h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /
Environmental
Microbiology
Variation
analysis
of
bacterial
polyhydroxyalkanoates
production
using
saturated
and
unsaturated
hydrocarbons
Saiqa
Tufail,
Sajida
Munir
∗,
Nazia
Jamil
DepartmentofMicrobiologyandMolecularGenetics,UniversityofthePunjab,Lahore,Pakistan
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:Received23February2016
Accepted11February2017
Availableonline29May2017
AssociateEditor:MiguelJ.
Beltran-Garcia
Keywords:
Wastefryingoil
Polyhydroxyalkanoates
Fluorescencemicroscopy
FTIR
a
b
s
t
r
a
c
t
Polyhydroxyalkanoates(PHA)areefficient,renewableandenvironmentfriendlypolymeric
esters.Thesepolymersaresynthesizedbyavarietyofmicrobesunderstressconditions.
Thisstudy wascarriedouttocheckthesuitabilityofwastefrying oilincomparisonto
other oils foreconomical bioplastic production.Sixbacterialstrainswereisolated and
identified as Bacillus cereus (KF270349), Klebsiella pneumoniae (KF270350),Bacillus subtilis
(KF270351),Brevibacteriumhalotolerance(KF270352),Pseudomonasaeruginosa(KF270353),and
Stenotrophomonasrhizoposid(KF270354)byribotyping.AllstrainswerePHAproducerssowere
selectedforPHAsynthesisusingfourdifferentcarbonsources,i.e.,wastefryingoil,canola
oil,dieselandglucose.ExtractionofPHAwascarriedoutusingsodiumhypochloritemethod
andmaximumamountwasdetectedafter72hinallcases.P.aeruginosaledtomaximum
PHAproductionafter72hat37◦Cand100rpmusingwastefryingoilthatwas53.2%PHA
incomparisonwithglucose37.8%andcookingoil34.4%.B.cereusproduced40%PHAusing
glucoseascarbonsourcewhichwashighwhencomparedagainstotherstrains.A
signif-icantlylesseramountofPHAwasrecordedwithdieselasacarbonsourceforallstrains.
SharpInfraredpeaksaround1740–1750cm−1 werepresentinFourierTransformInfrared
spectrathatcorrespondtoexactpositionforPHA.Theuseofwasteoilsandproductionof
poly-3hydroxybutyrate-co-3hydroxyvalerate(3HB-co-3HV)bystrainsusedinthisstudyisa
goodaspecttoconsiderforfutureprospectsasthistypeofpolymerhasbetterpropertiesas
comparedtoPHBs.
©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis
anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/
licenses/by-nc-nd/4.0/).
Introduction
Polyhydroxyalkanoic acids(PHA) presentinside prokaryotic
cellsarecarbonandenergyreservedgranulesstoredduring
environmentalstressconditions.About300differentbacterial
∗ Correspondingauthor.
E-mail:[email protected](S.Munir).
strainsareidentifiedtoaccumulatePHA.1AllGrampositive,
Gramnegative,archea,halophilic,halotolerant,rootnodule
bacteriaareabletoproducePHA.2,3
Bacteria has the ability toproduce PHA from a diverse
rangeofcarbonsourcessuchascomplexwasteeffluentsto
http://dx.doi.org/10.1016/j.bjm.2017.02.008
1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC
plantoils,carbohydrates,shortchainfattyacids,andalkanes.
Wastesaredischargedinlargeamountsfromfoodprocessing
industriesandagriculturalwastesthathavethepotentialto
beusedascarbonsourcesforPHAproduction.4
Carbohydratesareagoodsourcebuttheproductioncost
remainshighascomparedtosyntheticplastics,sothesearch
mustbecontinued.Carbonsubstratesincludingorganicfatty
acidshavealsobeenreportedforPHAproduction.5Therefore
tomakePHAproductionmoreeconomical,oneofthe
fore-moststepsistoisolatenaturallyoccurringbacterialstrains
thatutilizelessexpensivecarbonsourcessuchasmolasses
that are obtained either from sugar beets or sugarcane.
Molassesnormallysoldforabout33–50%ofthecostofglucose.
