Current World Environment Vol. 9(1), 192-202 (2014)

Studies on Fungal Strains of Selected Regions ofLudhiana and their Biochemical Characterization

DEEPIKA BHATIA, SIMRANJEET SINGH, ASHISH VYAS,HAKIM ISHFAQRASOOL, PARVINDER KAUR and JOGINDER SINGH*

Department of Biotechnology, Lovely Professional University, Phagwara (144401), Punjab, India.

http://dx.doi.org/10.12944/CWE.9.1.27

(Received: Feburary 13, 2014; Accepted: April 05, 2014)

ABSTRACT

Conservation methods often are focused on maintaining the biodiversityof a specificlandscape or ecosystem. Scientist’soften provide species richness as an indicator of biodiversity.However,species richness data are problematic when attempts are madeto enumerate microfungi,particularly those from the soil. Manysoil fungi fail to sporulate, making identification difficult.Othermeans of assessing the importance of fungi to ecosystempreservation must be developed.Otherwise, microfungi mightbe overlooked in discussions of ecosystem management andconservationissues. Herein, we have described the varieties of fungi was isolated from soils fromhigh and low yield areas of a field sites of selected regions of Ludhiana. Fungal Diversity wasanalyzed by isolation and purification of fungal cultures. In the present investigation a total FortyTwo Fungal strains have been isolated from fourteen sites of Ludhiana region. The morphologicalstudy revealed that these microbial forms have multiple occurrences at multiple sites. Finally ninefungal strains were purified and physicochemical characterized to check the effect of pH (3-9)and effect of temperature (25-45º C) on their growth. The colony diameter was measured regularlybetween 24 hr duration. Among all fungal strains maximum strains showed the maximum growthat pH-6; while in case of other samples the maximum growth was observed in pH range of 3-9. Allthe fungal samples were grown at their optimum pH which has been observed to check the effectof temperature on the growth. It was observed that all the fungal strains show maximum growth at25º C indicating their mesophilic nature. On the basis of morphological & enzyme productioncapacity, it was found that most of the fungal strains were of Aspergillus sp. and Fusariumsp. Theywere potential producers of amylase and cellulose. Some strains of Aspergillus were able toproduces both enzyme at the 4th day of incubation. The cellulose production capacities were moreas compared to rest of enzymes

Key words: Fungal Diversity, Sporulate, Mesophilic, Soil fungi,Physicochemical characterization.

INTRODUCTION

In a world dazzled by scientific discoveriesand technological advances leading to better livingstandards, the negative consequence of suchdevelopments are beginning to emerge and comeinto focus. Microorganisms perform their metabolicprocesses rapidly and with remarkable specificityunder ambient conditions, catalyzed by their diverseenzyme mediated reactions. Enzyme alternativesto harsh chemical technologies has led to intensiveexploration of natural microbial biodiversity todiscover enzyme which could function effectively

and generate pollution-free “dream technologies”in the immediate future. (Srinivasan et al., 1999).

We live on “a microbial planet” (Woese,1999) in the “Age of Bacteria” (Gould, 1996). Micro-organisms, the first cellular life forms, were activeon earth for more than 3.0 billion years before thedevelopment of multi-cellular, macroscopic lifeforms. During that time and continuing into thepresent, through the invention of a spectacular arrayof different metabolic and physiological capabilities,microbes evolved to exploit the multitude ofenvironments and microhabitats presented by the

193BHATIA et al., Curr. World Environ., Vol. 9(1), 192-202 (2014)

abiotic world. Rapidly accumulating evidenceindicates that microbe’s most likely account for thevast majority of kinds of organisms on earth.Microbes carry out a stunningly diverse array ofmetabolic activities, several of which wereinstrumental in creating conditions for the evolutionof other life forms.

Estimates of 1.7 million species have beendescribed to date; estimates for the total number ofspecies existing on earth at present vary from 5million to nearly 100 million. Fungi are secondlargest group of living biota available in the worldafter insects. The currently known fungal speciesare around 72,036 species and in India 27,000 fungiwere encountered (Manoharachary, 2002).

