SURFACTANT AND ADHESIVE FORMULATIONS FROM ALKALINE BIOMASS EXTRACTS · 2013-12-01 · representing...

SURFACTANTANDADHESIVEFORMULATIONSFROMALKALINEBIOMASSEXTRACTS

By

MatthewBaxter

AthesissubmittedinconformitywiththerequirementsforthedegreeofMasterofAppliedScience

DepartmentofChemicalEngineeringandAppliedChemistry

UniversityofToronto

©CopyrightbyMatthewBaxter2012

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SurfactantandAdhesiveFormulationsfromAlkalineBiomassExtracts

MatthewBaxter

MasterofAppliedScience

DepartmentofChemicalEngineeringandAppliedChemistryUniversityofToronto

2012

ABSTRACT

This work studies the ability to produce effective surfactant and adhesive

formulations using surface active biological material extracted from different

biomass sources using alkaline extraction methods. Two urban waste biomass

sourceswereusedtoproducesurfactants,ReturnActivatedSludge(RAS),andsolid

Urban Refuse (UR). The third biomass source investigated was isolated mustard

protein(MP).RASandMPextractswereinvestigatedforadhesiveproduction.

Theresultsindicatethatextractsfromthewastebiomasssources,RASandUR,can

becombinedwithacommercialsurfactant,sodiumdioctylsulfosuccinate(AOT),to

producesurfactantswithlowinterfacialtensionsagainstvariousoils.Thesehighly

surface‐activeformulationswereshowntobeusefulintheremovalofbitumenfrom

contaminatedsand.

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RAS and MP showed potential as protein‐based wood adhesives. These sources

wereusedinadhesiveformulationstoproduceastrongbondstrengthunderlow‐

pressure,ambientpressingconditions.

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ACKNOWLEDGEMENTS

I would first like to thank my supervisor, Dr. Edgar Acosta, for his guidance

throughout the course of this project. His advice, encouragement, and positive

attitudetowardsthisworkwerecrucialtothesuccessfulcompletionofthisthesis,

and to my progress as a researcher. His ability to stay calm during challenging

moments is a particular characteristic thatmade it a pleasure to have him asmy

advisor.IwouldalsoliketothankDr.FlorYunuenGarciaBecerraforherguidance

in starting the project, Dr. Grant Allen for use of his laboratory, and Dr. Levente

Diosady and Dr. Enzo Montoneri for providing essential materials for the

experimentalwork.

I want to acknowledge the Ontario Ministry of Research and Innovation for the

financialsupportinpursuingthisdegree.

I would also like to show my appreciation for my fellow researchers at the

LaboratoryofColloidandFormulationEngineering(LCFE)fortheirdiscussionand

assistance in the laboratory: Americo Boza, Johanna Chan, Oliver Chung, Sumit

Kiran, SuniyaQuraishi, andZihengWang. Mysummerstudents,BritonWellsand

FrancisChoi,deserveaspecialmentionfortheirdedicationandhardwork.Iwould

like to acknowledge the staff of the Chemical Engineering and Applied Chemistry

Departmentfortheirhelpwithalladministrativematters.

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TABLEOFCONTENTS

ACKNOWLEDGEMENTS......................................................................................................... iv

TABLEOFCONTENTS ............................................................................................................. v

LISTOFTABLES......................................................................................................................vii

LISTOFFIGURES .................................................................................................................. viii

CHAPTER1 Introduction ................................................................................................... 1

1.1 OverviewandObjectives .....................................................................................................11.2 References ................................................................................................................................6

CHAPTER2 Surfactantformulationsofalkalineextractsfromreturn

activatedsludgeandurbanrefuse,anduseinheavyoilremoval.......................... 8

2.1 Abstract .....................................................................................................................................82.2 Introduction.............................................................................................................................92.3 MaterialsandMethods...................................................................................................... 122.3.1 Materials ........................................................................................................................................... 122.3.2 Methods ............................................................................................................................................ 13

2.4 ResultsandDiscussion...................................................................................................... 192.4.1 Results ............................................................................................................................................... 192.4.2 Discussion ........................................................................................................................................ 34

2.5 Conclusion ............................................................................................................................. 42References ........................................................................................................................................ 44

CHAPTER3 Woodadhesiveformulationusingalkalineextractsfromreturn

activatedsludgeandmustardprotein ...........................................................................47

3.1 Abstract .................................................................................................................................. 473.2 Introduction.......................................................................................................................... 483.3 MaterialsandMethods...................................................................................................... 533.3.1 Materials ........................................................................................................................................... 533.3.2 Methods ............................................................................................................................................ 53

3.4 ResultsandDiscussion...................................................................................................... 563.4.1 Results ............................................................................................................................................... 563.4.2 Discussion ........................................................................................................................................ 66

3.5 Conclusion ............................................................................................................................. 743.6 References ............................................................................................................................. 75

CHAPTER4 ConclusionandRecommendations ......................................................784.1 Conclusion ............................................................................................................................. 784.2 RecommendationsforFuturework.............................................................................. 80

Nomenclature .........................................................................................................................83

APPENDIXCalculationsandDiagrams...........................................................................84

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CriticalMicelleConcentrationCalculation............................................................................ 84BitumenRemovalAnalysis ......................................................................................................... 87ElementalAnalysisConcentrations ......................................................................................... 89CalculationofBeta(β)ParameterforRASAOTMixture ................................................. 90References ........................................................................................................................................ 94

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LISTOFTABLES

Table2‐1ElementalanalysisofRASalkalineextractandUrbanRefuse(UR)extracts

............................................................................................................................................................... 19

