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. The isolates with optimum growth at neutral th e ac idi c an d alkaline pH range. It could be clearl y pH although did not necessaril y ex hibit maxim um elucidated th at pH has an effect on the predominance chitin degradati on at neutral pH but so me of them had of a paJ1icuiar group of orga ni sms relati ve to the habitat. better degradability at ac idic pH. Similarl y some 25 0 I DIAN J EXP SIOl, MARCH 2003 iso lates w ith optimal growth in th e acidic range chitinolysis were also preferred due to th e unabated ex hibited max imum chi tinolys is in neutral range and capab ility of streptomycete s to deg rade chitin. Iso lates so me w ith optimal growth in alkaline ran ge had w ith nea rly similar activities at the three pH va lues maximum chitinolys is ar pH 7.0. Thus, th ere were were al so selected for seco ndary sc reen ing. Thus, 200 so me va riations in th e optimum pH range for growth iso la tes were se lected for secondary sc ree ning which and chitinolys is. The gross colony morphology of iso enabled th e se lec ti on of strains for th e stud y of di ver lates could identify maj ority of them as act inomy sity of th eir ch itinascs. ce tes; however, iso lates with better chitin degradation at alkaline pH were mainly oth er bacteria. Secolldwy screenillg oi chitill degraders The iso lates we re further di vided into groups based The iso lates w ith potent chitinolyti c
Iso lates (6 1.2 %) had bi omass protein in th e range of 10- 15 mg and 11 .6 % iso lates had biomass protein above I S mg; most o f th ese iso lates were those w ith a greater colony size but (mll'ginal chitinolys is. Su ch 1.5 2.0 2.5 3.0 3.5 4.0 4.5 iso lates ca n be ve ry useful in producti on o f SCP from Ra ti o of zone of ch itin hyd rol ys is to colony chi ti nous was tes and thus as a so urce of protein or diameter animal feed. Sirepio/llyces sp. i"ormed the ll1aj or group o f po tent chitin degrader :.; whereas non-strepto I:ig. 1- !'1'C qU CIl CY distribu ti oll o r d ilTcI'C lll groups 01' cilitilloly tic mycetes and other bacteria comprised a relatively orgallisms ba se d Oil ratio o r cil itillolysis to colollY diamctcr (1) 1-1 sll1all proportion. The iso lates w ilh grea ter chitinase --lO D Ji ll 7.0 - . Ji ll 9. 5 0) NAWANI & KAPA DNI S: CHIT IN DEG RA DING BACTER IA FROM EXTREME AND MODERATE ENVIRON M E T 25 1
(lc ti vity (I ndio I' greater spec ifi c activity of chiti nase or Determinati ve Bacteriology' I. 36 isolates belonged to a hi gher ce ll protein were selected for furth er studi es. the genu s Streptomyces with a cell wa ll hav in g gly 49 iso lates were finall y selected to stud y the di ve rsity cine and L-DAP and a fa tt y acid profile with satu ex hibited by th eir chitinolytic systems whi ch we re rated, iso and anti -i so fatty ac id s. On th e bas is of id entified to th e genu s level fo r a comparati ve stud y growth , ph ys iol ogical and bi ochemical characteri stic s, of th eir chitinolytic systems. few isolates were found to be close to Streptomyces lIl acrosporlls, St replolll yces lJ yg roscopicus, Strepto Occllrrence oj chitin deg rading bacteria lIlyces jUl1l onjinensis, Streptomyces toruloslI s, Strep The forty-nine iso lates selected for further studies tOlllyces heterol1l ol'phus, Streptomyces jllipinensis, were identified to the genu s level. They were ass igned Streptolllyces cellulosae, St reptomyces roclJ ei. Strep ge notypes based on their chemotaxonom ic , growth , tomyces halstedii, Streptomyces e4oliatlls. Strepto physiological and biochemi cal characteri stics as per l1I yces albus, St reptol1l yces g ralllin ojaciens and St rep th e class ifi cation desc ri bed in th e Bergey's Manual of tOlllyces xant/lO cli rolllog enes. However, spec ies were Systematic Bacteriolog/ and Bergey 's Manual of not assigned to the isolates sin ce it was essential to
