Int.J.Curr.Microbiol.App.Sci (2015) 4(10): 605-622
ISSN: 2319-7706 Volume 4 Number 10 (2015) pp. 605-622 http://www.ijcmas.com
Review Article Aureobasidium pullulans - An Industrially Important Pullulan Producing Black Yeast
Ranjan Singh1*, Rajeeva Gaur2, Shikha Bansal1, Pritha Biswas1, Prabhash K. Pandey3, Farrukh Jamal3, Soni Tiwari2 and Manogya K. Gaur4
1Department of Botany and Microbiology, St. Aloysius College (Autonomous), Jabalpur, M.P., India 2Department of Microbiology (DST-FIST Supported, Center of Excellence), Dr. R. M. L. Avadh University, Faizabad, U.P., India 3Department of Biochemistry, Dr. R. M. L. Avadh University, Faizabad, U.P., India 4D.G.M. Environment (ETP and Biocompost), Balrampur Distillery, Bhabhanan, Basti, U.P., India *Corresponding author
A B S T R A C T
Aureobasidium pullulans, popularly known as black yeast, is one of the most widespread saprophyte fungus associated with wide range of terrestrial and aquatic habitats, in temperate and tropical environment. It is a polymorphic fungus that is K e y w o r d s able to grow in single yeast-like cells or as septate polykaryotic hyphae showing synchronous conditions, with budding cells. This fungus has been exploited Aureobasidium potentially for commercial production of various enzymes (amylase, xylanase, pullulans, pectinase, etc), single cell protein, alkaloids and polysaccharide, especially Pullulan, pullulan. Pullulan has been considered as one of the important polysaccharide for Single Cell production of biodegradable plastics. The fungus has excellent genetic make-up to Protein, produce various important metabolites at commercial production with limited Agro-Industrial species. Some of the A. pullulans have potential antagonistic activity against a Waste, number of phytopathogenic fungi used as bio-control agents of post-harvest Production diseases. It has been found to be tolerant to many metal ions which are common pollutants of soil and water. Several strains of this fungus have ability to degrade xenobiotic compounds. In the light of the above facts, this review article has emphasized on the orientation, morphology, biochemical characteristics, habitats and economic potentials of this industrially important yeast.
Introduction Aureobasidium pullulans (De Bary) G. fluctuating moisture content in phyllospere, Arnoud is a ubiquitous, polymorphic and and also isolated from damp indoor surfaces, oligotrophe black yeast like microfungus food and feed substances (65). . It is well that occurs frequently in wide range of known as a naturally occurring epiphyte or tropical and temperate environment with endophyte of a wide range of plant species 605 Int.J.Curr.Microbiol.App.Sci (2015) 4(10): 605-622
(e.g. apple, grape, cucumber, green beans, include fresh water, estuarine and marine and cabbage) without causing any symptom sediments and sea water. Occurrence and of disease (1). Chronic human exposure to distribution of A. pullulans by fluorescence A. pullulans via humidifiers or air dye and found it frequently on the conditioners can lead to hypersensitivity phylloplane of several plants was studied pneumonitis or humidifier lung . This (47). A. pullulans was isolated from green condition is characterized acutely by and senescent sugar maples, but it did not dyspnea, cough, chest infilterates and acute seem to be inducer and biodeteriorator (36). inflammatory reaction. The strains causing Seasonal occurrence of A. pullulans from infections in humans were reclassified to March to April in river Danube (77). This A.melanogenum (23) fungus has been isolated from several locations of Thailand (56, 57, 58). Pullulan It has also been found in the osmotically producing novel color variant strain of A. stressed environments like hyper saline pullulans was isolated from the phylloplane water in salterns (24) and the rocks (81). of Ficus sp in India (72). Similarly, A. This fungus disperses easily due to pullulans was isolated strain N13D from production of yeast-like propagules in large deep sea sediments of the Pacific Ocean quantity and found globally but rarely (28). A. pullulans was isolated from needles reported in the intense cold environment, as and twigs of pine plantation from northern investigations on fungal diversity are limited Spain (92). The morphological forms of this to frozen Antarctic soils and Siberian fungus are governed by many factors like permafrost where basidiomycetous yeasts temperature, pH, and oxygen concentration. were found (3). Several Aureobasidium like Nutritional factors mainly the source and black yeasts were isolated from glacial and concentration of carbon, nitrogen and sub-glacial ice in the coastal Arctic habitats mineral affect the morphology mainly yeast and the adjacent sea water (91). From the like cells which are responsible for the available records, it is apparent that this production of pullulan, an important fungus is most common in temperate zone polysaccharide. Morphological studies have with numerous records both from the British been well reviewed (17). This review has Isles and the United States including Alaska, concluded that A. pullulans, is an Antarctica, Denmark, Germany, omnipresent fungus which can survive in Netherlands, Poland, Austria, Czech different type of habitat. Some of the recent