The number of fungi recorded in Indiaexceeds 27,000 species, the largest bioticcommunity recorded after insects (Sarbhoyet.al.,1996). The true fungi belong to kingdom Eukaryotawhich has 4 phyla, 103 orders, 484 families and4,979 genera. A fungus is a member of a large groupof eukaryotic organisms that includesmicroorganisms such as yeasts and molds, as wellas the more familiar mushrooms.Fungi perform anessential role in the decomposition of organic matterand have fundamental roles in nutrient cycling andexchange. They have long been used as a directsource of food, such as mushrooms and truffles, asa leavening agent for bread, and in fermentation ofvarious food products, such as wine, beer, and soysauce. Since the 1940s, fungi have been used forthe production of antibiotics, and, more recently,various enzymes produced by fungi are usedindustrially and in detergents. Fungi are also usedas biological agents to control weeds and pests.Many species produce bioactive compounds calledmycotoxins, such as alkaloids and polyketides,which are toxic to animals including humans. Theirgrowth is probably related to their ability to grow at28°C and produce protein and fat hydrolyzingenzymes. Soil fungi have been recognized chieflyby two methods; direct examination and isolationby cultural methods. The limitations of the firstmethod are obvious, and although it has producedmany excellent results, the physical barriers in theway of such a method limit its usefulness, and inmost cases such observations as were made by itwere confirmed by resort to cultural methods. In the

second method, that of isolation of cultures, hasbeen more widely used and has produced thegreater numbers of species of recognized soil fungi.The substrate used in making the isolation is ofprime importance in determining the species thatwill be taken cannot be denied. The remarkablediscovery of Coker and his associates that manyspecies of saprolegniales are seemingly presentin a very wide range of soils and localities was theresult of the application of the techniques andmaterials used to isolate this group of fungi fromwater samples.

Biodiversity refers to the variability of lifeon Earth, all the living species of animals, plantsand microorganisms. According to Hawksworth(2002), fungi are a major component of biodiversity,essential for the survival of other organisms andare crucial in global ecological processes. Fungibeing ubiquitous organisms occur in all types ofhabitats and are the most adaptable organisms.The soil is one of the most important habitats formicroorganisms like bacteria, fungi, yeasts,nematodes etc. The filamentous fungi are the majorcontributors to the soil biomass (Alexander 1977).They form the major group of organotrophicorganisms responsible for the decomposition oforganic compounds. Their activity participates inthe biodeterioration and biodegradation of toxicsubstances in the soil (Rangaswamiet al., 1999). Ithas been found that more number of genera andspecies of fungi exist in soil than in any otherenvironment (Nagmaniet al., 2005). Contributingto the nutrient cycle and maintenance of ecosystemfungi play an important role in soil formation, soilfertility, soil structure and soil improvement (Hao-quinet al., 2008). Fungi take a very importantposition in structure and function of ecosystem. Theydecompose organic matter from humus, releasenutrients, assimilate soil carbon and fix organicnutrients. An intense study of abundance anddiversity of soil microorganisms can divulge theirrole in nutrient recycling in the ecosystem.

MATERIALS AND METHODS

Selection of Sample SitesFourteen different sites in various villages

of in and around vicinity of Ludhiana were selectedas shown in table-1 (given in supplementary sheets).

194 BHATIA et al., Curr. World Environ., Vol. 9(1), 192-202 (2014)

Collection of soil sampleDifferent types of soil samples viz. waste

soil (W), dump soil (D), Leaf litter (L), decomposingorganic manure (M) etc were collected. Samples(500gm) were suspended in sterile polythene bagand thoroughly packaged and kept at 4°C.

Isolation of FungiSamples were stored in the refrigerator at

4°C till the isolation of fungi. For isolating Fungi,the PDA media (solution containing all the nutrientsrequired for the growth of microorganisms) wasadded with 50 g/ml tetracycline to suppressbacterial and in the isolation plates. Mediumsupplemented with various chemicals componentswas inoculated and incubated at 28°C.

Media (g/l)Potato (Peeled) - 200Dextrose - 10Agar - 15pH - 5.6

Distilled Water - 1000 ml

Purification of fungal strainsAfter 4 days of incubation Fungal colony

started appearing as separated on PDA mediumand it was further incubated for 4 days at 28ÚC.The purified colonies were aseptically picked upand transferred to PDA slants. The slants wereincubated at 28°C for 4-5 days for maximum growth.The slants were then stored at 4°C in the refrigerator.

Maintenance of pure cultureSamples were stored in the refrigerator at

(4°C) for the identification of fungi.

Physico-chemical analysisEffect of pH on the growth of fungal colonies

The fungal samples were grown atdifferent pH ranges (3, 7 and 9) to check the effectof pH on their growth. The diameter (mm) of thefungal colonies was measured at different timeintervals.

