h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /
Industrial
Microbiology
Staphylococcus
xylosus
fermentation
of
pork
fatty
waste:
raw
material
for
biodiesel
production
Roger
Vasques
Marques
a,∗,
Matheus
Francisco
da
Paz
a,
Eduarda
Hallal
Duval
b,
Luciara
Bilhalva
Corrêa
a,
Érico
Kunde
Corrêa
aaLaboratoryofWastes,FederalUniversityofPelotas,Pelotas,RS,Brazil
bAnimalProductsInspectionLaboratory,FederalUniversityofPelotas,CampusCapãodoLeão,CapãodoLeão,RS,Brazil
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t
i
c
l
e
i
n
f
o
Articlehistory:
Received8October2015 Accepted17February2016 Availableonline21April2016 AssociateEditor:SolangeInes Mussatto Keywords: Sustainability Microorganism Staphylococcusxylosus Wasterecovery Slaughterhousewaste
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The needforcleanersourcesofenergyhasstirredresearchintoutilisingalternatefuel sourceswithfavourableemissionandsustainabilitysuchasbiodiesel.However,thereare technicalconstraintsthathinderthewidespreaduseofsomeofthelowcostraw materi-alssuchasporkfattywastes.Currentlyavailabletechnologypermitstheuseoflipolytic microorganismstosustainablyproduceenergyfromfatsources;andseveral microorgan-ismsandtheirmetabolitesarebeinginvestigatedaspotentialenergysources.Thus,theaim ofthisstudywastocharacterisetheprocessofStaphylococcusxylosusmediatedfermentation ofporkfattywaste.Wealsowantedtoexplorethepossibilityoffermentationeffectinga modificationinthelipidcarbonchaintoreduceitsmeltingpointandtherebyactdirectlyon oneofthemaintechnicalbarrierstoobtainingbiodieselfromthisabundantsourceoflipids. PorkfattywastewasobtainedfromslaughterhousesinsouthernBrazilduringevisceration ofthecarcassesandthekidneycasingofslaughteredanimalswasusedasfeedstock. Fer-mentationwasperformedinBHIbrothwithdifferentconcentrationsoffattywasteandfor differenttimeperiodswhichenabledevaluationoftheeffectoffermentationtimeonthe meltingpointofswinefat.Thelowestmeltingpointwasobservedaround46◦C,indicating thatthesechemicalandbiologicalreactionscanoccurundermilderconditions,andthat suchpre-treatmentmayfurtherfacilitateproductionofbiodieselfromfattyanimalwaste. ©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/
licenses/by-nc-nd/4.0/).
Introduction
Contemporarysociety reliesontheuseofvariousformsof energytomeetandachievethebestqualityoflife;andthe
∗ Correspondingauthor.
E-mail:rogermarquesea@gmail.com(R.V.Marques).
products and processes being used tothat end essentially relyonanuninterruptedsupplyofenergywhichalsoneeds tobeofhighquality, becompetitivelypricedandleave the leastpossibleimpactontheenvironment.Theglobalenergy matrixlargelydependsontheuseoffossilfuelsandisthus
http://dx.doi.org/10.1016/j.bjm.2016.04.018
1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
non-renewable.Thisisworrisomeasinternationalagencies monitoringenergysourcespredicta60%reductioninoiland coalsupplywithinthenext15years,whichwillinturnleadto aphenomenalriseinextraction,productandprocesscosts.1–3
Thispredicteddecreaseinenergyresources,constitutesone ofthegreatestchallengesofoursocietyandhasresultedin researchintoandimprovementofalternatetechnologiesthat ensureenergysustainability.Biofuelsareapertinent exam-plewhichwhilebeingobtainedfromrenewablerawmaterials suchasbiomass,effluentsandfattywaste,arealsoless pollut-ingbecausetheircombustionresultsinmuchloweremission levelsofcarbon monoxide,sulphurcompounds, particulate matterandaromatichydrocarbons.4–8 Therefore,the
