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     

Oxygen requirement in pullulan fermentation

Summary Oxygen was essential for the biosynthesis of pullulan by Aureobasidium pullulans. In a growth medium, pullulan yield and synthesis rate were proportional to the oxygen availability. However, under controlled oxygen environment in a non-growth medium, the synthesis rate and the yield of pullulan were inversely proportional to the oxygen tension. A relationship between melanin production and oxygen transfer conditions was also observed. The elapsed time prior to the appearance of the pigment was dependent upon the degree of oxygen availability.     

A new control strategy for yeast production based on the L/A* approach

Summary We studied the effect of temperature on the production of an extracellular neutral metalloproteinase of Bacillus megaterium in a laboratory fermentor under constant aeration and pH. The optimal temperature for growth (35–38° C) was higher than that for the synthesis of proteinase during exponential growth (below 31° C). The critical biomass concentration at which the exponential growth terminated decreased with increase in cultivation temperature. The specific rate of proteinase synthesis decreased when the critical biomass concentration was achieved. The observed decrease in proteinase synthesis was related to the cultivation temperature. The temperature also influenced the level of mRNA coding for proteinase. We formulated a mathematical model of cultivation describing the dependence of growth and proteinase synthesis on dissolved oxygen and temperature. The parameters of the model were identified for temperature intervals from 21 to 41° C using a computer. The optimum temperature for the enzyme production was 21° C. The productivity (enzyme activity/time) was maximal at 24–28° C. When optimizing the temperature profile of cultivation, we designed a suboptimal solution represented by a linear temperature profile. We have found that under conditions of continuous decrease in temperature, the maximal production of the proteinase was achieved at a broad range of temperature (26–34° C) when the rate of temperature decrease was 0.2–0.8° C/h. The initial optimal temperature for the enzyme productivity was in the range of 32–34° C. The optimum temperature decrease was 0.8° C/h. Offprint requests to: J. Chaloupka     

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 production from synthetic medium by a new mutant of Aureobasidium pullulans

Pullulan with different molecular-weight could be applied in various fields. A UV-induced mutagenesis Aureobasidium pullulans UVMU6-1 was obtained from the strain A. pullulans CGMCC3.933 for the production of low-molecular-weight pullulan. First, the obtained polysaccharide from A. pullulans UVMU6-1 was purified and identified to be pullulan with thin-layer chromatography, Fourier transform infrared, and nuclear magnetic resonance. Then, culture medium and conditions for this strain were optimized by flask fermentation. Based on the optimized medium and culture conditions (pH 4, addition of 4?g/L Tween 80 for 96?hr of cultivation), continuously fermentation was performed. The highest pullulan production and dry biomass was 109 and 125?g/L after fermentation for 114?hr, respectively. The average productivity was about 1?g/L/hr, which was intensively higher than the previous reported. This study would lay foundations for the industrial production of pullulan.     

Effect of pH on the batch fermentation of pullulan from sucrose medium

Two strains of the yeast-like fungus Aureobasidium pullulans 2552 and 140B have been used for the fermentative production of the polysaccharide pullulan from a sucrose synthetic medium. In the batch fermentation, either in Erlenmeyers or in the fermentor, the pH of the culture medium was decreased rapidly from its initial pH value of 5.5 to the self-stabilized final value of 2.5 within 24 h. Experiments on the effect of initial pH on the fermentation revealed that at very low initial pH values, such as at pH 2, the polysaccharide production was in-significant. However, the biomass concentration obtained was very high at this very low initial pH value. This interesting phenomenon was served as the basic principle for the development of the bistaged pH fermentation process for the production of pullulan. In this process the first stage of fermentation was conducted at the very acidic pH for the best production of biomass. When the biomass concentration reached its maximum value, the second stage of fermentation was initiated by adjusting the medium pH to a higher value for promoting the synthesis of the polysaccharide. Experiments conducted in Erlenmeyers and in the fermentor confirmed this concept. The bistaged pH process enhanced the polysaccharide concentration in the medium, influenced the rheological properties of the fermentation broth, and has a potential of operation under nonsterile and nonaseptic conditions.     

Effects of melanin on the accumulation of exopolysaccharides by Aureobasidium pullulans grown on nitrate

Aureobasidium pullulans produced pullulan and melanin when grown in medium containing low nitrate levels. With high nitrate concentrations, however, this fungus produced a mixture of exopolysaccharides (EPS) without melanin synthesis. At 0.78 g l(-1) N as nitrate, where no melanin synthesis occurred, maximum EPS yields reached 6.92 g l(-1) and then decreased to the final yield of 2.36 g l(-1). Following melanin addition (0.1 g l(-1)), yields reached 7.02 g l(-1) at 48 h and fell to a final yield of 5.21 g l(-1). The EPS produced in high nitrate medium contained both pullulan and (1-->3)-beta-glucan, but only pullulan was produced with melanin-supplementation. With melanin addition a doubling of (1-->3)-beta-glucanase activity was observed in high nitrate medium compared to that without supplementation. On the other hand amylolytic activities disappeared in medium with melanin production or addition. Culture filtrates sustained a higher reducing capacity (RC) when melanin was present. Low RC appeared to reduce (1-->3)-beta-glucanase activity and increase amylolytic activities. Thus, higher RC appears to inhibit production/activity of amylose-degrading enzymes capable of degrading pullulan, and stimulates (1-->3)-beta-glucanase synthesis/activity, leading to a preferential accumulation of pullulan.     

