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Pullulan The Magical Polysaccharide

By Ranjan Singh

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Dedecation

I dedecate my this work to my Late Grand Father Dr. Ram Pratap Singh, Father Mr. Madhuker Singh, Mother Mrs. Sudha Singh, my family and teachers and to my respected and learned guide Dr. Rajeeva Gaur.

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Acknowledgment Doing a work in the area of Science is tough and tideous and it is not possible to complete the work without the help of many peoples who are directly or indirectly involved in the work. In fact it is teamwork. Though it will not be enough to express my gratitude in words to all those people who helped me, I would still like to give my many, many thanks to all these people. First of all I would like to express my gratitude and thanks to the almighty who blessed and encouraged me to complete this work with an enjoyable experience. I would like to express my sincere and heartiest gratitude to Rev. Father Dr. G. Vazhan Arasu, Principal, St. Aloysius College (Autonomous), Jabalpur, M.P., India for allowing me to work in the college and for providing all the necessary facilities required to complete the research work. Without his support and blessings it would not have been possible to complete the work. I would also like to express my sincere gratitude to Rev. Father J. Ben Anton Rose, Vice-Principal, St. Aloysius College (Autonomous), Jabalpur, M.P., India for his motivation and blessings to complete the work. I would also like to thank Dr. (Mrs.) Shikha Bansal, Head, Department of Botany and Microbiology, St. Aloysius College, (Autonomous) Jabalpur, M.P., India for providing the necessary laboratory to carry out the work. Without her constant guidance and encourgment it would not have been possible to complete the work. I would like to thank my respected and learned guide Dr. Rajeeva Gaur, Associate Professor, Department of Microbiology, Dr. Ram Manohar Lohia Avadh University, Faizabad, U.P., India who gave me knowledge and guided me during my research work at the university. I extend my thanks to my student Ms. Pritha Biswas, who helped me to carry out many experiments and in analysis of data and culture maintance. Without her sincere efforts it would not have been possible for me to complete the work. I have no words to acknowledge my regards and sincere thanks to my all family members who have shown immense patience throughout my work and flourish me with their cordial support, blessings, constant encouragement and affection throughout the period of my work. I pay my humble dedication to my Grand Father, Late Mr. Vishnu Chandra Singh and Late Grand Mother Mrs. Leelawati Devi. I am sincerely thankful to my Father Mr. Madhuker Singh; Mother Mrs. Sudha Singh, Nana Late Dr. Ram ϯ 

Pratap Singh and Nani Late Mrs. Vansraji Devi, Tau Mr. Shivaji Singh, Tai Mrs. Mala Singh, Brothers Mr. Vijay Singh and Dr. Anshuman Singh and Bhabhis Mrs. Monica Singh and Mrs. Manika Singh. I am also thankful to my sister Miss Arunima Singh. I am also thankful to my betterhalf Mrs. Ranjana Singh, who always encouged me and always supported me during my work. Thanks are also extended to Siddharth and Samarth my two young nephews and to my niece Molu. I would like to extend my thanks to Dr. (Mrs.) Mamta Gokhale, Dr. (Mrs.) Sonali Nigam, Dr. (Mrs.) Femina Sobin, Mrs. Roshni Choubey and Ms. Shikha Gauri for their kind co-operation during the work. Lastly I would extend my thanks to lab attendants Mr. Ashish and Mr. Silas for their ever ready and helping attitude.

Date: Place:

(Dr. Ranjan Singh)

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Index

S. No. 1 2 3 4 5 6 7 8 9 10 11 12 13

Contents List of Tables List of Figures Introduction Review of Literature Objective Materials and Methods Result Discussion Summary Conclusion Future Prospects References Annexure

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Page No. 6 7 9-13 14-31 32 33-37 38-56 57-63 64-66 67 68 69-83 84

List of Tables

S. No. 1 2 3 4 5 6 7 8

List Of Tables Screening of isolates on solid media Effect of different incubation time on pullulan production Effect of different temperature on pullulan production Effect of pH on pullulan production Effect of different carbon sources on pullulan production Effect of different sucrose concentration on pullulan production Effect of different nitrogen sources on pullulan production Effect of different yeast extract on pullulan production

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Page No. 38 43 45 47 49 51 53 55

List of Figures

S. No. 1 2 3 4 5 6 7 8 9 10 11 12

List of Figures Chemical structure of the secondary (minor) repeating structure of pullulan Isolation of Aureobasidium pullulans from plant parts Colonies of Aureobasidium pullulans on basal agar plate Colonies of Aureobasidium pullulans (Yeast Form) on basal agar plate Motic images of Aureobasidium pullulans Effect of different incubation time on pullulan production Effect of different temperature on pullulan production Effect of pH on pullulan production Effect of different carbon sources on pullulan production Effect of different sucrose concentration on pullulan production Effect of different nitrogen sources on pullulan production Effect of different yeast extract on pullulan production

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Page No. 24 34 40 41 42 44 46 48 50 52 54 56

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INTRODUCTION Aureobasidium pullulans (De Bary) G. Arnound is a ubiquitous, polymorphic and oligotrophe black yeast-like microfungus that occurs frequently in a wide range of tropical and temperate environments. It is well known as a naturally occurring epiphyte or endophyte of a wide range of plant species (eg. apple, grape, cucumber, green beans, and cabbage) without causing any symptom of disease (Andrews et al., 2002). Chronic human exposure to A.pullulans via humidifiers or air conditioners can lead to hypersensitivity pneumonitis or “humidifier lung”. This condition is characterized acutely by dyspnea, cough, chest infilterates and acute inflammatory reaction. The strains causing infections in humans were reclassified to A. melanogenum (Gostincar et al., 2014) A. pullulans can occur in wide range with fluctuating moisture contents such as phyllosphere, damp indoor surfaces, degrading organic matters in soil and other substances (Samson et al., 2004). It has also been isolated from the osmotically stressed environments like hyper saline waters in salterns (Gunde-Cimerman et al., 2000) and the rocks. The fungus produces melanin and hence popularly known as ‘Black Yeast’. The formation of dark-coloured chlamydospores is the characteristic feature of this fungus (Ramos and Garcia-Acha, 1975). Earlier, A. pullulans was identified as Dematium pullulans De Bary (1884) and Pullularia pullulans (De Bary) Berkhout (1866). However, further reports include it under Ascomycota though; perfect stage has not yet been found (de Hoog and McGinnis, 1987). In recent classification, A. pullulans is regarded as Ascomycetous yeast and is placed in the Order Dothideales, Family Dothideaceae (Yurlova et al., 1999). Several Aureobasidium like black yeasts were isolated from glacial and subglacial ice in the coastal Arctic habitats and the adjacent sea water (Zalar et al., 2008). From the available records, it is apparent that this fungus is most common in temperate zone with numerous records both from the British Isles and the United States including ϵ 

Alaska, Antarctica, Denmark, Germany, Netherlands, Poland, Austria, Czech Republic, Russia and Japan. There are several reports of its occurrence in Mediterranean and arid zones including Italy, France, Egypt, Iraq, Pakistan and South Africa. It has also been extensively isolated from tropical and subtropical region namely, Brazil, India, China, Thailand, Malaysia, Jamaica (West Indies), etc. An extensive list of the habitats and geography from which strains of A. pullulans have been isolated is well documented by Leathers (1993) and Zalar et al. (2008). The fungus has been frequently isolated from moorland, peat bogs or peat podzol and forest soils. Therefore, it is considered as omnipresent fungus of the ecosystem and has been exploited potentially for the production of Pullulan, a polysaccharide. No other fungus has been reported to produce pullulan up to the level of A. pullulans. Beside this, there are several other genra and species viz. Tremella mesenterica, Cyttaria harioti, Cyttaria darwinii, Cryphonectria parasitica and Rhodotorula bacarum which have been known to produce pullulan in low quantity (Corsaro et al., 1998; Chi and Zhao, 2003). Recently, Gaur et al. (2010a) have isolated a novel fungus, Eurotium chevalieri (MTCC No. 9614) from the garden soil, which produced pullulan up to the level of A. pullulans. Pullulan comprises of the maltotriosyl units (poly-Į-1, 6 maltotriose) with the chemical formula as (C6H10O5).H2O. Three glucose units in maltotriose are connected by a Į-1, 4 glycosidic bonds, whereas consecutive maltotriose units are inter-connected to each other by Į-1, 6 glycosidic bond. This unique linkage pattern endows pullulan with two distinctive properties, the structural flexibility and enhanced solubility in water. Pullulan is a promising biomaterial and potentially used as a low-calorie ingredient in the foods, as a viscosity imparter and binder and as a packing agent because of its low oxygen permeability (Rekha and Sharma, 2007; Singh et al., 2008). It is a white powder that is odorless, flavorless and highly stable, water soluble but insoluble in organic solvents and non-hygroscopic. Its aqueous solution is stable and shows a relatively low viscosity as compared to the other polysaccharides. It forms thermo-stable, transparent, elastic, antistatic films with an extremely low oxygen permeability. It is moldable, ϭϬ 

spinnable, a good adhesive and binder. It is a non-toxic, non-mutagenic, edible, biodegradable and biocompatible, and has high resistance to temperature in the range of 200-220oC. Pullulan possesses several potential applications in cosmetics, diets, food, pharmaceutical and manufacturing industries as flocculants, adhesives, binder etc. (McNeil and Kristiansen, 1990; Leathers, 2003; Shingel, 2004). Being a non starch polysaccharide it can be used in food primarily as binder and thickener. Due to its low viscosity pullulan can also be used as low viscosity filler in beverages and sauces. Its adhesive nature is exploited for making of confectioneries like, chocolates, candies, nut cookies, lollipop toffee etc, and also as protective glazing agent in food product. In pharmaceutical its film forming ability is exploited for making oral strip where colours flavour and functional ingredient can be entrapped in the film matrix and effectively stabilized. Further this film forming ability is also utilized for making of capsule and soft gel capsule. It can also be used as bulking agent in tablets. Pullulan coating of dietary supplement tablets and soft gel helps to maintain the stability of active ingredient which is prone to oxidation. The regular introduction of Į-(1-6) linkage in pullulan usually interrupts, which would otherwise be a linear amylose chain resulting in distinct film-and fibre-forming characteristic that allow pullulan to mimic synthetic polymers derived from petroleum. Pullulan is also being used for the production of biodegradable plastics in Japan and U.S.A., because it resembles with polyethylene properties like tensile strength and ability to form thin transparent oxygen impermeable film. More than 300 patents for its applications have been worked out (Leathers, 2002). Hayashibara Co. Ltd. (Japan) is the main commercial producer of pullulan producing approximately more than 300 metric tons per year. The wholesale price of food-grade pullulan (PF-20) in Japan is nearly 20 US Dollars/kg, and pharmaceutical grade (deionozed) pullulan (PI-20) is sold for approximately 25 US Dollars. The other companies producing pullulan at commercial ϭϭ 

