Pur Paper Format

8/10/2019 Pur Paper Format

1/8

Trend and challenges in global scenarioEnvironmental pollution byplastic polymer

Polyester polyurethane andits biodegradation

B.Shaileshkumar

B.Tech Biotechnology

Department of Humanities and science

Arunai Engineering college

Tiruvanamalai

M.P.Tilak ra

B.Tech Biotechnology

Department of Humanities and science

Arunai Engineering college

Tiruvanamalai

Abstract Trend and challenges in global scenario, here weboth put forth the environmental pollution caused by plasticpolymer polyester polyurethane and providing solution that itcan be biodegraded and this paper is the review of an researcharticle and we put forth our discussion.

Index Terms I. Introduction II .Methods and materials inbiodegradation III Results IV iscussion.

I INTRODUCTION

Tremendous increases in the manufacture and

consumption of plastics over recent decades have led to

numerous ecological and economic concerns. The persistence

of synthetic polymers introduced into the environment by

human industry poses a maor threat to natural ecological

systems. The lo! cost and ease of manufacture have increased

global plastic demand more than "#$%fold& !ith theproduction of ".# million tons in "'#$ and ()# million tons

as of ($$* +(",. Despite recognition of the persistent

pollution problems posed by plastic& global production is still

increasing& !ith the largest increases e-pected in developing

nations. The sheer volume of plastics produced each year

presents a problem for !aste disposal systems. The scale of

this problem and the recalcitrance of some polymers to

degradation necessitate investigation into effective methods

for biodegradation of plastics. y gaining an understanding of

the mechanisms of polymer degradation& a more efficient

techni/ue for the biodegradation of plastic !aste can be

achieved. To accomplish this goal& researchers need greater

0no!ledge of ho! compounds are metaboli1ed by e-istingorganisms& an investigation of ne! organisms !ith

bioremediation potential& and the characteri1ation of novel

metabolic capabilities. 2 basic understanding of the biological

processes that lead to biochemical degradation !ill advance

the development of ne! bioremediation techni/ues.

3olyester polyurethane +3UR, is a plastic !idely used inindustry and manufacturing that has been sho!n to besusceptible to biodegradation +*& "$,. The polymer isgenerated by the condensation of a polyisocyanate and a

polyol. This results in a carbon polymer composed of a seriesof urethane lin0ages. 4ariations in the spacing bet!eenurethane lin0ages& as !ell as the nature of the substitutions&can change the properties of the resulting polymer from linearand rigid to branched and fle-ible. In a li/uid suspension 3URappears mil0y !hite and completely opa/ue. 5i0e other

polyurethanes& this product is synthesi1ed commercially forthe manufacture of te-tiles and te-tile coatings.

6n1ymatic degradation of 3UR by both fungi +)&*&"',and bacteria +""& "(& ")& "#& "7& "8& (9, has beendemonstrated. :oil fungi comprise the maority of organismsscreened for 3UR degradation activity +)& #,. ;ungi of thegenera 2lternaria& 2spergillus& 3homa& 3ennicilium&3lectosphaerella&


2/8


3/8

converting absorbance to percent clearance. :amples !eremeasured each day for ( !ee0s for optical absorbance todetermine a relative rate of clearance.

E. Sole car&on source assay

Organisms identified as having 3UR%degrading activity!ere tested for their ability to use 3UR as the sole carbonsource. ;or these studies& the substrate !as Impranil D5N&!hich contains 3UR suspended in only !ater +no N%methyl

pyrrolidone is present,. This isolates the Impranil as the solesource of carbon for metabolism and gro!th. The top fiveorganisms from the initial activity screens !ere gro!n onImpranil D5N !ith no other carbon sources +3UR%5min,. Thefungal samples !ere !ashed prior to inoculation to remove allresidual medium. These t!o considerationsMthe !ash and theD5N substrateMensure that the polymer is the sole source ofcarbon for fungal metabolism and gro!th. 3UR%5min !as

prepared in a manner similar to that for the 3UR%5 mediumbut in the absence of sodium citrate& thiamine& Casamino2cids& or agar. The organism 2spergillus niger !as tested as a

basis for comparison.