Table2‐2–ElementalandheavymetalanalysisforRASextractandURextracts,

expressedas(g/l).Allmeasurementsarenormalizedto1g/LofTOC ............... 20

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LISTOFFIGURESFigure2‐1‐Concentrationvs.SurfaceTensiongraphsforalkalinewasteextracts;

a)RAS,b)FORSUD,andc)CV365............................................................................................ 22

Figure2‐2–SurfaceTensionvs.concentrationcurvesforwastebio‐basedsurfactant

mixtures,containing40%bio‐basedsurfactant(a)RAS,b)FORSUD,c)CV365),

and60%AOTbyvolume ........................................................................................................... 25

Figure2‐3‐Interfacialtensionsforwastebio‐surfactantsmixedwithAOT,witha

constantmixtureconcentrationof1gTOC/L.Bio‐basedsurfactant%isinterms

ofvolume(v/v).NOTE:ThepHwasnotcontrolledforthesemixtures.ThepH

valuesat40%bio‐surfactantforRAS,FORSUD,andCV365were10.82,7.92,

and7.53respectively.TestsforRASwerelaterconductedat40%solution

adjustedtopH7,andtheIFTagainsttoluenewassimilar(0.18mN/m). ............ 27

Figure2‐4‐IFTof40%FORSUD,60%AOTatincreasedtotalmixture

concentrations................................................................................................................................ 30

Figure2‐5–OilremovaltestsforRAS‐AOT,FORSUD‐AOT,andAOTat1g/L.a)20µL

oftolueneused(T:B0.4,b)60µLtolueneused(T:B1.2).Soilaged1month ... 32

Figure2‐6–Soilaged12months,solutionmixedfor3minutes.60µLoftoluene

used(T:B1.2).Surfactantmixturesconcentrationsof1g/L...................................... 34

Figure3‐1‐Reactionbetweenglutaraldehydeandprotein(Wong,1993) ................... 50

Figure3‐2–ViscosityversusshearratecurvesforMPandRASsolutionsthatwere

treatedwithglutaraldehyde(GL)andheattreatment(HT).GL1indicates1%

wt.glutaraldehyde.a)MPsolutionsweremadewith7.5%MustardProtein,and

b)RASwith45%solidRAScontent...................................................................................... 57

Figure3‐3‐ShearstrengthofMPadhesivewith15%MPand0.5%GLandpre‐heat

treatmentforvariousperiodsoftime.HT1means1hour,HT2is2hours,etc.

Sampleswerepressedfor18hoursunderadeadweight,forapressureof

8.6kPa................................................................................................................................................. 59

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Figure3‐4–ShearstrengthforMPadhesivescontaining15%,25%,and40%

(labeledMP15,MP25,MP40),withglutarldehyde(GL)mixedin‐situ.Samples

werepressedfor1hourunderadeadweightwithapressureof8.6kPa. .......... 61

Figure3‐5–SheartestresultsforRASformulations.Theformulationsunderwent

pre‐heattreatment(HT),additionofglutaraldehyde(GL),orweremixedin‐situ

withglutaraldehyde(IM).Sampleswerepresseddownfor18hoursunder

8.6kPa................................................................................................................................................. 63

Figure3‐6‐DSCthermogramofunmodifiedMustardSeedProteinIsolate(MP)and

MPcross‐linkedbywith2%glutaraldehydew/w(MPGL2),andwith0.5%

glutaraldehydeand18hoursofheattreatmentat80°C(MPGL0.5HT18) .......... 65

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CHAPTER1 Introduction

1.1 OverviewandObjectives

Thisthesisfocusesontheuseofmultiplerenewablebiomasssourceswithsurface

active compounds to produce effective surfactant and adhesives formulations.

Surface active material can be recovered from renewable feedstock using simple

alkalineextractionmethods tohelpaddressagrowingneed fornatural,bio‐based

productsinthesurfactantandadhesivesindustries(McCoy,2008).Twosourcesof

waste biomass were explored for surfactant production and formulations

development; Return Activated Sludge (RAS), a by‐product of biologically treated

municipalwastewater,andUrbanRefuse(UR)fromsolidwastetreatmentfacilities.

Surfactantswereproduced fromRASandURusingalkalineextractionmethods to

collect a heterogenous mixture of surface active biopolymers. Adhesives were

produced from RAS, and mustard protein (MP) extracted from mustard seed, a

renewablefoodcrop.Theabilitytoextractthesurface‐activematerialsfromthese

sourcesusingalkalinemethodshasalreadybeenestablished(Garcia‐Becerraetal.

2010a;Quagliottoetal.,2006,Marnoch&Diosady,2006).Themainpurposeofthis

workwastouse thematerialsextracted fromrenewablebiologicalsources,waste

and non‐waste, and develop strategies for optimizing their formulations as

surfactantsandadhesives.

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Return Activate Sludge is an abundant source of organic matter, containing

microorganisms and exopolymeric substances that the microbes secrete. It is

primarily made up of proteins, polysaccarides, and lipids (Frolund et al., 1996).

Someof thematerials exhibit important surface‐activeproperties,whichhave the

potentialtobeharvestedforvariousindustrialapplications.Developingavarietyof

uses from thematerial extracted fromRAScanbeuseful in reducing the costand

environmental impactof itsdisposal (Kroiss,2004). Currentuses forRAS include

fertilizerandfuelproduction(Boocok,1992). RecentworkonRASwasconducted

todevelopa simpleandefficientalkalineextractionmethod for recoveringahigh

yield of the organic material. This extract was shown to have surface‐active

capabilities, with the potential to be used in surfactant or adhesive production

(Garcia‐Becerraetal.,2010b;Garcia‐Becerraetal.,2012).

