Screening for temperature tolerance of some. Aspergillus spp. Lahore-54590, Pakistan. *E. mail:

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1 Myopth (8) 6(&): 7- Sreening for temperture tolerne of some spergillus spp. *Neelm Nzir n Ghzl Nsim Lhore ompost (Pvt.) Lt. Mehmoo ooti, Ring Ro, Lhore, Pkistn. Institute of Myology n Plnt Pthology, University of the Punj, Lhore-559, Pkistn. *E. mil: neelmnzir@yhoo.om strt Three speies of spergillus viz.,. fumigtus,. niulns n. sprsus were isolte from ifferent sustrtes t high temperture (5 o ) n were sujete for thermotolerne sreening. Fresh n ry weight, sorne y extrt/spore suspension, spore ount n enzyme tivity were the prmeters use to evlute fungl speies uner ifferent tempertures regimes i.e. 5± o, ± o, 5± o n 6± o. Mximum thermotolerne ws oserve in. fumigtus then. sprsus n. niulns. Key wors: sorne, spergillus, enzymes n temperture. Introution The spergillus genus is vst group of orgnism of osmopolitn ourrene. Speies of spergillus re importnt meilly n ommerilly. Some speies n use infetion in humns n other nimls. Memers of the genus re lso soures of nturl prouts tht n e use in the evelopment of meitions to tret humn isese (Denning, 998). Some re lso employe in vriety of fermenttion proesses; loholi everges suh s Jpnese ske re often me from rie or other strhy ingreients rther thn from grpes or mlte rley using speies of spergillus (Du et l., 8). Some speies of genus re n importnt omponent of thermotolremt myoflor isolte from vriety of sustrtes from Lhore (Nzir et l., 7). The present stuy is n extension of the pervious work n is fous on evlution of thermotolernt ility of isolte spergillus speies. Mterils n Methos Isoltions Speies of spergillus were isolte from vrious sustrtes like ompost, lef mnure, frmyr mnure n ung n ulture on mlt extrt gr meium. Thermotolerne Sreening Thermotolerne of speies ws teste on liqui roth. Simple % mlt extrt roth ws prepre for this purpose n twelve flsks for eh fungus were tken whih were utolve with meium t o for min then inoulte with is of.5 mm of pure ulture of isoltes uner septi onitions. Flsks were inute on four tempertures viz., 5± o, ± o, 5± o n 6± o. Three replites for eh temperture were prepre. fter seven ys of inution fungl iomss ws seprte y filtrtion. Fungl extrt ws preserve for further iohemil stuies. Fresh weight of fungl iomss ws tken n then fresh iomss ws rie t 6 o for hours n fterwr ry weight ws note. sorne of fungl extrt ws etermine t 5 nm, 55 nm n 6 nm on spetrophotometer. Quntifition of spore numer The numer of the spore per ml ws etermine y hemytometer. Isolte of spergillus inoulte on Mlt extrt gr ulture meium n inute t 5± o, ± o, 5± o, n 6± o. fter ys the spores were hrveste y ing ml sterilize istille wter in the petriplte. 5 µl of spore suspension ws e on the hemytometer overe y glss over slip. Spores were ounte uner the mirosope n t ws reore. Enzyme tivity test Miroil strins Fungl ultures were mintine on the Mlt extrt gr (ME). For enzyme tivity test 7 ys ol ulture ws use.