Republic, Russia and Japan. There are studies on the behavior of this fungus clearly several reports of its occurrence in show two important characteristics of anti- Mediterranean and arid zones including fungal and antibacterial activity along with Italy, France, Egypt, Iraq, Pakistan and pullulan production. This approach makes South Africa. It has also been extensively such strain more economical for industrial isolated from tropical and subtropical region use. Earlier, A. pullulans was identified as namely, Brazil, India, China, Thailand, Dematium pullulans De Bary (1884) and Malaysia, Jamaica (West Indies), etc. An Pullularia pullulans (De Bary) Berkhout extensive list of the habitats and geography (1866). Using the criteria of conidiogenesis from which strains of A. pullulans have been that is, synchronous holoblastic conidial isolated is well documented. (39, 91). The production, A.pullulans was placed fungus has been frequently isolated from previously under Fungi Imperfecti, Order moorland, peat bogs or peat podzol and Moniliales, and Family Dematiaceae. forest soils. Other noteworthy habitats However, further reports included it under
606 Int.J.Curr.Microbiol.App.Sci (2015) 4(10): 605-622
Ascomycota though; perfect stage has not control and biodeterioration of xenobiotic yet been reported (14). In recent compounds has also been added in this classification, A. pullulans is regarded as review. Although the author has covered Ascomycetous yeast and is placed in the almost all the potential areas in brief, but Order Dothideales, Family Dothideaceae pullulan is little covered. However, after a (90). The fungus was taxonomically long gap, Leathers wrote a review entitled characterized on the basis of its Pullulan in 2002, emphasing on historical morphological and nutritional characteristics outline, chemical structures various comprising single species with three analytical and assay methods of pullulan different varieties such as A. pullulans var. including biosynthesis of pullulan in pullulans, A. pullulans var. melanogenum different nutrient sources. The genetics of A. and A. pullulans var. aubasidani Yurlova pullulans has been extensively covered in (15). Four strains of arctic Aureobasidium this review. Since then several recent reports have been discovered, that is, A. pullulans have come regarding novel method of var. pullulans, A. pullulans var. immobilization, use of cheaper carbon melanogenum, A. pullulans var. subglaciale sources and use of continuous fermentation and A. pullulans var. namibiae (91). for better yield and productivity. A novel fungus Eurotium chevalieri has also been A. pullulans exhibits polymorphism, for reported to produce pullulan which was at it can grow as budding yeast or as mycelia par to A. pullulans, commenced the depending upon environmental conditions. possibility of the isolation of newer strains The life cycle of A. pullulans was from natural ecosystem (20). Moreover, a thoroughly reviewed (15). The formation of review on pullulan emphasing on various dark-coloured chlamydospores is the factors namely; pH, various substrates, characteristic feature of this fungus (59). molecular weight distribution of native Some workers have described the vegetative pullulan along with molecular and cycle of A. pullulans as well as colony hydrodynamic property of pullulan and also characteristics on solid nutrient medium. mentioned, a novel area that is biomedical Colonies are initially smooth and eventually application of pullulan (70). A review become covered with slime. Starting as entitled Bioproducts from A. pullulans, a yellow cream, light pink or light brown, the biotechnologically important yeast has colonies finally become blackish due to been written elaborating some new production of a specific melanin at metabolites like Siderophores production by chlamydospore production stage. When A. pullulans and biocontrol efficiency observed under light microscope, the hyphae against post-harvest diseases. However, they look hyaline, smooth and thin-walled, 2 - 16 have also shown similar beneficial m wide with cells often wider than long metabolites like various enzymes namely; forming rough and compact mycelia. A. protease, amylase, lipase, cellulase, xylanase pullulans can be recognized by straight and single cell protein (SCP) producing conidia and by the presence of lobed chains capabilities of A. pullulans (11). of thick-walled chlamydospores. A review regarding the application of A. pullulans had In-spite of all the information up to 2009, been published emphasing the production of the commercial production of pullulan with various enzymes, single cell proteins and a proper fermentation technique regarding polysaccharide by this fungus (17). The use the better continuous production in solid of this fungus in environmental pollution state and submerged fermentation have not
