Biodegradation of Degradable Plastic Polyethylene by

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mr. 1991, p /91/3678-8$2./ Copyright 1991, Americn Society for Microbiology Vol. 57, No. 3 Biodegrdtion of Degrdble Plstic Polyethylene by Phnerochete nd Streptomyces Speciest BYUNGTAE LEE,' ANTHONY L. POMETTO III,1* ALFRED FRATZKE,1 AND THEODORE B. BAILEY, JR.2 Deprtment of Food Science nd Humn Nutrition nd Center for Crops Utiliztion Reserch' nd Deprtment of Sttistics,2 Iow Stte University, Ames, Iow 511 Received 24 August 199/Accepted 7 December 199 The bility of lignin-degrding microorgnisms to ttck degrdble plstics ws investigted in pure shke flsk culture studies. The degrdble plstic used in this study ws produced commercilly by using the Archer-Dniels-Midlnd POLYCLEAN msterbtch nd contined pro-oxidnt nd 6% strch. The known lignin-degrding bcteri Streptomyces viridosporus T7A, S. bdius 252, nd S. setonii 75Vi2 nd fungus Phnerochete chrysosporium were used. Pro-oxidnt ctivity ws ccelerted by plcing sheet of plstic into drying oven t 7 C under tmospheric pressure nd ir for, 4, 8, 12, 16, or 2 dys. The effect of 2-, 4-, nd 8-week longwve UV irrdition t 365 nm on plstic biodegrdbility ws lso investigted. For shke flsk cultures, plstics were chemiclly disinfected nd incubted-shken t 125 rpm t 37 C in.6% yest extrct medium (ph 7.1) for Streptomyces spp. nd t 3 C for the fungus in 3% mlt extrct medium (ph 4.5) for 4 weeks long with n uninoculted control for ech tretment. Weight loss dt were inconclusive becuse of cell mss ccumultion. For lmost every 7 C het-treted film, the Streptomyces spp. demonstrted further reduction in percent elongtion nd polyethylene moleculr weight verge when compred with the corresponding uninoculted control. Significnt (P <.5) reductions were demonstrted for the 4- nd 8-dy het-treted films by ll three bcteri. Het-treted films incubted with P. chrysosporium consistently demonstrted higher percent elongtion nd moleculr weight verge thn the corresponding uninoculted controls, but were lower thn the corresponding zero controls (het-treted films without 4-week incubtion). The 2- nd 4-week UV-treted films showed the gretest biodegrdtion by ll three bcteri. Virtully no degrdtion by the fungus ws observed. To our knowledge, this is the first report demonstrting bcteril degrdtion of these oxidized polyethylenes in pure culture. Reclcitrnt plstics ccumulte in the environment t rte of 25 million tons per yer. The fte of these orgnic polymers in the environment nd the time required for their totl minerliztion to CO2 hve yet to be fully understood. There is growing interest in the development of degrdble plstics to enhnce the biodegrdbility of the plstics in lndfills nd composts. One of the most commonly suggested uses for strch-bsed degrdble plstics is for composting of lwn, grden, nd shrub litter, which could reduce the volume of mteril entering the lndfills up to 2%. Therefore, we re investigting the bility of litter- or lignocellulose-degrding microorgnisms to ttck strch-contining degrdble plstics in pure culture. The degrdble plstic must still retin ll of the physicl properties expected by the consumer nd, then, when plced in the pproprite environment, degrde more rpidly thn conventionl disposble plstics. To enhnce the degrdtion of the polyethylene, chemicl or photo inititors or both re dded to the degrdble plstic films. For polyethylene films contining photo- nd pro-oxidnts, the primry inititors of oxidtion re light nd temperture, respectively. Both the pro-oxidnt nd the photo-oxidnt produce free rdicls on the long polyethylene chin, cusing the mteril to lose some of its physicl properties, to become oxidized, nd, possibly, to become more ccessible to microbil biodegrdtion (8, 9). * Corresponding uthor. t Journl Pper number J of the Iow Agriculture nd Home Economics Experiment Sttion, Ames. Project numbers 178 nd In this pper, we describe pure culture system for evluting the biodegrdbility of degrdble plstic films contining pro-oxidnt nd 