Work has also been done to demonstrate the potential surfactant properties of

solublebiopolymeric substances (SBP) thatwere isolated fromurban refuse (UR)

compost(Quagliottoetal.,2006;Montonerietal.,2009;Montonerietal.,2010).The

SBPsextracted frommunicipal solidwaste siteswere shown to lower surface the

tension of aqueous solutions, and possibly formmicelles. Similarly to RAS, these

wastes have a complex chemical composition, containing a mixture of molecules

representing the remnants of fats, proteins, polysaccharides and lignin. Green

urbanwasteswerecompostedforvariousperiodsoftime,andthedecayedorganic

matterwasextractedandtestedforsurfaceactivity(Montonerietal.,2010).

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Akeyobjectiveforthisworkwastoimprovethesurfaceactivityoftheextractsfrom

thesewastebiomasssources,RASandUR,bymixingwithahydrophobicsynthetic

surfactant to balance the hydrophilic‐lipophilic nature of themixture, and reduce

the interfacial tension (IFT) against oils that reflect a variety of industrial

applications. Biosurfactant and synthetic surfactant mixing had been explored

beforewithmicrobiallyproducedbiosurfactants,suchasRhamnolipids(Nguyenet

al., 2008; Nguyen & Sabatini, 2011). Previous work with UR waste extracts

indicated potential synergies with the synthetic surfactant, sodium dioctyl

sulfosuccinate (AOT), as indicated by interfacial tension results of the mixtures

againsthexane(Montoneri,2010).Thisworkwillexplorethepotentialsynergiesof

RAS and UR extracts against multiple oils with varying hydrophobicities, and in

theiruseintheremovalofheavyoil(bitumen)fromcontaminatedsandparticles.

Formulationsofwoodadhesivesweredevelopedusing twobiomass sources,RAS

andmustardprotein (MP).AlthoughMP isnotawastebiomass, thisworksimply

demonstrates another potential use for biological material extracted from a

renewablebiomasssourceviaasimplealkalineextractionmethod.

Adhesiveshavebeenproducedfromavarietyofbiologicalsources.Protein‐based

adhesiveswere commonly used in the early 1900s, with protein sources such as

animal bones and hides, milk (casein), blood, fish skins, and soybeans (Frihart,

2005). Carbohydrate‐based adhesives have also been produced (Baumann, &

Conner, 2003). Protein‐based adhesives have beenmodified to improve strength

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and moisture resistance, with much of the work being focused on soy proteins.

Thesemodificationsincludeproteindenaturingandcross‐linking(Hettiarachchyet

al.,1995;Kalapathyetal.,v1995;Wangetal.,2007).

Mustardprotein(MP)isisolatedfrommustardseed,afoodcropthatisproducedin

abundance, and contains a similar amino acidmake up to soy protein, indicating

similarabilitiestobeusedasanadhesive(Xuetal.,2003;Aideretal.,2012). RAS

andMPhavebeen shown tohavewoodadhesion capabilitiesunderhot, dry, and

high pressure bonding conditions (Garcia‐Becerra et al., 2012). However, hot

pressingisanenergyintensiveprocess,andadhesivesthatrequiretheseconditions

toformstrongbondshavelimitedapplications,suchasplywoodproduction.

In this work, modifications were introduced in the formulations of RAS and MP

basedadhesivestoenhancetheirabilitiestoformbondsunderambientconditions

and low pressure. These modifications included an optimization of the

concentration of protein and cross‐linking agent, and the introduction of heat

treatmentmethods. Protein cross‐linking is awell knownmethod for connecting

proteinstoformalargermolecule,whichcanimproveadhesivestrengthandwater

resistance(Wangetal.,2007;Reddyetal.,2008;Xiaoetal.,2010).Glutaraldehyde

is one of the most commonly used cross‐linking agents, and was used in the

formulations produced in this work. Heat treatment of formulations was

investigated to encourage the cross‐linking reaction within the adhesive

formulations.Adifferentapplicationmethodwasalsoexplored,wherebythecross‐

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linkerwasaddedtothewoodseparatelyfromtheproteinsolutiontoallowforin‐

situcross‐linkingandimprovedwoodpenetration.

Therewerepositivefindingsonsurfactantandadhesiveformulationsinthiswork.

RAS and UR extracts showed synergies with the commercial surfactant, AOT,

resulting in mixtures with low (<1mN/m) and ultralow (<0.1mN/m) interfacial

tensions.TheRASandURformulationswithAOTwerealsoshowntobeeffectivein

bitumenremovalfromcontaminatedsand.Intheadhesivework,RASandmustard

proteinformulationswereabletoformstrongbondsunderlowpressure,ambient

bondingconditions.

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1.2 References

Aider,M.,Djenane,D.,&Ounis,W.B.(2012).Aminoacidcomposition,foaming,emulsifyingpropertiesandsurfacehydrophobicityofmustardproteinisolateasaffectedbypHandNaCl. Int.J.FoodSci.Technol.,47(5),1028‐1036.

Baumann,M.G.D.,&Conner,A.H(2003).Carbohydratepolymersasadhesives.InA.Pizzi,&K.L.Mittal,(Eds.),HandbookofAdhesiveTechnology(pp.495‐510).NewYork:MarcelDekker.

Boocock,D.G.B.,Konar,S.K.,Leung,A.,&Ly,L.D.(1992).Fuelsandchemicalsfromsewagesludge.Fuel,71(11),1283‐1289.