;ungal cultures !ere gro!n for " !ee0 in potato de-trosebroth +3D,. :toc0 cultures !ere homogeni1ed by vigoroussha0ing& and " ml of each culture !as centrifuged at "(&"$$ Lg for " min. The supernatant !as removed& and the fungal

pellet !as resuspended in " ml of the 3UR%5min li/uidmedium. :amples !ere centrifuged and resuspended a secondtime to ensure removal of all residual 3D. The "%ml sampleof !ashed fungal material !as added to sterile culture tubes toa final volume of "$ ml 3UR%5min. The cultures !eremonitored for visual clearance of the opa/ue medium.:amples !ere measured every ( days for ( !ee0s for opticalabsorbance at *$$ nm to determine an appro-imate rate ofclearance. 2n increase in fungal mass correlating to 3URdegradation !as measured by lyophili1ing mycelial mass fromtriplicate cultures containing minimal medium !ith and

!ithout 3UR.

'. Anaero&ic degradation

2naerobic degradation of 3UR.Culture tubes containing '%ml ali/uots of 3UR%5min medium and " ml of !ashed fungalinoculums !ere incubated under anaerobic conditions. Theanaerobic environment !as generated using a D . Iodoacetate& a cysteine hydrolaseinhibitor& !as added to a final concentration of "$ >. 6DT2at a concentration of # m> !as used as a metallohydrolaseinhibitor. 6-tract that !as heat treated at '8BC for ($ min

served as a negative control. :amples !ere incubated in arotary incubator at 9$BC& and clearance !as observedmacroscopically after ( h.

ased upon initial observations that a serine hydrolase !asimplicated in 3UR degradation& activity%based probes !ereused to test for a serine hydrolase protein in crude cell%freefiltrates. The probe molecules contain a fluorophosphonatemoiety that reacts specifically !ith the active%site serines ofen1ymes in the serine hydrolase family and atetramethylrhodamine +T>R, tag +($,. Reactions of the crude

protein e-tract !ith the probe !ere carried out as previouslydescribed +($,. The resulting :D:%polyacrylamide gel !asvisuali1ed using a ;uifilm ;52%#"$$ fluorescent bed scanner!ith e-citation at #9( nm and a #7$%nm emission filter. Thesame gel !as silver stained to visuali1e all proteins in the

samples.

Crude protein from cell%free filtrate of 3estalotiopsismicrospora 6(7"(2 gro!n in "%liter cultures of 3UR%5minminimal medium and 3D rich medium !ere concentratedand purified to appro-imately '$H purity by gel filtrationchromatography +data not sho!n, using a :uperde- ($$column +


4/8

used as negative controls in the subse/uent studies. The hostplants& isolation media& and identities of the "8 active fungiand t!o inactive control fungi are listed in Table ".

;ig. ".

6-ample of 3UR%2 plates initially used to screen forpolyurethane%degrading activity after ( !ee0s of fungalgro!th. +2, Negative control. +, 3leosporales sp. strain6(7$#.

6ndophytes studied and the host plant species from !hichthe endophytes !ere isolated. :everal organisms of the

3estalotiopsis genus !ere represented among the activeorganisms in the first 3UR%2 clearance screen. ased uponinitial activity observed from this genus& all of the3estalotiopsis isolates in the collection !ere screened foractivity. :ome organisms !ithin the genus e-hibited highactivity +6(7"(2& 699"7& and 6(7""2,& some e-hibitedmoderate activity +699")2 and 6(7$82,& and others e-hibitedno detectable activity +6)""(2,. This variable e-pression !asobserved under identical gro!th conditions for each organism.@hile it is notable that some 3estalotiopsis microspora strainsdemonstrated some of the highest levels of activity& the level!as /uite variable among the various isolates.

The active organisms from the plate clearance test !ereassayed in t!o additional 3UR clearance assays& one using asolid medium and the second using a li/uid medium. In the

solid 3UR%2 clearance assay& fungal plugs !ere used toinoculate test tubes of 3UR%2 medium and vertical clearance!as measured after ( !ee0s of gro!th +;ig. (,. 2ll of the "8active fungi visibly cleared the 3UR%2 medium. :i-teen ofthese fungi demonstrated activity that !as at least t!ice that ofthe organism 3seudomonas chlororaphis& a positive control for3UR degradation +"9,. The top clearing organism in this solidmedium assay !as identified by IT: se/uencing as a5asiodiplodia sp. +strain 6(*""2,. ;our of the si- most activeorganisms belonged to the 3estalotiopsis genus& !ith highrelatedness at the species level to 3estalotiopsis microspora.These top si- organisms all cleared 3UR more efficiently thanthe positive%control fungus 2spergillus niger. The t!onegative%control organisms&


5/8

6-amples of 3UR%5 li/uid cultures at ( !ee0s afterinoculation. +, ;ungal cultures !ere gro!n in triplicate inli/uid 3UR%5 medium containing Impranil D5; for ( !ee0s.3ercent clearance !as determined by a decrease in light

:cattering at *$$ nm as measured by U4%visiblespectrophotometry. 3ercent clearance measurements !ereta0en after ( !ee0s of gro!th. 2ll data !ere normali1ed to thenegative control. The positive%control test organisms&3seudomonas chlororaphis and 2spergillus niger& arerepresented at the left. The # highlighted organisms+5asiodiplodia sp. strain 6(*""2& 3estalotiopsis microspora6(7"(2& 3estalotiopsis microspora 699"7& 3leosporales sp.strain 6(8"(2& and ionectria sp. strain 6('"$, !ereselected for further screening. 6rror bars represent thestandard deviation for each data set.