level are NOF Corporation (USA), Advance Enzyme technologies Ltd. (USA), Yushisein Co. Ltd. (Japan) and Sanging Biological Products (Japan) (Leathers, 2003). Despite of large number of uses, some of the problems associated with fermentative production of pullulan are (i) the formation of melanin pigment (ii) the inhibitory effects caused by high sugar concentrations in the medium and (iii) cost associated with pullulan precipitation and recovery (Oguzhan and Yangilar, 2013). Due to these problems, the application was actually limited due to the unavailability of better strains of A. pullulans, as well as, the maintenance of yeast-like cells during fermentation due to polymorphic nature of the organism. The quest for optimization of microbial metabolite is an essential requirement for the production of microbial metabolites such as, polysaccharide, enzyme, antibiotic etc. The major attention in the fermentation studies of A. pullulans was devoted to develop optimal cultivation conditions while maintaing a high productivity of the cells. The main objectives were high yield, short fermentation time, low cost, and high purity of the final product to meet-out the stringent requirements for food, cosmetics and pharmaceutical applications (Ponnusani and Gusasekar, 2014). Some important parameters that control the production of pullulan from A. pullulans are temperature, initial and final pH of the medium, oxygen supply, viscosity, concentration of nutrients along with kind of carbon and nitrogen sources (Lazaridou et al., 2002a; Chi and Zhao, 2003; Cheng et al., 2009). Most of the studies have been conducted on the production of pullulan by mesophilic A. pullulans while the fate of thermophilic species of A. pullulans for pullulan production has not yet been taken up sincerely. The morphological forms of a thermotolerant A. pullulans for pullulan production are an important aspect during fermentation. The yeast phase of growth in the fermentor at specific temperature and pH was reported to produce higher pullulan yield. However, some reports have also indicated that chlamydospores and swollen cells are responsible for pullulan production. (Simon et al., 1995; Li et al., 2009). Therefore, optimization of all the said parameters for pullulan production by the newer isolate is essential viz., carbon sources (sucrose, ϭϮ 

glucose, fructose, lactose, maltose, galactose etc.), nitrogen sources (ammonium sulphate, yeast extract, sodium nitrate, sodium nitrite, histidine, glycine etc.) and micronutrients (copper sulphate, zinc sulphate, magnesium sulphate, sodium chloride etc.) at different incubation period and temperature are being used in production of pullulan under aerobic conditions (Chi and Zhao, 2003; Cheng et al., 2009). The present investigation was concentrated on the optimization of physicochemical and nutritional parameters for pullulan production by A.pullulans in flask type fermentation system.

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REVIEW OF LITERATURE (I) Classification, Morphology and distribution of A. pullulans Aureobasidium pullulans (De Bary) G. Arnoud is a ubiquitous, polymorphic and oligotrophe black yeast-like microfungus that occurs frequently in a wide range of tropical and temperate environments. Earlier, A. pullulans was identified as Dematium pullulans De Bary (1884) and Pullularia pullulans (De Bary) Berkhout (1866). The genus Aureobasidium was established in 1891 by Viala and Boyer who described A. vitis as a common colonizer of the sugary grape surfaces. Using the criteria of conidiogenesis i.e., synchronous holoblastic conidial production, A. pullulans was placed previously under Fungi Imperfecti, Order Moniliales, Family Dematiaceae (Ramos and Garcia-Acha, 1975; Hermanides-Nijhof, 1977). However, further reports include it under Ascomycota though; perfect stage has not yet been found (de Hoog and McGinnis, 1987). In recent classification, A. pullulans is regarded as Ascomycetous yeast, and is placed in the order Dothideales, Family Dothideaceae (Deshpande et al., 1992; Yurlova et al., 1999). The fungus was taxonomically characterized by de Hoog and Yurlova (1994) on the basis of its morphology and nutritional physiology. Because of the variability in morphological and physiological characteristics, the species has been described with three different varieties such as A. pullulans var. pullulans (Viala and Boyer, 1891), A. pullulans var. melanogenum (Hermanides-Nijhof, 1977) and A. pullulans var. aubasidani Yurlova (Yurlova and de Hoog, 1997). Zalar et al., (2008) have found four strains of artic Aureobasidium i.e. A. pullulans var. pullulans, A. pullulans var. melanogenum, A. pullulans var. subglaciale and A. pullulans var. namibiae. A. pullulans is found in fluctuating moisture contents such as phyllosphere (Andrews et al., 1994), damp indoor surfaces, degrading organic matters in soil and food and feed substances (Samson et al., 2004). It can also be found in osmotically stressed environments like hyper saline waters in salterns (Gunde-Cimerman et al., 2000), and ϭϰ 

the rocks (Urzi et al., 1999). This fungus disperses easily due to production of yeast-like propagules in large quantity and found globally though, it has rarely been reported in cold environments because the investigations on fungal diversity are limited to frozen Antarctic soils and Siberian permafrost where basidiomycetous yeasts were found (Babjeva and Reshetova, 1998; Golubev, 1998). In the recent past a number of Aureobasidium like black yeast were isolated from glacial and sub glacial ice in coastal artic habitats and the adjacent sea water (Zalar et al., 2008). Multilocus molecular analysis revealed lower CFU numbers of Aureobasidium from these ice samples. A .pullulans exhibits polymorphism for it can grow as budding yeast or as mycelia depending upon environmental conditions. The life cycle of A. pullulans was thoroughly reviewed by Cooke (1959), Hermanides-Nijhof (1977) and de Hoog and Yurlova (1994). The formation of dark-coloured chlamydospores is the characteristic feature of this fungus (Ramos and Garcia-Acha, 1975). Ramos and Garia-Acha (1975) have described the vegetative cycle of A. pullulans and the optimal conditions for the conversion of one morphological form to other. The colonies are initially smooth and eventually become covered with slime. Starting as yellow cream, light pink or light brown, the colonies finally become blackish due to the production of chlamydospores. When observed under light microscope, the hyphae look hyaline, smooth and thinwalled, 2-16 ȝm wide with cells often wider than long forming rough and compact mycelia. It can be recognized by straight conidia and the presence of lobed chains of thick-walled chlamydospores. It is a fast growing fungus, the colonies of which develop up to 3 cm in diameter within 3 days of incubation period. At 25oC, when grown on Sabouraud’s Dextrose Agar medium containing plates, the colonies appear as creamy, moist, initially white but later becoming black (often in sectors) with colouration on reverse side of the plates. A. pullulans is differentiated from other dark pigmented fungi by its initial white-pink colonies later turning black with blastoconidia produced synchronously in tufts, and formation of dark chlamydospores. ϭϱ 

Guterman and Shabtai (1996) revealed five basic morphologies of A. pullulans which are yeast like cells, young blastospores, swollen blastospores, chlamydospores and mycelia in batch fermentation. The swollen blastospores are of the similar shapes but large (12-15 ȝm) with a granular texture. The chlamydospores are round, large (1725 ȝm), heavily pigmented and in most cases appear bacillary. Filamentous mycelia are long and partially composed of pigmented chlamydospores (Kumar et al., 2003). A. pullulans is commonly isolated from the environmental samples (Cooke, 1959; Hermanides-Nijhof, 1977). From the available records it is apparent that this fungus is most common in temperate zone with numerous records both from the British Isles and the United States of America including Alaska, Antarctica, Denmark, Germany, The Netherlands, Poland, Austria, Czech Republic, Russia and Japan. There are several reports of its occurrence in Mediterranean and arid zones including Italy, France, Egypt, Iraq, Pakistan and South Africa. It has also been extensively isolated from the tropical and subtropical regions viz. Brazil, China, India, Thailand, Malaysia and Jamaica (West Indies) etc. An exhaustive list of the habitats and geography from which A. pullulans had been isolated and had been documented by Domsch et al. (1980) and Zalar et al. (2008). The fungus has been frequently isolated from moorland, peat bogs or peat podzol and forest soils. Other noteworthy habitats include fresh waters, esturine and marine sediments and sea water (Domsch et al., 1980; Leathers, 1993). Lis and Andrews (1997) studied the occurrence and distribution of A. pullulans by fluorescence dye, and found it frequently on the phylloplane of several plants. Kuter (1986) isolated A. pullulans from green and senescent sugar maples, but it did not seem to be the inducer and biodeteriorator. Slavikova and Vadkertiova (1997) have also reported the seasonal occurrence of A. pullulans from March to April in the river Danube. Punnapayak et al. (2003) have also isolated this fungus from the air in several locations of Thailand while Prasongsuk et al. (2005, 2007) have reported the strains of this taxon from different habitats including plant leaves to painted surfaces in Thailand. Similarly, Haifeng et al. ϭϲ 