6% strch. Biodegrdbility ws evluted by weight loss, tensile strength loss, chnges in percent elongtion, nd chnges in polyethylene moleculr weight distribution. Chemicl degrdtion of the plstic ws initited by 7 C het pretretment or 365-nm UV irrdition pretretment of the film. Biologicl nd chemicl trnsformtions in the degrdble plstic mteril upon incubtion under shking with erobic lignin-degrding microorgnisms nd with uninoculted culture broth were demonstrted. MATERIALS AND METHODS Microorgnisms. The lignocellulose-degrding microorgnisms used were the bcteri Streptomyces viridosporus T7A (ATCC 39115), Streptomyces bdius 252 (ATCC 39117), nd Streptomyces setonii 75Vi2 (ATCC 39116) nd the fungus Phnerochete chrysosporium (ME 446). All cultures were mintined on gr slnts t 4 C (13). The strch-degrding bility of ech microorgnism ws determined by using culture streks onto strch gr plte contining 1% (wt/vol) ntive corn strch,.5% (wt/vol) yest extrct, nd minerl slts solution (13). Ech culture ws incubted t 37 C for 1 to 2 weeks. Strch utiliztion ws confirmed by flooding the pltes with iodine (1). The results demonstrted strch clering by S. bdius nd S. viridosporus only. P. chrysosporium nd S. setonii did grow on the culture plte, but no clering of the strch ws detected. Downloded from on April 9, 218 by guest

2 VOL. 57, 1991 BIODEGRADATION OF DEGRADABLE PLASTIC POLYETHYLENE 679 Bcteril Cultures C O >' < t C~ ). c c II - Dys for het tretment Fungl Cultures 12 1 V zero S control V P.chryiosporium 8 * s 4 2- O~~~~~~~~~~ Dys for het tretment FIG. 1. Chnge in percent elongtion for -, 4-, 8-, nd 12-dy het-treted (7 C) degrdble plstic films fter 4 weeks of incubtion in culture medi, with nd without (control) ligninolytic microorgnisms. Zero control is het treted but not incubted. Ech dt point represents n verge of four replictes. CA ) D U, = C >o C S C zodd ) C o. D x- ) CO OD ) CO ".. UD CQ C - C -O3 m - - t,. O C 8,o I'C C- ~.. mc C o CL _ D o. A o B D X._ z o _' 'IC u 11-4 k) -4j ~-c (A (A (A \1 14 () A ) \ 'IC (-A W U4 W > (AA ( ) -P C-4-4 -'-j -4. oo (A -o 4 -j I.- P. (A t.i " (-A A. " \ \ \O.)th (Ao\o ON " (-A w1-) j ta \C -~t >- C) ui Ooj \ (-A (A" (A& ~-& (A - -i \ " (- so.)o. o 'IC t') t4 " (A (A w --j (-A (-A -PI w so -. <- ~-o ~ w (A (.A o A A Ut w. C ) o (A I o ~ 4> ) t_ -4j \C,, () ON (A w (A ) t-41-4 (A (A ~- -4 o (.A O 'I. P. CN oo t t'4 c o (A -4-4 oo ) \ )... I ~I 4 1~ Q) 9. 4I zi 1~ ~I 1l zi I.i (IQ 5 o ) ; IE _ OC X-tQ ) _t _-. U) Degrdble plstic film pretretment nd culturing. (i) Het (7 C)-treted films. Archer-Dniels-Midlnd POLYCLEAN msterbtch degrdble plstic films mde with liner lowdensity polyethylene contining pro-oxidnt nd 6% strch were used. Pro-oxidnts re mixtures of trnsition metls (i.e., Fe, Zn, Ni, nd/or Mn) nd lipids (i.e., corn or soyben oil) which re compounded into the finl polyethylene product t very low levels. Films were commercilly prepred ccording to Archer-Dniels-Midlnd-recommended specifictions. To ccelerte the pro-oxidnt ctivity, sheets of degrdble plstic were plced in 7 C forced-ir oven, with both sides exposed to ir for mximum of 2 dys. Sheets were removed every 4 dys. The sheets were then cut, following the sme orienttion (opposite to mchine direction), into strips (4 by 1 in. [1.16 by 2.54 cm];.6 to.7 mm thick). (ii) UV (365 nm)-irrdited films. The sme strting films just described were cut into strips (6 by 1 in. [15.4 by 2.54 cm]) nd irrdited in chmber under long-wve UV lmp (365 nm; 115 V, 6 Hz,.6 A, 2/15 W; Spectronics Corp., Westbury, N.Y.) for 2, 4, or 8 weeks, with equl time exposure on both sides of the film. Films were 17 cm from the lmp, with n irrdition re of 39 by 32 cm t 33 C nd reltive humidity of 4.5%. The films were then recut into 4 by 1-in. strips fter irrdition. (iii) Chemicl disinfection of films. The disinfection proce- ),- _F-, p) ;K, DQQ C.). r -X U,.k..) Downloded from on April 9, 218 by guest