devel-opmentofbiofuelsfromagro-industrialwasteconstitutesan importantalternative asthese can be furthertransformed into second-generation biofuels that do not compete with agriculturefor resources, havenegligible costcompared to vegetablesourcesandareplentifulincountrieswith agricul-turalpotency.9–13Technologicalproblemscontinuetoprevent
the economic, attractive and sustainable use ofpork fatty wastefortheproductionofbiodiesel,oneofthemisthehigh coldfilter pluggingpoint, which precludes its use in vehi-clesoperatinginregionswithtemperaturesbelow15◦Cdue to crystal formation, leading to low or none viscosity.14–16
The high melting point of pork fat (>70◦C) demands sig-nificant energy consumption to liquefy this waste, which contributes to an increase in the final price of biodiesel fromsuchsources.Thus,eventhoughtherawmaterialhas a lower cost compared to vegetable oils, improvements in thetechnologytotransformporkfattywaste intobiodiesel arerequiredtomakethisaneconomicallycompetitivefuel source.17
Biofuelproductionnow increasinglyreliesontheuse of microorganisms,eitherforthedirectorfortheassisteduse oflipidsasbiofuels.18–22Therefore,aprocessthatuses
lipoly-ticmicroorganismsforbothtreatmentandrecoveryofanimal fattywaste showpromise asaworkablestrategythatuses agro-industrial waste for biodiesel production with desir-ablequalitycharacteristicsandundereconomicallyfeasible conditions.23–26 Staphylococcus xylosus,a Gram-positive
coc-cus,isamicroorganismthatfitstheserequirementsasithas GRAScertification(GenerallyRecognisedAsSafe),knownto haveproteolyticand lipolyticactivity,andregularlyusedin thefoodindustryespeciallyforfermentationofmeat prod-ucts as well as for the production and intensification of flavour,colourstabilisationandperoxidedecomposition.27–29
Importantly, apart from these in vivo applications, S. xylo-sus and other bacteria from the same genus also secrete exoenzymes that catalyse lipid-based reactions.22,30–35 A
useful example of such secreted enzymes is the serine acetyltransferase(E.C.3.1.1.3),whichiscapableofcatalysing alcoholysis,esterification,transesterificationandhydrolysis, amongothers.36,37Additionally,theeasewithwhichS.xylosus
canbeisolated andculturedmakesit apotential enhance-ment agent in the use of pork fatty wastes for biodiesel production.38
Giventhese favourable characteristics, the mainaim of this study wasto investigatethe process characteristics of porkfattywastefermentationbyS.xylosus.Wealsowantedto explorethepossibilityofthisfermentationeffectingachange
inlipid carbonchainstoreduceits meltingpointand thus actdirectlyononeofthemaintechnicalbarrierstoobtaining biodieselfromthisabundantsourceoflipids.
Material
and
methods
Collectionofsamples
The fatty waste was collected from freshly slaughtered animalspriortothewashingofthecarcassesfroma slaugh-terhouseinsouthernBrazil.Inordertocollectthefatwith the leastblood contaminationaspossible,the fatty kidney sheathsof25carcasses,eachweighingapproximately0.6kg wereremoved,collectedintosterilebags,transportedonice, andimmediatelysubjectedtofermentation.Allanalyseswere performedintriplicate.
Bacterialculturesandgrowth
S.xylosusNRRLB-14776wasculturedinBrain-HearthInfusion broth(BHI–Acumedia–NeogenCorporation®7116A,Brazil) aspreviouslydescribedbyMaurielloetal.27untilrequired
ini-tialcellmassconcentration(8.0logCFUmL−1),afterwhicha loopofbacteriumwastransferredtotheBHIbrothand incu-batedat35±0.1◦Covernight.
Porkfattywastefermentation
The experiment followed a fully randomised two-factorial designwiththreerepetitionswherein thetwofactors were fermentation time(1.5, 3.0,4.5, 6.0and 7.5h)and fat con-centrationofthe BHIbroth(2,4,6,8,10,50,60,70,80 and 90%).