(2007) isolated A. pullulans strain N13D from deep sea sediments of the Pacific Ocean. In India, Singh and Saini (2007) have also isolated A. pullulans from phylloplane of Ficus sp., while Zamora et al. (2008) isolated A. pullulans from needles and twigs of the pine plantation from the northern Spain. Aureobasidium pullulans has been employed for different useful purposes as it produces a variety of important metabolites, viz., enzymes, antibiotics, single cell protein (SCP) and polysaccharides (Leathers, 2003). Tests have shown it to be safe for the use as SCP (Han et al., 1976; Zajic et al., 1979). Due to ubiquitous nature of this fungus on phylloplane, any change in its occurrence might prove to be an indicator of environmental perturbations generated by chemicals or other biological organisms (including genetically enginnered organisms) landing on the leaf surfaces. Zacchi and Martini (2003) reported that the distribution of A. pullulans within a population of soft scale insects (Saissetia oleae, Rhyncota coccidae) varied significantly as a function of development age. This observation may be consistent with the hypothetical role of microbial endosymbionts in favoring the host growth and development. Some of the isolates of A. pullulans showed antagonistic activity against a number of phytopathogenic fungi (Andrews et al., 1983) and were considered as possible biocontrol agents of the postharvest diseases (Leibinger et al., 1997). The strain L47 of A. pullulans was isolated in south Italy from corpospere of table grape berries and was successfully applied to control postharvest diseases of different fruits and vegetables. This strain provided high protection levels against P. digitatum on grape fruit, Botrytis cinerea, Aspergillus niger and Rhizopus stolonifer on grape, B. cinerea and Monilia laxa on sweet cherry and B. cinerea on kiwi fruit, cherry, tomato, apples and strawberries (Schena et al., 1999, 2002). (II) Economic importance of A. pullulans A. pullulans has been employed for different useful purposes as it produces a variety of important metabolites, enzymes, antibiotics, single cell protein (SCP) and polysaccharides (Chi et al., 2009). Tests have shown it to be safe for the use as SCP ϭϳ 

(Chi et al., 2008). A. pullulans has shown the potential in controlling and monitoring environmental pollution. Due to ubiquitous nature of this fungus on phylloplane, any change in its occurrence might prove to be an indicator of environmental perturbations generated by chemicals or other biological organisms (including genetically engineered organisms) landing on the leaf surfaces. Some isolates of A. pullulans showed antagonistic activity against a number of phytopathogenic fungi and were considered as possible biocontrol agents of post harvest diseases (Mounir et al., 2007). Some of the recent studies on the behavior of this fungus clearly show two important characteristics of anti-fungal and antibacterial activity along with pullulan production. Almost all the A. pullulans strains showed activity against Gram-negative bacteria, but none of them inhibited Staphylococcus aureus and Bacillus spp. Strikingly, Pseudomonas seem to apparently much more sensitive to A.pullulans (Kalantar et al., 2006). Yoshikawa et al., (1993) reported a group of antifungal antibiotics, named aureobasidins, from A. pullulans. A. pullulans appears to be a promising organism for the development of newer antimicrobial agents, both for chemotherapy as well as non-medical applications. Dake and Beniwal (2014) showed the extracellular antimicrobial activity of Aureobasidium pullulans against several Gram positive and Gram negative bacteria. A large number of bioactive and structurally diverse fungal metabolites have been used for the development of valuable pharmaceuticals (Singh et al., 2015). Aureobasidium pullulans is considered as a probable source for antagonistic activity against a number of phytopathogenic fungi and possible bio-control agent of post harvest diseases. The work aims at the antibacterial compounds produced by Aureobasidium pullulans (NCIM 1049). Extraction of antimicrobial compounds was carried out using solvent extraction and ammonium sulfate precipitation method. Antibacterial activity of resultant extracts was studied using agar disc diffusion method. Extraction using ethyl acetate as well as by salt precipitation method exhibited significant intracellular and extracellular antibacterial activity against Staphylococcus aureus, Escherichia coli and Bacillus subtilis. The antibacterial activity resulting from salt precipitation method showed ϭϴ 

stability from pH 7-9, thermostability upto 60ºC. It was insensitive to SDS, EDTA and enzymes like trypsin, papain and Serratia peptidase.The strain L47 of A. pullulans was isolated in South Italy from corpospere of table grape berries and was successfully applied to control post harvest diseases of different fruits and vegetables (Schena et al., 2002). This strain provided high protection levels against P. digitatum on grape fruit, Botrytis cinerea, Aspergillus niger and Rhizopus stolonifer on grape, B. cinerea and Monilia lax a on sweet cherry and B. cinerea on kiwi fruit, cherry, tomato, apples and strawberries .This approach makes such strain more economical for industrial use (Schena et al., 1999). (III) Enzymes from A. pullulans Different strains of A. pullulans isolated from different environments can produce amylase, protease, lipase, cellulose, xylanase, etc, which have great potential application in industries. Proteases have been shown to have many applications in detergents, leather processing, silver recovery, medical purposes, food processing, feeds, chemical industry as well as waste treatment. Proteases also contribute to the development of high-added applications or products using the enzyme-aided digestion of protein from different sources (Kurmar and Tagaki, 1999). However, little has been known about protease from marine-derived yeasts (Chi et al., 2007). Amylases have many applications in bread and baking industry, starch liquefaction and saccharification, textile designing, paper industry, detergent industry, analysis in medical and clinical chemistry, and food and pharmaceutical industries. Because most yeasts from environments are safe (GRAS, generally regarded as safe) compared to bacteria, interest in amylolytic yeasts has been increased in recent years as their potential value for conversion of starchy biomass to single cell protein and ethanol has been recognized (Gupta et al., 2003). Recently, some amylases from terrestrial yeasts also have been found to have the ability to digest raw starch. However, very few studies exist on the amylase-producing marine yeasts (Li et al., 2007 d). Although ϭϵ 

amylase activity produced by bacteria is much higher than that produced by A. pullulans, the bacteria only can produce Į-amylase (Nidhi et al., 2005). Lipases catalyze a wide range of reactions, including hydrolysis, interesterification, alcoholysis, acidolysis, esterification and aminolysis. Although lipases from Candida rugosa and Candida antartica have been extensively used in different fields, very few studies exist on the lipase produced by yeasts isolated from marine environments (Chi et al., 2006). Lipases from A. pullulans are extracellular. It was also found that the crude lipase produced by A. pullulans HN2.3 has high hydrolytic activity towards olive oil, peanut oil and soybean oil (Liu et al., 2008 a). Cellulose is the most abundant organic material on the earth consisting of glucose units linked together by ȕ-1, 4-glycosidic bonds. It has been observed that most of the cultures of A. pullulans have usually failed to show any cellulolytic activity (Buzzini and Martini, 2002). Zhang and Chi (2007) reported the ability to produce cellulose by different strains of A. pullulans isolated from different marine environments. Xylanases have many applications in paper, fermentation and food industries, as well as in waste treatment. The fungus A. pullulans Y-2311-1 was shown to be among the most proficient of the xylan-degrading fungi, secreting extremely high levels of xylanolytic enzymes into culture media. (IV) Bio-control with A. pullulans Currently, fungicide treatments represent the primary method for the control of post-harvest diseases of fruits and vegetables. However, public concern about fungicide residue and development of fungicide resistant isolates of post-harvest pathogens have promoted the search for alternative means, less harmful to human health and to the environment. In recent years, considerable success has been achieved utilizing microbial antagonists to control post-harvest diseases. Because the infection of fruits by postharvest pathogens often occurs in the field prior to harvest, it may be advantageous to apply antagonists before harvest. For this approach to be successful, putative biocontrol strains must be able to tolerate low nutrient availability, UV-B radiation, low ϮϬ 

temperatures, and climatic changes. The yeast-like fungus A. pullulans is one of the most widespread and well-adapted saprophytes, both in the phyllosphere and in the carposphere. A. pullulans has a high tolerance to desiccation and irradiation and has been considered as an effective biocontrol agent against post-harvest diseases (Mounir et al., 2007). A. pullulans is well known for its biotechnological importance particularly for the production of a biodegradable extracellular polysaccharide i.e. pullulan comprising of maltotriosyl units. Three glucose units in maltotriose are interconnected by Į-1, 4 glycosidic bonds, whereas consecutive maltotriose units are connected to each other by Į-1, 6 glucosidic bonds. This unique linkage pattern endows pullulan with distinctive physical traits. Pullulan is a promising biomaterial and is potentially used as a low calorie ingredient in the foods, and as a viscosity imparter and binder and as a packaging agent because of its low oxygen permeability (Rekha and Sharma, 2007; Singh et al., 2008). (V) A. pullulans as single cell protein (SCP) A variety of microalgae such as Spirulina and Chlorella and brown algae are extensively used as feed for cultured marine animals (Chi et al., 2006; Ravindra, 2000). However, they have some limitations for animal consumption. Some yeasts such as Saccharomyces cerevisiae, Candida utilis and Candida tropicalis also have been used for their single-cell protein (Ravindra, 2000). They have many advantages over algae and bacteria (Ravindra, 2000; Gao et al., 2007). Unfortunately, little is known about the marine yeasts that have high protein content and can be used as aqua-feed. A total of 327 yeast strains from seawater, sediments, mud of salterns, guts of the marine fish, and marine algae were obtained. Chi et al. (2009) estimated the crude protein of the yeast and found that eight strains of the marine yeasts grown in the medium with 20 g/l glucose contain more than 30.4 g protein per 100 g of cell dry weight. With the exception of one strain of A. pullulans with nucleic acid of 7.7% (w/w), all other yeast strains contain less than 5% (w/w) of nucleic acid. Analysis of fatty acids shows that all Ϯϭ 

the yeast strains tested have a large amount of C18:0 and C18:1 fatty acids, while analysis of amino acids indicates that the yeast strains tested have a large amount of essential amino acids, especially lysine and leucine which are very important nutritive components for marine animals (Chi et al., 2008). Therefore, A. pullulans that contains high content of protein may be especially important in single cell protein production by transforming the waste products such as starch, protein, cellulose and xylan into cell protein in A. pullulans (Gaur et al., 2010 b). (VI) Siderophore from A. pullulans Siderophores are low-molecular-weight, iron-chelating ligands produced by nearly all the microorganisms. Siderophores can affect microorganisms in the environments in several ways as result of their role as iron scavenging compounds, especially marine microorganisms because iron is an essential nutrient for virtually all forms of life and is difficult to obtain due to its low solubility in marine environments. It has been confirmed that yeasts produce only hydroxamate-type compound, while bacteria produce hydroxamate as well as catecholate siderophores (Riquelme, 1996). Siderophores are also found to have many applications in medical and environmental sciences. They can be used to control growth of the pathogenic bacteria in marine fish and the complexing ability of siderophores can be used to develop the processes for metal recovery or remediation of waste sites, including radioactive waste as they are extremely effective at solubilizing actinides and other metals from polluted environments (Li et al., 2008). Most bacterial infections in marine animals are found to be caused by Vibrio parahaemolyticus, Vibrio anguillarum and Vibrio harveyi. Therefore, it is very important to find some antibacterial agents against these pathogens. Although many antibacterial peptides and killer toxins have been found to be active against some pathogens in marine animals, they are not stable in marine environments and easily attacked by proteases produced by marine micro-organisms (Li et al., 2007d; Wang et al., 2007). Over 300 yeast strains isolated from different marine environments were screened for their ability to produce siderophore. Among them, one yeast strain ϮϮ 