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.     

Polysaccharide production by a reduced pigmentation mutant of Aureobasidium pullulans NYS-1

AIMS: To isolate a reduced pigmentation mutant of Aureobasidium pullulans NYS-1 and characterize its cellular pigmentation plus its polysaccharide and biomass production relative to carbon source. METHODS AND RESULTS: Cellular pigmentation, polysaccharide levels and biomass production by the isolated mutant NYSRP-1 were analysed relative to carbon source. Cellular pigmentation of the mutant was lower than its parent strain using either carbon source. The mutant elaborated higher polysaccharide levels on sucrose than on corn syrup. The pullulan content of the polysaccharide synthesized and biomass production by the mutant rose as the carbon source concentration was increased. CONCLUSION: It is feasible to isolate a reduced pigmentation mutant from strain NYS-1 that exhibits elevated polysaccharide production using corn syrup as a carbon source. SIGNIFICANCE AND IMPACT OF THE STUDY: The mutant provides an advantage for commercial pullulan production because of its reduced pigmentation and enhanced polysaccharide synthesis.     

High‐pressure systems for gas‐phase free continuous incubation of enriched marine microbial communities performing anaerobic oxidation of methane

Novel high‐pressure biotechnical systems that were developed and applied for the study of anaerobic oxidation of methane (AOM) are described. The systems, referred to as high‐pressure continuous incubation system (HP‐CI system) and high‐pressure manifold‐incubation system (HP‐MI system), allow for batch, fed‐batch, and continuous gas‐phase free incubation at high concentrations of dissolved methane and were designed to meet specific demands for studying environmental regulation and kinetics as well as for enriching microbial biomass in long‐term incubation. Anoxic medium is saturated with methane in the first technical stage, and the saturated medium is supplied for biomass incubation in the second stage. Methane can be provided in continuous operation up to 20 MPa and the incubation systems can be operated during constant supply of gas‐enriched medium at a hydrostatic pressure up to 45 MPa. To validate the suitability of the high‐pressure systems, we present data from continuous and fed‐batch incubation of highly active samples prepared from microbial mats from the Black Sea collected at a water depth of 213 m. In continuous operation in the HP‐CI system initial methane‐dependent sulfide production was enhanced 10‐ to 15‐fold after increasing the methane partial pressure from near ambient pressure of 0.2 to 10.0 MPa at a hydrostatic pressure of 16.0 MPa in the incubation stage. With a hydraulic retention time of 14 h a stable effluent sulfide concentration was reached within less than 3 days and a continuing increase of the volumetric AOM rate from 1.2 to 1.7 mmol L^?1 day^?1 was observed over 14 days. In fed‐batch incubation the AOM rate increased from 1.5 to 2.7 and 3.6 mmol L^?1 day^?1 when the concentration of aqueous methane was stepwise increased from 5 to 15 mmol L^?1 and 45 mmol L^?1. A methane partial pressure of 6 MPa and a hydrostatic pressure of 12 MPa in manifold fed‐batch incubation in the HP‐MI system yielded a sixfold increase in the volumetric AOM rate. Over subsequent incubation periods AOM rates increased from 0.6 to 1.2 mmol L^?1 day^?1 within 26 days of incubation. No inhibition of biomass activity was observed in all continuous and fed‐batch incubation experiments. The organisms were able to tolerate high sulfide concentrations and extended starvation periods. Biotechnol. Bioeng. 2010; 105: 524–533. © 2009 Wiley Periodicals, Inc.     

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.     

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.     

Viability and cellulose synthesizing ability of Gluconacetobacter xylinus cells under high-hydrostatic pressure

The effect of pressure on viability and the synthesis of bacterial cellulose (BC) by Gluconacetobacter xylinus ATCC53582 were investigated. G. xylinus was statically cultivated in a pressurized vessel under 0.1, 30, 60, and 100 MPa at 25°C for 6 days. G. xylinus cells remained viable and retained cellulose producing ability under all the conditions tested, though the production of cellulose decreased with increasing the pressure. The BCs produced at each pressure condition were analyzed by field emission scanning electron microscopy (FE-SEM) and Fourier Transform Infrared (FT-IR). FE-SEM revealed that the widths of BC fibers produced under high pressure decreased as compared with those produced under the atmospheric pressure. By FT-IR, all the BCs were found to be of Cellulose type I, as the same as typical native cellulose. Our findings evidently showed that G. xylinus possessed a piezotolerant (barotolerant) feature adapting to 100 MPa without losing its BC producing ability. This was the first attempt in synthesizing BC with G. xylinus under elevated pressure of 100 MPa, which corresponded to the deep sea at 10,000 m.     

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.     