Inordertoreducetheproductioncostresearchersare
try-ing to produce PHA from plant oils. These oilshave been
projectedtobemoreefficientcarbon sourcesforindustrial
PHAproductionthansugars.6 Industrial-scaleprocessesfor
the production ofPHA from plantoils are currentlybeing
developed.7Differentplantoilssuchaspalmkerneloil,palm
olein,crudepalmoil,palmacidoil,canolaoil,soyabeanoil,
cornoilandoliveoilhavebeenreported.8,9Comamonas
testo-steronihasbeenstudiedforitsabilitytosynthesizemedium
chainlengthmcl-PHAfromvegetableoilssuchascastorseed
oil,coconutoil,mustardoil,cottonseedoil,groundnut oil,
oliveoilandsesameoil.Kaharandcoworkersreportedthat
therecombinantstrainsofRalstoniaeutrophaareabletouse
plantoilsuchassoybeanoil,10althoughtheuseofplantoils
forplasticproductionmaycreateasenseofcompetitionfor
foodsources.Variousagro-industrialwasteslikewheatbran,
potatostarch,sesameoilcake,groundnut oilcake,cassava
powder,jackfruitseedpowderandcornflourweretestedfor
PHAproductionbyBacillussphaericuswhichshowedthat
jack-fruitseed powder led tothe production ofmaximum 19%
PHBs.Manyotheragriculturalandindustrialwasteshavealso
beenreported ascarbonsourcesincludingcassavabagasse
hydrolyzate,babassu,soycake,canemolassesandwheyfor
PHBproduction.11Somescientistshavetriedwasteplantoils
assubstrate.IthasbeenshownthatCupriavidusnecator
pro-duced1.2g/LPHAfromwastefryingoilthatisinaccordance
withpreviousresultsobtainedwithglucose.12
TheaimofthisresearchworkwastocheckthePHA
pro-ductionabilityofpurifiedstrainsusingcheapcarbonsources.
Weanalyzedproductionabilityoflipaseproducingbacterial
strainsusingfourdifferentcarbonsourcesincludingglucose,
wastefryingoil,dieselandcookingoil.Studyofgrowth
kinet-ics of bacterial strains and product accumulation kinetics
using different oils parallel with glucose as standard
car-bonsourcewas carriedout. Thenchemical analysisofthe
producedPHAusingFouriertransforminfraredspectroscopy
(FTIR)wasperformedtoidentitymonomertypeofPHAour
bacterialstrainsare able toproducebyusingthesecarbon
sources.
Materials
and
methods
Bacterialstrains
StrainswereobtainedfromDepartmentofMicrobiologyand
MolecularGenetics,UniversityofthePunjab,Lahoreandwere
named accordingly using the abbreviation for strain (STN)
STN-6toSTN-11.AllthestrainswerestreakedonL-agarplates
andincubatedat37◦Cfor48htogetisolatedcolonies.PHA
detectionagar(PDA)13containing5g/mLNilebluewasused
forthedirectscreeningofPHAproducers.Medium(PDA)was
supplementedwith20g/Lglucose.Plateswerestreakedand
incubatedat37◦Cfor24–48h.14
OptimizationofbacterialstrainsforpH
InordertofindoptimalpHforbacteriatogrowandaccumulate
PHAgranules,strainsweregrownonPDAmediumhavingpH
range3–9.Opticaldensity(O.D.)wasrecordedafterevery24h
till72hat600nmandgraphswereplotted.
Analysisoflipaseenzymeactivityofbacterialisolates
Purifiedstrainsweretestedfortheirabilitytoutilizeoilsand
theactivityoflipaseenzymewascheckedbygrowingthemon
tributyrineagar.15
SudanblackBstaining
Sudan blackB(0.3%)in(ethyl alcohol)wasusedfor
detec-tion ofPHA inclusionbodies incells.Heat fixed smearsof
strains were preparedand stained withSudan black Bfor
15min.14SlideswereexaminedunderOlympusCX31
micro-scope(Olympus,Inc,USA)using100×objectivelenswithoil
immersion. Fluorescentstaining
Forfluorescentmicroscopy,10Lof72holdbacterialculture
and50Lofacridineorange(0.1%)weretakeninaneppendorf
tubeandincubatedat30◦Cfor30min.Pelletwascollectedby
centrifugationat4000×g for5min.16 Oncleanmicroscopic
slidesmearwaspreparedandobservedunder100×objective
lensofLeicaBZ-01fluorescencemicroscope(Leica
Microsys-tems,Germany).
Growthkineticstudiesandgrowthconditions
Shake flask fermentation process was carried out using
250mLErlenmeyerflaskscontaining100mLofPDAmedium
withglucose,waste fryingoil(WFO),cookingoiland diesel
as carbon substrates at 2% concentration and sterilized
by autoclaving at 121◦C and 15lb/in.2 for 20min. The oil
moleculeswereseparatedusingsonicator12andthenshaking
inincubator sothatbacteria wereable toutilizeit.
