J. Phytol. Res. 30 (1) : 7-15, 2017 ISSN 0970-5767

ISOLATION AND IDENTIFICATION OF FUNGAL FLORA ON DEGRADING POLYTHENE HEAPS AROUND WATER BODIES OF JHANSI

ANKITA SAXENA and ARVIND PAREEK* Department of Microbiology, Mewar University, Gangrar, Chittorgarh, 312901, India * Department of Botany, Maharshi Dayanand Saraswati University, Ajmer, 305009, India * Corresponding author : E-mail: [email protected]

Polythene is an important part of daily life, that’s why demand of polythene production is increasing worldwide each year but its final non-treated waste is making earth polluted. Polythene is becoming major threaten element for environment, seems lack of awareness and poor waste management in last two decade. Fungi are ubiquitously found as reported from in each and every habitat, even can survive in adverse environment. Biodegradation is a best way, if possible, to overcome this problem. Still fungal flora of decaying polythene waste is not much worked out. This piece of work was conducted in order to find out a microbial consortium for the biodegradation of polythene. Samples were collected from various water bodies of Jhansi and screened for their fungal flora. In present study 10 fungal strains were isolated from different natural decaying sites.

Keywords: Biodegradation; Fungi; Land pollution; LDPE; Persistent organic pollutants; Polythene.

Introduction sheets or bags do not allow water and air to Polythene is the most versatile synthetic go into earth which causes infertility of soil, manmade substance created out of the fossil preventing degradation of other normal fuel and other organic material extracted substances, depletion of underground water from coal, oil and natural gas1. Polythene source, danger for animal life and the main are nonmetallic moldable compounds that cause of blocked drains4. Promiscuous and can be pushed into almost any desirable frequently deliberate release of plastics is shape and then retain in that shape 2. As the responsible for escalating environmental consumption is growing, parallel waste pollution. To remuneration this management by chemical and physical environmental damage, there is an urgent methods becoming a challenging task as it need of potential method. produces persistent organic pollutants Biodegradation executed by the (POP’s) known as furans and dioxins3. To microorganisms has environmental-friendly get rid of such a menace, commonly landfill benefit and this method is less expensive and burning has done that is the core origin and alternative of decaying (Physical and of pollution. On the other hand, Plastic chemical changes) organic pollutant5.

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Among all biological agents, microbial Present piece of work was focused enzymes are one of the most powerful tools on isolation of fungal flora grown on for the biodegradation of polythene and rich polythene debris around water bodies in microbial flora easily originates near water Jhansi due to potential of fungal strains and bodies because of its friendly environmental their degradability, because fungi are condition. Sometime submerged polythene versatile organisms, able to grow and found with heavy fungal growth, slime and degrade a variety of compounds, organic soft textured, tearing easily with brittle contaminants and polymeric materials9. nature. Fungi are vigorously found, easily Enzyme activity of biodegradation is higher grown and a diverse group of organisms in fungi than in bacteria. Isolation of belonging to saprophytic fungi. Fungi are different fungal strains were made from evolved to adapt almost every environment, submerged polythene with heavy fungal specially marine and freshwater source. This growth at decaying sites in Jhansi. A variety property drives fungi to grown on polythene of species were found. Fungal isolation and even in adverse environment. A verity of identification from polythene is never heterotrophic fungi associated with testified before in Jhansi. polythene, is reported decaying sites near Research location water bodies6. Saprophytic fungi are quickly Degraded polythene sample were collected germinated and vital for the maintenance of from different water bodies of Jhansi as natural ecosystem by breaking down of given below (Table 1). Which is located in decomposes into small organic molecules, 25°26’55,03”N, 78°34’10,63”E, Lat: which can be absorbed by decomposers or 25.4441, Long: 78.5676. It has an average nearby plants and other microbial flora7. It elevation of 284 meters (935 feet). Jhansi was reported that Aspergillus spp. is most lies on the plateau of central India, an area dominantly contributing and the other dominated by rocky relief and minerals dominant spores are Fusarium spp., underneath the soil. Average annual Curvularia spp. and Cladosporium spp. in minimum and maximum temperature range environment8. is from 4°C to 46°C.

Table 1. List of different water bodies

S. No. Habitats No. of sample No. of fungal isolates 1 Parichha dam 2 3 2 Antia pond 4 6 3 Pahuch dam 2 4 4 Betwa river 4 5 5 Orachha river 3 2 6 Sukamadukama dam 4 3 7 Pahuch canal 4 2

Methodology isolation. Samples were inoculated and Samples were collected from different water plated on PDA plates and sub cultured for bodies (dumping sites) of Jhansi city. These pure fungal isolates. Fungi were identified samples were further treated for fungal by microscopic examination10. J. Phytol. Res. 30 (1) : 7-15, 2017 9