HN6.2 (2E00149) which was identified to be A. pullulans was found to produce high level of siderophore. Under the optimal conditions, this yeast strain could produce 1.1 mg/ml of the siderophore. L-Ornithine was found to enhance the siderophore production, while Fe3+ could greatly inhibit the siderophore production. The crude siderophore produced by the yeast strain HN6.2 is able to inhibit cell growth of V. anguillarum and V. parahaemolyticus, the common pathogenic bacteria isolated from diseased marine animals. (VII) Physical and chemical structure of pullulan Polysaccharides which have been produced by microorganisms been have property that are very useful in various industrial application (Choudhury et al., 2011) A new fungal exopolysaccharide (EPS) with interesting industrial properties are well known (Singh et al., 2008). Pullulan is an extracellular and neutral microbial polysaccharide produced by A.pullulans in starch and sugar cultures. (Cheng et al., 2010; Karim et al., 2009; Wu et al., 2012; Xiao et al., 2012 a, b). Pullulan is a white powder which is odorless, flavourless and highly stable. It is water soluble but insoluble in organic solvents and non-hygroscopic. Its aqueous solution is stable and shows a relatively low viscosity as compared to other polysaccharide. It is non toxic, non-mutagenic, odorless, tasteless and edible. It forms films easily which is thermostable, transparent, elastic, antistatic films with extremely low oxygen permeability. It is biodegradable and biocompatible. Pullulan decomposes at 250oC-280oC and is moldable, spinnable, act as good adhesive and binder. The discovery of the enzyme pullulanase provided a critical tool for the analysis of the structure of pullulan (Bender and Wallenfels, 1961). Pullulanase specifically hydrolyzes Į-(1-6) linkage of pullulan and converts the polymer almost quantitatively to maltotriose (Bender and Wallenfels, 1961; Wallenfels et al., 1961, 1965). Based on this result, pullulan frequently is described as a polymer of Į-(1-6) linked maltotriose subunits. Ϯϯ 

Catley and coworkers established that pullulan contains maltotetrose subunits in addition to the predominant maltotriose subunits (Catley et al., 1966; Catley, 1970; Catley and Whelan, 1971). The frequency of maltotetraose subunits appears to vary on a strain-specific basis, from about 1% to 7% of total residues (Taguchi et al., 1973; Catley et al., 1986). Evidence suggests that maltotetraose subunits are distributed randomly throughout the molecule (Carolan et al., 1983). Unlike the maltotriose subunits in pullulan, maltotetraose residues are substrates for many Į-amylases, and it has been proposed that hydrolysis of pullulan at these sites accounts for the decrease in molecular weight commonly observed in late cultures.

Figure 1: Chemical structure of the secondary (minor) repeating structure of pullulan.

Ϯϰ 

(VIII) Estimation Methods General methods applicable to polysaccharide can be used to detect and quantitate pullulan. The basic structure of pullulan was deduced in part from its specific optical rotation and IR absorption characteristics. Periodate analysis; methylation analysis and partial acid hydrolysis were also important. However, studies using pullulanase, which specifically hydrolyzes Į-(1-6) linkages in pullulan were definitive in resolving the fundamental structure of the polymer (Bender and Wallenfels, 1961). Assays based on pullulanase remain the principal method for the specific detection and measurement of pullulan. Catley (1971) developed a sensitive radioassay for pullulan. A. pullulans cultures were fed 14C-glucose to produce labeled pullulan. After pullulanase digestion, maltotriose and maltotetrose were separated by paper chromatography and measured by liquid scintillation counting. Finkelman and Vardanis (1982b) described a simplified radioassay based on the solubilization by pullulanase of radioactive counts into ethanol. Leathers et al. (1988) described a quantitative pullulan assay based on the reducing sugars (maltotriose equivalents) released by pullulanase digestion. Israilides et al. (1994) suggested the complete hydrolysis of pullulan by means of pullulanase and glucoamylase, followed by the specific measurement of glucose using glucose oxidase. The primary method for the estimation of pullulan remains the same that is ethanol precipitation method and pullulanase assay method. Kelly and Catley (1977) used ethanol to determine the biomass of pullulan and pullulanase for pullulan estimation. Similarly, Israilides et al. (1998) followed the same method for pullulan estimation as followed by Kelly and Cately. Barnett et al. (1999) estimated the crude pullulan by weighing the ethanol precipitate from the cell-free fermentation broth. Sena et al. (2006) estimated pullulan by precipitation of one volume of 2propanol per volume of supernatant. Purity of pullulan thus produced was estimated by enzymatic hydrolysis to maltotriose with pullulanase from Klebsiella pneumoniae. Singh and Saini (2007) estimated pullulan by ethanol precipitation method. Similarly Thirumavalavan et al. (2008, 2009) estimated dry weight of total biomass by Ϯϱ 

centrifuging the fermentation broth in a high speed centrifuge. The collected cell mass was washed twice with saline and distilled water and dried at 90oC till the mass reaches constant weight. Pullulan was estimated by adding two volume of ethanol to the supernatant at 4oC for one hour. The precipitate obtained was washed with acetone and filtered through a pre-weighed Whatman number 1 filter and dried at 90oC for constant weight. (IX) Factors affecting pullulan production Multiple factors interact in the regulation of pullulan biosynthesis. Some of the important parameters controlling the production of pullulan are temperature, pH, kind and concentration of carbon and nitrogen sources, oxygen supply, agitation rate etc. Aureobasidium pullulans strains typically produce pullulan when cultured on sucrose, glucose, fructose, maltose, starch or maltooligosaccharides (Catley, 1971; Leathers et al., 1988; Chi and Zhao, 2003; Cheng et al., 2009). Sucrose often has been described as the optimal substrate. Less frequently, sugars such as xylose, arabinose, mannose, galactose, rhamnose, and lactose have also been reported to support pullulan production, usually in the reduced yields (Imshenetskii et al., 1981; LeDuy et al., 1983). Several workers have used glucose as the carbon source for pullulan production (Kim et al., 2000; Lee et al., 1999; 2001; Campbell et al., 2004; Prasongsuk et al., 2007; Thirumavalavan et al., 2008, 2009). Cheng et al. (2009) reported the production of pullulan by A. pullulans when grown on sucrose. Gaur et al. (2010a) reported the maximum pullulan production from a novel pullulan producing fungus Eurotium chevalieri, when grown on sucrose. Singh et al. (2012) reported maximum pullulan production by A. pullulans on sucrose media. Different nitrogen sources have also been evaluated for the growth and production of pullulan. Kim et al. (2000) used glucosamine as nitrogen source for the pullulan production in shake-flask culture. Optimum temperature for the pullulan production appears to vary slightly from strain to strain, usually in the range of 24oC to 30oC. Futhermore, few of the strains even grow well at a higher temperature range of 35oC to Ϯϲ 

40oC (Zajic, 1967; Imshenetskii et al., 1981; McNeil and Kristiansen, 1990). Kelly and Catley (1977) incubated the medium at 25oC for pullulan production while Auer and Seviour (1990) produced higher pullulan at 28oC. Few workers have incubated their medium at 28oC for the pullulan production (Kim et al., 2000; Lee et al., 2001; Lazaridou et al., 2002b). Chi and Zhao (2003) reported the production of pullulan at 28oC. They concluded that pullulan yields decreased rapidly when the incubation temperature was higher than 28oC. This means that pullulan production by this yeast strain was sensitive to higher temperature. Goksungur et al. (2004) incubated the medium at 28oC while Sena et al. (2006) incubated the medium at 26oC. Thirumavalavan et al. (2008, 2009) incubated the medium at 30oC for optimum pullulan production. Similarly Cheng et al. (2009) incubated the medium at 30oC for better pullulan production. Gaur et al. (2010a) reported the maximum pullulan production at 35oC from a novel pullulan producing fungus, Eurotium chevalieri. This may suggest that the optimal temperature for pullulan production also vary from strain to strain. Singh et al (2012) reported maximum pullulan production at 42oC by A. pullulans. Hydrogen ion concentration also plays an important role in the production of pullulan. Different pH has different effects on the production of pullulan in varying media. Lee et al. (2001) concluded that in shake-flask culture, the maximum growth of A. pullulans can be obtained with an initial pH 3.5, while pullulan production was optimal at the pH 6.5. Lazaridou et al. (2002a) reported that during the first 24 hour of the fermentation, the pH decreased and then increased at later stages of the fermentation. This could be due to deamination of amino acids present in molasses by the microorganism and the production of ammonia, which increase the pH of the medium. Similarly, Lazaridou et al. (2002b) concluded that the pullulan concentration and biomass dry weight of the solids slightly decreased when the initial pH of the culture medium increased from pH 4.0 to pH 6.0. All the fermentation parameters subsequently increased by a further rise of pH from 6.0 to 7.0 and decreased thereafter, the highest pullulan concentration, biomass dry weight, pullulan yield and the rate of sugar Ϯϳ 