Experi-mentaldesignincludesfourflasksforeachcarbonsourceat
2%concentrationandwasinoculatedwith24holdbacterial
culture from seed culture medium. One flaskwas kept as
control and all were incubated at 37◦C and 100rpm in a
shaker.Thealiquots(2mL)ofincubatedmediumweretaken
out atregularintervalsof8htill72hfromboththecontrol
and inoculated medium. Bacterial growth was determined
byrecordingopticaldensityat600nm.Alongwithit,15mL
sample was collected every 24htill 72h and biomass was
obtainedbycentrifugationat4000×gfor20min.Pelletwas
ExtractionofPHAusingsodiumhypochloritemethod
PHA was extracted from dried biomass after 72h using
sodium hypochlorite digestion method.17 In 8g biomass,
100mLsodiumhypochlorite(30%)wasaddedandincubated
for90minat37◦C.Pelletwascollectedbycentrifugationand
washedwithalcohol,dissolvedinchloroformandtransferred
tocleanandpre-weighedserumtubes.Laterchloroformwas
allowedtoevaporateandweightofPHAwascalculated.
Fouriertransforminfraredspectroscopy(FTIR) spectroscopy
ForFTIRanalysisbiomasswascollectedasepticallyafter72h
ofincubation.Mediumwasremovedbywashingitthricewith
autoclaveddistilled waterand centrifugation.Theresulting
washed biomasswas lyophilizedand further driedby
pla-cingit in a hotair oven for3h. Onepart of biomasswas
combinedwiththreepartsofKBrandgrinded wellsothat
atransparentpelletwasobtained.Thispelletwasplacedin
IRspectrometerandpeakswereobtained.Theselectedrange
was400–4000wavenumbercm−1.
IsolationofgenomicDNA
IsolationofbacterialDNAfrom5mLcultureofselectedstrains
wascarriedoutusingDNAisolationmethod,18suspendedthe
obtainedpelletin100LTEbufferandstoredat−20◦C.Gel
electrophoresiswascarriedoutfordetectionofDNAby
load-ing5LofgenomicDNAinwellsofthegelandallowedtorun
at70V.
Polymerasechainreaction(PCR)amplificationofPhaC genesequences
GeneamplificationwasperformedusingPCRforPhaCgene.
Forthispurpose,extractedgenomicDNAwasusedas
tem-plate.PCRreactionmixturewaspreparedinsterilecondition.
This PCR PhaC gene amplification program was run for
30 cycles. Annealing temperature was 60◦C. PCR product
was purified by using liquid PCR product purification kit
(Fermentas, Inc.). All steps were performed according to
instructionsgiven inmanufacturer protocol.Purified
prod-ucts were run on 1% agarose gel. PCR purified products
weresubmittedtoCenterofExcellenceinMolecularBiology
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 STN-STN-9 STN-8 STN-7 STN-6 10 STN-11 O.D (600nm ) Strains pH 3 pH 5 pH 7 pH 9
Fig.1–pHOptimizationofbacterialstrainsgrowninPHA biosynthesismediumat37◦Cwithconstantshakingat 100rpm.
(CEMB), UniversityofthePunjab,Lahore,Pakistanfor
anal-ysis. Deoxyribonucleic acid DNA sequencing was carried
out by dideoxy method (chain termination) using
appara-tus applied biosystem DNA sequencer. All the sequence
resultsforamplifiedfragmentswere analyzedusingBLAST
algorithm(http://www.ncbi.nlm.nih.gov/blast/Blast.cgi).
Mul-tiple sequence alignments, and sequences were
sub-mitted to NCBI GenBank at http://www.ncbi.nlm.nih.gov/
genbank/submit.html.
Results
Bacterialstrains
PreviouslyisolatedstrainswereobtainedfromDepartmentof
MicrobiologyandMolecularGenetics,Universityofthe
Pun-jab,Lahore,Pakistanandidentifiedbysequencing.Thesewere
grownonPDAmediumwithpHrange3–9whichshowedthat
neutralpH(7)istheoptimalpHforgrowthandPHA
produc-tion(Fig.1).NogrowthwasobservedatpH3whileverylittle
atpH9whichindicatedthatthemicroorganismscouldnot
surviveatacidicorbasicpH.