607 Int.J.Curr.Microbiol.App.Sci (2015) 4(10): 605-622 yet been taken up properly because applied to control post harvest diseases of commercial production technology with different fruits and vegetables (67). This specific design of fermentor technology is strain provided high protection levels still lacking behind and must be developed against P. digitatum on grape fruit, Botrytis for better pullulan production. Moreover, cinerea, Aspergillus niger and Rhizopus immobilization in solid state as well as stolonifer on grape, B. cinerea and Monilia submerged fermentation using suitable solid lax a on sweet cherry and B. cinerea on kiwi matrix must be discussed. However, in fruit, cherry, tomato, apples and strawberries present review an approach has been (66). incorporated in the light of these aspects. Enzymes from A. pullulans Economic importance of A. pullulans Different strains of A. pullulans isolated A. pullulans has been employed for different from different environments can produce useful purposes as it produces a variety of amylase, protease, lipase, cellulose, important metabolites, enzymes, antibiotics, xylanase, etc, which have great potential single cell protein (SCP) and applications in industry. Proteases have been polysaccharides (11). Tests have shown it to shown to have many applications in be safe for the use as SCP (12). A. pullulans detergents, leather processing, silver has shown the potential in controlling and recovery, medical purposes, food monitoring environmental pollution. Due to processing, feeds, chemical industry as well ubiquitous nature of this fungus on as waste treatment. Proteases also contribute phylloplane, any change in its occurrence to the development of high-added might prove to be an indicator of applications or products using the enzyme- environmental perturbations generated by aided digestion of protein from different chemicals or other biological organisms sources (35). However, little has been (including genetically engineered known about protease from marine-derived organisms) landing on the leaf surfaces. A. yeasts (10). Amylases have many pullulans appears to be a promising applications in bread and baking industry, organism for the development of newer starch liquefaction and saccharification, antimicrobial agents, both for chemotherapy textile desiging, paper industry, detergent as well as non-medical applications. industry, analysis in medical and clinical Extracellular antimicrobial activity of chemistry, and food and pharmaceutical Aureobasidium pullulans was demonstrated industries. Because most yeasts from against several Gram positive and Gram environments are safe (GRAS, generally negative bacteria (13). A large number of regarded as safe) compared to bacteria, bioactive and structurally diverse fungal interest in amylolytic yeasts has been metabolites have been used for the increased in recent years as their potential development of valuable pharmaceuticals value for conversion of starchy biomass to (76). Some isolates of A. pullulans showed single cell protein and ethanol has been antagonistic activity against a number of recognized (25). Recently, some amylases phytopathogenic fungi and were considered from terrestrial yeasts also have been found as possible biocontrol agents of post harvest to have the ability to digest raw starch. diseases (50). The strain L47 of A. pullulans However, very few studies exist on the was isolated in South Italy from corpospere amylase-producing marine yeasts (45). of table grape berries and was successfully Although amylase activity produced by
608 Int.J.Curr.Microbiol.App.Sci (2015) 4(10): 605-622 bacteria is much higher than that produced material. A transfructosylating reaction by by A. pullulans, the bacteria only can -fructofuranosidases (FFases; EC 3.2.1.26) produce -amylase (51). from A. pullulans has typically been used to produce FOS. A. pullulans DSM 2404 was Lipases catalyze a wide range of reactions, found to form at least five kinds of FFases in including hydrolysis, inter-esterification, a sucrose medium. The fructosyltransferase alcoholysis, acidolysis, esterification and (FTase) produced by A. pullulans CCY-27- aminolysis. Therefore, lipases, especially 1-94 is stable in a broad range of pH and microbial lipases, have many industrial temperature up to 65°C, with an optimum applications (29). Although lipases from pH 4.4 and temperature of 65°C (53). In Candida rugosa and Candida antartica have addition, it has been found that - been extensively used in different fields, fructofuranosidase and maltosyltransferase very few studies exist on the lipase produced from A. pullulans have many advantages by yeasts isolated from marine environments over those from the bacterial strains and (9). Lipases from A. pullulans are Aspergillus sp. due to no repression of its extracellular. It was also found that the expression by glucose and high crude lipase produced by A. pullulans transfructosylating activity (89). HN2.3 has high hydrolytic activity towards olive oil, peanut oil, soybean oil and lard Mannan and heteromannans are widely (48). Cellulose is the most abundant organic distributed in nature as part of the material on the earth consisting of glucose hemicellulose fraction in plant cell walls. units linked together by -1, 4-glycosidic Mannan consists of -1, 4-linked d- bonds. It has been observed that most of the mannopyranose residues. Mannanases are cultures of A. pullulans have usually failed useful in many fields including bio- to show any cellulolytic activity (6). bleaching of pulp in paper industry, However, some isolates of A. pullulans of bioconversion of biomass wastes to the tropical origin produced CMCase (endo- fermentable