Effect of Temperature on the growth of fungalcolonies

The fungal samples were grown atdifferent temperature ranges (25°C, 35°C, 45°C) tocheck the effect of temperature on their growth.

Bio-Chemical Screening of SamplesAmylase assay

Screening was done as per the method ofBehlet al., (2006) and Rele (2004). Screening offungal culture for enzyme activity was carried outon agar media on petri plates containing 1 %soluble starch. After solidification of medium around10 mm well was cut out aseptically with the help ofcork borer. The well was filled with crude enzymeextract and incubated at 28°C for 96 hrs in anupright position. The plates were flooded withGram’s iodine solution and observations were madeto see clear zone around the well. A negative controlwas maintained by adding heat denatured crudeenzyme sample. Denaturation was done by boilingenzyme extract at 110°C for 20 min and then cooledsuddenly in an ice bath for 5 min. After flooding withGram’s iodine solution a clear area around the lineof growth indicates starch hydrolysis

Cellulase assayScreening was done as per the method of

Waksman and Fred (1922). Screening of fungalculture for enzyme activity was carried outonCzapekDox Agar Medium petri plates. Aftersolidification of medium around 10 mm well wascut out aseptically with the help of cork borer. Thewell was filled with crude enzyme extract andincubated at 28°C for 96 hrs in an upright position.The plates were flooded with 0.1% Congo red dyesolution andobservations were made to see clearzone around the well. A negative control wasmaintained by adding heat denatured crudeenzyme sample. Denaturation was done by boilingenzyme extract at 110°C for 20 min and then cooledsuddenly in an ice bath for 5 min. After flooding with0.1% Congo red dye solution a clear area aroundthe line of growth indicates starch hydrolysis.

Protease assayScreening was done by sub culturing of

the fungus produced on skimmed milk medium oneby one onto the PDA medium and were finallytransferred to PDA slants and maintained at 4°C.Proteases were screened using with fungalselective media (PDA: potato dextrose agar, maltextract, Czapek and Dox medium), containing twodifferent protein substrate, soya meal (20 g/l) andCasein Peptone (10 g/l). These ingredients wereadded to induce the protease synthesis by the fungi

195BHATIA et al., Curr. World Environ., Vol. 9(1), 192-202 (2014)

and the growth was measured after 24hrs, 48hrs,72hrs and 96hrs.

Identification of CulturesThe cultures were identified by studying

their macroscopic appearance, pigmentation andgrowth rate (table-5 given in supplementarysheets). Visual examination was done to study theimportant characters such as colour, texture,macroscopic structures, growth zones, aerial andsubmerged hyphae and colony topography. Themicroscopic examination was made by observingthe slide cultures after staining with Lacto phenolCotton Blue. Based on these feature identificationwas made following Gilman’s Manual for Fungusidentification (Gilman J.C 1995).

RESULTS

Physico-chemical characterization of purifiedfungal strainsEffect of pH on the growth of fungal colonies

The fungal samples were grown atdifferent pH ranges (3, 7 and 9) to check the effectof pH on their growth. The diameter of the fungalcolonies (mm) was measured at different timeintervals.

The pH is the symbol for the logarithm ofthe reciprocal of the hydrogen ion concentration(Log/H+) in grams atoms per liter. The measure ofthe relative acidity or alkalinity of a solution is calledpH.

pH is one of the important factor thatdetermines the growth & morphology ofmicroorganisms as they are sensitive to theconcentration of hydrogen ion present in themedium. Each enzyme system of organisms has aparticular pH range in which it can function. The pHvalue also serves as a valuable indicator of theinitiation and end of the enzyme synthesis (Friedrichet al., 1989).

Effect of Temperature on the growth of FungalColonies

The fungal samples were grown atdifferent temperature ranges (25°C, 35°C, 45°C) tocheck the effect of temperature on their growth. Thediameter of the fungal colonies (mm) was measuredat different time intervals.