Frihart,C.R.(2005).Woodadhesionandadhesives.InR.M.Rowell(Ed.),Handbookofwoodchemistryandwoodcomposites(pp.15‐273).BocaRaton,FL:CRCPress,Inc.

Frolund,B.,Palmgren,R.,Keiding,K.,&Nielsen,P.H.(1996).Extractionofextracellularpolymersfromactivatedsludgeusingacationexchangeresin.WaterRes.,30(8),1749‐1758.

GarciaBecerra,F.Y.,Acosta,E.J.,&Allen,D.G.(2010a).Alkalineextractionofwastewateractivatedsludgebiosolids.Bioresour.Technol.,101(18),6983‐91.

GarciaBecerra,F.Y.,Allen,D.G.,&Acosta,E.J.(2010b).Surfactant‐likeproperties

ofalkalineextractsfromwastewaterbiosolids.J.SurfactantsDeterg.,13,261‐271.

Garcia‐Becerra,F.Y.,Acosta,E.J.,Allen,D.G.(2012).Woodadhesivesonalkaline

extractsfromwastewaterbiosolidsandmustardprotein.J.Am.OilChem.Soc.,89(7).1315‐1323.

Hettiarachchy,N.S.,Kalapathy,U.,&Myers,D.J.(1995).Alkali‐modifiedsoyproteinwithimprovedadhesivesandhydrophobicproperties.J.Am.OilChem.Soc.,72(12),1461‐1464.

Kalapathy,U.,Hettiarachchy,N.S.,Myers,D.,&Hanna,M.A.(1995).Modificationofsoyproteinsandtheiradhesivepropertiesonwoods.J.Am.OilChem.Soc.,72(5),507‐510.

Kroiss,H.(2004).Whatisthepotentialforutilizingtheresourcesinsludge?WaterSci.Technol.,49(10),1‐10.

Marnoch,R.,&Diosady,L.L.(2006).Productionofmustardproteinisolatesfromorientalmustardseed(BrassicajunceaL.).J.Am.OilChem.Soc.,83(1),65‐69.

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McCoy,M.(2008,January21,2008).Greenercleaners.ChemicalandEngineeringNews,86(3),15‐23.

Montoneri,E.,Boffa,V.,Savarino,P.,Perrone,D.G.,Montoneri,C.,Mendichi,R.,Acosta,E.J.,&Kiran,S.(2010).Behaviourandpropertiesinaqueoussolutionofbiopolymersisolatedfromurbanrefuse.Biomacromolecules,11,3036‐3042.

Montoneri,E.,Boffa,V.,Savarino,P.,Perrone,D.G.,Musso,G.,Mendichi,R.,Chierotti,M.R.,&Gobetto,R.(2009).Biosurfactantsfromurbangreenwaste.ChemSusChem,2(3),239‐247.

Nguyen,T.T.,&Sabatini,D.A.(2011).Characterizationandemulsificationpropertiesofrhamnolipidandsophorolipidbiosurfactantsandtheirapplications.Int.J.Mol.Sci.,12(2),1232‐1244.

Nguyen,T.T.,Youssef,N.H.,McInerney,M.J.,&Sabatini,D.A.(2008).Rhamnolipidbiosurfactantmixturesforenvironmentalremediation.WaterRes.,42,1735‐1743.

Quagliotto,P.,Montoneri,E.,Tambone,F.,Adani,F.,Gobetto,R.,&Viscardi,G.(2006).Chemicalsfromwastes:compost‐derivedhumicacid‐likematterassurfactant.Environ.Sci.Technol.,40(5),1686‐1692.

Reddy,N.,Tan,Y.,Li,Y.,&Yang,Y.(2008).Effectofglutaraldehydecrosslinkingconditionsonthestrengthandwaterstabilityofwheatglutenfibers.Macromol.Mater.Eng.,293(7),614‐620.

Wang,Y.,Mo,X.,Sun,S.,Wang,D.(2007).Soyproteinadhesionenhancebyglutaraldehydecrosslink.J.Appl.Polym.Sci.,104(1),130‐136.

Xiao,Z.,Xie,Yanjun,X.,Militz,H.,Mai,C.(2010).Effectofglutaraldehydeonwaterrelatedpropertiesofsolidwood.Holzforschung,64,483‐488.

Xu,L.,Lui,F.,Luo,H.,&Diosady,L.L.(2003).Productionofproteinisolatesfromyellowmustardmealsbymembraneprocesses.FoodRes.Int.,36(8),849‐856.

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CHAPTER2 Surfactantformulationsofalkalineextractsfromreturnactivated

sludgeandurbanrefuse,anduseinheavyoilremoval

2.1 AbstractThepotentialsynergismbetweenbio‐basedsurfactants,producedfromthealkaline

extraction of waste biomass, and a synthetic surfactant was assessed. This

synergismwasexplored in termsof surfaceand interfacial tension reduction, and

the ability of mixtures to remove heavy oil from oil‐bearing sand. The waste

biomass sources investigatedwere return activated sludge (RAS) frommunicipal

wastewater fromToronto,Canada, andUrbanRefuse (UR)matter frommunicipal

solidwaste compost treatment facilities inPiemonte, Italy. Surfactants fromboth

sourceswereextractedusingalkalineextractionmethods.ThereweretwoURbased

surfactantsinvestigated,whichcamefromdifferenttypesofurbanwastefeedstock.