sugars, upgrading of animal glucanase) and alphacellulase feed stuff, and reducing the viscosity of (exoglucanase) activity (34). The ability to coffee extracts. They also have potential produce cellulose by different strains of A. applications in the production of manno- pullulans isolated from different marine oligosaccharides, which are utilized environments (93). Unfortunately, it is still selectively by intestinal Bifibacterium completely unknown about species and used as valuable food sweetener cellobiohydrolases in A. pullulans, and the or additive (46). In screening for producers gene encoding -glucosidase in A. pullulans of extracellular -1, 4- mannanase among has not been cloned. Xylanases have many yeasts and yeast-like microorganisms, the applications in paper, fermentation and food best producers were found among strains of industries, as well as in waste treatment. The A. pullulans (33). However, the gene fungus A. pullulans Y-2311-1 was shown to encoding mannanase in A. pullulans have be among the most proficient of the xylan- not been cloned yet. degrading fungi, secreting extremely high levels of xylanolytic enzymes into culture A. pullulans as single cell protein (SCP) media. A variety of microalgae such as Spirulina Fructo-oligosaccharides (FOS) are a class of and Chlorella and brown algae are prebiotics widely used as a functional food extensively used as feed for cultured marine
609 Int.J.Curr.Microbiol.App.Sci (2015) 4(10): 605-622 animals (9; 60). However, they have some several ways as result of their role as limitations for animal consumption. Some ironscavenging compounds, especially yeasts such as Saccharomyces cerevisiae, marine microorganisms because iron is an Candida utilis and Candida tropicalis also essential nutrient for virtually all forms of have been used for their single-cell protein life and is difficult to obtain due to its low (60). They have many advantages over algae solubility in marine environments. It has and bacteria (60; 19). Unfortunately, little is been confirmed that yeasts produce only known about the marine yeasts that have hydroxamate-type compound, while bacteria high protein content and can be used as aqua produce hydroxamate as well as catecholate feed. A total of 327 yeast strains from siderophores (61). Siderophores are also seawater, sediments, mud of salterns, guts of found to have many applications in medical the marine fish, and marine algae were and environmental sciences. They can be obtained. The crude protein of the yeast was used to control growth of the pathogenic estimated and it was found that eight strains bacteria in marine fish and the complexing of the marine yeasts grown in the medium ability of siderophores can be used to with 20 g/l glucose contain more than 30.4 g develop the processes for metal recovery or protein per 100 g of cell dry weight (11). remediation of waste sites, including They belong to Metschnikowa reukaui radioactive waste as they are extremely (2E00001), Cryptococcus aureus effective at solubilizing actinides and other (2E00002), A. pullulans (2E00060), Y. metals from polluted environments (44). lipolytica (2E00004), and Hanseniaspora Most bacterial infections in marine animals uvarum (2E00007), respectively. With the are found to be caused by Vibrio exception of A. pullulans 4#2 (2E00003) parahaemolyticus, Vibrio anguillarum and with nucleic acid of 7.7% (w/w), all other Vibrio harveyi. Therefore, it is very yeast strains contain less than 5% (w/w) of important to find some antibacterial agents nucleic acid. Analysis of fatty acids shows against these pathogens. Although many that all the yeast strains tested have a large antibacterial peptides and killer toxins have amount of C18:0 and C18:1 fatty acids, been found to be active against some while analysis of amino acids indicates that pathogens in marine animals, they are not the yeast strains tested have a large amount stable in marine environments and easily of essential amino acids, especially lysine attacked by proteases produced by marine and leucine which are very important micro-organisms (43; 83). Over 300 yeast nutritive components for marine animals strains isolated from different marine (11). Therefore, A. pullulans that contains environments were screened for their ability high content of protein may be especially to produce siderophore. Among them, one important in single cell protein production yeast strain HN6.2 (2E00149) which was by transforming the waste products such as identified to be A. pullulans was found to starch, protein, cellulose and xylan into cell produce high level of siderophore. Under the protein in A. pullulans. optimal conditions, this yeast strain could produce 1.1 mg/ml of the siderophore. L- Siderophores from A. pullulans Ornithine was found to enhance the siderophore production, while Fe3+ could Siderophores are low-molecular-weight, greatly inhibit the siderophore production. iron-chelating ligands produced by nearly all The crude siderophore produced by the yeast the microorganisms. Siderophores can affect strain HN6.2 is able to inhibit cell growth of microorganisms in the environments in V. anguillarum and V. parahaemolyticus, the