:ole carbon source assay. The five most active organisms!ere tested for their ability to degrade 3UR in li/uid cultureusing 3UR as the sole carbon source +3UR%5min,. ;or thesestudies !e used Impranil D5N& !hich is e/uivalent to theImpranil D5; +an anionic aliphatic 3UR dispersion in !ater,used for the previous studies but does not contain N%methyl

pyrrolidone. If an organism gro!s in Impranil D5N& it can use3UR as the sole carbon source for metabolism and gro!th. 2ll

fungal inocula !ere !ashed !ith minimal medium to removeresidual carbon and en1ymes from the stoc0 culture. The topfive organisms from the t!o screen assays !ere tested for thisactivity& and all five !ere capable of 3UR degradation activityunder this minimal medium condition. 6ach fungus sho!edsignificant mycelial gro!th as monitored by visual inspection.;or 3estalotiopsis microspora 6(7"(2& cultures gro!n !ith3UR sho!ed increased gro!th of $.""$ g $.$9" g of fungalmaterial over that of a control culture gro!n under identicalconditions !ith no 3UR added. In addition& the positive%control fungus 2spergillus niger !as able to slo!ly degrade3UR as a sole carbon source& as previously reported +7,.

In order to compare the rates at !hich 3UR !as degradedunder this condition& the top five organisms in the solid andli/uid screen assays !ere gro!n in duplicate and trac0ed for

"* days +;ig. )2,. T!o organisms& 3estalotiopsis microspora6(7"(2 and 699"7& demonstrated the highest rate of 3URclearance. The appro-imate half time for clearance for theset!o organisms !as # days. The cultures !ere visuallytransparent by the end of the "*%day period& consistent !ithcomplete degradation of the Impranil D5N +;ig. ),. The onlynon translucent material remaining in the flas0 consisted offungal gro!th. In comparison to other standards& the half timefor clearance by the control organism 2spergillus niger !as "#days& and the bacterium 3seudomonas chlororaphis did notreach a state of half clearance !ithin the "*%day time course.6-periments performed using Impranil D5; as the substratesho!ed that the presence of N%methyl pyrrolidone did notaffect the gro!th rate for the organisms tested. Thisestablished that 3UR alone is sufficient for fungal gro!th forthese organisms. 2s a result& Impranil D5N !as used forsubse/uent studies to characteri1e anaerobic gro!th and thenature of the degradation reaction.

;ig. ).

+2, Degradation of 3UR as a sole carbon source by the fivemost active organisms !as monitored over a "*%day timecourse. Cultures containing 3UR%5min medium !ith ImpranilD5N as the sole carbon source !ere inoculated !ith !ashedfungal inoculums. The assay !as performed in duplicate& andthe values sho!n represent the averages for the t!o cultures ateach time point. 2spergillus niger +green, and 3seudomonaschlororaphis +red, served as the positive controls. +,3estalotiopsis microspora 6(7"(2 +left, degrading 3URImpranil D5N as a sole carbon source after a "*%day timecourse. The remaining material in the flas0 is the result offungal gro!th.

2naerobic degradation of 3UR.:ome of the organismstested during the li/uid culture assays& including 3estalotiopsismicrospora 6(7"(2 and 3estalotiopsis microspora 699"7&!ere observed gro!ing from the bottom of the culture tuberather than from the top. This suggested that these fungi arecapable of anaerobic degradation of the polyurethane. To testthis possibility& the assay using 3UR%5min medium !ithImpranil D5N substrate !as repeated under anaerobicconditions. The top five organisms from the previous assay!ere inoculated in triplicate and gro!n for " and ( !ee0s in aD


6/8

;ig. #.