Mixturesofthesewastebio‐basedsurfactantswiththesyntheticsurfactant,sodium

dioctyl sulfosuccinate (AOT), at a total concentration of 1gTOC/L, were able to

achieve low interfacial tensions (<1mN/m)with toluene and hexane,without the

addition of electrolytes. The mixtures generally achieved IFTs an order of

magnitude below that of the pure bio‐based surfactant. At an increased total

concentrationof10gTOC/L,aURextract(labeledFORSUD),mixedat40%withAOT,

reachanultra‐low IFTof0.019mN/magainsthexane. Furthermore, theRAS‐AOT

and UR‐AOT mixtures were examined for their use in the removal of heavy oil,

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bitumen,fromcontaminatedsand.ThelowIFTsofthemixturesprovedtobeuseful

inremovingoilfrombitumen‐coatedsandparticles.

2.2 IntroductionThe need for the development of surface‐active compounds from renewable

biological sources is growing in importance as sustainability and environmental

concernsofproducingcommercialsurfactantsfrompetroleumfeedstockcontinues

to grow. Bio‐based surfactants can be produced in a variety ofways and have a

variety of industrial applications (Lin, 2006; Rahman & Gakpe, 2008; Schramm,

Stasiuk, &Maragoni, 2003; Singh, VanHamme,&Ward, 2007; VanHamme et al.,

2006). Traditional biosurfactants can provide some advantages to synthetic

surfactants,suchaslowertoxicity,higherbiodegradability,andhighertoleranceto

pH,temperature,andsalinity(Chenetal.,2010).Theyhavebeenderivedfromthe

extraction of triglycerides, lecithin, lysolecithins, and other phospholipids from

plant and animal tissue. Traditional biosurfactants have been produced from

secretedmaterialsofmicro‐organisms.Thesematerialsincludeglycolipids,suchas

Rhamnolipidsandsophorolipids,andsaponins (Garcia‐Becerraetal.,2010a;Chen

etal.,2010).However,thereareconcernsoverthetruesustainabilityofsurfactants

derived from biomass like plants and animals, such as deforestation, and

competitive land use for food crops. Biosurfactant production from micro‐

organisms often requires relatively expensive carbon sources, which can be

alternativelyusedasfoodsourcesforanimals. Therearealsolimitationsin large‐

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scaleproductionandpurificationefficiency(Nitschkeetal.,2005;Garcia‐Becerraet

al.,2010a).

Waste biomass is an alternative renewable source for surface‐active materials.

Surfactantsextractedfrombiologicalsourcesusingsyntheticmeansarereferredto

as bio‐based surfactants. Bio‐based surfactants can be extracted from waste

biomassthroughvariouschemicaltreatments,suchasalkalinetreatment,pyrolisis,

and simple extraction and separation (Garcia‐Becerra et al., 2010b). One of the

sources of waste biomass that this chapter explores is Return Activated Sludge

(RAS), a by‐product of biologically treated municipal wastewater, from the

Ashbridges Bay Wastewater Treatment Plant in Toronto, Canada. This is an

abundant source of organicmatter, containing various constituents such as cells,

proteins, polysaccharides, lipids, and humic substances (Garcia‐Becerra et al.,

2010c; Frolund, Keiding, & Nielsen, 1996). Identifying and exploiting a range of

value‐addedproducts from sludge canhelp reduce the environmental impact and

net cost of its disposal,which accounts for approximately 50% of total operating

costsofatypicalwastewatertreatmentplant(Kroiss,2004,Odegaardetal.,2002).

The extraction process used for RAS was developed by Garcia‐Becerra et al.

(2010b).Thiswasasimplealkalineextractioncarriedoutatroomtemperatureina

vesselexposedtoair. AtextractionpHlevelsgreaterthan12,thecellwallsofthe

micro‐organismsaredisrupted,causingcelllysis,andmostoftheorganiccontentis

liberated into the alkaline solution. The procedure collects up to 75% of the

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sludge’s organic matter, and yields a liquor with total organic carbon (TOC)

concentrationsbetween3‐4gTOC/L(Garcia‐Becerraetal.,2010b).

Another source ofwaste biomass examined in thiswork is Urban Refuse (UR), a

sourceofdecayedorganicmaterialfrommunicipalsolidwastecompost.Surfactant

extracts from UR have been produced and examined for surface activity at the

UniversityofTorinobytheMontonerigroup,usingalabscaleprocedurecarriedout

at65°Cfor24hoursatapHof10,underanitrogenblanket(Quagliottoetal.,2006,

Montonerietal.,2010).Thefeedstockforthiscompostextractionofsurface‐active

biopolymers comes from solid urban refuse (UR) that underwent different

treatments in waste treatment facilities located in Piemonte, Italy. The extracts

wereshowntobesurfaceactive(Quagliottoetal.,2006;Montonerietal.,2009).To

further reduce interfacial tensions, UR extracts were tested in mixtures with

synthetic surfactants. Synergismswereobservedwith the commercial surfactant,

sodiumdioctylsulfosuccinate(AOT),asindicatedbylowIFTs(≈0.2mN/m)against

hexane(Montonerietal.,2010).

Thepurposeofthisworkwastofurtherevaluatethepotentialsynergismbetween

RAS and UR extracts in mixtures with the synthetic surfactant, sodium dioctyl

sulfosuccinate (AOT). This synergism was explored in terms of surface and

interfacial tension. Since RAS extract is hydrophilic in nature, and may contain

similar biopolymers to UR, it was hypothesized that it would experience similar

synergieswiththemorehydrophobicAOT.AsforURextracts,althoughinitialsigns

of synergismwith AOT have already been observed, themixtureswill be further

12

investigatedunderconditionsthataremorerelevanttoindustrialapplications.For

example, the UR extracts tested in this work were produced from a pilot scale

process, whereas previouswork used a lab scale extraction processwith slightly

differentconditions(Quagliottoetal.,2006,Montonerietal.,2010). Furthermore,

testswillbeconductedatlowerconcentrations,andwillconsideroilsthatrepresent

arangeofpotentialindustrialapplications.