610 Int.J.Curr.Microbiol.App.Sci (2015) 4(10): 605-622 common pathogenic bacteria isolated from an effective biocontrol agent against post- diseased marine animals. This is the first harvest diseases (Mounir et al., 2007). It was time to report that the crude siderophore found that two of A. pullulans (SL250 and produced by the marine-derived yeast can SL236), plus a proven antagonist (isolate inhibit growth of the pathogenic bacteria L47), are able to control Penicillium isolated from marine animals (84). The first digitatum on grapefruit, Botrytis cinerea, step in siderophore biosynthesis is the N5- Rhizopus stolonifer, and A. niger on table hydroxylation of ornithine catalyzed by grape and B. cinerea and R. stolonifer on orinithine N5- oxygenase. The further cherry tomato. reactions of siderophore biosynthesis are catalyzed by non-ribosomal peptide The yeast-like fungus A. pullulans strain synthetases (27). The presence of Fe3+ in the Ach1-1 was applied to control mold growth medium can greatly repress the expression on apple caused by Penicillium expansum of the gene encoding ornithine N5- (5). The competition for apple nutrients, oxygenase (11), while the presence of L- most particularly amino acids, may be a ornithine can enhance the exression of the main mechanism of the biocontrol activity gene encoding ornithine N5-oxygenase in A. of A. pullulans strain Ach1-1 against blue pullulans HN6.2.( 84). mold caused by P. expansum on harvested apple fruit. Micromycetes capable of Biocontrol from A. pullulans developing on lubricants was identified of various origin and nature, used in various Currently, fungicide treatments represent the industrial applications to investigate the primary method for the control of post- reaction of micromycetes on different oil harvest diseases of fruits and vegetables. products and to discuss the possibilities to However, public concern about fungicide use the obtained species of micromycetes for residue and development of fungicide environment protection from intensive resistant isolates of post-harvest pathogens pollution with lubricants and their wastes. have promoted the search for alternative Different micromycetes reacted differently means, less harmful to human health and to to the impact of various lubricant the environment. In recent years, components. They found that A. pullulans considerable success has been achieved responded somewhat poorly as compared to utilizing microbial antagonists to control other fungus (49). post-harvest diseases. Because the infection of fruits by post-harvest pathogens often Pullulan from A. pullulans occurs in the field prior to harvest, it may be advantageous to apply antagonists before Pullulan, which is a linear -D-glucan made harvest. For this approach to be successful, mainly of maltotriose repeating units putative biocontrol strains must be able to interconnected by (1, 6) linkages, is a tolerate low nutrient availability, UV-B water-soluble homopolysaccharide produced radiation, low temperatures, and climatic extracellularly by the polymorphic changes. The yeast-like fungus A. pullulans micromycete A. pullulans (Sutherland, is one of the most widespread and well- 1998). The regular alternation of -1, 4 and adapted saprophytes, both in the -1, 6 bonds results in two distinctive phyllosphere and in the carposphere. A. properties of structural flexibility and pullulans has a high tolerance to dessication enhanced solubility (38). This and irradiation and has been considered as polysaccharide is of economic importance
611 Int.J.Curr.Microbiol.App.Sci (2015) 4(10): 605-622 with increased application in food, has been commercially produced since 1976 pharmaceutical, agricultural and chemical by the Hayashibara Company Ltd industries (17; 78; 70; 73; 21; 75). Pullulan (Okayama, Japan), which remains the produces a high viscosity solution at a principal supplier. Recent arrangements with relatively low concentration and can be used Pfizer for production of oral care strips may for oxygen impermeable films and fibers, result in expanded markets for pullulan (21). thickening or extending agents, or adhesive or encapsulating agents (73). Despite being Despite of large number of uses, some of the a -D-glucan, pullulan is resistant to -D- problems associated with fermentative amylolysis and may be used in low-calorie production of pullulan are (i) the formation food formulation. The chemical formula of of melanin pigment (ii) the inhibitory effects pullulan is (C6H10O5).H2O. Many authors (2; caused by high sugar concentrations in the 69) have reported that sulfated pullulan and medium and (iii) cost associated with phosphorylated pullulan have an pullulan precipitation and recovery. (52). anticoagulant, antithrombotic and antiviral Due to these problems, the application was activities, and chlorinated, actually limited due to the unavailability of sulphinylethylated, etherified, carboxylated, better strains of A. pullulans, as well as, the acetylated and esterified pullulan can be maintenance of yeast-like cells during used as important materials for chemical fermentation due to polymorphic nature of industries. So it becomes very important to the organism. The quest for optimization of search for better pullulan-producing yeast microbial metabolite is an essential strains. It is now widely accepted that requirement for the production of microbial pullulan