Thefermentationwascarriedoutundersemi-solid-state conditions and consisted ofpork fatty waste asthe water insolublecomponentthatprovidednutrients(primarily car-bon)andanchorageformicroorganisms,apartfromBHIbroth andtheinoculum(1%,v/v).Thefattywastewasmelted,dried, filtered (filter paper Whatman 12.5cm), ground and sieved througha0.5mmsieve(32mesh).Next,thefatwassterilised bymembranefiltrationandasepticallyweighedinsterile plas-tic bags. Afterthe addition ofthe required volume ofBHI brothwiththenecessaryinitialcellconcentrationofboththe strainsofS.xylosus,thebagswereplacedina“Stomacher” homogeniser(SPLabour–modelSP-190-type)for2minand thentransferredtosterileErlenmeyerflasks;thetotal fermen-tationvolumewas200mL.Theflaskswerekeptinashaker incubator(35±0.1◦C,bufferpH7.0,100rpm)forinitiationof fermentation,andformaintenanceofaerobicconditionsin thereactor.Dissolvedoxygenwasmeasuredwithaportable oximeter (LUTRON DO-5519) and dissolved oxygen satura-tionwasmaintainedat100%throughoutfermentation.The choiceofgrowthconditionsandculturemediumusedwere basedonpreviouslyreportedoptimalgrowth conditionsfor
S.xylosus.27Fermentationwasinterruptedatthementioned
targetanalyticalpoints,whichcorrespondedtotendifferent concentrationsoftesting;andsamplesofapproximately1g wereasepticallycollectedfromeachErlenmeyerflaskand sub-jectedtomeltingpointanalysis.
Meltingpointanalysis
Testtubescontainingapproximately1gofsamplewere par-tially immersedin avolumetricflask withwater and kept onaheatingmantlethatslowlyincreasedthetemperature from30◦Cto80◦C.39Thismethoddetectsthreetemperatures
thatcharacterisethemeltingpointsofthreedifferentfatty acidsasdescribedbyDouareetal.40AsKnotheandDunn41
havereportedthatinitialandfinalmeltingtemperaturesare affectedbythequantityofthesamplebeinganalysed,only coretemperatureofthesamplewasconsidered.Meltingpoint ofthenon-fermentedfattywastewasdeterminedaccording toAOCSmethodology.39
Statisticalanalysis
ThenormalityofdatawastestedusingShapiro-Wilk’stest, homoscedasticitywastestedwiththeHartleytest,andresidue independence was tested using graphical analysis. After ensuringthat the assumptions were met,analysis of vari-ance (ANOVA) was performed using F test on values with
p<0.05.Experimentaldatawereanalysedusingresponse sur-facemethodologytofitasecond-orderpolynomialequation thatwasgeneratedusingregression.Theresponsewas ini-tiallyfit tofactorsusingmultipleregression andthe initial modelselectionwas basedon: (a) lowresiduals;(b)low
p-value;(c)lowstandarddeviation;and(d)highR2.Asecond orderpolynomialequation was thenfitted tothe response data(Eq.(1)). y=ˇ0+
ˇixi+ ˇii·x2i + ˇijxixj (1)wherey isthe response factor (fattywaste melting point),
xi is the first independent factor (fermentation time), xj is
thesecondindependentfactor(fatconcentration),ˇ0 isthe intercept,ˇiand ˇj arethefirst-ordermodelcoefficients,ˇii
and ˇjj are the quadratic coefficients for the factors i and
j and ˇij is the linear model coefficient of the interaction
betweenfactorsiandj.AlldatawereanalysedusingTheR Projectsoftware(version3.1.0,TheRFoundation®).Statistical parametersshowingap-valuelowerthan0.05wereconsidered significant.
Results
Meltingpoint
Theestimatedvaluesforthevariousfactorsarepresentedin
Table1,andthep-valuesindicatedthatmostofthecoefficients
weresignificantandthaterrorinmodeldesignwasminimised whenallofthesecoefficientswereconsideredtogether. Addi-tionally,thefinalestimatedresponseofthemodeladequately representedrealvaluesandtheestimatedvaluesofthese fac-tors.
Thefinalresponsemodelequationwasderived(Eq.(2))and theresponsesurfaceforthisequation(Fig.1)showsthatthe meltingpointtendstodecreasewhenintermediate fermenta-tiontimeswerereachedandformedacellpointbetweenfour andfivehoursofthefermentationprocess.Atthistime-point, italsoappearsthatthefatconcentrationofthemedium sig-nificantlycontributes(linearly)totheobservedphenomenaas thelevelcurvesatthispointhaveanearnullslope.
100 90 80 Melting point (ºC) Fat concentr ation ( %) Ferm entation t ime (h) 70 60 50 40 80 70 60 50 40 30 20 10 0 1 2 3 4 5 6 7 8
Fig.1–Responsesurfaceplotrepresentingtheeffectsoffat concentration,fermentationtimeandtheirreciprocal interactiononporkfattywastemeltingpoint.