2 8 Nzir n Nsim Growth onitions To mintin the fungl growth mei use h: Yest extrts =. g (NH ) SO =. g, MgSO 7H O =. g, l H O =. g, FeSO 7H O =.5 g, KH PO = g n Min ron soure = g. Petin n ellulose were use s min ron soure. roth ssy roth meium ws prepre for enzyme tivity test. Petin n ellulose were use s ron soures. oniil suspension of isolte fungi ws juste y 6 spore ml -. This oniil suspension ws poure into eh 5 µl whih were inute t 5±. fter ys of inution perio, 5 ml roth ws tken, for petin stine with 5 µl,.5% ruthenium re solution for ½ hour n hnge in olor ws heke. Sme the proeure ws one with ongo re ye for ellulolyti enzymes. Results n Disussion Isoltion Three speies of spergillus viz.. fumigtus,. niulns n. sprsus were isolte from ifferent sustrtes t high temperture (5 o ). Fresh n ry weight omprison The growth of spergillus spp vrie gretly t ifferent tempertures n their inution perio. The fungl iomss erese t elevte temperture. Thermotolerne ereses with inrese of temperture: mximum growth ws etermine t o for. fumigtus n. sprsus while for. niulns it ws reore t 5 o. ove 5 o fungl iomss erese n t 6 o negligile growth ws reore. Mximum vlue ws onsiere s optimum vlue for other temperture regimes for every spergillus sp. In. fumigtus % reution ourre thn the optimum vlue ( o ) wheres 79.6% reution in fresh iomss of fungl isolte t 6 o note when ompre with optimum tht kept t o while t 5 o the fresh iomss exhiite n inrese of.78% n 76.% when ompre with 5 o n 6 o fresh iomsses respetively. reution of.5% form the optimum oserve t this (5 o ) temperture. Growth of. sprsus when ompre with its optimum tempertures myelil growth inrese with inrese in temperture up to º followe y eline. The optimum temperture for fungl myelil growth onsiere s º (. sprsus) sine the mol showe optimum to norml growth t 5 º where the perentge reution from Myopth (8) 6(&): 7- optimum ws 5.5% while t 5 º the fungus showe 77.6% growth whih is.8% less from the optimum ( º) moreover t 6 º. regulr erese in weight of iomss reore when temperture inrese in. niulns. Mximum growth ws reore t 5 º n t º 6.% from optimum ( o ). Men iomss weight of mol note t 5 º n 6 º with reution 7.97% n 89.% from optimum ws oserve these tempertures (Fig. ). sorne omprison Thermotolerne of fungi ws heke y sorne t 5, 55 n 6 nm wvelengths. The sorne of fungl extrt inrese when the temperture ws inrese n 5 º extrt onsiere s optimum for. fumigtus n. sprsus. The rte of sorne of. fumigtus t 55 n 6nm inrese with n inrese in temperture. n sorne of. sprsus,.58 note for 5 o extrt whih rekone s optimum followe y 6 º extrt (.97), 5 º extrt (.77) n º extrt (.7) similrly. sprsus t 55nm lso exhiit sme tren of sorne s in 5nm while 6nm. sprsus extrts exhiit eresing tren of sorne vlues with the highest sorne (.55) reore for 5 º extrt, followe y 5 º extrt (.87), º extrt (.79) n 6 º extrt, whose sorne vlue ws.67. tren of inrese followe y eline in sorne ws visulize in. niulns. Mximum sorne ws note for 6 º extrt t ll wve lengths (5, 55 n 6 nm). The rte of sorne t this wvelength initilly erese followe y inrese (Fig. ). Quntifition of spore numer Spore proution epene on temperture, perio of inution n ulture meium, when provie ll given ftors to spergillus spp. it me to knowlege tht numer of spores is epenent on temperture; with the inrese in temperture spore unne erese. The most suitle temperture for mximum spore proution rekone 5 o (.5 8 ) for. fumigtus while t this temperture. sprsus n. niulns generte.5 6 n 9. 6 numer of spores respetively. t o. sprsus generte.5 7 numers of spores wheres 8 lulte for. fumigtus, whih is, less then other tempertures exhiit tren of reution in numer of spores. t 5 o. fumigtus generte 7 spore while t 6 o no growth n sporultion ourre of ll isoltes of spergillus spp. (Fig. ).

3 Sreening for temperture tolerne of some genus spergillus Temperture ( o ) Temperture ( o ) Temperture ( o ) Fig.: Fresh weight n ry weight omprison of :. fumigtus; :. sprsus; :. niulns sorne (nm) Temperture (o) sorne (nm) nm 55nm 6nm sorne (nm) Temperture ( o ) nm 55nm 6nm 5nm 55nm 6nm Fig. : sorne omprison of :. fumigtus; :. sprsus; :. niulns Myopth (8) 6(&): 7-