is a linear polysaccharide with metabolites such as, polysaccharide, maltotriosyl repeating units joined by -(1, enzyme, antibiotic etc. The major attention 6)-linkages. Alternatively, the structural in the fermentation studies of A. pullulans formula of pullulan may be presented as a was devoted to develop optimal cultivation regular sequence of panoses bonded by -(1, conditions while maintaing a high 4)-linkages. Minor structural abnormalities productivity of the cells. The main are reported in pullulan. A careful objectives were high yield, short hydrolysis of pullulan by exo-and fermentation time, low cost, and high purity endoenzymes showed chain fragments of the final product to meet-out the stringent resistant to the action of enzymes. Such requirements for food, cosmetics and resistance was attributed to the presence of pharmaceutical applications (55). maltotetraose residues distributed randomly along the pullulan chain. However, these Biosynthesis of pullulan structural abnormalities should not affect the overall physico-chemical properties of Exopolysaccharide from A. pullulans pullulan (8). Even in the first work on pullulan The producer of pullulan, A. pullulans, is a biosynthesis, researchers observed that the black yeast widely spread in all ecological culture produced two different niches including forest soils, fresh and sea exopolysaccharides. One of these polymers water, plant and animal tissues, etc. corresponds to pullulan, and the second is Generally, the culture of A. pullulans is frequently described as a water-insoluble classified as non pathogenic; however, some jelly-like material. An electron microscopy strains may cause health problems. Pullulan study revealed that both pullulan and the
612 Int.J.Curr.Microbiol.App.Sci (2015) 4(10): 605-622 insoluble polysaccharide are localized on an overwhelming number of studies, pullulan outer surface of the chlamydospores, the elaboration was found to occur only with the cells that were considered as the main yeast-like morphology of A. pullulans (7), polysaccharide producer on non growth whilst in other several papers the ability to media. The highly dense peripheral layer synthesize the polysaccharide was the was ascribed to the chain of pullulan characteristic of the chlamydospore arranged in a network covering the inner population (71). At least there is convincing layer of -(1, 3)-glucan composed of agreement among researchers that pH glucose and mannose (71). Very little is provokes morphological changes of cells, known up to now explaining how the which in turn may additionally differentiate mechanism of biosynthesis of these jelly- biosynthesis routes. The yeast-like cells at like glucans is associated with the pullulan neutral pH produce pullulan of a very high elaboration, though there were indications molecular weight (42), whilst combined that the elaboration of insoluble cultivation of the mycelial and the yeast-like exopolysaccharide is dependent on genetic cellular forms can be beneficial for high type of A. pullulans. In particular, it is not pullulan concentration (63). clear yet whether environmental conditions, for example, pH or morphological changes Mechanism of pullulan biosynthesis of the cells are responsible for its extracellular elaboration (30). Although many investigations on biochemical mechanisms of Influence of pH and cell morphology exopolysaccharide biosynthesis in bacteria have been carried out (16), relatively little is Relatively low initial pH (pH 2.5) understood about the mechanisms of suppresses synthesis of pullulan but pullulan biosynthesis in A. pullulans. If the stimulates elaboration of the insoluble pullulan biosynthesis and regulation in A. glucan. An optimal value of pH for the pullulans are elucidated, it will be very easy pullulan production lies in the range to enhance pullulan yield using molecular between 5.5 and 7.5 (42). There were only a methods. Pullulan can be synthesized from few reports where highest pullulan content sucrose by cell-free enzymes of A. pullulans was achieved by cultivating the when both adenosine triphosphate (ATP) microorganism in acidic pH. It is of interest and uridine diphosphate (UDP)-glucose are to note that the optimal pH established for added to a reaction mixture (70). The size of the biomass growth is 4.5 or lower. This UDP-glucose pool and glucosyltransferase difference in optimal values of pH for the activity in the cell of A. pullulans Y68 pullulan synthesis and cell mass growth obtained in their laboratory may be indirectly correlates with the independent correlated with high pullulan production character of these two processes (32). (11). Therefore, effects of different sugars However, the relationship between on pullulan production, UDP-glucose morphology and the polysaccharide- (UDPG) pool, and activities of - producing capacity of the culture cannot be phosphoglucose mutase, UDPG- ignored since the polysaccharide elaboration pyrophosphorylase, and glucosyltransferase is known to be associated with the specific in the cells of A. pullulans Y68 were cell morphology (71), though the exact investigated (18). It was found that more cellular type responsible for pullulan pullulan is produced when the yeast strain is synthesis is still a matter of debate. In an grown in the medium containing glucose