Table1–Estimativeresponsemodelregressioncoefficients,standarderror,tvalueandsignificanceoftheresponse surfacemodelforthemeltingpointbehaviourofporkfattywasteobtainedthroughS.xylosusfermentation.
Factor* Parameterestimate DF Standarderror Fvalue*
tvalue** p>t Generalmodel – 5 2.817 155.854* <0.001 Intercept 81.873 5 1.283 63.836** <0.001 X1 −15.650 5 0.574 −27.279** <0.001 X2 −0.128 5 0.036 −3.607* <0.001 X12 1.648 5 0.061 26.978** <0.001 X22 0.0005 5 <0.0001 1.452** 0.149 X1X2 0.022 5 0.0032 6.680** <0.001
Y=81.873−15.650X1−0.128X2+1.648X21+0.0005X22
+0.022X1X2 (2)
Discussion
Theresponsesurfaceplotofthefermentationprocessshows thattheinitialestimatedmeltingpointwas81.87◦C(X1=0;
X2=0),whichsignificantlydeclinedasfatconcentrationand fermentationtimeincreasedandthemaximumdecreasein estimatedmeltingtemperature(Y=44.8◦C)occurredbetween 4and5hoursoffermentationandat38–42%fat concentra-tion.Further,duringthisperiod,themeltingpointappearsto beinfluencedneitherbyfermentationtimenorbyfat concen-trationofthemedium,indicatingastabilisationinbacterial activitywhichinturnledtotheobserveddecreasein physi-cochemicalparameters.Fatconcentrationofthemediumdid notindividuallyinfluencemetabolicactivity asthemelting pointshowedanon-significantvariationovertherangeoffat concentrationusedintheexperiments.However,Fig.1also showsthatthereisapossibilityofaninteractionbetweenthe twoprocessvariables(i.e.,fatconcentrationandtime),with fermentationtimebeingthemostprominentfactorcapable ofeffectingamodificationinporkfattywaste.Theseprocess characteristicsareespeciallypromisingbecausearelatively cheapfeedstockwasused forfermentation. Wefoundthat after reaching the lowest estimated melting point, the fat waste then exhibitedan increasein melting pointas both fermentationtimeand fat concentrationincreased;similar processcharacteristicshavebeenpreviouslyreported.42
Zhou et al.43 have reported that sudden changes in
theoperationalconditionsofindustrialandlaboratory pro-cessesinvolvingmicroorganismscausesdisturbancesintheir metabolismand physiologicalactivity which inturn accel-erate or halt microbial growth. This reflects a period of adaptationthatmicrobesrequiretoadjustDNAsynthesisand RNAreplicationtosuitthechangesinculturemediumandto resumetheirgrowth.
Accordingly,ourdatasuggestthatoptimaloperating con-ditions were achieved at fat concentrations between 55% and62%andbetween3.8and4.5hoursoffermentation,as theseconditionsreducedthemeltingpointtoapproximately 45–47◦C.Theseinferencesarebasedontheassumptionsthat thebestprocessconditionsarethosethatresultinthe great-estdecreaseinmeltingpoint,usethehighestconcentration offattywastepossibleandrequiretheshortestfermentation timesoastoincreasetheeconomicfeasibilityoftheoverall process.
Conclusion
Basedontheresultspresentedherein,itispossibleto con-cludethat semi-solid fermentationofporkfatty wastes by
S.xylosuscauses adropupto36◦Cinmeltingpointunder idealprocessconditions.Thisimpliesthataliquidstate fer-mentationprocess,whichisclosertoambienttemperature, mayfacilitateareductioninreactioncostsandalsoproduce anincreaseinreactionyield.Further,processenhancements
suchasflowconditionsduringfermentation,wouldensure thatmilderprocessconditionsaresufficienttoachieve effi-ciency.Therefore,pre-treatmentofthefattywastecanhelp optimisetheproductionofbiodieselfromsuchagro-industrial wastesinaprocessthatisbotheconomicallyattractiveand sustainable.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
TheauthorsthanktheCoordinationfortheImprovementof Higher Education(CAPES–fromPortuguese)forthefinancial supportgiventothisresearch.
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