3 68 LEE ET AL. APPL. ENvIRON. MICROBIOL. CL C C CO 9 eli ON.eN o H o r- ON ) Bcteril Cultures -o ) -o CZ) CcO c Ctu CA ) -z CZ C C._ X CO o,. OCO: Y,3O )I > CvsO ) CO.. CO4 (A ;. r. 'tt 'o CO e CN cn tf CF cro C^ r-c e.i o o N -4 o CN r N ref) f4c N '-4 'IC r~o ) I,N z en.t x N r- Cl4 N Cl r- m O n I~ ; I~c Nrf- C It en 11 N1 r--q -4 o It o-4 CI I/n t ll ON Al - r- ef 1, C)1t n. CO gco o e' -') '. =. *o E > CZ). CO 3 CO. E sn Y Q u u Q7;.3 co n C.) cn cn :z *E Z )c -.Q 1 o Dys for het tretment Fungl Cultures o zero \* control v v P. chrysosporium Dys for het tretment FIG. 2. Weight-verge moleculr weight (Mw) for -, 4-, 8-, 12-, 16-, nd 2-dy het (7 C)-treted degrdble plstics films fter 4 weeks of incubtion in culture medi, with nd without (control) ligninolytic microorgnisms. Zero control is het treted but not incubted. Ech dt point represents n verge of four replictes. dure used with ech pretreted film consisted of plcing the strips into covered beker (no more thn 15 to 2 strips), dding fresh solution of universl disinfectnt (1) contining 7 ml of Tween 8, 1 ml of blech, nd 983 ml of sterile wter, nd stirring for 3 to 6 min. Ech film ws removed with sterile forceps nd plced into covered beker of sterile wter, where it ws stirred for 6 min t room temperture. The films were then septiclly trnsferred into stnding 7% (vol/vol) ethnol solution nd left for 3 min. Ech film ws then plced into preweighed sterile petri dish. The dishes with films were plced into n incubtor t 45 to 5 C to dry overnight, llowed to equilibrte to room temperture, nd weighed to +.1-mg ccurcy; the weight of the film ws then determined. (iv) Film culturing nd medi. Preweighed disinfected films were septiclly dded to sterilized culture medium. The medium contined either.6% (wt/vol) yest extrct (Difco Lbortories, Detroit, Mich.) in nitrogen-free minerl slts solution (5.3 g of N2HPO4, 1.98 g of KH2PO4,.2 g of MgSO4. 7H2,.2 g of NCl,.5 g of CCl2-2H2, plus 1 ml of trce element solution [14] per liter of deionized H2, ph 7.1 to 7.2) or 3.% (wt/vol) mlt extrct (Difco) in deionized wter (ph 4.5) for the Streptomyces spp. nd P. chrysosponum, respectively. Films in culture medium were incubted with shking for 24 h before inocultion to ensure III Downloded from on April 9, 218 by guest

4 VOL. 57, 1991 BIODEGRADATION OF DEGRADABLE PLASTIC POLYETHYLENE 681 sepsis. Culture medium ws inoculted with spores from specific ligninolytic microorgnism nd ws incubted with shking t 125 rpm for 4 weeks t 37 nd 3 C for the bcteri nd fungus, respectively (7, 15, 16). Four replictes were prepred for ech different pretreted film. (v) Film hrvest. Plstic strips were hrvested, wshed in 7% ethnol to remove s much cell mss from the residul film s possible, dried t 45 C s just described for 24 h, nd equilibrted, nd the weights were determined. Ech of the different films with nd without het or UV pretretment ws compred with the corresponding uncultured mteril (zero controls) s well s with uninoculted incubted films (uninoculted controls) in either.6% yest extrct or 3% mlt extrct medium. Test used to evlute chnges in degrdble plstics. Biodegrdtion ws followed by weight loss, chnges in tensile strength (the stress mesured t frcture of the specimen), percent elongtion (the extension of the mteril under lod [ASTM D882-83]), nd chnges in polyethylene moleculr weight distribution. The dt nlyses were determined by SAS progrm (17) by using n nlysis of vrince to scertin differences between corresponding zero control nd uninoculted control (chemicl degrdtion) nd for differences between the corresponding uninoculted control nd ech of the microbil tretments (biologicl degrdtion). Vlues with P <.5 were considered significntly different. (i) Tensile strength nd percent elongtion determintions. Chnges in tensile strength nd percent elongtion were determined on n Instron model 111 t room temperture nd 5 mm/min with 5-cm gp. All smples were equilibrted to 5% reltive humidity for t lest 4 h preceding nlysis (ASTM D882-83, Stndrd Test Method for Tensile Properties of Thin Plstic Sheeting). (ii) Polyethylene moleculr weight distribution. A Wters model 15-C (Wters/Millipore