Effect of cycling dissolved oxygen concentrations on product formation in penicillin fermentations

Summary Limitations in mass and momentum transfer coupled with high hydrostatic pressures create significant spatial variations in dissolved gas concentrations in large fermenters. Microorganisms are subjected to fluctuating environmental conditions as they pass through the zones in a stirred vessel or along a closed loop fermenter.A 7-litre fermenter was modified to simulate the dissolved gas and hydrostatic pressure gradients in large vessels.The effect of cycling dissolved oxygen tension (DOT) on penicillin production by Penicillium chrysogenum P1 was investigated. The fermentation was affected by evironmental conditions such as medium composition, pH, size of inoculum, stirrer speed and DOT. Inoculum size below 10% (v/v) and stirrer speeds above 850 rpm caused significant reductions in specific prenicillin production rates (q_pen). q_pen values were measured at different constant DOT levels. Below 30% air saturation q_pen decreased sharply and no production was observed at 10%. Penicillin synthesis was impaired irreversibly below 10% DOT. The same profile was observed at higher stirrer speeds and air flow rates indicating that the effect was a physiological one. Oxygen uptake of the culture was affected significantly below 7% DOT, demonstrating that the critical DOT values for penicillin production and oxygen uptake are two distinct parameters. Carrying out the fermentation at one atmosphere over pressure was found to have no effect. When the dissolved oxygen concentration of the culture medium was cycled around the critical DOT for penicillin production, a considerable decrease in the specific penicillin production rate was observed. The effect was reversible but not transient, indicating a shift in cell metabolism.These results demonstrate the unfavourable effect of fluctuating environmental conditions on culture performance in stirred tanks. They suggest that these effects should be accounted for during strain selection, process development and scale up stages of an industrial process if the productivities in small scale vessels are to be obtained.     

Fed-batch cultivation of Saccharomyces cerevisiae in a hyperbaric bioreactor

Fed-batch is the dominating mode of operation in high-cell-density cultures of Saccharomyces cerevisae in processes such as the production of baker's yeast and recombinant proteins, where the high oxygen demand of these cultures makes its supply an important and difficult task. The aim of this work was to study the use of hyperbaric air for oxygen mass transfer improvement on S. cerevisiae fed-batch cultivation. The effects of increased air pressure up to 1.5 MPa on cell behavior were investigated. The effects of oxygen and carbon dioxide were dissociated from the effects of total pressure by the use of pure oxygen and gas mixtures enriched with CO(2). Fed-batch experiments were performed in a stirred tank reactor with a 600 mL stainless steel vessel. An exponential feeding profile at dilution rates up to 0.1 h(-)(1) was used in order to ensure a subcritical flux of substrate and, consequently, to prevent ethanol formation due to glucose excess. The ethanol production observed at atmospheric pressure was reduced by the bioreactor pressurization up to 1.0 MPa. The maximum biomass yield, 0.5 g g(-)(1) (cell mass produced per mass of glucose consumed) was attained whenever pressure was increased gradually through time. This demonstrates the adaptive behavior of the cells to the hyperbaric conditions. This work proved that hyperbaric air up to 1.0 MPa (0.2 MPa of oxygen partial pressure) could be applied to S. cerevisiae cultivation under low glucose flux. Above that critical oxygen partial pressure value, i.e., for oxygen pressures of 0.32 and 0.5 MPa, a drastic cell growth inhibition and viability loss were observed. The increase of carbon dioxide partial pressure in the gas mixture up to 48 kPa slightly decreased the overall cell mass yield but had negligible effects on cell viability.     

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 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.     

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.     

Responses to precipitation treatment for <Emphasis Type="Italic">Haloxylon ammodendron</Emphasis> growing on contrasting textured soils

The responses to precipitation of Haloxylon ammodendron (C.A. Mey.) Bunge (Chenopodiaceae), a small xerophilous tree growing on contrasting textured soils, were evaluated under no, natural, and double precipitation treatments during the entire growing season of 2006. The contrasting textured soils are sandy and heavy textured, and both are the original habitat of H. ammodendron at the south edge of Gubantonggute Desert, Central Asia. Photosynthesis, leaf water potential, transpiration, water use efficiency and leaf biomass production were monitored throughout the growing season. Root distribution of H. ammodendron was evaluated at the end of the experiment. Overall, this small tree did not show significant response to a large summer precipitation pulse or precipitation treatments, in terms of photosynthetic carbon assimilation on either soil. The leaf water potential, transpiration, and water use efficiency appeared to be highly sensitive to a large precipitation pulse and precipitation treatments in sandy soil; and leaf biomass production was also much higher for plants in sandy than that of heavy-textured soil. In sandy soil, defoliation occurred when pre-dawn leaf water potential dropped below −3.0 MPa, while in heavy-textured soil, defoliation occurred when pre-dawn leaf water potential dropped below −3.75 MPa. For similar above-ground parts, the small trees at the sandy site developed much deeper root systems and had nearly double the surface area of feeder roots compared to those at the heavy-textured site. Partially owning to the deeper and larger root system, H. ammodendron growing at coarse-textured site was in better water conditions than those at heavy-textured site under the same climatic conditions.