows: glucose 20, g/L; peptone, 40?g/L; yeast extract, 35?g/L; NaCl, 10?g/L; MgSO₄, 1.2?g/L; L-methionine, 1?g/L; rotate speed, 220?rpm; loaded volume, 30?mL; inoculation, 1%; temperature, 37°C; and initial medium, pH 7.5. The recombinant strain produced 128.2?mg/L of SAM under the above conditions in shake flask. The production of SAM in a 5?L fermentor was also investigated. The maximal biomass of the recombinant strain was 60.4?g/L after the cells were cultured for 20?hr, and the highest yield of SAM was 300.9?mg/L after induction for 8?hr in a 5?L fermentor. This study provides a good foundation for the future production and use of SAM.     

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     

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.     

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.     

Downstream processing and characterization of pullulan from a novel colour variant strain of Aureobasidium pullulans FB-1

Exopolysaccharide produced by a new novel colour variant strain of Aureobasidium pullulans FB-1 was purified by cell harvesting and precipitation of the polymer. Various organic solvents were screened for pullulan precipitation. Isolation and purification of pullulan from fermentation broth was carried out using single-step purification strategy by isopropyl alcohol precipitation. Ratio of culture supernatant to isopropyl alcohol and time of precipitation were optimized for pullulan precipitation. Maximum yield (4.47%, w/v) of polysaccharide was obtained when two volumes of ice-cold isopropyl alcohol were added to one volume of supernatant with precipitation time of 12 h. IR spectra as well as carbon-13 and proton NMR spectra in aqueous solution of intact polysaccharide obtained from A. pullulans FB-1 and commercially available pullulan (Sigma, USA) revealed solely α-(1 → 6) linked maltosyl units, in accord with the generally accepted structure of pullulan. Maximum hydrolysis (94.25%) of purified pullulan at 50 °C by pullulanase was achieved under agitation (150 rpm) after 360 min.     

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

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

Enhanced doxorubicin production by Streptomyces peucetius using a combination of classical strain mutation and medium optimization

Abstract

Doxorubicin (DXR), which is produced by Streptomyces peucetius, is an important anthracycline-type antibiotic used for the treatment of various cancers. However, due to the low DXR productivity of wild-type S. peucetius, it is difficult to produce DXR by one-step fermentation. In this study, a DXR-resistance screening method was developed to screen for DXR high-producing mutants. Then, S. peucetius SIPI-11 was treated several times with UV and ARTP (atmospheric and room temperature plasma) to induce mutations. Treated strains were screened by spreading on a DXR-containing plate, isolating a mutant (S. peucetius 33-24) with enhanced DXR yield (570?mg/L vs. 119?mg/L for the original strain). The components of the fermentation medium, including the carbon and nitrogen sources, were optimized to further enhance DXR yield (to 850?mg/L). The pH of the fermentation medium and culture temperature were also optimized for effective DXR production. Finally, DXR production by S. peucetius 33-24 was investigated in flask culture and a fermenter. The yield of DXR was as high as 1100?mg/L in a 5-L fermenter, which is the highest DXR productivity reported thus far, suggesting that S. peucetius 33-24 has the potential to produce DXR by direct fermentation.     

出芽短梗霉Aureobasidium sp. SRF的菌株特性分析及胞外多糖结构鉴定

菌株SRF是1株从意大利树莓(Rubus corchorifolius)果实表面分离、可产胞外多糖的新菌株。在鉴定其分类归属的基础上,对其产生的胞外多糖进行了结构分析和发酵条件优化,为寻找微生物多糖提供新的菌株,为开发利用资源微生物提供借鉴。通过形态学和ITS序列对比分析进行菌株鉴定;通过薄层层析和红外光谱分析,确定胞外多糖结构;通过单因素检测试验,确定影响产糖量的主要因素;响应面Plackett-Burman和Box-Behnken设计筛选发酵产胞外多糖的最优条件。结果表明,出发菌株SRF隶属于出芽短梗霉属,命名为Aureobasidium sp. SRF;SRF所产胞外多糖为普鲁兰多糖;单因素检测表明,对多糖产量影响最大的因素为碳源浓度、氮源浓度、无机离子浓度,其次是碳源、氮源、无机离子、pH值;根据响应面结果确定最优发酵条件为麦芽糖8%(质量分数)、酵母提取物3%(质量分数)、钙离子0.3 g/L、pH 6,产糖量达5.93 g/L。SRF是1株来源于树莓浆果表面,可产胞外普鲁兰多糖的出芽短梗霉新菌株,是1株产微生物多糖的候选菌株。     

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.     

Efficient butanol production under aerobic conditions by coculture of Clostridium acetobutylicum and Nesterenkonia sp. strain F

Clostridium acetobutylicum is widely used for the microbial production of butanol in a process known as acetone–butanol–ethanol (ABE) fermentation. However, this process suffers from several disadvantages including high oxygen sensitivity of the bacterium which makes the process complicated and necessitate oxygen elimination in the culture medium. Nesterenkonia sp. strain F has attracted interests as the only known non-Clostridia microorganism with inherent capability of butanol production even in the presence of oxygen. This bacterium is not delimited by oxygen sensitivity, a challenge in butanol biosynthesis, but the butanol titer was far below Clostridia. In this study, Nesterenkonia sp. strain F was cocultivated with C. acetobutylicum to form a powerful “coculture” for butanol production thereby eliminating the need for oxygen removal before fermentation. The response surface method was used for obtaining optimal inoculation amount/time and media formulation. The highest yield, 0.31 g/g ABE (13.6 g/L butanol), was obtained by a coculture initiated with 1.5 mg/L Nesterenkonia sp. strain F and inoculated with 15 mg/L C. acetobutylicum after 1.5 hr in a medium containing 67 g/L glucose, 2.2 g/L yeast extract, 4 g/L peptone, and 1.4% (vol/vol) P2 solution. After butanol toxicity assessment, where Nesterenkonia sp. strain F showed no butanol toxicity, the coculture was implemented in a 2 L fermenter with continual aeration leading to 20 g/L ABE.     

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