613 Int.J.Curr.Microbiol.App.Sci (2015) 4(10): 605-622 than when it is cultivated in the medium studies of A. pullulans was devoted to supplementing other sugars. However, when developing optimal cultivation conditions more pullulan is synthesized, less UDP- while maintaining a high productivity of the glucose is left in the cells of A. pullulans cells. The main objectives were high yield, Y68 (11). High pullulan yield is positively short fermentation time, low cost, and high related to high activities of - purity of the final product to meet the phosphoglucose mutase, UDPG- stringent requirements for food, cosmetics pyrophosphorylase, and glucosyltransferase and pharmaceutical applications (70). in A. pullulans Y68 grown on different Although the literature on pullulan sugars. A pathway of pullulan biosynthesis biosynthesis is contradictory because of in A. pullulans Y68 was proposed based on differences among the numerous strains of different studies (18; 11). It is thought that A. pullulans, it was clearly demonstrated the lower amount of pullulan produced by A. that the yield of pullulan strongly depends pullulans Y68 from fructose and xylose may on the rate of substrate conversion (32). be caused by the longer biosynthetic Moreover, the concentration of the pathway leading from fructose and xylose to polysaccharide produced by A. pullulans is UDP-glucose. It is thought that most of dependent on the carbon source. It is UDP-glucose is used to synthesize pullulan important to note that the exopolymer when the glucosyltransferase activity is very synthesis was observed on glucose, sucrose, high, leading to very low UDP-glucose level fructose and maltose. On a medium in the yeast cells. This may imply that very containing maltose as a carbon source, the high glucosyltransferase activity is the wild fungus intensively grew, but had low unique characteristic of A. pullulans Y68 pullulan producing activity. The problem of which can produce high yield of pullulan. low pullulan producing activity was solved Because the phosphoglucomutase and later by the development of several mutant UDPG-pyrophosphotylase activity in the strains of A. pullulans with improved ability yeast cells grown in the medium containing to synthesize pullulan (54; 86). By using glucose is also very high, UDP-glucose is these cultures, it became possible to perform synthesized continuously to supply the large-scale fermentation processes under precursors for high pullulan synthesis when well-controlled conditions. Progress was the very high glucosyltransferase activity particularly stimulated by the development occurs in the cells of A. pullulans Y68. of new fermentation reactors designed to However, high level of UDP-glucose is left maintain high productivity of the culture. In when the yeast cells are grown in the addition, visual inspection methods (26) and medium containing xylose and fructose, several analytical techniques, including respectively, due to low glucosyltransferase capillary electrophoresis and high activity. Therefore, it is believed that the performance liquid chromatography (4; 87), proposed pathway of pullulan biosynthesis were applied successfully to monitor will be helpful for the metabolism changes in the cell morphology and engineering of the yeast strain to further carbohydrate composition of the cultivation enhance pullulan yield. broth. In order to reduce the cost of the fermentation product, pullulan biosynthesis Substrates and efficiency of pullulan from the hydrolyzates of potato starch waste fermentation was studied (4). Fermentation of A. pullulans on a medium containing 20% The major attention in the fermentation maltose-rich hydrolyzates yields 115%
614 Int.J.Curr.Microbiol.App.Sci (2015) 4(10): 605-622 higher concentration of pullulan than that pullulan was obtained with the use of obtained on glucose syrup, indicating that agarose-entrapped culture. Other researchers maltose is a better substrate than glucose for noticed that this method is inconvenient for pullulan production by the studied strain of pullulan production because of the several A. pullulans. Other wastes from the undesirable events, including the restriction agricultural and food industries such as of polysaccharide diffusion through deproteinized whey (62), beet molasses (37), microporous sorbents and the destruction of sugar cane juice, and even peat hydrolyzate the immobilization system due to a rapid (41) are also considered as economical and increase of the entrapped biomass (80). efficient substrates for the pullulan production. An exhaustive literature survey Another approach to stabilize growth devoted to the use of different industrial conditions and thereby increase pullulan wastes for pullulan production and the yield is the use of continuous fed-batch problems associated with the recovery and cultivation. An optimization of fed-batch characterization of the final product has cultivation was performed by the been presented (37). investigation on the effect of feed mode and composition of the feed solution on the Fermentation techniques efficiency of pullulan fermentation. The fed- batch culture gives high pullulan yields; The influence of aeration on vital activity of however, the higher rates of pullulan