It was also expected that the improved interfacial tensions of the surfactant

mixtureswouldtranslatetoimprovedperformanceintheremovaloftheheavyoil,

bitumen,fromcontaminatedsand.

2.3 MaterialsandMethods

2.3.1 MaterialsThefollowingreagentswerepurchasedfromSigma‐Aldrich(Oakville,Canada)and

used without further purification: 50% wt NaOH solution (reagent grade), for

alkaline extraction, HCl (35‐37% wt., reagent grade), hexane (HPLC grade),

hexadecane (99%+), toluene (99%+), hexadecane (99%+), NaCl (99%+), acetone

(reagent grade). Sodium Dioctyl sulfosuccinate(AOT, 96%). QC4 and high purity

Sulfur standard solutions, for ICPAESAnalysis,werepurchased fromSCPScience

(BairD’urfe,QC,Canada).

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Cokerfedbitumen(CFB)wasdonatedbySyncrudeCanadaLtd.Thecompositionof

bitumenhasbeenreportedelsewhere(Akbarzadehetal.,2005;Kiranetal.,2009).

Washedplaysandwassievedtoameshsizeof70‐100.

2.3.2 MethodsReturnActivatedSludge(RAS)Extraction:

TheprocedureforextractingtheorganiccontentofReturnActivatedSludgefollows

that developed by Garcia‐Becerra et al. (2010b). The aerobically treated return

activated sludge (RAS) was collected from the Ashrbidges Bay Wastewater

Treatment Plant (1400 population equivalent; average capacity: 725,000m3/day;

sludge retention time: 2.5 days; aeration time: 6‐8h). The RAS sampling and

extraction tookplace inMayof 2011. RASwas collected, placed into an icebath,

transported to the lab, and left to settle for 2 hours. The clear supernatantwater

wasremovedtoreducetheamountofNaOHrequiredtoraisethepHofthesolution,

and allow for amore concentrated extract. The pH of the concentratedRASwas

thenraisedto12.6,using50%NaOHsolution,andmixedforfourhoursat500rpm

at room temperature. ThealkalineRASextractwas thenstoredat4°Candsolids

wereallowedtosettletothebottom.

UrbanRefuse(UR)Extraction:Bio‐basedsurfactantsextractedfromurbanrefusewereproducedatapilotplantin

Regione Piemonte, Italy, by the Montoneri research group at the University of

Torino. TheseURextractsarelabeledFORSUD,CV365,andash‐freeFORSUD. The

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feedforFORSUDisaby‐productofsolidurbanwastethatwasfermentedthrough

ananaerobicdigestionprocess.CV365comesfromamixtureoffoodresiduesand

publicparkgreentrimmings,compostedaerobicallyfor365days.

The surface‐active organic material was extracted from the UR wastes using an

alkalineextraction. Thesolidwaste feedwasreactedwithNaOHfor4hat60°C,

and centrifuged. The supernatant liquidphasewas run through anultrafiltration

membranewitha5kDacutoff.Theproductwasthendriedtoyieldasolidsample

containing about 25% ash. To produce the ash‐free sample, the ash‐containing

productwas furtherwashedwithHCl ,water, hydrofluoric acid, and finallywash

withwater.Theash‐freeproductwasthendriedat60°C.

Theash‐containingandash‐freedriedsolidswere thenre‐dispersed intoaqueous

solutionsforanalysis.

ElementalAnalysis:TheTotalOrganicCarbon(TOC)contentandTotalNitrogenforRASandURextracts

weremeasuredusingaShimadzuTOC‐VCHSanalyzer(MandelScientificCompany,

Inc., Guelph, Canada). This equipment uses a non‐dispersive infrared (NDIR) gas

analyzer tomeasure the total carbon concentration in liquid samples. Potassium

hydrogenphthalatewasusedasthestandardfortheTotalCarbon(TC)calibration

curve, andwas prepared by being heated at 105°C for one hour and cooled in a

desiccator. Amixture of sodium hydrogen carbonate and sodium carbonatewas

used as the standard for developing the Inorganic Carbon (IC) calibration curve.

15

Reagent grade potassium nitrate solutionwas used as the standard for the Total

Nitrogen(TN)calibrationcurve.

The sulfur and trace metals content of the RAS and UR extracts were measured

directlyusingInductivelyCoupledPlasmaAtomicEmissionSpectrometry(ICPAES)

usingaPerkinElmerModelOptima3000DVICPAESapparatus.

SurfaceTensionMeasurements:The surface tension of RAS and UR extracts, and their mixtures with AOT, were

measuredwithaSigma700tensiometer(KSVInstruments,Helsinki,Finland)using

theDuNuoy ringmethod. Measurementswere carriedout at room temperature,

usingastabilizationtimeof10minutes.Toensureinstrumentaccuracy,thesurface

tension of deionized water (3µS/cm) was repeatedly measured under the same

conditionstoensureasurfacetensionreadingof71‐72mN/m.Thesurfacetension

ofextractsandvariousmixturesofextractandAOTwereanalyzedasafunctionof

extract concentration, in terms of mass of Total Organic Carbon (gTOC/L). The

dilutionswerepreparedusingdeionizedwater. Thecriticalmicelleconcentration

was calculated at the break point in the surface tension‐surfactant concentration

isotherm(Rosen,2004).RefertotheAppendixforfurtherexplanationandsample

calculations.