Thestrains were analyzedforPHAproductionby
grow-ing themon PHAdetectionagar (2% glucose)with5g/mL
Nilebluedye,outofsix,threestrainscouldproduceblue
flu-orescence underUV-transilluminator.All isolatescontained
black granules in them when observed with Sudan Black
B (Fig. 2). Genomic DNA was successfully isolated from
all 6 strains. Thesewere identifiedas STN-6Bacillus cereus
(KF270349), STN-7 Klebsiella pneumoniae (KF270350), STN-8
Bacillus subtilis(KF270351),STN-9Brevibacteriumhalotolerance
(KF270352), STN-10 Pseudomonas aeruginosa (KF270353), and
STN-11Stenotrophomonasrhizoposid(KF270354)byribotyping.
PhaCgenewassuccessfullyamplifiedfromP.aeruginosa.
Analysisoflipaseenzymeactivityofbacterialisolates
Strains STN-8, STN-10and STN-11were abletoformclear
haloafter24hofincubationwhileSTN-7andSTN-6formed
slightlyvisiblehaloafter48hofincubationwhichconfirmed
thatstrainshaveactivelipaseenzymes andhenceare able
toutilizeoils.Oneoftheisolates,STN-9wasunableto
indi-catelipaseenzymeactivityasitdidnotformhaloaroundthe
growthevenafter48hofincubation.
Fluorescentmicroscopy
AcridineorangestainedslidesofthreeselectedstrainsSTN-7,
STN-8andSTN-10,showedorangefluorescencewithallthe
givencarbonsources.Resultsshowedthatlessernumberof
cells wasfluorescing underUVlightascomparedtowhite
light.Maximumnumbersoffluorescentcellswereobserved
forSTN-10whenwastefryingoil(WFO)wasusedinthemedia.
Howevernumberoffluorescentcellsforallthreestrainswas
comparablewithdifferentcarbonsources.Resultsindicated
acleardifferenceinnumberoffluorescentcellswhen
incu-bationtimeincreasedfrom24to72h.Anincreaseinsizeof
fluorescentcellswasnoticedanditseemedthatlargercells
A
B
C
Fig.2–BlackgranulesinstrainSTN-10observedwithSudanblack(0.3%)stainingafter(A)24h;(B)48h;(C)and72h. Examinedtheslidewithoilimmersionundermicroscopeat100×objectivelens.
inexternalmorphologyorshapeoffluorescentcellswasalso
noticed.
Growthkineticsstudiesofbacterialstrains
Growthcurvesindicatedthatallstrainsproliferatedeasilyon
allsourcesexceptdiesel.Inalmostallcasesgrowthsteadily
increasedwithincreasingtimeofincubationfrom2to60h.
Thestrainslogandlagphaseswereobservedatvariable
incu-bation time however; stationary phase for all strains was
around 60–72h. Strain STN-7, STN-8 and STN-10produced
maximumgrowthwithWFOafter48h.BothSTN-6andSTN-9
showedmaximumgrowthafter56hofincubationwith
glu-cose and cooking oil as carbon source respectively. Strain
STN-11indicated maximum absorbance at40th hour with
WFOsupplementedPHAmediaandutilizedwastefryingoil
andcookingoilequallywellforitsgrowth(Fig.4).
Kineticsofproduct(PHA)accumulation
AlmostallstrainsshowedsimilarpatternofPHA
accumula-tion,i.e.,amountofPHAincreasedgraduallythrough24,48
and 72hofincubation. Among all theisolates strain
STN-10producedmaximumamountofPHA53.2%(23.7g/L)using
WFOascarbonsourceafter72h.TheamountofPHA
accumu-lationwithcookingoilascarbonsourcewas34.4%(13.7g/L)
shownbystrainSTN-10andprovedtobethebeststrainto
uti-lizeoils.StrainSTN-8accumulated35.7%(9.0g/L)fromglucose
and 38.4%from WFO(12.3g/L) after72hincubationperiod.
Strain STN-7 produced maximum 9.6g/L(27.8%) PHA from
WFOandstrainSTN-6accumulated40%PHAusingglucose
ascarbonsourceandwasnotedastheonlystraintoutilize
glucosethemostascomparedtoallothersourcesused.The
maximumamountobtainedwithdieselwas0.02g/Lwhichis
10%oftotalbiomasscontentproducedbystrainSTN-10after
72h.Biomassproductionandthecomparativeaccumulation
A
B
B
Fig.3–FluorescencemicroscopyofstrainSTN-10with0.1%acridineorangeshowingpresenceofPHAgranulesafter72hof incubation(A)onPHAbiosynthesismediasupplementedwithWFOascarbonsource,(B)onmediasupplementedwith cookingoilascarbonsourceand(C)onmediasupplementedwithglucoseasacarbonsource(magnification100×).