cells producing pullulan was studied in production and substrate uptake are detail (17). Under anaerobic conditions, the characteristics of the traditional batch cell population neither grows nor produces cultivation. Recently, a novel fermentor pullulan. An intense aeration during system for continuous production of fermentation leads to a significant increase pullulan. This novel design could serve as an of pullulan concentration. This effect is excellent fermentor system in industries for especially pronounced on a nitrogen-rich large scale continuous production of media. An inverse effect of aeration was pullulan (74). detected upon fermentation on the media deficient in nitrogen source, where intense Production of pullulan from agro- aeration suppressed pullulan production. An industrial wastes increase in oxygen transfer rate achievable by increasing a gas partial pressure may Pullulan can be synthesized from a variety improve the polysaccharide producing of carbohydrate substrates incorporated into activity of A. pullulans. High airflow rates either defined (synthetic) or non-defined and high working pressure is beneficial for media. Within the latter are several agro- the growth of cell mass and pullulan industrial wastes which have been shown to synthesis. In order to prevent cell be suitable for pullulan production. disintegration, cell immobilization Utilization of these substrates would seem to procedures were applied to pullulan be ecologically sound and economically production (80). The elaboration of pullulan advantageous as they have low or even using cells of A. pullulans entrapped in negative costs. In this way the potential of agarose and carrageenan was studied (85). pullulan production from agro-industrial Both immobilized systems were found wastes is expected to lower the cost of technically acceptable for pullulan production and seems to be a very promising production; however, the highest content of ecologically and economically sound way of
615 Int.J.Curr.Microbiol.App.Sci (2015) 4(10): 605-622 bioconversion. However, the pullulan which besides playing an important role in bio- is produced from different substrate may deterioration and in controlling vary in purity and other physical and environmental pollution. More than 300 chemical characteristics. This is more patents describing the production and use of pronounced when agro-industrial wastes are pullulan and pullulan derivatives are known. used as a carbon source for the fermentation. Although a detail study has been done Some of these characteristics like the regarding the morphology, distribution and molecular weight are very important when economic importance of this fungus still, we commercialization of the production of think that, a lot of investigations are needed pullulan from these wastes is under for better exploitation of this economically consideration (31). important fungus.
The cost of pullulan primarily depends on Acknowledgement the raw materials, especially of carbon source, which play a major role in the The corresponding author (Ranjan Singh) is economics of pullulan production. The sugar thankful to the Head, Department of Botany such as sucrose, glucose, fructose, maltose, and Microbiology, St. Aloysius College starch, or malto oligosaccharides support (Autonomous), Jabalpur, M.P., India for the pullulan production by A. pullulans. There necessary laboratory facility provided to are various reports on the production of carry out the research. Thanks is also pullulan from different sources such as extended to Principal, St. Aloysius College sweet potato (88), soyabean pomace (68), (Autonomous), Jabalpur, M.P., India, for potato starch waste (4), deproteinized whey providing the necessary chemicals and glass (62), agro-industrial waste such as grape wares required to carry out the research skin pulp extract, starch waste, olive oil work. waste effluents and beet molasses (31), brewery waste (64), jagerry which is Reference concentrated sugar cane juice (82), carob pods (63) and Jerusalem artichoke (22; 52). Andrews, J.H., Spear, R.N., Nordheim, E.V. The production pullulan was investigated Population biology of Aureobasidium using various coconut by-products and it has pullulans on apple leaf surface. advantages such as reduction of production Canadian J. of Microbiol. 2002; 48(6): cost and recycling of natural sources, in 500-13. addition the effect of initial pH, Alban, S., Schauerte, A., Franz, G. fermentation time and nitrogen sources were Anticoagulant sulfated investigated in batch fermentation. This is polysaccharides: part I. Synthesis and the first report using coconut by-products for structure-activity relationship of new the production of pullulan (79). pullulan sulfates. Carbohydr. Polym. 2002; 47: 267-276. Conclusion Babjeva, I., Reshetova, I. Yeast resources in natural habitats at polar circle latitude. Aureobasidium pullulans is an industrially Food Technol. Biotechnol. 1998; 36: important fungus which produces a number 8-13. of by-products including several enzymes, Barnett, C., Smith, A., Scanlon, B., single cell protein and an industrially Israilides, C.J. Pullulan production by important polysaccharide namely pullulan Aureobasidium pullulans growing on
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