Co., Milford, Mss.) hightemperture gel permetion high-pressure liquid chromtogrph (HT-GPC) ws used to determine chnges in the moleculr weight distribution for the residul polyethylene. Three identicl Wters,u-Styrgel HT-liner columns, with functionl moleculr weight (Mw) rnge of 5 to 8,,, were used in series. A mobile phse of 1,2,4-trichlorobenzene (GC/GPC grde; Burdick & Jckson/Bcter Inc., Mrkeson, Mich.) ws used without ntioxidnt. A flow rte of 1 ml/min nd n injection volume of 2 uli were used. Totl run time ws 55 min per injection, followed by 5-min equilibrtion dely. A refrctive index detector ws used. Injector, columns, nd detector were ll held t 14 C, nd the solvent pump ws held t 5 C. A moleculr weight clibrtion curve ws constructed bsed on nine different nrrow-moleculr-weight distribution polystyrene stndrds, with pek moleculr weights rnging from 2,7 to 2,7,. Smples were prepred in 1,2,4-trichlorobenzene contining 2 ppm (2,ug/ml) of Sntnox R (Monsnto, Akron, Ohio) s ntioxidnt nd contined.15% (wt/vol) polyethylene. Initilly, 45-mg polyethylene smples were dded to mber jrs long with 3 ml of 1,2,4-trichlorobenzene with ntioxidnt. The jrs were cpped nd plced in 15 to 155 C convection oven for 4 h with occsionl swirling. The dissolved smples were trnsferred to Wters filter vils, mnully filtered through the integrl, Teflon housed, sintered stinless-steel filter (.5,um) nd immeditely plced into the HT-GPC utosmpler t 14 C. Duplicte injections were run from ech smple. Mxim 82 computer softwre (Wters/Millipore Co.), ws used to determine weight-verge moleculr weight (Mw), numberverge moleculr weight (M), nd polydispersity (M,/fM) of the polyethylene smples. RESULTS Het (7 C)-treted films. Weight loss dt were inconclusive becuse of bcteril or fungl cell mss ccumultion on the films. Usully, weight gin ws mesured, but slight loss of weight by S. bdius 252 nd S. setonii 75Vi2 ws detected for 12-dy het-treted films (1.4 nd.73%, respectively) nd for S. bdius 252 for 16-dy het-treted films (1.2%). Uninoculted-controls generlly hd weight gin (verge, 2.%), which is consistent with oxidtion of the polyethylene (4, 5) nd wter bsorption by the strch. The 16- nd 2-dy het-treted smples for the inoculted nd uninoculted controls were too brittle for tensile strength nd percent elongtion mesurements. Tensile strength vlues for bcteri nd fungus indicte little chnge compred with the zero control nd uninoculted control smples. The only exception ws for the 4-dy het-treted smples with S. viridosporus T7A, which hd 5% reduction in tensile strength compred with the uninoculted control nd zero control. All bcteril strins demonstrted reductions in the percent elongtion vlues with ech of the het tretments, wheres the fungus cused n increse in percent elongtion vlues (Fig. 1). The initil mteril (zerotime film with no het tretment), fter 4-week incubtion (uninoculted controls) in the bcteril nd fungl medium, hd 47 nd 12% reductions in percent elongtion, respectively, when compred with the zero control. Differences in percent elongtion between the uninoculted controls nd zero controls for 4-, 8-, nd 12-dy 7 C het-treted smples were slightly different (rnge, to 11%). The bcteril cultures generlly showed bout 16% reduction (rnge, 13 to 23%) in percent elongtion when compred with the corresponding uninoculted controls. Only the initil mteril incubted with S. bdius 252 demonstrted reltively no chnge compred with the controls. However, fungus-cultured het-treted films consistently demonstrted n increse in percent elongtion (rnge, 16 to 21%) when compred with the corresponding uninoculted controls (Fig. 1). The HT-GPC dt were used to evlute chnges in moleculr weight distribution of the residul polyethylene films for the different microorgnisms nd physicl tretments (Tbles 1 nd 2). Chnges in weight-verge moleculr weight (Mw) due to het (7 C) tretment or corresponding microbil degrdtion prlleled number-verge moleculr weight (Mn) vlues. During the 7 C het tretment of the initil degrdble film (zero control), the polyethylene