0 0.5 1 1.5 2 2.5 3 3.5 80 72 64 56 48 40 32 24 16 8 0 O.D 600n m STN-6 0 0.5 1 1.5 2 2.5 3 3.5 80 72 64 56 48 40 32 24 16 8 0 O.D 600n m STN-7 0 0.5 1 1.5 2 2.5 3 3.5 80 72 64 56 48 40 32 24 16 8 0 O.D 600n m STN-8 0 0.5 1 1.5 2 2.5 3 3.5 80 72 64 56 48 40 32 24 16 8 0 O.D 600n m STN-9 0 0.5 1 1.5 2 2.5 3 3.5 80 72 64 56 48 40 32 24 16 8 0 O.D 600n m Time (h)
Glucose Waste frying oil Diesel oil Cooking oil
STN-10 0 0.5 1 1.5 2 2.5 3 3.5 80 72 64 56 48 40 32 24 16 8 0 O.D 600n m Time (h) Time (h) Time (h) Time (h) Time (h) STN-11
Fig.4–GrowthpatternofstrainsgrowninPHAbiosynthesismediasupplementedwithdifferentcarbonsourceshavingpH 7,cultivatedat37◦Cand100rpminashakingincubator.
ofPHAin100mLmediaafter72hofincubationareshownin
Fig.5.
ChemicalanalysisbyFTIRspectroscopy
ResultsforFTIRanalysisshoweddifferentpeaks
represent-ingthepresenceofdiversefunctionalgroups.Thepresence
ofdifferentpeaksshowed thatbacterialbiomasshas
com-plexaccumulations.Manysharppeaks werepresentinthe
range of 1000 wave number (cm−1) to 3000 wave number
(cm−1).ThisisthespecificregionofPHAspecificfunctional
groups.ThespectraforBacillussubtilus(STN-8)P. aeroginosa
(STN-10)andKlebsiellapneumoniae(STN-7)revealedthatsharp
absorptionbandsat1741cm−1,1635cm−1and1249cm−1were
present which correspond to ester carbonyl group (C O),
estergroupstretchingC–O and–CH grouprespectivelyand
correspond to the peak positions for PHA (Fig. 6). Bacillus
strainshowed thesefunctional groupsbyutilizing glucose,
wastefryingoilandcookingoil.IthasbeenreportedthatB.
cereusshowedalmostthesamepeakpositionsbygrowingon
nutrientbrothand thesepeaksare characteristicpeaks for
PHBs.19
Discussion
PurifiedcarbonsourcesusedforPHAaccumulationarecostly
thathindersPHAproductionatlargescale,thereforewetried
to identifycheapand readily availablecarbon sources
glu-cose,wastefryingoil,dieselandcookingoil.Weusedcooking
oilandwastefryingoilspecificallyofcanolaasit ishardly
reportedforthiskindofbiological(microbial)transformation.
Althoughstudiesusingwastefryingoilhaveexposedthatit
isagoodcarbonsourcebutlessworkhasbeendone.
ThehighestPHA producing strainP. aeruginosa (STN-10)
produced53.2%PHAusingwastefryingoilthatishigherthan
thatproducedusingglucose(37.2%),astandardcommercially
availablecarbonsource.Pseudomonasspp.hasbeenshownto
produce5and23.52%PHAoftheirDCWbyutilizing waste
fryingoil20,21andhashighgrowthrateamongoildegrading
bacteria byfollowing-oxidation pathway.22Waste sesame
oilhasalsobeenreportedascarbonsourceforPHB
accumu-lation byC.necator withmaximum yieldof4.6g/L.23 Other
strainsusedinthisstudy,Klebsiellapneumoniae(STN-7)andB.
subtilis(STN-8)alsoproducedgoodyieldswith27.8and38.4%
0 10 20 30 40 50 60 CO DL WFO Glucose STN-6 0 10 20 30 40 50 60 CO DL WFO Glucose STN-7 0 10 20 30 40 50 60 CO DL WFO Glucose STN-8 0 10 20 30 40 50 60 CO DL WFO Glucose STN-9 0 10 20 30 40 50 60 CO DL WFO Glucose STN-10 0 10 20 30 40 50 60 CO DL WFO Glucose STN-11 DCW (g/L) PHA wt.