Temperature is a vital biochemical factorwhich controls the enzymatic activities.Ninefungalstrains were selected for temperaturecharacterization study. Optimum temperature for theactivity of Protease produced by Aspergillusnigersp., Aspergillusflavus sp., Fusarium sp.,was at 25-45°C respectively. The data presented in tablesclearly reveals that fungal strains show maximumprotease activity at 25-45°C.Ali (1992) also recordedoptimum temperature of 30°C for proteaseproduced by A. fumigatus.Fungal strains producedproteases beyond 30°C but in lesser yields thanthat produced at optimal temperature. Thesetemperatures might not havebeen suitable forEnzyme production. This is in accordance with thereview of Daniel et al., (2010) whostated thatincrease in temperature led to increase inactivity

Table 1: Selection of Samples Sites

S. No. Name of The Site Site Code

1 Vill. KoharaTeh: KODistt Ludhiana

2 Vill.JandialiTeh: JADistt Ludhiana

3 Vill. RamgarhTeh: RADistt Ludhiana

4 Vill. TajpurTeh: TADistt Ludhiana

5 Vill. SuneetTeh: SUDistt Ludhiana

6 Vill. LaltoTeh: LADistt Ludhiana

7 Vill. JhabewalTeh: JHDistt Ludhiana

8 Vill. AyaliKhurdTeh: AKHDistt Ludhiana

9 Vill. Ayali KalanTeh: AKADistt Ludhiana

10 Vill. MundianKhurdTeh: MKHDistt Ludhiana

11 Vill. Mundian KalanTeh: MKADistt Ludhiana

12 Vill. MangliucchiTeh: MUCDistt Ludhiana

13 Vill. BastijhodewalTeh: BJODistt Ludhiana

14 Vill. Sunder nagarTeh: SUDistt Ludhiana

196 BHATIA et al., Curr. World Environ., Vol. 9(1), 192-202 (2014)

but that there was limit to the increase inactivitybecause higher temperatures led to a sharpdecrease in activity. This could be due to thedenaturing of protein structure.

Purified fungal strains werephysicochemical characterized to check the effectof pH (3-9) and effect of temp. (25-45ºC). Effect ofpH on the growth of fungi indicates that the fungalstrainswere shown the maximum growth at acidicpH i.e3. Some strains of Aspergillus were shownmaximum growth at alkaline pH-9. They show themaximum growth at 4th day of incubation with slightdecrease afterwards. A pH gradient agar plates withsurface pH ranging from about 3-9 was easilyprepared. The gradient was sufficiently stable topermit different growth characteristics amongvarious fungal species at different pH value to bedistinguished. The pH effects on the growth rate,

conidiogenisis and pigment formation wereconsistent with the results obtained by otherworkers. Wheeler et al., (1990) reported that theeffect of pH on growth rate of 61 isolates of fungi.Four species of Aspergillus grows over the pH range3-9 and temperature 37ºC. in general Aspergilluswere the most tolerant of alkaline pH. Thetemperature effect on fungal strains was studied attheir observed optimum pH values from 3.0 - 9.0and at different temperature ranging from (25-45ºC). Optimum temperature was found to be 25ºCfor all the fungal strains. The result clearly indicatesthe presence of mesophilic strains of Aspergillusand Fusarium sp. The maximum growth was foundto be occurs at the 4th day of incubation. Graduallyon increasing temperature up to 45ºC, the growthdecreases afterwards. The colony diameter wasmeasured regularly after the gap of 24 hrs atdifferent temperature range.

Table 2: Effect of different pH range on the growth of fungal sp

Aspergillus glaucus Aspergillus fumigatus Fusarium spTime pH- pH- pH-

pH-3 pH-7 pH-9 pH-3 pH-7 pH-9 pH-3 pH-7 pH-9

24 hrs 0 2 2 0 1.5 0.5 0 2.5 2.548hrs 0 3.5 3 0 2.5 2.5 0 3.5 5.872hrs 0.5 4.8 7.5 0 4.5 4.5 0 4.8 796hrs 1 9.5 10.5 0 11.5 6.5 1.5 9 8.5

Fusarium sp. Aspergillus nidulans Fusarium spTime pH pH pH

pH-3 pH-7 pH-9 pH-3 pH-7 pH-9 pH-3 pH-7 pH-9

24 hrs 0 0 1.5 0 2 2.5 0 2.5 2.548hrs 0 0 3 0 2.8 5.5 0 3.5 5.872hrs 0 2 5 0.8 6 6.5 0 4.8 796hrs 2.5 3.5 7.5 2 9.5 8 1.5 9 8.5

Fusarium sp. Aspergillus flavus Aspergillus nigerTime pH- pH- pH-

pH-3 pH-7 pH-9 pH-3 pH-7 pH-9 pH-3 pH-7 pH-9

24 hrs 0 0 1.5 0 1.5 0.5 0 2 148hrs 0 0 3 0 3 3 0 2.8 2.572hrs 0 2 5 1.5 7.5 7.5 0 6 496hrs 2.5 3.5 7.5 1.8 10.5 11 0.5 11 7