utilization were obtained in cultures grown at an initial pH of 7.0 with this initial pH, the fermentation broth seemed to be maintained between 5.0 to 6.0 throughout a long period of fermentation. This range of pH seems to favour mostly the growth of yeast-like cells as well as some mycelial production. Chi and Zhao (2003) reported the maximum production of pullulan in the batch cultivation at an initial pH 7.0. Goksungur et al. (2004) concluded that polysaccharide and pullulan concentrations increased when the initial pH of the culture medium increased from 4.5 to 7.5, and decreased thereafter. However, the biomass dry weight decreased with each unit increase of the pH. Highest pullulan concentration was obtained in the culture grown at an initial pH of 7.5 while the highest biomass in dry weight was obtained at pH 4.5. Thirumavalavan et al. (2008) found an optimal initial pH of 6.5 for pullulan production. Cell mass and pullulan concentration obtained at pH levels was minimum when compared to higher pH level. The same workers in the year 2009 had reported that the pullulan concentration gradually increased with increasing the initial pH up to 7, and then decreased when grown on the coconut milk. The highest pullulan concentration was achieved at pH 7.0. A lower production of cell mass and pullulan was obtained at lower pH level when compared to higher levels of pH. This is probably due to an influence of acidic pH on morphological character of the organism. Cheng et al. (2009) reported the effect of plastic composite support and pH profiles on pullulan production in a biofilm reactor. Gaur et al. (2010a) have reported a maximum pullulan production at pH 5.5 from a novel pullulan producing fungus, Eurotium chevalieri. Similarly, Singh et al. (2012) reported maximum pullulan production at pH 5.5 by A. pullulans. (X) Mechanism of pullulan synthesis Although many investigations on biochemical mechanisms of exopolysaccharide biosynthesis in bacteria have been carried out (Degeest and Vuyst, 2000), relatively little is understood about the mechanisms of pullulan biosynthesis in A. pullulans. Pullulan can be synthesized from sucrose by cell-free enzymes of A. pullulans when both adenosine triphosphate (ATP) and uridine diphosphate (UDP)-glucose are added to a Ϯϴ 

reaction mixture (Shingel, 2004). Chi et al. (2009) reported that the size of UDP-glucose pool and glucosyltransferase activity in the cell of A. pullulans Y68 obtained in their laboratory may be correlated with high pullulan production. Therefore, effects of different sugars on pullulan production, UDP-glucose (UDPG) pool, and activities of phosphoglucose mutase, UDPG-pyrophosphorylase,and glucosyltransferase in the cells of A.pullulans Y68 were investigated (Duan et al., 2008). It was found that more pullulan is produced when the yeast strain is grown in the medium containing glucose than when it is cultivated in the medium supplementing other sugars. However, Chi et al. (2009) concluded that when more pullulan is synthesized, less UDP-glucose is left in the cells of A. pullulans Y68. High pullulan yield is positively related to high activities of Į-phosphoglucose mutase, UDPG-pyrophosphorylase, and glucosyltransferase in A. pullulans Y68 grown on different sugars. A pathway of pullulan biosynthesis in A. pullulans Y68 was proposed based on different studies (Duan et al., 2008; Chi et al., 2009). It is thought that the lower amount of pullulan produced by A. pullulans Y68 from fructose and xylose may be caused by the longer biosynthetic pathway leading from fructose and xylose to UDP-glucose. It is thought that most of UDP-glucose is used to synthesize pullulan when the glucosyltransferase activity is very high, leading to very low UDP-glucose level in the yeast cells. This may imply that very high glucosyltransferase activity is the unique characteristic of A. pullulans Y68 which can produce high yield of pullulan. Because the phosphoglucomutase and UDPGpyrophosphotylase activity in the yeast cells grown in the medium containing glucose is also very high, UDP-glucose is synthesized continuously to supply the precursors for high pullulan synthesis when the very high glucosyltransferase activity occurs in the cells of A. pullulans Y68. However, high level of UDP-glucose is left when the yeast cells are grown in the medium containing xylose and fructose, respectively, due to low glucosyltransferase activity. Therefore, it is believed that the proposed pathway of pullulan biosynthesis will be helpful for the metabolism engineering of the yeast strain to further enhance pullulan yield. Ϯϵ 

(XI) Production of pullulan from agro-industrial wastes Pullulan can be synthesized from a variety of carbohydrate substrates incorporated into either defined (synthetic) or non-defined media. Within the latter are several agro-industrial wastes which have been shown to be suitable for pullulan production. Utilization of these substrates would seem to be ecologically sound and economically advantageous as they have low or even negative costs. In this way the potential of pullulan production from agro-industrial wastes is expected to lower the cost of production and seems to be a very promising ecologically and economically sound way of bioconversion. However, the pullulan which is produced from different substrate may vary in purity and other physical and chemical characteristics. This is more pronounced when agro-industrial wastes are used as a carbon source for the fermentation. Some of these characteristics like the molecular weight are very important when commercialization of the production of pullulan from these wastes is under consideration (Israilides et al. 1994). The cost of pullulan primarily depends on the raw materials, especially of carbon source, which play a major role in the economics of pullulan production. The sugar such as sucrose, glucose, fructose, maltose, starch, or malto oligosaccharides support pullulan production by A. pullulans. There are various reports on the production of pullulan from different sources such as sweet potato (Wu et al., 2009), soyabean pomace (Seo et al., 2004), potato starch waste (Barnett et al., 1999), deproteinized whey (Roukas, 1999 b), agro-industrial waste such as grape skin pulp extract, starch waste, olive oil waste effluents and beet molasses and brewery waste (Roukas 1999 a), jagerry which is concentrated sugar cane juice (Vijayendra et al., 2001), carob pods (Roukas and Biliaderis, 1995)and Jerusalem artichoke (Goksungur et al., 2011; Oguzhan and Yangilar, 2013). Production of pullulan was investigated in batch fermentation using coconut by-products by Thirumavalavan et al. in 2009. This method has advantage such as reduction of production cost and recycling of natural sources. ϯϬ 

Oliveira et al (2015) used white granulated sugar as carbon and energy source by two Aureobasidium pullulans strains (IOC 3467 and IOC 3011) was studied aiming cost reduction and maximization of the process yield. For this purpose, different sources of nitrogen – NaNO3, (NH4)2SO4, NH4NO3, urea and residual brewery yeast – were added to the medium in different concentrations to a carbon/nitrogen ratio of 5 and 150. All nitrogen sources in the results showed that the use of white granulated sugar and residual brewery yeast as carbon and nitrogen source, respectively, increased the pullulan production. The use of these carbon and nitrogen sources allows the cost reduction of pullulan production since they are renewable feedstock, low-cost, abundant and available worldwide. Israilides et al 2013 reported pullulan production from Waste potato starch from the manufacture of potato crisps or from other potato processing industries.

ϯϭ 

Objective The prime objective of the study was to isolate a thermotolerant strain of this fungus from environmental samples, and to optimize various physico-chemical parameters for the production of pullulan in non- stirred flask fermentation system. 



ϯϮ 

MATERIALS AND METHODS

I. Collection, isolation, screening and identification of pullulan producing Aureobasidium pullulans from different habitats The samples were collected from garden soil, agriculture field soil, as well as, from different plant parts viz. leaves and flowers from the college campus and the different areas of Jabalpur by random sampling method in sterile polyethylene bags. The samples were collected in the month of January and February in 2015. All the 5 samples collected (1 from garden soil, 1 from agriculture soil, and 3 from different plant parts) were subjected to isolate Aureobasidium on its respective medium after enrichment. A. Isolation of Aureobasidium pullulans from different samples Isolation was done by using selective enrichment method as followed by Pollock et al. (1992). The samples, 25 g of the soil, and plant each were mixed in 250 ml sterile distilled water. The samples were kept on a magnetic stirrer for 2-4 hours to obtain maximum dispersion. Serial dilution of the samples (10-2 to 10-4) were placed on petriplates having basal agar medium formulated by Prasongsuk et al. (2007) having composition Glucose-2.0%, Ammonium Sulphate-0.06%, Magnesium Sulphate-0.04%, Sodium Chloride-0.1%, di-Potassium Hydrogen Orthophosphate-0.5%, Yeast Extract0.04% and having pH 5.0. These plates were subsequently incubated at 37±2o C, for 2-4 days to isolate colonies. The colonies appeared black colored after 4 days of incubation, and thereafter lysis of mycelia occurred showing yeast-like cells. Further, the isolates were maintained on the same medium at 4oC in slants and sub-cultured monthly. The isolates were identified on the basis of morphology and cultural characteristics using the standard identification manuals (Gilman, 1967).