(g/L
)Fig.5–ComparisonofdrycellweightandPHAweightforallstrainsobtainedfrom100mLcultureafter72hofflask fermentationat37◦Cand100rpm(WFO,wastefryingoil;DL,diesel;CO,cookingoil).
2345.021 24 1743.338 13 1643.0 5 1550.490 97 1095.371 83 539.9719 85 2931.2 7 2306.4 5 1751.05 2 1465.6 3 1403.9 2 1103.0 8 601.6 8 2869. 5 1349.9 3 0 5 10 15 20 25 30 35 40 45 50 55 60 4000 3600 3200 2800 2400 2000 1600 1200 800 400 Tr ansmission (% ) Wavenumber (cm-1) 8-G 8-WF0 8-C0
Fig.6–Fouriertransform-infraredspectroscopy(FTIR)ofbiomassproducedbyBacillussubtilis(STN-8)growninPHA biosynthesismediasupplementedwithglucose(G),wastefryingoil(WFO)andcookingoil(CO)ascarbonsourcesshowing peaksforPHBat1743–1751cm−1.
inB.subtilisdecreasedafter48hasitmaybeconsumedfor
sporulation process.24 The DCW did not follow the same
trendas acleardecrease inthe amount ofDCW occurred
after48hwhichmaybeduetonutrientsdepletion.
StrainSTN-7showedthatforallthecarbonsourcesused
PHAaccumulationincreasedwithincreaseinDCWovertime
exceptforWFOwhereDCW remained constant.
Compara-tivelytheutilizationofWFOwaslessincaseofSTN-6and
STN-11thatproduced18.1and5.7%PHAusingwastefrying
oilrespectivelybut produced good yieldusingglucose and
CO.These strains may lackthe enzymes forconversion of
fatty acids into PHAasthe environment oftheir
prolifera-tion werefreeofoilsbut enrichedwithcarbohydrates and
othernutrients.StrainSTN-9showedavariablepattern for
PHAproductionwithallcarbonsources.Byutilizingglucose
anddieselascarbonsourceitproducedmaximumPHAafter
48hbutdecreasedafterwardswhichmaybebecauseofthe
re-utilizationofthesestoragegranules.Ithasbeenreported
thatatcessationoflogphaseorearlystationaryphasethePHA
accumulationreachesitsmaximumbutafterthatitdecreases
duetosporulation.24
Allstrainsdidnotutilizedieselandnotevenaccumulated
itintheformofPHA.Thereasonmaybeitscomposition
con-taininghighconcentrationsofmanyaliphaticandaromatic
hydrocarbons. These hydrocarbons change the membrane
structure by changing the membrane fluidity and protein
conformations. This overall alteration disrupts membrane
integrity, energytransduction mechanisms and membrane
associatedenzymeactivity.Thisishowdieselkills
microor-ganismsandprovestobetoxicforthemicrobes25whileother
reasonmaybeitsvolatilenaturewhichmakesitunavailable
throughoneweekexperimentalprocedure.
OnthewholetheresultsofkineticsofPHAaccumulation
showedthatstrainsSTN-10,STN-7andSTN-8growbestby
utilizingwastefryingoil(WFO)ascarbon sourceand
accu-mulatemorePHAusingWFOthancookingoil.Thismaybe
duetochangeincompositionofoilafterfryingasithasbeen
reportedthatafterbeingused,achangeincompositionoccurs
andhaveverylessquantityofpolarcompounds.26Afterfrying
thecanolaoilithas78%ofoleicacidlevelandverylowlevel
oftotalpolarcompounds.Otherthanoleicacid,20%linoleic
acidwasalsopresent.27Thecompositionofoilbeforeandafter
fryingchangesthepercentageofdifferentcompounds(lauric
acid,palmiticacid,oleicacid,linoleicacid andstearicacid,
etc.)eitherincreases ordecreases,and overallfried oilhas
lowmolecularweightacids,28 freefatty acidsand the
con-stituents(proteins,carbohydratesandlipids)ofthefriedfood
gettransferredtothatoil,23whichseemstobebeneficialfor
theefficientproductionofPHA.Ourstrainsnotonlyproduced
PHBsbuttheyproducedPHBsandPHVscopolymers.Thecost
effectiveproductionofco-polymersusing waste oilas
car-bonsourcehasbeen achievedpreviously23 and ithasbeen
reportedthatapolymericmaterialwhichisablendofPHBs
andPHVsismorestrongandflexiblethanPHBs29hencemore
suitableforpracticalapplications.
