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Chitin Degrading Potential of Bacteria from Extreme and Moderate Environment

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Chitin Degrading Potential of Bacteria from Extreme and Moderate Environment In diJn Journ al or Experi mentJI Biology Vol. 4 1. March 2003. pp. 248-254 Chitin degrading potential of bacteria from extreme and moderate environment N N Nawani & B P Kapadni s* Department of Microbiology. Uni versi ty or Pune. Pune 4 11 007. In dia Recei ved 22 Nove lllber 2002; revis ed 19 j allllclI] 2003 Five hundred chitin-degrad ing bacteria we re iso lated rrom 20 different locations. Hi gh percenta ge of potent chitin­ degraders was obtain ed fro m po llu ted regions. Potent chit in- degrading bacteri a were selected by primary and ,eeondary scree nin g. Among th e se lected isolates. 78% we re rcprcsc nt cd by the gcn us STreplUlllyces. Majority of th c iso lat es had good chitinolys is relati ve to the growt h although iso lates with beller growth were al so seen. Such isolat es arc important 1'0 1' the producti on of SCP from chitinous wastes. The potent iso lates be longed to th e genera STrepf()/.'lyces. KirasalUsporia. Sacc/wwpolyspol'{[. Nocardioides. Nocardiollsis. I-Ierbidospora. MicrOIllOIlOSP0/'{f. Micwbispo/'{f. I ICTill oplalle..... SerraTia. iJacilllis and Pse lidolllollas. Thi s study form s J comprehensive base for the study of divcrsity of chitinolytic syste ms of bacte ri J. Ch itin , the ~-I , 4 linked polymer of N-acetylgluco­ Materials and Methods samine is the second most ab undant polysaccharide in The locati ons of sa mpling included habitats classi­ nature. Chitin is a structural component of th e ce ll fied as ex treme and moderate as shown in Tab le I, wa ll s of fungi as we ll as of shell s or cuti cles of arthro­ fro m where soi I and water samples were co ll ec ted pods, cru staceans, insects and mollusks. The major using poi nt-integratin g samplers and by th e proce­ con tri buti on of chi tin to so il is in the form of anim al dures recommended in APHA :l . Hand -held scoops and biomass. Similarly, in the marine enviro nment more shovels were used for sampling near-surface so il s. th an lO ll metri c tons of chitin is annually produced l. Soil and aquatic systems both act as major harbors of Isolation and enllllleration o/chiTin degraders chitin degraders. The biological conversion of chitin by Isolation of chitin degraders was done on co ll oid al mea ns of microbial chitinases is mainly responsibl e for chitin agar (CCAt Isolation was performed by serial the repl eni shment of carbon and nitrogen to th e atmos­ diluti ons of the sampl es and plating on buffered) phere , th ereby maintaining the ecological balance. Due CCA. The number of isolates capable of chitin degra­ to multiple app li ca ti ons of chitinases in biocontrol, dation was enumerated at three pH va lu es (4.0. 7.0 waste management, medicine and biotechnology, th ey and 9.5) and isolates hav in g good zo ne of clea rance become interestin g enzymes fo r study. on CCA were preserved and subj ected to primary and The present stud y was aimed to isolate chitin­ second ary screening fo r selection of potent isolates. degrad in g bacteria and understand the diversity of th eir chitinolyti c sys tems. For thi s purpose, soil and Primary screening of chitin degraders water samples were obtain ed from various locations, Primary screening was performed by spo t in oc ul at­ both ex treme and moderate. considering the fact th at in g th e iso lates on CCA buffered to pH 4.0, 7.0 and it is not necessary to go to th e ex treme environments 9.5 to class ify them into groups based on max imum to encounter exoti c diversit/. This stud y primarily chitinolys is at ac idi c, neutral or alk aline pH. Zone of gives an idea of th e ecological effects on chitin degra­ chitin hydrolysis and colony diameter were recorded dation att ribute of bacteria. A detailed comparison of up to 7 days at 40°C. Wh ether th e isolates ha ve a the chitinolyti c systems, keeping in view the habitat similar range of pH for growth as fo r chitin degrada­ of the iso lates can further reveal some interestin g eco­ tion was determined by monitorin g th eir growth in logical effects. nutri ent broth or Bennett 's medium at pH 4.0, 7.0 and 9.5 . *Correspondent author: Secondary screenin g 0/ chiTin degraders Tel: 9 ! 0205690643 Secondary screenin g was performed to check th e Fax:9 ! 