ϯϯ 

Sample 1: Isolation of A.pu ullulans from Marigold petals

Sample 2: Isolation of o A.pullulans from Rose petals

Sample 3: Isolation of A.pu ullulans

Sample 4: Isolation of o A.pullulans

from Nerium leaves

from Nerium stem

p Figure 2: Isolation of A.pullulans from plant parts

ϯϰ 

B. Inoculum Preparation Cell suspension was prepared by inoculating 1 ml of 48 hours grown culture in 200 ml basal nutrient broth, and then incubated at 37oC for 24 hours to achieve active exponential phase of culture. In the non-stirred flask experiments, 0.5% inoculum was used. C. Screening of A. pullulans for pullulan production on quantitative basis in liquid medium The isolates were screened for pullulan production in the basal broth medium. The cultures of the isolates were grown in 250 ml Erlenmeyer flasks containing 100 ml medium and incubated at 37oC. The similar culture medium was used for maintenance of the cultures except that agar was omitted and sucrose concentration was varied between 2% to 9% as indicated in each case. A series of experiments were carried out in order to study the effect of different concentrations of carbon and nitrogen sources for pullulan production. II. Estimation of Pullulan and Biomass Isolation and purification of extracellular polysaccharide After fermentation, the culture medium was heated at 100oC in water bath for 15 minutes, cooled to room temperature and centrifuged at 12,000 rpm at 4oC for 10 minutes to remove cells and other precipitates. Three ml of the supernatant were transferred into a test tube, and then 6 ml of cold ethanol (99% ethanol) was added to the test tube and mixed thoroughly and held at 4oC for 12 hours to precipitate the extracellular polysaccharide. After removal of the residual ethanol, the precipitate was dissolved in 3 ml of deionized water at 80oC and the solution was dialyzed against deionized water for 48 hour to remove small molecules in the solution. The exopolysaccharide was precipitated again by using 6 ml of the cold ethanol and the ϯϱ 

residual ethanol was removed, the precipitate was then dried at 80oC to a constant weight (Badr-Eldin et al., 1994). Pullulan weight was measured using electronic balance (Sartorius, USA) and expressed in gram/liter. III. Physico-chemical and nutritional parameters for pullulan and biomass production in non-stirred flask fermentation conditions A total of 5 isolates were isolated from different soil samples and plant parts from different regions of Jabalpur. All the 5 isolates were purified and identified on the basis of cultural and morphological characteristics. Further all the 5 isolates were subjected to different media on petriplates. Out of these 5 isolates 1 best isolate was selected which showed the best growth on the solid media in 3 weeks. Further this isolate was subjected to various physico-chemical and nutritional parameters to check the production of pullulan. A. Effect of different pH, temperature, incubation periods on the pullulan and biomass production The basal medium for A. pullulans with different pH viz. 2.0, 3.0, 5.0, 7.0, and 9.0 and temperature viz. 30 oC, 37 oC, 43 oC, 50oC, 60oC were used for the production of pullulan and biomass. The initial pH (5.0) was varied in the medium by adding either 1N HCL or 1N NaOH as required. The basal fermentation medium was then inoculated with 0.5% inoculum and incubated for different incubation for 72 hours at 37oC. Further, the pullulan and biomass production was optimized at different incubation period viz. 24, 48, 72, 96 and 120 hours at different temperatures. Futher, the pullulan and biomass production were estimated quantitatively. B. Effect of different carbon and nitrogen sources and their concentrations on the pullulan and biomass production Various carbon sources viz. glucose, fructose and sucrose, (at a concentration of 2%, 5% and 7%) were individually added in the basal medium and inoculated with 0.5% A. pullulans culture along with 0.5% ϯϲ 

of different nitrogen sources and

incubated at 37oC for 48 hours to observe the effect of these carbon and nitrogen sources on pullulan and biomass production. Further, different concentrations of sucrose (2, 5, 7 and 9%) were used for pullulan and biomass production. In another experiment different nitrogen sources viz. ammonium sulphate, yeast extract, sodium nitrate, (at a concentration of 0.5%) were individually added into the basal medium and inoculated with 0.5% of active culture of A. pullulans in the exponential phase of growth and incubated at 37oC for 72 hours to observe the effect of these carbon sources on pullulan and biomass production. In another experiment, different concentrations of yeast extract (0.1, 0.2, 0.3, 0.4, 0.5, 0.6 and 0.7%) were evaluated for the pullulan and biomass production. C. Fermentation conditions Fermentation was carried out in an Erlenmeyer flask having total volume of 250ml and with working volume of 100ml. The fermentation medium was sterilized at 121oC for 15 minutes and incubation was done at 37oC with all other conditions at the optimal levels determined previously. Statistical Analysis: Karl Pearson method (Variability) was followed for statistical analysis. All the experiments were done in triplicate and mean values were calculated using standard deviation.

ϯϳ 

RESULTS I. Isolation and characterization of Aureobasidium pullulans A. Isolation and screening of the isolates A total of 5 isolates were isolated from soil and plant parts from different regions of Jabalpur. All the 5 isolates were purified and identified on the basis of cultural and morphological characteristics. Further all the 5 isolates were subjected to different media on petriplates. Out of these 5 isolates 1 best isolate was selected which showed the best growth on the solid media in 1 week (I-3). Further this isolate was subjected to various physico-chemical and nutritional parameters to check the production of pullulan.

Table 1: Screening of isolates on solid media Basal

Diameter of fungus after one week incubation (in cm)

media with I-1

I-2

I-3

I-4

I-5

different sugars Sucrose

3.2± 0.02

6.0± 0.01

14.8± 0.02

7.9±0.01

2.8±0.01

Glucose

3.0±0.01

5.8±0.03

11.2±0.02

6.3±0.02

1.9±0.01

Maltose

2.8±0.02

4.4±0.03

5.3±0.01

5.5±0.02

1.1±0.01

I= Isolates

B. Description of A. pullulans on basal agar medium plate 1. The colonies were young and resembling unicellular budding yeast cells (Blastoconidia). 2. The colonies were fast growing, smooth, covered with slimy masses of conidia, cream or pink hyaline and septate, frequently becoming dark-brown with age and undergoing

hallothallic

transfer

celled,

thick-walled,

anthroconidia commonly known as chlamydoconidia. ϯϴ 

darkly

pigmented

3. The colonies were hyaline, smooth-walled, single-celled and ellipsoidal. The colonies grew rapidly. 4. The diameter of colony increased at a rate of 1-2 cm per day on basal agar medium plate. 5. The colonies were flat, smooth, moist, yeast-like, mucoid appearances. 6. The surface was white, pale, pink or yellow at beginning and become brown to black by aging. 7. With aging, white colony got black and velvety, hyphae became visible. Blastoconidia showed pale colour.

C. Microscopic description of A. pullulans 1. The conidia were one-celled, hyaline and form clusters or located along the hyphae. 2. Blastospores and chlamydospores may also be observed. 3. Hyphae are hyaline, smooth, and thin-walled, with transverse septa. 4. The hyphae form thick-walled, large phacoid-shaped chlamydospores (brownblack in colour).

ϯϵ 

Figure 3: Colonies off Aureobasidium pullulans on basal agar plate

ϰϬ 

Figure 4: Colonies of Aureoobasidium pullulans (Yeast form) on basal agar plate

ϰϭ 

Figure 5: Motic im mages of Aureobasidium pullulans.

ϰϮ 

II.

Screening of isolate (I-3) on the basis of pullulan production in the basal

broth medium A. Effect of different pH, temperature, incubation period on pullulan production 1. Effect of different incubation time on pullulan production Effect of different incubation time viz. 24 hours, 48 hours, 72 hours, 96 hours and 120 hours was used for quantitative yield of pullulan and biomass by A. pullulans in the basal medium. These experiments were conducted to screen the best incubation period for highest pullulan and biomass production. The highest production of pullulan (5.6±0.05 g/100ml) was observed at 72 h. When the incubation was further increased the production became steady. Similarly highest biomass production (3.4± 0.02 g/100ml) was observed at 72h because pullulan production is directly related to biomass production that is more the biomass production more is pullulan production. Table 2: Effect of different incubation time on pullulan production Incubation

Pullulan

production Biomass

period (hours)

(g/100ml)

(g/100ml)

24

0.0

0.0

48

3.2± 0.04

2.8± 0.03

72

5.6± 0.05

3.4± 0.02

96

5.5± 0.03

3.4± 0.04

120

5.5± 0.04

3.3± 0.03

ϰϯ 

production

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ϰϴ

ϳϮ

ϵϲ

ϭϮϬ

dŝŵĞ;ŝŶŚŽƵƌƐͿ

Figure 6: Effect of different incubation time on pullulan production

ϰϰ 

2. Effect of different temperature on pullulan production Effect of different temperature viz. 30oC, 37oC, 43oC, 50oC, 60oC was used for quantitative yield of pullulan by A. pullulans in the basal medium. These experiments were conducted to screen the best temperature for highest pullulan production. The isolate was incubated at different temperature to see the effect of the temperature on pullulan production. The highest production of pullulan (3.1± 0.03 g/100 ml) was obtained at 37°C. Further when the temperature was increased the production dropped significantly.

Table 3: Effect of different temperature on pullulan production

Temperature

Pullulan production (g/100ml)

30°C

1.3± 0.01

37°C

3.1± 0.03

43°C

2.3± 0.01

50°C

0.6± 0.02

60°C

0.4± 0.01

ϰϱ 

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ϯϳΣ

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ϱϬΣ

ϲϬΣ

dĞŵƉĞƌĂƚƵƌĞ;ΣͿ

Figure 7: Effect of different temperature on pullulan production

ϰϲ 

3. Effect of different pH on pullulan production The effect of different pH viz. 2 to 9 in the basal medium was evaluated for pullulan production by A.pullulans (Table 4). Out of all the pH, best result was seen at pH 5.5 (Table 4). A slight increase (7.0) or decrease (3.0) in the pH level, slightly affected the production, but higher increase or decrease of pH sharply reduced the pullulan production.

Table 4: Effect of pH on pullulan production pH

Pullulan Production (g/100ml)

range

In different sugars Maltose

Glucose

Sucrose

2.0

0.0

0.0

0.0

3.0

0.8±0.02

0.81±0.02

1.5±0.03

5.0

1.4±0.01

1.6±0.02

3.1±0.01

7.0

0.5±0.04

0.9±0.03

2.0±0.03

9.0

0.2±0.02

0.3±0.02

0.6±0.02

ϰϳ 

ϯ͘ϱ

WƵůůƵůĂŶƉƌŽĚƵĐƚŝŽŶŐͬϭϬϬŵů

ϯ Ϯ͘ϱ Ϯ DĂůƚŽƐĞ ϭ͘ϱ

'ůƵĐŽƐĞ ^ƵĐƌŽƐĞ

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ϯ

ϱ

ϳ

ϵ

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Figure 8: Effect of pH on pullulan production

ϰϴ 

B. Effect of different carbon and nitrogen sources and their concentration on pullulan production 1. Effect of different carbon sources on pullulan production Various carbon sources viz. sucrose, glucose, and maltose (at a concentration of 2%, 5% and 7%) were individually added in the basal medium and inoculated with 0.5% A. pullulans culture and incubated at 37oC for 2-4 days to observe the effect on pullulan production. Out of these carbon sources, sucrose was found best for pullulan production followed by glucose and maltose, respectively (Table 5).