Non Extreme 69%
Moderate ~ ~ Extreme
Compos t 6% Non- Acid Saline Pollu ted Soils lake 38% 35% 46%
Hot Springs 13%
Non-Polluted Polluted
Khadak- Huday- was ala dah Laundry Pas han Agri cul - 4% 15% 10% 15%
~~r~ /\~ ~ Jeddah Pavana 1\\l ~ Sasoon LJ~ S-J 15% 17% ~~ Docks 13% Kale-wadV 1\V, \lD 15% U Aden Muth~ Mecca 13% 22% Mula 23% 17%
Fi g. 2 - Habilal-wise di slri buli on of e ffi cient chilin degraders selecled fo r secondary screening 252 I 'DIAN J EXl' BIOL, MARCH 2003 confirm th e same using molecular phylogeny abundantly found in so il and water, th e recycling of approaches. Of th e forty-nine potent chitin-deg rad ing which is esse ntial for an ecological ba lance. Chitin bacteri a, th e genu s Slreplolllyces was found to be pre degradation is mediated in so il and water by several sent in almost every hab itat. L onar Lake, M ecca and types of microorgani sms, of wh ich act inomycetes ag ri cultural land had maximum percentage of potent playa major role. M ost studies on chitinases indicate chitinolytic streptomycetes, whereas none of th ese th at th ese enzy mes generally ex hibit maximum ac ti v were obta ined from Sassoon Docks and Pas han Lake. ity at pH ranging from 5.0 to 7.0 13 like th e chitinase Besides streptomycetes, Lonar Lake was inhab ited by from th e well-studied bacterium, Ser ratia 1/1Cl r CeSCeIlS, chitin deg rad in g genera of Bacil/lls and MiCrDIl IOII O which produces chitinase w ith optimum activity nea r SPOJ'(l . 'fhe iso late class ified as MicrolllO llospo m was pH 6.0 and w ith stab ility in a wide pH rangel-I. How char, cteri zed by si ngle non-mot i Ie sess i Ie spores and ever, chitinases w ith pH optima of 4.0 have bee n ce ll wa ll containing amino ac ids IIleso-DAP, glyc ine found in streptomycetes 5 and Microbispom Sp. 12 and and xylose and arabinose as whole ce ll sugars. The those with pH optima in th e alkal ine range have been iso lates from ac id so ils and hot springs were of genus reported for Streptolllyces al bido.flavlIsl 5 and Nocar- SlreplOlllyces however, Microbisporrt sp., which has ( IIOPS· 1..\' a 1/JIlS I Ii an d BaC I' 11 liS sp. 17 been recen tl y reponed for its chitinoly ti c potential, A predominance of chitin degraders in moderate was iso lated in this stu dy from th e hot springsl2. regions th an in ex treme locati ons could be due to th eir Of th e polluted habitats, fJ acil/lIs sp. was isolated deviation from the normal ones in terms of pH and from Sassoon Docks an d strains or the genera Psell temperature so th ey allow th e survi va l of limited or dOlllOllas, MicrDlllO llOSpom, NOc(lU/iopsis and Se r m gani sms capab le of thri ving under th ese co nditions. li(l were obtained from Mula, Mutha an d Pav an a ri v T he seco nd reason could be a relati ve ly lesser avail ers respec tively. The genus Nocardiopsis was charac ab ility of chitin in th ese environmcnts. This wa s how terized by the presence of IIleso-DAP in th e ce ll wa ll ever, not see n for ac id so ils from Sana, where a good and no diag nost ic suga rs in vvh ole ce ll hydrolysa tcs. number of chitin deg rad ers were found. Most