M, dropped drmticlly fter 4-dy het tretment. This ws followed by slight rise nd then continul reduction. This undulting pttern in M, is ttributed to cross-linking between polymer chins (6, 9). The sme trend, consisting of slight rise for the dy 8 treted film in Mw, ws observed for the uninoculted controls nd for ech of the inoculted films (Fig. 2). The MBs for both the fungl nd bcteril uninoculted controls were consistently lower thn for the zero control, with significnt (P <.5) differences being determined between the - nd 4-dy controls. In lmost every tretment, the Streptomyces spp. effected reduction in polyethylene weight-verge moleculr weight, with significnt (P <.5) reduction being determined for the 4- nd 8-dy het-treted films (Tble 1, Fig. 2). S. viridosporus ws the overll best, with n verge reduction mong tretments of 21% (rnge, 11.8 to 67.8%), but there ws no significnt (P <.5) difference mong bcteril Downloded from on April 9, 218 by guest

5 682 LEE ET AL. APPL. ENVIRON. MICROBIOL ) C', Elution time (min) Elution time (min) FIG. 3. Comprison of HT-GPC chromtogrms for - nd 8-dy het (7 C)-treted degrdble plstics fter 4 weeks of incubtion with ligninolytic microorgnisms. (Top) Zero ( dys) is initil mteril (zero-time control), zero (8 dys) is 8-dy het-treted mteril, nd control is uninoculted 8-dy het-treted mteril incubted for 4 weeks t 37 C (uninoculted control). (Bottom) Control is 8-dy het-treted uninoculted control, nd T7A (8 dys) is the 8-dy het-treted film incubted with S. viridosporus t 37 C for 4 weeks. Downloded from on April 9, 218 by guest tretments. For the 2-dy 7 C het-treted films, S. bdius, S. setonii, nd P. chrysosporium showed slight increse in Mw, wheres S. viridosporus effected no chnge compred with the control (Tbles 1 nd 2). Generlly, the films incubted with the Streptomyces spp. demonstrted reduction in polydispersity (Mf/M.), which signifies nrrowing in the overll moleculr weight distribution (Tble 1). However, this ws not the cse for the fungus-cultured films (Tble 2). Almost ll of the fungl tretments demonstrted higher Mw when compred with the corresponding uninoculted controls, with significnt (P <.5) differences for -, 12-, nd 2-dy het-treted films. This reduction in polydispersity is illustrted by Fig. 3, which displys the ctul shift in the HT-GPCs for the initil mteril zero-time controls, the 8-dy het-treted zero control, nd the 8-dy uninoculted controls nd S. viridosporus-cultured films. For ech of the controls, the right side of the chromtogrm (round 28 min) is essentilly the sme line, with seril reduction in both polydispersity (M,/Mn) nd pek height being evident (Fig. 3, top). However, the bcteril film chromtogrm not only is nrrower thn the controls but is shifted completely to the right (Fig. 3, bottom). This shift to the right is indictive of brekdown of the polyethylene to smller-chin-length molecules. UV-irrdited films. Weight loss dt were inconclusive becuse of cell mss ccumultion on the films. All percent elongtion vlues bruptly incresed fter 2 weeks of UV irrdition nd dropped fter 4 nd 8 weeks of tretment

6 VOL. 57, 1991 BIODEGRADATION OF DEGRADABLE PLASTIC POLYETHYLENE 683 c :p l 2 cs -i Bcteril Cultures S O zero * control // v \\ v S.vlridosporuB / /\ \\ v S.bdius v \ [3.suetonti UV(365nm) tretment(wk) Fungl Cultures UV(365nm) treted(wk) FIG. 4. Chnge in percent elongtion for -, 2-, 4-, nd 8-week UV (365 nm)-irrdited degrdble plstics films fter 4 weeks of incubtion in culture medi, with nd without (control) ligninolytic microorgnisms. Zero control is het treted but not incubted. Ech dt point represents n verge of two replictes. (Fig. 4). This increse hs been ssocited with UV-initited cross-linking between polyethylene chins (6, 9, 11). Generlly, the bcteril nd fungl strins showed reduction in percent elongtion for the 2-week UV-irrdited films when compred with the zero control nd the uninoculted control, but generlly no chnge for the 4- nd 8-week UV-treted films (Fig. 4). Furthermore, for the 2-week UV-treted film, the