16

InterfacialTensionMeasurements:

The interfacial tension (IFT) (against hexane, toluene, and hexadecane) was

measured for the RAS extract and the Urban Refuse (UR) extracts mixed with

sodiumdioctylsulfosuccinate(AOT)atvariousratios. Nosaltwasadded,andthe

pH of the stock solutions was not adjusted. The pH for the stock RAS, FORSUD,

CV365, and ash‐free FORSUD extractswere 12.6, 8.6, 8.5, and 10.13 respectively.

For IFTsabove2mN/m, theDuNuoyringmethodwasused. For IFTs lower than

2mN/m,aspinningdroptensiometerwasused(TemcoInc.,Model500,Texas).In

thistechnique,aborosilicateglasstubewasfilledwithsurfactantmixture,anda2µL

dropletofoil(hexane,toluene,orhexadecane),wasinjectedusingaHamiltonglass

syringe(701N,10µL). Theglasstubewasspunatanappropriatespeed(RPM),at

whichtheoildropletexpandedtoapointwhereitslengthwas4timesgreaterthan

itswidth.ThewidthandRPMwerethenrecordedandusedtocalculatetheIFT(γ,

mN/m)withthefollowinggeneralequation:

(1)

In thisequation,Δρ is thedensitydifferencebetween theheavyphase (surfactant

solution) and light phase (toluene, hexane, or hexadecane) (g/mL), ω is the

rotationalvelocity(rad/s),andristhewidthoftheoildroplet(cm).Theequationis

thenconvertedtouseparametersthataredirectlyrecordedfromtheequipment:

17

(2)

WhereTistherotationalvelocityinRPM,Dmeasisthemeasuredwidthofoildroplet

(mm),andCrisacorrectionfactorusedtocalculatetheactualdropletwidth(to

accountforrefractionoftheglasstubeandsolution).Cr=Dactual/Dmeas.

OilRemoval:Sand contamination,washing, andanalysis experimentsweredevelopedbasedon

theworkofQuraishietal.(2012).

SandContamination:

The sand was contaminated in the lab to ensure consistent bitumen content

throughout all sand used for washing experiments. The clean sand was

contaminatedwith5wt%bitumenbycoatingthesandwitha50%(w/w)bitumen‐

toluenesolution. This levelofoilcontent isreflectiveof typicaloilconcentrations

found on oil‐bearing solids and oil contaminated sands from oil spills (Alberta

Research Council Oil Sands Research Department, 1983). The solution was

preparedtoensureevencoating,whichcannotbeaccomplishedwithpurebitumen

becauseof itshighviscosity. 50gofsandwasmixedwith5gofthe50%bitumen‐

toluenemixture.Thecoatedsandwasthenplacedunderafumehoodtoevaporate

thetoluene,andwasagedforvariousperiodsoftime.

18

SurfactantWashingExperiments:1g of contaminated soil was placed in a 20mL glass scintillation vial. Varying

amountsoftoluenewereadded(5,10,15,20,or60µL)tofacilitatetheremovalof

bitumen.Thetoluenewasaddedtothevialandthenmixedfor10secondsusinga

mini vortexmixer. Then, 4mLof surfactantmixturewas added, and the vialwas

thenmixed in a VWR VX‐2500multi‐tube vortexer, for different periods of time,

ranging between 10 seconds and 3 minutes. These parameters were determined

based work by Quraishi et al. (2012), as well as preliminary tests that were

conducted to identify the challenging oil removal conditions using water. After

washing,thesampleswerecentrifugedfor20minutes,andallowedtositfor24.

OilRecoveryAnalysis:

Theamountofoil removed fromthecontaminatedsandwasdeterminedusingan

UltrospecPlusvisiblelightspectrophotometer.Afterwashing,therecoveredoiland

surfactantwashing fluidwere removedusingaglassPasteurpipette (9”), and the

remainingsandwasdriedunderafumehoodforaminimumof48hours.Thedried

sandwithresidualoilthatwasnotremovedbythesurfactantwasthencleanedwith

4mLofToluene,whichwasmixedwiththesandfor15minutes.Theassumptionis

that toluene removes all of the bitumen (Quraishi et al., 2012). For absorbance

readings, 20µL of the toluene containing the residual oilwasmixedwith another

1.5mL of toluene in an acrylic cuvette for further dilution that would allow for

absorbancereadings. Thedilutedbitumenandtoluenemixturewasthenanalyzed

forabsorbanceatawavelengthof400nm(Quraishietal.,2012). Theoil removal

19

percentagewasdeterminedbasedonpositivecontrol tests,where4mLof toluene

was used to remove all of the oil from uncleaned, contaminated sand, and the

absorbancereading fromthese tests corresponded to100%oil removal. Refer to

theappendixforaschematicofthisprocessandoilremovalequation.

2.4 ResultsandDiscussion

2.4.1 ResultsElementalAnalysis:AbasicchemicalanalysiswasperformedontheReturnActivateSludge(RAS)and

UrbanRefuse(UR)extracts;FORSUD,ash‐freeFORSUDandCV365.Table2‐1shows

the concentrations of Carbon, Nitrogen, and Sulfur (C,N,S), as well as the ratios

normalized to 1g/L of carbon. Although the overall carbon concentrations of the

concentratedextractsarequitedifferent,theN/CandS/Cratiosdonotvarymuch

betweensamples,althoughtheN/CratioappearstobeslightlyhigherforRAS.Also

note that the ratios between the filtered (ash‐free) and unfiltered FORSUD are

similar.