or th e Its substra te mycelium was dcnse ly branched and ac tinoillycetes ca n tolerate pH up to 5.0 Acidophiles appear to be confined to acid so ils and larvae, few c1ad oceran and copepod cru sta ceans in litters and neutrophiles are numerous in soils close to th ese habitats. neutrality but also occur in low numbers in acidic Most efficient chitin degraders were from Mula so il slH. It has been reported th at p H optimum for hy and Mutha ri ve rs, ag ri cultural land , Jeddah and drolys is of co lloidal chitin is broadly related not only M ecca . The so il and water samples from pol luted lo to the pH optimum for growth but also to th e pH of ca tions and garden and ag ri cultural so ils had more th e so il from wh ich the iso lates comes, which seemed number of efficient chitinolytic bacteria. Thus, so ils in agreement to our observations, although so me de and water bodies with a high organ ic content and thu s viati ons could be observed. The iso lates capable of high competition see m to favor the predominance of max imum chitinolys is at ac idic pH can be important iso lates expressing good chitinolytic activity. This fo r recycling of chitinous matter in ac idic so ils. Chiti fact may also be app licab le to other characteristi cs of nases from these iso lates ca n be co mmercially ex th ese microorganisms. ploited in biologica l control of funga l pl ant path ogens The identifica tion of most important chitin deg rad as we ll as in fungal protoplas ting. i ng bacteria revealed streptomycetes to be th e major A lkalophilic bacteria are found with highes t fre contributors to chitin deg radation irres pecti ve of ex quency in alka line so ils although they are also present treme or moderate habitat. They are also predom inant in neutral and acidic so ils but in limited numbers. in th e cultivable reg ions further suggesting their im Most enzy mes from alkalophiles are characteri zed by portance in the bi oconversion of chitin i!l th ese hab i an optimum pH on th e alkaline side. A good number tats. The ability of AClilloplancs to survive and grow of iso lates capab le of chitin degradation in the alka in a wide pH ran ge ex plains th eir occurrence in alka line range co uld be iso lated in thi s study. M ost of line reg ions of laundry. Further, their ab ility to de th ese were bac illary but few ac tinomycetes al so ex grad e chitin in a wide pH range ex pl ains th eir po ss ible pressed ac ti vity and grew optimally at pH 9.0. These role in chitin degradati on in alka line habitats. How iso lates can be eco logica lly important as being most ever, potent chitinolytic streptomycetes were not respo nsible members of chitin degradation in hab itats found in Sassoo n Docks and Pashan Lake. Their rel::t with alkaline pH and al so ca n be industrially impor tive in frequent occurrence in Sassoon Doc ks could be tant. Chitinases with alkaline pH optima and stab ility as this habitat is of marine origin. Con tradictory to 19 2 1 ca n be applicat ive in biological controI . . The peri Lonar Lake, Pashan Lake did not harbor potent chiti trophi c memb rane of insect gut lining is chitinous and nolytic streptomycetes despite being polluted, a fact th e alkali active chitinases ca n be used in sy nergism that needs further ex pl orat ion. The non-streptomycete with other biocontrol agents. Chitinases acti ve and actinomycetes were mainly fo und in moderate loca stab le at alka line pH are ex pec ted to have a better ti ons and their contribution