bcteril uninoculted controls demonstrted very little loss compred with the zero control, wheres the fungl control displyed significnt reduction in percent elongtion (Fig. 4). The moleculr weight distribution for the UV-treted films prlleled the percent elongtion ptterns, showing significnt increse in verge moleculr weight for the 2-week treted film, followed by stedy decrese for the 4- nd 8-week UV tretments (Fig. 5). Unexplinbly, the zero control Mw nd M. vlues were generlly lower thn those of the uninoculted controls for both the bcteril nd fungl medi (Tbles 3 nd 4). Compred with the corresponding uninoculted controls, ll three bcteril cultures demonstrted loss in Mw nd M. for the 4-week UV-treted films, wheres only S. viridosporus nd S. bdius effected reduction for the 2-week UV-treted films (Tble 3). No degrdtion ws displyed by ny of the bcteril cultures for the 8-week UV-treted films. The fungus effected essentilly no reduction in Mw for the UV-treted films. Further O , Bcteril Cultures /irn zero v * control V S.viridosporus v Vv S.bdius 3 S.setonui V UV(365nm) treted(wks) Fungl Cultures UV(365nm) treted (wk3) FIG. 5. Weight-verge moleculr weight (Mw) for -, 2-, 4-, nd 8-week UV (365 nm)-irrdited degrdble plstics films fter 4 weeks of incubtion in culture medi, with nd without (control) ligninolytic microorgnisms. Zero control is het treted but not incubted. Ech dt point represents n verge of two replictes. more, the uninoculted controls nd the microbe-incubted 2- nd 4-week UV-treted films demonstrted n increse in polydispersity (M,/nM), nd the 8-week UV-treted zero control, inoculted, nd uninoculted control films remined reltively constnt (Tbles 3 nd 4). DISCUSSION 1 v~~~~~~~~~ O~~ zero control V P.chrysosporium One of the key dvntges of pure culture biodegrdtion ssy is the bility to identify wht portion of the degrdtion is due to chemicl degrdtion nd wht cn be ttributed directly to biologicl degrdtion. Pro-oxidnt ctivity due to culture medium, temperture, nd shking is evident by compring the zero controls with the corresponding uninoculted controls for ech of the different het tretments nd different culture medi (Tbles 1 nd 2). These differences could be ssocited with temperture, dissolved oxygen, medium composition, nd ph. There is higher trnsition metl concentrtion in the bcteril medium (see Mterils nd Methods). Furthermore, for the P. chrysosporium-incubted films, fungl growth on the plstic film ws extensive nd difficult to remove. In ll fungl tretments, n increse in percent elongtion (verge, 2%) nd M, (verge, 62%) ws detected when compred with the corresponding uninoculted controls (Tble 2). How- Downloded from on April 9, 218 by guest

7 684 LEE ET AL. APPL. ENVIRON. MICROBIOL. TABLE 3. Weight-verge moleculr weights (Mw), number-verge moleculr weights (M), nd polydispersity (MfhWM) vlues for UV (365 nm)-irrdited degrdble plstic with pro-oxidnt nd 6% strch before nd fter 4-week shke flsk incubtion t 37 C with ligninolytic Streptomyces spp. o wksb 2 wks 4 wks 8 wks Streptomyces sp. - 79w Mn. 7FM-.M R, Mn 79w/Mn 7gw Mn 79,/Mn Rmw n Mw Zeroc 181,379 37, ,717 18, ,955 1, ,756 11, Controld 96,828 22, ,495 23, ,215 21, ,749 1, S. viridosporus 88,71 2, ,11 21, ,563 2, ,69 12, S. bdius 86,33 24, ,212 22, ,292 19, ,583 11, S. setonii 86,98 24, ,397 24, ,524 18, ,374 1, Vlues were determined by HT-GPC. All vlues represent verges obtined from duplicte plstic strips ech obtined from duplicte HT-GPC runs. b Weeks of UV (365 nm) irrdition. c Zero vlues represent UV (365 nm)-irrdited smple without disinfection or culturl incubtion (zero control). d Control vlues represent UV (365 nm)-irrdited smples tht were chemiclly disinfected nd incubted-shken but were uninoculted (uninoculted control). ever, degrdtion ws evident when compred with the corresponding zero control vlues. This reduction in prooxidnt ctivity for the fungl cultures could be due to the fungl mt insulting the film from environmentl fctors such s dditionl trnsition metls in the medium nd, possibly, oxygen. The