Table 5: Effect of different carbon sources on pullulan production isolates Carbon source

Pullulan production (g/100ml) (2% concentration)

Maltose

0.9±0.01

Glucose

1.7±0.02

Sucrose

2.5±0.02

ϰϵ 

Pullulan production (g/100ml)

ϯ Ϯ͘ϱ Ϯ ϭ͘ϱ ϭ Ϭ͘ϱ Ϭ DĂůƚŽƐĞ

'ůƵĐŽƐĞ

^ƵĐƌŽƐĞ

Different carbon sources (2%)

Figure 9: Effect of different carbon sources on pullulan production

ϱϬ 

2. Effect of different sucrose concentration on pullulan production

In another set of the experiment, different concentrations of sucrose (2, 5, 7, and 9) in the medium were tested at 37oC and pH 5.5 for pullulan production (Table 6). The isolates produced different amount of pullulan. The isolate produced higher pullulan at 5% sucrose concentration beyond which their amount decreased (Table 6).

Table 6: Effect of different sucrose concentration on pullulan production

Sucrose concentration Pullulan production (grams/100ml) (%) 2

2.5±0.05

5

2.8±0.04

7

1.6±0.04

9

0.6±0.03

ϱϭ 

ϯ

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ϭ

Ϭ͘ϱ

Ϭ Ϯ

ϱ

ϳ

ϵ

^ƵĐƌŽƐĞŽŶĐĞŶƚƌĂƚŝŽŶ;йͿ

Figure 10: Effect of different sucrose concentration on pullulan production

ϱϮ 

3. Effect of different nitrogen sources on pullulan production

Similarly, in other set of experiment, different inorganic and organic nitrogen sources viz. ammonium sulphate, yeast extract, sodium nitrite at the rate of 0.5% were used in the growth medium for pullulan and biomass production by (Table 7). The isolates produced different amount of pullulan, but the best result was reported by yeast extract at pH 5 and temperature 37oC for 3 days of the incubation (6.2±0.04 g/100ml).

Table 7: Effect of different nitrogen sources on pullulan production

Nitrogen sources (0.5 %)

Pullulan Production (grams/100ml)

Sodium nitrate

4.9±0.04

Yeast extract

6.2±0.04

Ammonium sulphate

3.2±0.05

ϱϯ 

ϳ

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EŝƚƌŽŐĞŶƐŽƵƌĐĞƐ;Ϭ͘ϱйͿ

Figure 11: Effect of different nitrogen sources on pullulan production

ϱϰ 

4. Effect of different yeast extract concentration on pullulan production Different concentrations of yeast extract were also studied, and highest pullulan production was recorded at 37°C at the same concentration of yeast extract (0.5%), while above or below this concentration, the production decreased significantly.

Table 8: Effect of different yeast concentration on pullulan production

Yeast Extract concentration (%)

Pullulan production (g/100ml)

0.1

0.2±0.01

0.2

1.6±0.02

0.3

2.7±0.02

0.4

5.3±0.03

0.5

6.5±0.02

0.6

4.7±0.01

0.7

3.3±0.01

ϱϱ 

ϳ

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Figure 12: Effect of different Yeast Extract Concentration on pullulan production

ϱϲ 

DISCUSSION

I. Isolation, distribution and morphogenetic characteristics and optimization of the fermentative parameters for pullulan and biomass production by the Aureobasidium pullulans (A) Morphological characters of A. pullulans Aureobasidium pullulans was previously classified under Fungi Imperfecti, Order Moniliales, Family Dematiaceae (Ramos and Garcia-Acha, 1975; Hermanides-Nijhof, 1977). However, further reports include it under Ascomycota, though a perfect stage has not been found (de Hoog and McGinnis, 1987). In recent classification, A. pullulans is regarded as Ascomycetous yeast and is placed in the Order Dothideales, Family Dothideaceae (Deshpande et al., 1992; Yurlova et al., 1999).

The fungus was

taxonomically characterized by de Hoog and Yurlova (1994) on the basis of its morphology, nutrition and physiology. However, owing to variability in morphological and physiological characteristics the species has been described under three different varieties namely; A. pullulans var. pullulans (Viala and Boyer, 1891), A. pullulans var. melanogenum (Hermanides-Nijhof, 1977) and A. pullulans var. aubasidani Yurlova (Yurlova and de Hoog, 1997). A. pullulans exhibits polymorphism for it can grow as budding yeast or as mycelia depending upon the environmental conditions. The formation of dark-coloured chlamydospores is the characteristic feature of this fungus (Ramos and Garcia-Acha, 1975). Ramos and Garcia-Acha (1975) have described the vegetative cycle of A. pullulans and the optimal conditions for conversion of one form to other. Although the organism actually being polymorphic, studies usually have been focused on dimorphic transition between yeast-like cells and mycelia, or between yeast-like cells and chlamydospores (large resting cells). Guterman and Shabtai (1996) revealed five basic morphologies of A. pullulans which are yeast-like cells, young blastospores, swollen blastospores, chlamydospores and mycelia in batch fermentation. ϱϳ 

Aureobasidium pullulans is well-known for its biotechnological importance particularly for the production of a biodegradable extracellular polysaccharide namely, pullulan. Various physico-chemical and nutritional factors affect the production of this polysaccharide in shake-flash conditions. The polymorphic fungi require appropriate physical and chemical conditions for metabolite production. Richards (2002) evaluated the pullulan of A. pullulans as safe consumption for human. Hayashibara industry in Japan is commercially producing pullulan and recommending this product under the substances generally recognized as safe (GRAS). Hayashibara discusses the fate of the pullulan in the digestive tract, referring to a published study using digestive enzyme In vitro and fecal culture digestion experiments. Based on this study, Hayashibara concluded that the salivary enzyme and other enzymes in the upper gastrointestinal tract hydrolyze pullulan only to a limited extent, therefore, concluded that typical bacteria of the distal intestinal tract in humans hydrolyzes the pullulan further and fermented the hydrolysis products to short-chain fatty acids. Human consumption study reported that no symptoms other than abdominal fullness were recorded. Therefore, commercial production of this polysaccharide is need of the hour. This can be used for the preparation of biodegradable plastics due to its physical behaviour resembling polyethylene and transparent oxygen impermeable film forming capability. In view of the importance of this polysaccharide, an attempt was made to isolate a thermotolerant, strain of A. pullulans from different environmental samples viz., soil and aerial parts of different plants, and to optimize physico-chemical, as well as, nutritional parameters for higher pullulan production. The growth condition requires higher water activity, mesophilic temperature range and agricultural residue used as complex organic compounds. This fungus grows luxuriantly on the general fungal media having glucose, sucrose and starch. However, growth medium without containing monosaccharide does not favour fast growth as well as yeast-like phase, and only allows the filamentous growth. Further, it has been ϱϴ 

observed that the addition of glucose/ sucrose accelerates the fermentation process by producing higher yeast-like cells mainly responsible for pullulan formation. The yeastlike form on the agar medium containing sucrose or glucose shows cream to pink color in the early stage of growth and later dark-brown color. This fungus is found in all types of ecosystem, for example, normal soil, forest soil, and fresh water, aerial portion of plant wood, fruits and marine estuary sediments. It is mostly saprophytic, but a very few strains are mild plant pathogens. Industrial use of this fungus is very important because it produces varieties of metabolites, like pullulan, antibiotics, organic acids, enzymes etc. Identification of A. pullulans is difficult because of the four morphological forms existing during fermentation. A potential isolate of A. pullulans having thermotolerant nature was selected after screening and optimization at different physico-chemical and nutritional levels in the fermentor system for pullulan production. The selected wild strain produced 6.2±1.5 g/100 ml pullulan at 37oC, which is the highest amount of pullulan produced by any strain of this fungus till to date. This polysaccharide has more than 300 patents for application in different area like medical, pharmaceutical, foods, cosmetics, agriculture etc. (B)

Effect of temperature, pH and incubation period on pullulan and biomass

production in non-stirred flask fermentation system For evaluation of pullulan production fermentation conditions were evaluated in flask fermentation system. Mostly yeast-like phase is found in the broth at a temperature range of 25oC to 28oC. Till date this fungus, has been reported to produce yeast-like cells at 38oC to 42oC, which is mainly responsible for pullulan production. India is a temperate as well as subtropical country, and thus A. pullulans diversity in different climatic conditions may be a reason of achieving a thermotolerant strain of this fungus from the ecosystem. Till now no report is available about thermotolerant strain of A. pullulans for pullulan production from Central India. Furthermore, the mechanism of pullulan production by thermotolerant strain has not yet been cleared. ϱϵ 

Incubation period is a common factor for pullulan production during fermentation and it varies from strain to strain (Chi and Zhao, 2003). As mentioned earlier, A. pullulans has mainly four different growth phases, yeast-like cells; young blastospores; swollen blastospores and chlamydospores in its life cycle (Guterman and Shabtai, 1996). Yeast-like cells is mainly responsible for pullulan production (Catley, 1980; Campbell et al., 2004). Maximum pullulan production was seen at 72 hours of incubation. The present finding also shows similar type of effects with the strain regarding the pullulan production by yeast-like cells. Similar trend of pullulan production was also observed by Chi and Zhao (2003), even though nutrient was present in the medium. Similarly, maximum biomass was also produced at 72 hours of incubation, since the formation of biomass directly depends on pullulan formation which indirectly depends on some of the factors like sucrose concentration and other physico-chemical parameters. Maximum pullulan production was achieved when the cells reached to stationary phase at 72 hours of incubation, and beyond this, no further growth in the cells were observed and the production of pullulan became stable due to limited nutrition and accumulation of toxic metabolites. Fermentation temperature is one of the most important factors which alter the morphological forms of A. pullulans from yeast-like cells to blastospores or filamentous, as change in the morphology of A. pullulans at elevated temperature adversely affect pullulan production. Yeast phase of growth in submerged system in the presence of suitable carbohydrate and its concentration has shown maximum pullulan production. (McNeil and Kristiansen, 1990). It was observed that 43oC was found suitable for higher pullulan and biomass production by this strain. On the other hand, highest yeast-like cells could be attained at 43oC since yeast cells are mainly responsible for pullulan production. Thermotolerant strain is required for industrial production of pullulan as temperature goes high in industry and only those strains can work well and sustain high temperature. ϲϬ 