Results and Discussion average length 9-11µm; elongated to Sub-cultured isolates were identified using polyhedric12. microscopic observation and using 3. Chaetomium murorum Lactophenol cotton blue stain. Ten isolates Division: were identified; on the basis of their Class: morphological and cultural characteristics, Order: Sordariales observed and noted after 7 days of Family: Chaetomiaceae incubation. These fungi were identified as Genus: Chaetomium Penicillium chrysogenum, Rhizopus Species: murorum nigricans, Chaetomium murorum, Colony was bluish gray to black in color, Memnoniella echinata, Aspergillus with the surronding agar was yelloish fumigatus, chartarum, brown. Perithecia dark colored, globose to Aspergillus niger, Chaetomium globosum, subglobose, 260-320 X 250-300 µ ostiolate. Aspergillus flavus and Fusarium oxysporum Terminal hair dark olive brown, slender, 1. Penicillium chrysogenum gracefully flexed, with open circinate, blunt Division: Ascomycota tips, 5.5-6 µ wide, smooth septate (Plate-1, Class: Eurotiomycetes Fig. C). Lateral hairs olive brown below, Order: Eurotiales fading above, straight to flexed, with pointed Family: Trichocomaceae tips, smooth, septate, 4.5-5 µ wide at the Genus: Penicillium base. Asci was club-shaped, 8-spored. Species: chrysogenum Ascospores dark olive green, ellipsoid, 12- Colony is cottony, rapid growth, conidium 13.5 X 6-7.5µ, subapiculate at 1 or both green and diameter of colony was around ends 13.The genus Chaetomium was 35mm. Conidia 2.8x2.5 µm, Subglobose, established by Kunze and Schmidt14 but Smooth, Finely roughened ( Plate-1,Fig. A) further redefined on several occasions and Stripe was 258x3.4 µm. Phialide was flask discovers many other species13, 15-17. shaped and branching pattern was 4. Memnoniella echinata quarteverticillate11. Division: Ascomycota 2. Rhizopus nigricans Class: Sordariomycetes Division: Zygomycota Order: Class: Mucormycotina Family: Hypocreales Order: Mucorales Genus: Memnoniella Family: Mucoraceae Species: echinata Genus: Rhizopus Colonies small, thick, white-black composed Species: nigricans of hyaline septate mycelium. Conidiophores Colony is white, cottony, rapid growth. were blackish, erect, hyaline at the base 55- Hyphae broad, scarcely septate, rhizoids and 85µm, long, 3-4µm wide. Conidiophores stolons present. Sporangiophores were bear one or two compact whorls of about ten brown. Rhizoid length was 300-350. phialides (Plate-1, Fig. D). Phialides sub Sporangiophore length was 1500-4000. hyaline, one celled, about 7-9 µm long, 2µm Sporangium length was 150-350 (Plate-1, wide, slightly diverging. Conidia opaque Fig. B). Columellae were almost round. black, globose rough or somewhat angular Sporangiospores were variable in size, 6 µm to 8 µm wide and 4 µm thick, forming

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persisting long chain of conidia up to 150 Class: Eurotiomycetes µm long dark in color 18. Order: Eurotiales 5. Aspergillus fumigatus Family: Trichocomaceae Phylum: Ascomycota Genus: Aspergillus Class: Eurotiomycetes Species: niger Order: Eurotiales Colonies start white to pale yellow but Family: Trichocomaceae quickly formed jet-black conidia, the reverse Genus: Aspergillus color is usually buff or yellow – gray. Species: fumigatus Characteristically obscurus metulae and Colonies cottony, velvety, green-brown with phialides, conidia are spherical, 3-5 µ, and a white apron at the margin. The reverse roughen with maturity (Plate-1, Fig. G)21. color is usually white to tan unlike most 8. Chaetomium globosum aspergillus species. A. fumigatus grown at Division: Ascomycota 45° and conidiophores are long 300-500 µ, Class: Sordariomycates have club-shaped vesicles that are 30-50 µ Order: Sordariales in diameter(Plate-1, Fig. E). Vesicles are Family: Chaetomiaceae uniseriate and are covered by conidia on Genus: Chaetomium only the distal half conidia arise in chain and Speices: globosum tend to sweep toward the central axis19. Colony size is 7-8 mm, with pale or Konduri et al.20 was also worked on many olivaceous aerial mycelia. Ascomata mature Aspergillus spp. and reported that within 7-9 days, measured 175-280µm, and Aspergillus spp. can grow to 72% reduction were olivaceous, gray- green or brown in in percentage of elongation in polythene. reflected light and tended to be superficial, 6. spherical, ovate or obovate and ostiolate.The Division: Ascomycota ascomatal wall was brown in color and Class: Sordariomycates composed of textura intricate. The cells Order: Hypocreales were 2.0-3.5 µm in breadth and ascomatal Family: Stachybotryaceae hairs were numerous, typically unbranched, Genus: Stachybotrys flexuous, undulate or coiled, often tapering, Species: chartarum septate, brownish, 3-4.5 µm in breadth at Stachybotrys chartarum is also known as base and up to 500 µm in length. The asci Stachybotrys atra and stachybotrys were clavate or slightly fusiform, stalked, alternans. Colonies are thick dark black. evanescent, measured, 30-40 X 11-16 µm, Mycelium hyaline, almost hyaline towards and contained eight ascospores (Plate-1, Fig. base, septate, fuliginous near tip, 73-91 µ H). Ascospores were limoniform, typically long, 13-16 µ wide. Conidiophore biapiculate, bilaterally flattened, brownish terminating in 3-5 cluster of phialides. when mature, thick walled, contained Phialides 70-90 µ long X 4-6 µ in diameter numerous droplets, measured 9-12 X 8-10 X (Plate-1, Fig. F). Conidia single, aseptate, 6-8 µm, and featured an apical germ pore14. smooth, ellipsoidal to browdly ellipsoidal, 9. Aspergillus flavus dark colored 8-16 X 3-9 µ diameter13. Division: Ascomycota 7. Aspergillus niger Class: Eurotiomycetes Division: Ascomycota Order: Eurotiales Subphylum: Pezizomycotina Family: Trichocomaceae J. Phytol. Res. 30 (1) : 7-15, 2017 11