degrdble plstics used in this reserch re not designed to be photodegrdble. However, UV light is known inititor of polyethylene oxidtion, nd photo-oxidnt ctivity is enhnced by the ddition of trnsition metls such s coblt, mngnese, nickel, nd zinc (8), which re lso used s pro-oxidnt ctlysts. Almost no chnge in Mw ws observed for the zero control fter 2 weeks of UV irrdition, wheres n verge 2.5-fold increse in Mw ws demonstrted for ll incubted films (inoculted nd uninoculted) (rnge, 1.7 to 3.3-fold increse) when compred with zero time smples (Tbles 3 nd 4). However, the 4-week UV tretment did ccelerte bcteril biodegrdtion when compred with its corresponding control (Tble 3). For the UV-treted films, S. bdius nd S. setonii effected the highest percent reductions in Mw of 31 nd 36%, respectively, for the 4-week UV-treted films, nd S. viridosporus effected the highest degrdtion rte (68% reduction in Mj) for the 8-dy het-treted films, when compred with the corresponding uninoculted controls (Tbles 1 nd 3). This suggests tht the het nd UV tretments both generte very different residul oxidized polyethylene products, which hd direct effect on the biodegrdbility of the polymer. Generlly, the UV-treted films were more reclcitrnt thn the het-treted films, which lso suggests difference between the two residul polyethylenes. Lignocellulose-degrding bcteri, common to composting systems, cn biologiclly cleve the wter-insoluble, high-mw, chemiclly oxidized polyethylene residue of degrdble plstics. Furthermore, the dt indicte tht prooxidnt ctivity is essentil for inititing polyethylene biodegrdtion. This prllels previous soil degrdtion reserch on polyethylene-contining photo-oxidnts (4, 5). Also, het tretment t 7 C nd UV tretment t 365 nm did ccelerte oxidtive ctivity, mking the plstic more rpidly biodegrdble by breking up long polyethylene chins. Previous studies hve shown only the low-moleculr-weight portion of polyethylene to be biologiclly degrded (3-6). The culturl conditions used re considered to be conducive to lignin degrdtion nd the induction of bcteril lignin peroxidses (1, 2, 7, 15, 16). Mny of the P. chrysosporium culture broths (3% mlt extrct) becme colorless during incubtion. Fungl ligninse vertryl lcohol ssy (18) reveled tht culture flsks with colorless medium hd positive ligninse ctivity while the flsks with no chnge in medium color were negtive (unpublished dt). If the bcteril ligninses re involved in polyethylene biodegrdtion nd the Phnerochete lignin peroxidses re not, this would indicte distinct difference between the two degrdtion systems, which hs not been documented previously. Using strch gr ssy, we determined tht S. setonii nd P. chrysosporium were unble to utilize ntive corn strch, which is component of the degrdble plstic film. S. setonii did demonstrte polyethylene degrdtion, while P. chrysosporium did not. Therefore, the requirement for strch s cosubstrte in polyethylene biodegrdtion by microorgnisms will need further study. However, the degrdtion of strch in degrdble plstics hs lredy been documented (12). On the other hnd, non-plstic-degrding Downloded from on April 9, 218 by guest Weight-verge moleculr weights (Mw), number-verge moleculr weights (M), nd polydispersity (MwfMn) vlues TABLE 4. for UV (365 nm)-irrdited degrdble plstic with pro-oxidnt nd 6% strch before nd fter 4-week shke flsk incubtion t 3 C with P. chrysosporium Fungus O wksb 2 wks 4 wks 8 wks MwMn MW/Mn Mw M~~7n?9w/Mn?9n MwlMn Rw M9n VwwMn Zeroc 181,379 37, ,717 18, ,955 1, ,756 11, Controld 13,24 26, ,419 23, ,735 2, ,461 11, P. chrysosporium 143,697 34, ,391 24, ,82 2, ,558 12, Vlues were determined by HT-GPC. All vlues represent verges obtined from duplicte plstic strips ech obtined from duplicte HT-GPC runs. b Weeks of UV (365 nm) irrdition. c Zero vlues represent UV (365 nm)-irrdited smple without disinfection or culturl incubtion (zero control). d Control vlues represent UV (365 nm)-irrdited smples tht were chemiclly disinfected nd incubted-shken but were uninoculted (uninoculted control).