197BHATIA et al., Curr. World Environ., Vol. 9(1), 192-202 (2014)

Table 3: Effect of temperature on the growth of fungal sp. at selective pH

Aspergillus glaucus Aspergillus fumigatus Fusarium spTime Temp at pH-9 Temp at pH-8 Temp at pH-6

pH-3 pH-7 pH-9 pH-3 pH-7 pH-9 pH-3 pH-7 pH-925°C 35°C 45°C 25°C 35°C 45°C 25°C 35°C 45°C

24 hrs 9 4.5 1.5 6.5 6.5 2.5 5 4 448hrs 15 7.5 3 12 11 4 13 7.5 4.572hrs 22 15 5.5 20.5 15.5 6.5 24 13 8.596hrs 30 17.5 12 30 22 11.5 33.5 18.5 13.5

Fusarium sp. Aspergillus nidulans Fusarium spTime Temp at pH-8 Temp at pH-6 Temp at pH-6

pH-3 pH-7 pH-9 pH-3 pH-7 pH-9 pH-3 pH-7 pH-925°C 35°C 45°C 25°C 35°C 45°C 25°C 35°C 45°C

24 hrs 7 3.5 3.5 8 2.5 4 3.5 3.5 448hrs 13 11.5 4.4 15.5 6.5 5 13.5 7.5 572hrs 25 15 10 17.5 10 7.5 18 15 6.596hrs 40 16.5 12.5 24 14 14.5 26 18.5 11

Fusarium sp. Aspergillus flavus Aspergillus nigerTime Temp at pH-8 Temp at pH-6 Temp at pH-6

pH-3 pH-7 pH-9 pH-3 pH-7 pH-9 pH-3 pH-7 pH-925°C 35°C 45°C 25°C 35°C 45°C 25°C 35°C 45°C

24 hrs 8.5 5 5 8.5 4.5 6.5 10.5 4.5 7.548hrs 12.5 11.5 7.5 15 6.5 8.5 12.5 6.5 9.572hrs 24 13.5 9 27 11 11.5 25 9.5 12.596hrs 36.5 16 14 32 15.5 15.5 34 14 16.5

Table 4: Enzymatic screening of purified fungal strains [Colony diameter (mm) was measured]

S. Identified Amylase Screening Cellulase Screening Proteaseno. Fungal Strain Starch Hydrolysis Test Congo Red Dye Test Screening

1 Aspergillus glaucus + (1.5 mm) + (2.0 mm) -2 Aspergillus fumigatus _ + (1.0 mm) + (8.5 mm)3 Fusarium sp. + (1.5 mm) + (2.0 mm) + (9.5 mm)4 Fusarium sp. + (1.0 mm) + (2.0 mm) + (9.0 mm)5 Aspergillus nidulans _ _ + (10.5 mm)6 Fusarium sp. + (1.5 mm) + (2.0 mm) + (8.0 mm)7 Aspergillusf lavus + (1.5 mm) + (1.5 mm) + (7.5 mm)8 Aspergillus niger + (1.0 mm) + (2.0 mm) + (8.0 mm)9 Fusarium sp. + (2.5 mm) + (2.0 mm) + (8.0 mm)

198 BHATIA et al., Curr. World Environ., Vol. 9(1), 192-202 (2014)Ta

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199BHATIA et al., Curr. World Environ., Vol. 9(1), 192-202 (2014)

Fig. 1

Fig. 2

200 BHATIA et al., Curr. World Environ., Vol. 9(1), 192-202 (2014)

Enzymatic screening of purified fungal strainsEnzymes are the most important products

obtained for human needs through microbialsources. Enzymes are highly efficient environmentalfriendly protein catalyst synthesized by livingsystems. They have significant advantage over

chemical catalyst in terms of catalytic activity, abilityto work at moderate temperature and the ability toget produced in large amounts (Chand & Mishra,2003). Dr. JockichiTakamine (1914) was the firstindiviual to realize the mechnical possibility ofcultivated enzymes and presented them to society.Although he was mainly concerned with fungal

Protease screening of purified fungal strains

Cellulase screening of purified fungal strains

Amylase screening of purified fungal strains

201BHATIA et al., Curr. World Environ., Vol. 9(1), 192-202 (2014)

enzymes, Boidin and Effront (1917) were pioneersin the output of enzymes produced by bacteria.Since that time microbial enzymes have taken theplace of enzymes from plants and animals(Underkofler, et al. 1957).