Thefindings of this research work recommend the use
ofwastefryingoilsbecausePHAproductionusingwasteoil
washigh ascomparedtocooking oilandglucose. Bacteria
under research not onlyproduced PHB but also produced
3(PHB-co-PHV) which more strongly states the potential of
thesebacteriaandthewaste fryingoilassourcefor
indus-trialuse17asthiscopolymerismorepromisingmaterialfor
practicalapplications.Italsoshowedthatutilizationofwaste
fryingoilnotonlymakesbioplasticmaterial(PHA)economical
andimprovesitsmarketbutitwillalsomakemanagement
of oily waste very easy.Future prospects includethe
opti-mization of these bacterial strains for PHA production at
largescalefermentationforvolumetricproductionofthis
Eco-Greenmaterialusingwastefryingoilascheapsubstrate.30
Conflicts
of
interest
Itis statedthat the authors havenoconflictofinterest in
eitherwaysuchaspersonal,political,financial,academic,or
religious.
Acknowledgement
TheauthorswouldliketothankUniversityofthePunjaband
HigherEducationCommission,Pakistan(ProjectNo:
NRPU-20-3443)forprovidingfinancialassistanceinthiswork.
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e
s
1.TeekaJ,ImaiT,ChengX,etal.ScreeningofPHA-producing
bacteriausingbiodiesel-derivedwasteglycerolasasole
carbonsource.JWaterEnvironTechnol.2010;8(4):373–381.
2.QuillaguamanJ,GuzmanH,VanT,HattiKR.Synthesisand
productionofpolyhydroxyalkanoatesbyhalophiles:current
potentialandfutureprospects.ApplMicrobiolBiotechnol.
2010;85(6):1687–1696.
3.VanTD,HuuPT,ThiBN,ThiTN,MinhLD,QuillaguamanJ.
Polyesterproductionbyhalophilicandhalotolerantbacterial
strainsobtainedfrommangrovesoilsampleslocatedin
NorthernVietnam.Microbiologyopen.2012;1(4):395–406.
4.CheeJY,YogaSS,LauNS,LingSC,AbedRM,SudeshK.
Bacteriallyproducedpolyhydroxyalkanoate(PHA):
convertingrenewableresourcesintobioplastics.In:Current
Research,TechnologyandEducationTopicsinAppliedMicrobiology andAppliedBiotechnology.;2010:1395–1404.
5.ShahidS,MosratiR,LedauphinJ,etal.Impactofcarbon
sourceandvariablenitrogenconditionsonbacterial
biosynthesisofpolyhydroxyalkanoates:evidenceofan
atypicalmetabolisminBacillusmegateriumDSM509.JBiosci
Bioeng.2013;116(3):302–308.
6.BuddeCF,RiedelSL,HubnerF,etal.Growthand
polyhydroxybutyrateproductionbyRalstoniaeutrophain
emulsifiedplantoilmedium.ApplMicrobiolBiotechnol.
2011;89(5):1611–1619.
7.SudeshK.Plantoilsandagriculturalby-productsascarbon
feedstockforPHAproduction.In:Polyhydroxyalkanoatesfrom
PalmOil:BiodegradablePlastics.Springer;2013:37–46.
8.AkiyamaM,TsugeT,DoiY.Environmentallifecycle
comparisonofpolyhydroxyalkanoatesproducedfrom
renewablecarbonresourcesbybacterialfermentation.Polym
DegradStab.2003;80(1):183–194.
9.TsugeT.Metabolicimprovementsanduseofinexpensive
carbonsourcesinmicrobialproductionof
polyhydroxyalkanoates.JBiosciBioeng.2002;94(6):579–584.
10.KaharP,TsugeT,TaguchiK,DoiY.Highyieldproductionof
eutrophaanditsrecombinantstrain.PolymDegradStab.
2004;83(1):79–86.
11.RamadasNV,SinghSK,SoccolCR,PandeyA.
Polyhydroxybutyrateproductionusingagro-industrial
residueassubstratebyBacillussphaericusNCIM5149.Braz
ArchBiolTechnol.2009;52(1):17–23.
12.VerlindenRAJ,HillDJ,KenwardMA,WilliamsCD,Piotrowska
SZ,RadeckaIK.Productionofpolyhydroxyalkanoatesfrom
wastefryingoilbyCupriavidusnecator.AMBExpress.
2011;1(1):11.
13.RamsayJ,BergerE,VoyerR,ChavarieC,RamsayB.Extraction
ofpoly-3-hydroxybutyrateusingchlorinatedsolvents.