Genus: Aspergillus Fungi are wildly found organism, grown Species: flavus rapidly. Fungi have the strong proficiency to Conidiophores were heavy walled, degrade lignin, cellulose, starch and other uncoloured, coarsely roughened, usually less organic material and consumed it as than 1mm in length. Vesicles are elongate nutrition26. This blissful nutrient strategy is when young, later becoming subglobose or base of biodegradation. Natural decaying globose, varying from 10-65 mm in site in Jhansi are good source of microbial diameter (Plate-1, Fig. I). Phialides were flora, environmental condition is favorable uniseriate or biseriate. The primary branches for rapid fungal growth. Samples were were up to 10 mm in length and secondary collected with heavy fungal growth. 10 5mm. Conidia were typically globose to strains were isolated, out of which 9 were subglobose, varying from 3.5 to 4.5 mm from Ascomycota and one wasof diameter22. Zygomycota. Similar to this study, Gilan et 10. Fusarium oxysporum al. 27 was also worked on different fungal Division: Ascomycota species with starch blend polythene and Class: Sordariomycetes found that degradation rate is directly Order: Hypocreales proportional to starch content present in Family: Nectriaceae polythene. Konduri et al. 20 was also Genus: Fusarium investigated on many Aspergillus spp. and Species: oxysporum reported that Aspergillus spp. can grow to The coloration of Fusarium oxysporum 72% reduction in percentage of elongation mycelium was white. Their conidiophores, in polythene. Chaetomium spp., Rhizopus means through which Fusarium oxysporum spp., Penicillium spp., Aspergillus spp. are asexually reproduce, were short, single, saprophytic fungi and economically lateral monophialides (flask-shaped beneficial. The genus Chaetomium was projections) in the aerial mycelium, later established by Kunze and Schmidt28 but arranged to densely branched clusters. Their further redefined on several occasions and macro-conidia were fusiform, slightly discover many other species13,15-17. C. curved, pointed at the tip, mostly three Globosum is recognized as a celluloltic and septate, basal cells pedicellate, 23-54 x 3-4.5 endophytic , known for having great µm (Plate-1, Fig. J). Micro-conidia were potential in agricultural, medicinal and abundant, never in chains, mostly non- industrial fields29. Some collected samples septate, ellipsoidal or cylindrical, straight or were even found fragile, brittle and in curved, 5-12 x 2.3-3.5 µm. chlamydospores crumble situation with very heavy fungal were terminal or intercalary, hyaline, growth. Fungi can grow everywhere except smooth, 5-13 µm23. in under water. The atmosphere of Jhansi is Materials those are most susceptible to appropriate for fungi. Previously, Raaman et mold attacks such as wood jute etc., al.6 conduct the similar study near Chennai were water damaged, in which molds and reported Aspergillus niger, A. japonicus, most frequently encountered A. terreus, A. flavus and Mucor sp.and were Penicillium (68%), Aspergillus (56%) reported 8% degradation in one month and Chaetomium (22%)24. Water ecosystem period by A. niger. Some excellent studies is very rich in microbial diversity,only less was also carried out the tremendous variety than 5% of species have been described25. of fungal flora grown on polythene on 12 Saxena & Pareek

lignocellulose, PHB, PCL, PBS, PBS/A, Cryptococcus Laurentii, Penicillium Spp., Poly[3HB-co-(12 mol%) 3HV] in last two Curvularia Spp., Aspergillus fischeri, decates- Chaetomium globosum, Rhizopus Gliocladium virens etc 9,29-34. Environment spp., Fusarium oxysporum, Mucor spp., of Jhansi is highly suitable due to

A B C

D E F

G H

I J

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