+2, 3UR degradation measurements of fungal culturesgro!n under aerobic and anaerobic conditions. Triplicate setsof fungal cultures !ere gro!n in 3UR%5min mediumcontaining Impranil D5N as the sole carbon medium underaerobic or anaerobic conditions. 2t the end of ( !ee0s ofgro!th& samples !ere removed from the sealed chamber andthe percent clearance of the 3UR in solution for each set of

cultures !as measured by decreased light scattering at a!avelength of *$$ nm using U4%visible spectrophotometryThe results for the controls 3seudomonas chlororaphis and2spergillus niger are sho!n at the left. T!o strains of3estalotiopsis microspora +6(7"(2 and 699"7, maintained

polyurethanase activity !hen gro!n under anaerobicconditions& !hile 3seudomonas chlororaphis& 2spergillusniger& 5asiodiplodia sp. strain 6(*""2& 3leosporales sp. strain6(8"(2& and ionectria sp. strain 6('"$ all sho!edsignificant reductions in the magnitude of 3UR degradationactivity. 6rror bars represent the standard deviation for eachdata set. +, The rates of degradation !ere compared bymeasurement in triplicate of cultures of 2spergillus niger and3estalotiopsis microspora 6(7"(2 after " !ee0 and ( !ee0s ofgro!th. 3estalotiopsis microspora 6(7"(2 sho!s e/uivalentrates of degradation activity under aerobic and anaerobic

conditions. 6rror bars represent the standard deviation foreach data set.

IR analysis of 3UR degradation. The mechanism of 3URdegradation !as initially investigated by infrared spectroscopy+"*& (),. 3UR samples of Impranil D5N display a largeabsorption pea0 at "&79# cmE" corresponding to the C+O,%Oester lin0age in the polyurethane polymer +;ig. *2,. ;ungalmaterial !as added to the 3UR substrate and spectra collectedthroughout the course of the degradation e-periment. 2

progressive reduction in the relative intensity of the pea0 at"&79# cmE" !as observed and !as accompanied by moresubtle changes at other !ave numbers. y the time the culturehad became visually transparent& there !as a complete loss ofthe absorbance pea0 at "&79# cmE" +;ig. *,. 5oss of this

pea0 is consistent !ith hydrolysis of the ester bond in theurethane lin0age.

;ig. *.

+2,


7/8

activity of 3UR clearance. ?eat%treated e-tract !as used as anegative control. 2ddition of 3>:;& a serine hydrolaseinhibitor& resulted in inactivation of the biodegradationactivity. 2ddition of iodoacetate and 6DT2 had no effect onthe rate or e-tent of activity +data not sho!n,.

The reaction of the crude cell%free filtrate of 3estalotiopsismicrospora 6(7"(2 !ith serine hydrolase inhibitor probesefficiently labelled one protein in the fraction gro!n in 3UR%5min medium. :pecific protein labelling !as absent in thecell%free filtrate from 3D li/uid culture& the condition in!hich no 3UR degradation activity !as observed +;ig. 7,.These results suggest that an appro-imately ("%0Da serinehydrolase%li0e en1yme is responsible for 3UR degradation.;urthermore& gro!th in 3UR%5min induced secretion of thisen1yme +;ig. 72,. The ("%0Da protein !as purified from thecrude filtrate to appro-imately '$H purity& and it retained theability to degrade 3UR +data not sho!n,.

;ig. 7.

3robing for serine hydrolases in crude protein from cell%free filtrate. :D:%32


8/8

ester hydrolysis by a serine hydrolase is responsible for 3URbiodegradation.

This investigation established the robust polyurethanedegradation activity under anaerobic conditions in !hich thesynthetic polymer served as the only carbon source for thefungus. 2 cell e-tract of the active culture containing a criticalserine hydrolase is able to clear the polymer in under " h usingthe 3UR concentrations reported here. This !or0 establishesthat endophytes are a useful source of biodiversity !ith

potential application for bioremediation. The relative ease !ith!hich organisms can be isolated and screened ma0es this ahighly accessible and environmentally relevant proect forengaging undergraduate students in scientific research. It is

possible that activities against other& more recalcitrantpolymers could be discovered using this abundant source ofbiodiversity and this can be proceed only in the lab conditionand our aim is to proceed this e-periment in open atmosphericenvironment. ut there is a critical issue that the 3estalotiopsismicrospora can biodegrade the both plastic +3UR, !hich is ine-istent condition and as !ell as the !aste !e planned to dothis e-periment in forth coming years !ith basic core0no!ledge.

R6;6R6NC6:

Q" Sonathan R. Russell"&&

Q( Seffrey ?uang"&&

Q9 3ria 2nand"&&

Q) Gaury Gucera"&

Q# 2manda olecular iophysics and iochemistry& Aale University&Ne! ?aven& Connecticut $*#($

Q( Universidad Nacional :an 2ntXnio 2bad del Cusco& 3eru 6scuela 3ost

Documents

Pur Paper Format