BiotechnolTech.1994;8(8):589–594.
14.MunirS,JamilN.Characterizationofpolyhydroxyalkanoates
producedbycontaminatedsoilbacteriausingwastewater
andglucoseascarbonsources.TropJPharmRes.
2015;14(9):1605–1611.
15.RashidN,ShimadaY,EzakiS,AtomiH,ImanakaT.
Low-temperaturelipasefrompsychrotrophicPseudomonas
sp.strainKB700A.ApplEnvironMicrobiol.2001;6(9):4064–4069.
16.KumarB,PrabakaranG.ProductionofPHB(bioplastics)using
bio-effluentassubstratebyAlcaligenseutrophus.IndianJ
Biotechnol.2006;5(1):76–79.
17.RamsayJ,BergerE,RamsayB,ChavarieC.Recoveryof
poly-3-hydroxyalkanoicacidgranulesbya
surfactant-hypochloritetreatment.BiotechnolTech.
1990;4(4):221–226.
18.SambrookJ,RussellDW.Invitromutagenesisusing
double-strandedDNAtemplates:selectionofmutantswith
DpnI.MolClon.2001;2:13.19–13.25.
19.ValappilSP,BoccacciniAR,BuckeC,RoyI.
PolyhydroxyalkanoatesinGrampositivebacteria:insights
fromthegeneraBacillusandStreptomyces.Antonievan
Leeuwenhoek.2007;91(1):1–17.
20.ChanPL,YuV,WaiL,YuHF.Productionof
medium-chain-lengthpolyhydroxyalkanoatesby
Pseudomonasaeruginosawithfattyacidsandalternative
carbonsources.In:Twenty-SeventhSymposiumonBiotechnology
forFuelsandChemicals.Springer;2006.
21.HwanSJ,JeonCO,ChoiMH,YoonSC,ParkW.
Polyhydroxyalkanoate(PHA)productionusingwaste
vegetableoilbyPseudomonassp.strainDR2.JMicrobiol
Biotechnol.2008;18(8):1408–1415.
22.SuriyamongkolP,WeselakeR,NarineS,MoloneyM,ShahS.
Biotechnologicalapproachesfortheproductionof
polyhydroxyalkanoatesinmicroorganismsandplants–a
review.BiotechnolAdv.2007;25(2):148–175.
23.ObrucaS,MarovaI,SnajdarO,MravcovaL,SvobodaZ.
Productionofpoly(3-hydroxybutyrate-co-3-hydroxyvalerate)
byCupriavidusnecatorfromwasterapeseedoilusing
propanolasaprecursorof3-hydroxyvalerate.BiotechnolLett.
2010;32(12):1925–1932.
24.ValappilSP,PeirisD,LangleyG,etal.Polyhydroxyalkanoate
(PHA)biosynthesisfromstructurallyunrelatedcarbon
sourcesbyanewlycharacterizedBacillusspp.JBiotechnol.
2007;127(3):475–487.
25.KangYS,ParkW.Protectionagainstdieseloiltoxicityby
sodiumchloride-inducedexopolysaccharidesinAcinetobacter
sp.strainDR1.JBiosciBioeng.2010;109(2):118–123.
26.HabaE,EspunyM,BusquetsM,ManresaA.Screeningand
productionofrhamnolipidsbyPseudomonasaeruginosa47T2
NCIB40044fromwastefryingoils.JApplMicrobiol.
2000;88(3):379–387.
27.WarnerK,OrrP,ParrottL,GlynnM.Effectsoffryingoil
compositiononpotatochipstability.JAmOilChemSoc.
1994;71(10):1117–1121.
28.VidalMJ,ResinaPO,HabaE,ComasJ,ManresaA,VivesRJ.
Rapidflowcytometry–NileredassessmentofPHAcellular
contentandheterogeneityinculturesofPseudomonas
aeruginosa47T2(NCIB40044)growninwastefryingoil.Ant VanLeeuwen.2001;80(1):57–63.
29.RasheedR,LathaD,RamachandranD,GowriG.
CharacterizationofbiopolymerproducingStreptomyces
parvulus,optimizationofprocessparametersandmass
productionusinglessexpensivesubstrates.IntJBioassays.
2013;2(4):649–654.
30.KungS,ChuangY,ChenC,ChienC.Isolationof
polyhydroxyalkanoatesproducingbacteriausinga
combinationofphenotypicandgenotypicapproach.Lett