Pullulan production by A. pullulans at different pH range has shown different trends of pullulan production, therefore, this aspect has been discussed in the light of inorganic nitrogen sources and their concentrations in the medium. Production of pullulan was studied at different range of pH (2.0-9.0) by various workers and have reported that the change in pH of the medium is due to the presence of inorganic and organic sources of nitrogen for different rate of ammonium and hydrogen ions may be released in the medium resulting in increase or decrease of the pH of the medium affecting the rate of production of microbial metabolites (Lee et al., 2001; Chi and Zhao, 2003; Cheng et al., 2009). Maximum pullulan production was recorded at pH 5. This implies that the optimal initial pH values for pullulan production depend on strain to strain, change in the composition of the fermentation medium and growth conditions. Therefore, the physiological function of A. pullulans varies from strain to strain in case of pH. (C) Effect of carbon and nitrogen sources on pullulan and biomass production Carbon and nitrogen sources and their concentrations are also very important parameters affecting production of pullulan by A. pullulans in fermentor. Maximum pullulan production was observed in sucrose as carbon source, and ammonium sulphate as nitrogen source, in the medium. Sucrose showed best result among all the carbon sources used at the level of 2%, while optimal sucrose concentration at which maximum pullulan was observed at 5%. A similar result was also found by Cheng et al. (2009). The pullulan production increased with the increase in initial sugar concentration from 1-5%, where as further increase of sugar concentration resulted in reduction of pullulan yield. The decline in polysaccharide production encountered with high sugar concentration in the medium is probably due to osmotic effects, a lower level of water activity as well as plasmolysis events along with regulatory mechanism of the cells of the organisms.

ϲϭ 

Among different nitrogen sources (organic and inorganic), the highest pullulan production was reported with yeast extract at the level of 0.5%. The polysaccharide production commended on reaching nitrogen limiting condition, and the yield of pullulan fell when excess yeast extract was present, even under condition which otherwise supported its synthesis. Various nitrogen sources were optimized by different workers for pullulan production (Cheng et al., 2009; Chi and Zhao, 2003). Perhaps, high nitrogen content may be responsible for the synthesis of pullulan or in production of higher biomass resulting in higher pullulan yield. One significant result of the study was that the pullulan was melanin free. Melanin is not required in industry as it damages the final product. Our fungal strain produced melanin-free pullulan (white pullulan) which is needed for commercial producton of pullulan. In recent years, a wide range of microbial metabolites have been used for industrial production worldwide for human welfare. Pullulan is one of the important microbial metabolite produced by Aureobasidium pullulans all over the world. This fungus has only been used for production of pullulan commercially. Pullulan, an exopolysaccharide, is a linear homopolymer composed of maltotriose subunits interconnected with Į-1, 6 glycosidic linkages. The regular alternation of Į-1, 4 and Į-1, 6 bonds results in two distinctive properties, the structural flexibility and enhanced solubility in water. This polymer is being exploited in food, cosmetic, medical and pharmaceutical industries. There are more than three hundred patents for its applications. Pullulan has been used for manufacturing of biodegradable plastics, because of its elastic and film forming capability which resembles with plastics in order to reduce the hazardous risk of polyethylene bags and therefore, the packing material prepared by the pullulan for this purpose is current need of the hour. Indian climatic conditions favour diversified microflora and it has been found that A. pullulans is ubiquitous and has been found in different ecosystems. Therefore, isolation of a better pullulan producing strain from the natural ecosystem and its optimization at ϲϮ 

different physico-chemical and nutritional parameters for higher yield of pullulan are very essential. A very scant work has been done all over the world regarding the pullulan production. Recently, only Japan and USA have started the commercial production and its application. Hayashibara Co. Ltd., Japan has estimated that the daily intake of pullulan for its general use in food is 9.4 gram per person per day (g/p/d) as the intake of pullulan is self-limiting due to its organoleptic properties. In India, a very little attention has been paid to isolate such beneficial fungus and its production at commercial level. Further, the fermentation technology especially immobilization of microbial cells for continuous pullulan production and isolation of newer strain from the natural ecosystem as well as through mutational technique are still open for a wide research in order to achieve economic production.

ϲϯ 

SUMMARY

In view of the above facts, the aim of the present investigation was to isolate and screen A. pullulans having ability to produce pullulan. Furthermore, the fungus was optimized for higher yield of this polysaccharide at different physico-chemical and nutritional parameters viz., different temperature, pH, incubation period, different concentrations of carbon sources (sucrose, glucose and maltose) and nitrogen sources (ammonium sulphate, yeast extract, sodium nitrate etc.). (I) Isolation, screening and characterization of Aureobasidium pullulans from different sources A total of 5 isolates were isolated from soil of different parts of Jabalpur. All the 5 isolates were purified and identified on the basis of cultural and morphological characteristics. All the isolates were screened for pullulan and biomass production. These strains were characterized for their growth and morphology on the agar plates along with microscopic examination. The following morphological characteristics were reported. (A) Growth and colony morphology on the basal agar medium (1)

The colonies were fast growing, smooth, covered with slimy masses of conidia, cream or pink hyaline and septate and maturing within 4 to 6 days of incubation.

(2)

The colony diameter attained 6.9 cm at 35oC on the 4th day on potato dextrose agar.

(3)

The surface was white, pale, pink or yellow at beginning and became brown to black by aging.

(B) Microscopic description of Aureobasidium pullulans (1)

The conidia (4-6×2-3 ȝm in size) were one-celled, hyaline and form clusters and located along the hyphae.

(2)

Blastoconidia and chlamydospores were also observed in the late stage. ϲϰ



II. Physico-chemical and nutritional parameters for pullulan production in fermentor A. Effect of temperature, pH and incubation period on pullulan and biomass production Temperature, pH and incubation period play important role in pullulan and biomass production. Different incubation periods were evaluated for pullulan and biomass production. Maximum pullulan and biomass production were seen at 72 hours of incubation and beyond this no further growth in the cells were recorded, thus production of pullulan became stable. Similarly, different temperatures were evaluated for pullulan and biomass production. Maximum pullulan and biomass production were recorded at 37oC. Further increase or decrease in temperature reduced the yield of pullulan in the medium. Different pH was also evaluated for pullulan production. Maximum pullulan were produced at pH range of 5 and thereafter increase or decrease in the pH of the medium decreased the pullulan yield.

B. Effect of different carbon sources and their concentrations on pullulan production Different carbon sources were tested for pullulan production by this fungus at a particular concentration (2%). Sucrose was found best for pullulan and biomass production followed by glucose and maltose. Furthermore, different concentrations of sucrose (2-9%) were also tested for pullulan and biomass production. Optimal concentration of sucrose for pullulan production by this strain was 5%. Further increase or decrease in sugar concentration reduced the pullulan yield.

C. Effect of different nitrogen sources and their concentrations on pullulan production Different nitrogen sources (0.5%) viz. sodium nitrate, yeast extract, ammonium sulphate, was tested individually for pullulan production. Yeast extract was found best ϲϱ 

for pullulan production. After that, different concentrations of yeast extract (0.1-0.7%) were also tested for pullulan production. Optimal concentration of yeast extract for pullulan production by this strain was 0.5%. Further increase or decrease in ammonium sulphate concentration reduced the pullulan yield significantly.

ϲϲ 

CONCLUSION Aureobasidium pullulans strains, having ability of pullulan production, are abundantly present in the environment and can easily be isolated and characterized on the basis of their polymorphic nature from different ecosystem. They have been exploited for pullulan production world-wide. Pullulan is an important polysaccharide used for the manufacturing of biodegradable plastics, having unique physical and chemical properties resembling plastics. Therefore, isolation, optimization of various physicochemical and nutritional parameters for pullulan production by this organism are very essential. The yeast-like cells predominanted during pullulan production. The optimum pH 5, temperature 37oC, 5% sucrose and 0.5% yeast extract concentration were found best for pullulan production. The isolated strain produced higher amount of pullulan in flask shake culture. Therefore, A. pullulans can be isolated from natural ecosystem for commercial production of pullulan.

ϲϳ 

FUTURE PROSPECTS A. pullulans produce pullulan which is a biodegradable polysaccharide processed into fibers and used for packaging food and drug products. Pullulan appears as an ingredient in cosmetics, pharmaceuticals, food and beauty products because of its solubility in water, and adhesive properties.

Pullulan, it also provides a smoother texture to

formulas, provides foam retention, is anti-static and oil resistant, and water soluble and therefore easily rinsed away. At present no pullulan production is done in India. Hayashibara Co. Ltd. (Japan) is the main commercial producer of pullulan worldwide. Further, the fermentation technology especially immobilization of microbial cells for continuous pullulan production and isolation of newer strain from the natural ecosystem as well as through mutational technique are still open for a wide research in order to achieve economic production.

ϲϴ 

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ANNEXURE • Basal broth Medium ¾ Glucose/ Sucrose/ Maltose – 2gm ¾ Ammonium Sulphate- 0.06gm ¾ Magnesium Sulphate- 0.04 gm ¾ Sodium Chloride- 0.1gm ¾ Di-potassium hydrogen orthophosphate- 0.5gm ¾ Yeast Extract- 0.04 gm ¾ pH- 5 ¾ Distilled water- 100ml • Sabouraud Dextrose Agar ¾ Dextrose- 40 gm ¾ Peptone- 10 gm ¾ Agar agar- 15 gm ¾ Distilled Water- 1000 ml • Potato Dextrose Agar ¾ Potato infusion- 200 ml ¾ Dextrose- 20 gm ¾ Agar agar- 20 gm ¾ Distilled water- 1000ml 

ϴϰ