A total of 9 fungal strains were purifiedfrom different soil samples and enzymatic screeningwas performed at optimum pH and temperature.The results indicate that number of microbial formshas multiple occurrences and additionally, theyhave the capacity to produce amylase underoptimum environmental conditions. All the purifiedfungal strains were screened for the enzymeproduction capacity viz. amylase, cellulase andprotease at their recorded optimum pH andtemperature. Most of the strains were potentialproducer of cellulase among all. Maximum enzymesynthesis occurred at the 4th day of incubation withgradual decrease afterwards. The pH andtemperature effects the enzyme activity of fungalstrains. The strains of Aspergillus and Fusarium sp.were the maximum producer of enzymes.

Chellapandi (2009) reportedAspergillusflavusand Aspergillusterreusas thepotential strains for production oftannery proteasein submerged fermentation. To improve theproductivity of protease enzyme in liquid broth,various media ingredients have been optimized.The crude and partially purified proteasespreliminarily characterized and used for unhairingprocesses at lab scale in tannery. The proteaseobtained from these strains showed the goodactivity in wide alkaline condition at 50 ºCsuggesting the possibility of using in leather anddetergent industry.

Result also raises hope for purification andapplications in various industrial processes. Thiscan have important implications in proteolyticbreakdown of proteins by these fungal strains forthe establishment of a robust and cost effectiveprocess in various industries like brewing,baking,textiles and laundry etc.

The present study raises the hope tofurther characterize these fungal strains forenzymatic characterization and to be harnessedfor industrial applications.

DISCUSSION

The major goal of microbial ecology is tounderstand microbial diversity in natural habitats;therefore knowledge of both microorganisms andhabitats is essential. A Total of 14 Sites wereselected for sample collection for the isolation ofthe fungal strains, quantitatively the number offungal cultures were large has been shown in table1 and 2 respectively. A total of forty two fungalcultures were isolated from fourteen sample sites.After critical morphological observation (Colonycolour, Colony shape, pigmentation, pattern ofgrowth in five days) and microscopic observation,many of the fungal cultures were found to be similarand found to be present at multiple locations andindicated by different superscripts. Identified fungalstrains are, Aspergillus fumigatus, Aspergillusglaucaus, Aspergillus nidulans, Fusarium sp.,Aspergillus niger. The results show that the regionLudhiana is flourished with microbial flora. Theabove results also confirm the isolation studiesearlier done by Goyalet al 2008, Jain et al 2005.The effect of pH on the growth of Aspergillusglaucaus and Aspergillus fumigatus showedmaximum growth at pH-9 (alkaline pH). The colonydiameter was measured i.e 11.5mm. The growthincreases after 48hrs. Sporulation was observedafter 96hrs. Aspergillus flavus shows that theoptimum pH was recorded i.e acidic pH-6. Thecolony diameter was measured 12.5 after 96 hrs.Green colored spores was observedmicroscopically after 48 hrs. Similar results wereobserved with the fungal strains. The optimum pHwas 6 & the colony diameter measured i.e 12.5.Sporulation was observed after 96 hrs. The datapresented in Table 1 & 2 indicates thatAspergillusfumigatus grows maximally at 25ºC atalkaline pH i.e 8. The colony diameter wasmeasured after the gap of 24 hrs. The maximumcolony diameter recorded 30 mm after 96 hrs at25ºC. The mesophilic fungal strain,Aspergillusfumigatus which grows at 25ºC at acidicpH (i.e 6). The growth rate decreases whentemperature increases up to 45ºC. It clearly depictsthat the optimum temperature was found to be 25ºCat pH 8 for the growth of Fusarium sp. The highestcolony diameter was measured i.e 40 mm after 96hrs. the presence of mesophilic fungal strain ofAspergillusnidulans. The fungal sample were

202 BHATIA et al., Curr. World Environ., Vol. 9(1), 192-202 (2014)

allowed to grow at its optimum pH (i.e 6). Theoptimum temperature found to be recorded was25ºC and highest colony diameter was 24 mm. Thestrain of Aspergillusflavus&Aspergillusniger. The

optimum temperature was found to be 25ºC atacidic pH (i.e 6). Very less growth was observedafter increasing temperature up to 45ºC.

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2. Ali gao, Formation of protease byAspergillusfumigatusand Penicilliumspp. J.King Saud Univ. Sci., 4(2):127 (1992).

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