to chitin degradati on in hal f-life in th e gut of th e insec ts, which has an alka th ese hab itats needs further studies. line pH, in co mpari so n to chitin:1ses from neutrophi les Thus from th is st ud y it could be ascert ain ed that or ac idophiles, which may ex hibit a shorter half-li fe at chitin-degrading bacteri a may be ubiquitous relative th at pH. to th e abundance of thi s polymer in nature. Chitinoly The possible role of chitin deg raders in alka line sis ca n occur in a wide pH range and pH as an envi habitats like Lonar Lake is qu es ti onabl e from th e ronmental factor seems to have an impact on th e dis poi nt of view of the so urce of thi s poly mer in this en tribution and activity of chitinolytic bacteri a, up [ 0 a vi ronment. Sal ine lakes are genera lly dominated by certain ex tent. Chitinases of bacteria from extreme blue-gree n algae and are poor in plankton ic and be n loca tions might ex hibit some unique properties with thic fauna . T hi s is particularly true for Lonar Lake, res pect to activity, stability or oth er characters, wh ich where Spirlllilla platellsis is found ex pl aining cert ain might be indu strially important. Pollution may ill gen leve l of eutrophi ca ti on in the lake . Haloa lkalophilic eral affect surviva l or enrichment of chitinolyti c bac archea ca n so metimes be a part of hypersa line and teri a depending 011 th e nutrient ava ilab ility, a fa ct that alkaline lakes . These lakes generally function ,IS se lf st References 12 Nawan i N N, Kapadni s B P, Da~; A D, Rao A S & Mahaj;1I1 I Kehya ni N 0 & Roseman S, lJiocililll Biopllvs AC/o, 1473 S K, J Appl Microbilll, 93 (2 002 ) 965 . ( 1999) 108. 13 Hara S, Yamamura Y, Fuji i Y, Mega T & Ike naKa T. J /Jill 2 Pace N R, Sciellce, 276 ( 1997) 734. cll elll , 105 ( 1989) 4R4. 3 Cl ese ri L S, Greenberg A E & Trussell R R, Alllericall Pllblic 14 Nawa ni N N & Kapadni s B p, J Appl Microliiol, 90 (200 I) Ncol/II Associ(lfiOIl (A PNA ) (Ameri can Water Works As so 803 . ciat ion and Water Polluti on Control Federation. Eel. 17th IS Broadway R M, Williams D L, Ka in C W. Ha rm an G E. APHA, Washin gton DC) 1989 . Lorito M & Labeda D P, Lell API'I Microbiol, 20 ( 1995) 27 1. 4 Hsu S C & Lockwood J L, Appl Microbial, 29 ( 1975) 422. 16 Tsujibo 1-1 , Yoshida Y, Miyamota K, J-bsegawa T & Inam ori 5 Williams S T & Rob in so n C S, J Cell Microbiol, 127 ( 19R 1) 55 . Y, /Jios ci BiOleclll101 Biocllelll. 56 ( 1992) 1304. 6 Monreal J & Rcese E T, Call J Microbial. IS ( 1969) 689. 17 Bhu shan B. J Appl Microbiol, 88 (2000) 800. 7 Willi ams S T, Sharpe M E & Holt J G, Berge.I"s MOllllal of 18 Williams S T & Mayficld C I, Soil Bioi Bioc/wlII. 3 ( 197 1) Svs/cl/ /(/ /i c BOCie riology. (Vol. 4. Willi ams and Wilkins, 197. London) 1989. 19 Wiwat C, Lert ca nawaniclraku l M, Siwayapram P, Pantu wa B Shirling E B & Gottlieb D. III/ J Sys/ BaC/eriol. 16 ( 1966) tan a S & Bhumirata na A, Celi e 179 ( 1996) 11 9. 311. 20 & 144 ( 1998) 9 Stancck. J A & Robcrts G D, Apl)1 Mi crobiol, 28 ( 1974) 226. Sampson M N Gooclay G W. Mi crobiology. 10 Lan gham C D, Williams S T, Snea th P H A & Mortimer A 2 189 . M , J Cell MicruiJiol, 135 ( 1989) 121. 2 1 Bhushan B & Hoondal G S, Wo..td J Microbiol /Jio/ ec/lIlOl. II Holt J G. Kri eg N R. Sncath P H A, Staley J T & Williams IS ( 1999) 403. S T, Bergev 's MClllllol of De/eollill o/ive BOCieriolagv (N inth 22 Na ndy N & Deo V, Tata Iron and Steel Company, hmshecl Ed iti on, Williams and Wilkins, London) 1994 . pur, 8 ( 1961) 1.