4 Nzir n Nsim No. of spore/ml ( 6 ) Temperture ( o ) No.of spores/ml( 6 ) No. of spores/ml ( 6 ) Temperture ( o ) Temperture ( o ) Fig.: Spore proution omprison of :. fumigtus; :. sprsus; :. niulns Enzyme tivity test Enzyme tivity teste for two enzymes, petinolyti n ellulolyti enzyme. Petin ws egre y petinolyti enzyme t o n on stining solution with ruthenium re; isolortion of ye ourre whih showe the presene of petinolyti enzyme in the solution. When the retion of ye ws one for 6 o extrt isolortion ws not ourre for ll teste mols (. fumigtus,. niulns n. sprsus). Disolortion intensity ws low t 5 o n 5 o tht emonstrte tht t this temperture petinolyti enzyme ws proue ut not t tht level whih n isolor the stin ut isolortion of 5 o n o extrt of. niulns ws lerly visulize. For the etetion of ellulolyti enzyme solutions of ifferent tempertures stine with ongo re ye, mximum isolortion ws visulize t o n 5 o for. fumigtus,. niulns n. sprsus whih show slight hnge in ye olor t 5 o extrt while no visile hnge in ye olor oserve in 5 o n 6 o extrts of oth fungi tht emonstrte ellulolyti enzyme proution ut not in etetle quntity, whih n isolor the ye. The egring enzymes might present t every temperture ut working epens on temperture n other prmeters. Three ifferent speies of spergillus (. fumigtus. sprsus n. niulns) were isolte from ifferent sustrtes viz. ompost, lef mnure, frmyr mnure n ung t 5 o. Thermotolernt rnge of these speies heke t ifferent tempertures viz., 5± o, ± o, 5± o n 6± o. Mximum growth ws reore t o in se of. fumigtus n. sprsus while. niulns generte mximum Myopth (8) 6(&): 7- growth t 5 o n very minute growth ws oserve t 6 o while stisftory growth ws note t 5 o. Whih mens spergillus speies n tolerte temperture up to 5 o. sorne is nother prmeter whih ws onsiere for the evlution of thermotolerne of these selete fungi (rin, et l., 6). This ws opte s iniret mesure to ompre sporultion ility t vrile regimes. sorne ws heke t three wvelengths i.e., 5 nm, 55 nm n 6 nm. 5 nm, whih omes uner the rnge of visile light, n result, hve shown mximum sorne y ll selete fungi, t 55 nm. The rte of sorne ws muh less thn 6 nm. sorne rte revels interesting vrition in the growth rte (Hsio n jörn, 98). Numer of spores per ml ws n itionl prmeter to emonstrte the thermotolerne of selete fungi. Numer of spores ws inversely relte to the growth of the mol (Wolk ). s the temperture inrese the rte of spore formtion eline. The ron soures use in experiment were ellulose n petin for etetion of ellulolyti n petinolyti enzymes. Metoli tivity hs lwys een onsiere s vitl fetures to elevte the performne of n iniviul. The proution of extrellulr enzyme is effete y environmentl onitions; temperture ffets the proution of enzyme whih ws muh fster. When the egrtion of the sustrte i.e. petin ws stuie its isintegrtion shown t every temperture exept 6 o Hnsen (). Mximum isolortion of the ye (ruthenium re) ws one t o. Similrly in the se of ellulolyti enzyme the egrtion ws one t every temperture exept 6 o.

5 Sreening for temperture tolerne of some genus spergillus Referene rin K, Ekhrt Lori G, So hi-leung, Shupe, To F, Groom LH, Mihel S, 6. The response of visile/ner infrre sorne to woo-stining fungi. J. Woo Fier Si., 8: Denning DW, 998. Invsive spergillosis (Stteof-the-rt linil rtile). lin. Infet. Dis. 6: Du, Lin KS, Koutins, Wng R, Doro P, We, 8. whet iorefining strtegy se on soli-stte fermenttion for fermenttive proution of suini i. ioresour. Tehnol., 8: 5-5. Hsio K, jörn LO, 98. spets of photoinution n rotenogenesis in the fungus Vertiillium griinum. Physiol. Plntrum, 5: 5-8. Hnsen K M, Thuesen, Soererg J R,. Enzyme ssy for ientifition of petin n petin erivtives, se on reominnt pette lyse. J. O Inter., 8: Nzir N, Mirz JH, khtr N, jw R, & Nsim G, 7. Some stuies on thermophili n thermotolernt fungi from Lhore, Pkistn. Myopth, 5: 95-. Wolk DM, Johnson H, Rie EW, Mrshll MM, Grhn KF, Plummer, Sterling R,. Spore ounting Metho n ell ulture Moel for hlorine Disinfetion Stuies of Enephlitozoon syn. Septt intestinlis. ppl Environ Miroiol., 66: Myopth (8) 6(&): 7-