0205690087 Em ail : bpkapadn [email protected] ab ility of iso lates to degrade practical grade chitin NA WAN I & KAPADNIS : CHITI N DEG RADIN G BACTERI A FROM EXTREME AN D MODERATE EN VIR ONMENT 249 T:.Jb lc I - Ci :J ssifi c:J tion of s:J mpli ng locati ons and occurrcncc of chi tin olytic bactcria Extrcmc locati ons* Modcratc locati on_s __..- ____ Po ll ut cdt No n'-pollu tcdtf Sali nc Lake, Lonar Sassoon Docks, MUlllbai Hudayduh, Saud i Arab ia Acid so il s, Sana , Ycmcn Mu la rive r, Punc Mecca, Saudi Arabia Ac id so il s, Ranchi , Indi a Mu tha ri ve r, Punc Adcn, Yemcn Hot sp rin gs, Vrajrc shwa ri , Mum b:l i P:.Jva na ri ve r, Punc Jcddah, S:J udi Arabia Compost, Pune Pash:J n Lakc, Punc Pcacock Bay, Pune Ag ri cultu ra l land, Punc Compost, Mum ba i La un dry Spots, Punc Kalewad i, Punc Potcnt ch itinolyti c b:Jctcri a fo und in - *Ex trcme locations: Sl replolllyces, Baci llus, MicroIIlOllo.lp om, Microbispora tPolluted locations: SlreplOlllyces, Bacillus, Pseudolllullas, Serr(l{ia, Micl'OlIIOII Ospom. Nucardiopsis, KilasalU­ sporia, ACl illop/olles ttNo n-polluted loc:J ti ons: SlreplUlllyces, H erbiduspom , Nocardiuiiles, Sacclwl'Opo/yspora flakes and to produce ex tracellul ar chitinase. 0. 1 ml The sampl es from Lonar Lake with pH in th e alkal in e cell or spore suspension of 00600 0.1 was inoculated range ex hibited the predominance of isolates capable in SO ml of chitin medium at optimum pH va lu e and of degrading chitin in th e alk alin e range. Soil samples was in cubated in a rotary in cubator shaker (I SO rpm) from ag ricultural lands at Pune, Mecca, at 40°C for 6 days. Ex tracellul ar chitinolytic acti vity Hudaydah, and Sana had more number of ch itin in th e culture mediu m was determined usin g swoll en degraders in compa ri son to the so il sampl es from chitin as th e substrate as described by Monreal and Lonar, Vrajreshwa ri hot springs and laundry spots. Reese6 at optimum pH of chitinolys is as determined Whereas, an intermedi ate count was seen for soi I sam­ by primary screening. One unit of chitinase ac ti vity pl es fro m Aden, Jeddah and Pashan and lowest cou nts was defin ed as the amount of enzy me required to re­ were for so il sampl es from Peacock Bay. In general, ., lease one flm ol of Gl cNAc in I min un der th e above­ th e number of chitin degraders in water sampl es was menti oned conditi ons of enzy me reacti on. lesser than for so il sampl es, where culti vated and garden so il s had a hi gher number of chitinolyti c Identification oj chitinolytic bacteria bac teri a. The sampl es with ac idi c pH lik e th ose from The identi ficati on of ac tin omycetes and other bac­ Sana and Ranchi had more number of isolates capable teri a was based on th e methods recommend ed in th e of degrading chitin in the ac idi c pH range. Most of the Bergey's Manu al of Sys tematic Bac teri olog/ in clud­ iso lates in samples with neutral pH lik e th ose from in g other reco mmended references and Intern ati onal Mula, Mutha, Mecca and Jeddah etc. displ ayed mo re Streptomyces Projects. The chemotaxonomi c stu dies number of bacteri a degrading chitin in th e neutral p H were ca rri ed out fo r actin omycetes onl y by detennina­ range. Thus, pH as a selecti on fac tor may not be ti on of cell wa ll amino ac id 9 and wh ole cell suga rs. so lely restri cted to chitin degradati on but may also be Probabili sti c identificati on matri ces we re used fo r ex tended to other acti viti es of the isolates. Five hu n­ identificati on of Streptomyces spec ies based on dred isolates di sp lay ing good chi ti nolys is were pre­ ph ys iological and bi ochemi cal charac teri sti cs lO and served fo r screenin g and subsequ ent selec ti on of po­ other genera were manu all y id entified. tent chitin degraders. Results Primary screening oj chitin degraders Primary screenin g enabled the classifi cati on of" Isolation and enumeration oJchitin deg ra ders isolates as those exhibiting max imum chitinolys is at The adva ntage of simultaneous isolati on at three acidi c, neutral or alkaline pH va lu es . Most of the pH va lues th an isolati on on medium of sin gle p I-I isolates with acidi c or alkaline pH optima for growth va lu e was th at thi s method gave th e probable relati ve had better chitinolys is at ac idi c or alk alin e p H respec­ abundance of bac teri a capabl e of degrading chitin in tively.
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