8 VOL. 57, 1991 BIODEGRADATION OF DEGRADABLE PLASTIC POLYETHYLENE 685 microorgnisms, growing solely on the strch, could form n insulting film on the surfce similr to P. chrysosporium, reducing the pro-oxidnt degrdtion rte. This insultion effect could explin some of the vrition in degrdble plstic performnce tht hs been observed in field studies. This sitution could be corrected by dding dditionl ctlyst (trnsition metl) to the films nd not relying on the immedite environment to provide supplementl ctlyst. Conclusions. From the results presented, we cn conclude tht there is strong evidence to support reduction in plstic integrity cused by microbil biodegrdtion of degrdble plstics contining pro-oxidnts nd 6% strch. Furthermore, use of pure culture system permits the distinction between chemicl nd biologicl degrdtion of these novel mterils by providing the necessry controls. It lso fcilittes the experimentl repliction needed to obtin sttisticl evlutions of the dt. To our knowledge, this is the first pure culture study to demonstrte tht lignin-degrding microorgnisms cn ctully degrde the oxidized polyethylene component of degrdble plstics, s indicted by moleculr weight reductions. ACKNOWLEDGMENTS This reserch ws supported by the Iow Stte University Center for Crops Utiliztion Reserch, U.S. Deprtment of Agriculture, the Iow Stte Legislture, Iow Deprtment of Agriculture nd Lnd Stewrdship, nd the Iow Agriculture nd Home Economics Experiment Sttion. REFERENCES 1. Adhi, T. P., R. A. Korus, nd D. L. Crwford Production of mjor extrcellulr enzymes during lignocellulose degrdtion by two streptomycetes in gitted submerged culture. Appl. Environ. Microbiol. 55: Adhi, T. P., R. A. Korus, A. L. Pometto III, nd D. L. Crwford Lignin degrdtion nd production of microbilly modified lignin polymers by Streptomyces viridosporus in slurry biorector. Appl. Biochem. Biotechnol. 18: Albertsson, A. C Biodegrdtion of synthetic polymers. II. A limited microbil conversion of "'C in polyethylene to "'CO2 by some soil fungi. J. Appl. Polym. Sci. 25: Albertsson, A. C., S.. Andersson, nd S. Krisson The mechnism of biodegrdtion of polyethylene. Polym. Degrd. Stbil. 18: Albertsson, A. C., nd Z. G. Bnhidi Microbil nd oxidtive effect in degrdtion of polyethylene. J. Appl. Polym. Sci. 25: Albertsson, A. C., nd S. Krlsson The three stges in degrdtion of polymers-polyethylene s model substnce. J. Appl. Polym. Sci. 35: Asther, M., C. Cpdevil, nd G. Corrieu Control of lignin peroxidse production by Phnerochete chrysosporium INA-12 by temperture shifting. Appl. Environ. Microbiol. 54: Chnd, M., nd S. K. Roy Plstic technology hndbook. Mrcel Dekker, Inc., New York. 9. Cornell, J., A. M. Kpln, nd M. R. Rogers Biodegrdbility of photooxidized polylkylenes. J. Appl. Polym. Sci. 29: Gerhrdt, P., R. G. E. Murry, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg, nd G. B. Phillips (ed.) Mnul of methods for generl bcteriology. Americn Society for Microbiology, Wshington, D.C. 11. Grssie, N., nd G. Scott Polymer degrdtion nd stbiliztion. Cmbridge University Press, Cmbridge. 12. Imm, S. H., nd J. M. Gould Adhesion of n mylolytic Arthrobcter sp. to strch-contining plstic films. Appl. Environ. Microbiol. 56: Pometto, A. L., III, nd D. L. Crwford Ctbolic fte of Streptomyces viridosporus T7A-produced, cid-precipitble polymeric lignin upon incubtion with ligninolytic Streptomyces species nd Phnerochete chrysosporium. Appl. Environ. Microbiol. 51: Pridhm, T. G., nd G. Gottlieb The utiliztion of crbon compounds by some ctinomycetles s n id for species determintion. J. Bcteriol. 56: Rmchndr, M., D. L. Crwford, nd G. Hertel Chrcteriztion of n extrcellulr lignin peroxidse of the lignocellulolytic ctinomycete Streptomyces viridosporus. Appl. Environ. Microbiol. 54: Rmchndr, M., D. L. Crwford, nd A. L. Pometto III Extrcellulr enzyme ctivities during lignocellulose degrdtion by Streptomyces: comprtive study of wild type nd geneticlly mnipulted strins. Appl. Environ. Microbiol. 53: SAS Institute Inc SAS user's guide: sttistics, version 5 ed. SAS Institute Inc., Cry, N.C. 18. Tien, M Properties of ligninse from Phnerochete chrysosporium nd their possible pplictions. Crit. Rev. Microbiol. 15: Downloded from on April 9, 218 by guest