Table21ElementalanalysisofRASalkalineextractandUrbanRefuse(UR)extracts

ElementalConcentration(g/L) RatioSample

C N S N/C S/CRAS 3.2±0.11 0.85±0.07 0.08 0.26±0.02 0.026FORSUD 25.4±4.0 4.8±0.55 0.54 0.19±0.04 0.021CV365 32.2±5.5 4.1±0.43 0.91 0.13±0.02 0.028FORSUDash‐free 22.1±2.8 3.3±0.10 0.33 0.15±0.02 0.015

20

FurtherchemicalcharacterizationofwasteextractsurfactantscanbefoundinTable

2‐2,withamoredetailedelementalanalysisnormalizedtoaconcentrationof1g/L

oftotalorganiccarbon(TOC).ThecalciumconcentrationintheunfilteredFORSUD

isanorderofmagnitudehigherthanforash‐freeFORSUD.TheCa2+oftheunfiltered

FORSUDrelativetocarboncontentisroughlysimilartoRAS,andCV365hasamuch

higherratiothanallsamples.

Table22–ElementalandheavymetalanalysisforRASextractandURextracts,expressedas(g/l).Allmeasurementsarenormalizedto1g/LofTOC

NormalizedtoTOCcontentConsituent RAS FORSUD FORSUDash

freeCV365

TOC 1 1 1 1TN 2.6E‐01 1.9E‐01 1.5E‐01 1.3E‐01Na 6.8E‐01 1.4E‐01 9.5E‐02 1.1E‐01S 2.6E‐02 2.1E‐02 1.5E‐02 2.8E‐02K 2.2E‐02 9.7E‐03 5.5E‐04 2.9E‐02Si 1.6E‐02 2.1E‐02 2.1E‐02 6.4E‐02Fe 1.6E‐02 8.5E‐03 8.6E‐05 3.7E‐02Al 1.6E‐02 1.1E‐02 3.5E‐04 2.3E‐02Ca 9.8E‐03 2.2E‐02 1.1E‐03 1.0E‐01B 5.5E‐03 1.2E‐02 1.3E‐02 9.1E‐03Mg 2.0E‐03 3.0E‐03 3.5E‐04 2.0E‐02Cu 1.3E‐03 2.8E‐04 1.1E‐04 9.3E‐04Zn 7.2E‐04 6.9E‐04 3.6E‐05 2.0E‐03Ti 6.3E‐04 3.2E‐04 6.8E‐05 1.8E‐03Ba 1.3E‐04 1.3E‐03 1.4E‐05 5.8E‐04Ni 8.7E‐05 8.6E‐05 0.0E+00 2.4E‐04Mn 8.1E‐05 3.7E‐04 0.0E+00 2.7E‐03Cr 4.4E‐05 5.1E‐05 1.8E‐05 2.0E‐04

21

SurfaceTension:Garcia‐Becerra et al. (2010b)observed thatRASextractdisplayed surface activity

similar to that of commercial surfactants. Upon increasingRAS concentration, the

surface tension reduced from 72mN/m to about 35‐40mN/m beyond the critical

micelle concentration (CMC). Surface activity has also been reported for Urban

Refuseextracts(Montonerietal.,2010).ThesurfacetensionforRAS,FORSUD,and

CV365extractsstudied in thisworkarepresented inFigure2‐1,whichshowsthe

surfacetensionversustheconcentrationoftheextract,measuredingramsperlitre

ofTotalOrganicCarbon(TOC).

22

Figure21Concentrationvs.SurfaceTensiongraphsforalkalinewasteextracts;a)RAS,b)FORSUD,andc)CV365

y=‐8.742ln(x)+42.078R²=0.98079

303540455055606570

0.1 1 10 100SurfaceTension(mN/m

)

Concentration(gTOC/L)

a)RAS

303540455055606570


)


b)FORSUD

303540455055606570


)


c)CV365

23

The concentration of RAS extract was 3.2±0.11gTOC/L, and was diluted with

deionizedwater. ThepHwasnotcontrolled in thesemeasurements,butprevious

testshaveshownthattheextractmaintainsitssurfaceactivityoverawiderangeof

pH(Garcia‐Becerraetal.,2010c).Asreportedinthemethodssection,thepHforthe

stock RAS, FORSUD, and CV365 extracts were 12.6, 8.6, and 8.5 respectively.

FORSUDandCV365werealsodiluted,butfrommuchhigherstockconcentrationsof

TOC,asreportedinTable2‐1. Thecriticalmicelleconcentration(CMC)oftheRAS

solution was calculated as the concentration beyond which, the surface tension

becomes constant (Rosen, 2004), and in this case, equal to 39.5mN/m (Refer to

AppendixforCMCcalculation). TheCMCforRASwascalculatedtobe1.34gTOC/L.

Thesurfacetensions forFORSUDandCV365solutionscontinuedtodecreasewith

increasing concentration, but without a clear sign of becoming constant, thus no

CMCwasreportedfortheseURextracts. Theminimumsurfacetensionsrecorded

were 47.7 mN/m at 24.5 gTOC/L for FORSUD, and 50.0 mN/m at 32.2 gTOC/L for

CV365. Note that these measurements were based on dynamic surface tensions

measuredovera10minuteperiod. Previousreports forsimilarURextractswere

abletoreportCMCs,basedonsurfacetensionsmeasuredovera200minuteperiod

(Quagliottoetal.,2006;Montonerietal.,2010).Thisadditionaltimeallowsforthe

extracttoadsorbtotheair‐waterinterfaceandreachequilibrium,whichcanleadto

lower surface tension and CMC values. The 10‐minute surface tension

measurements in this work was selected to produce measurements that are

relevant to the time scale of cleaning processes, and use amethodology thatwas

24

consistentwith the surface tensions and CMCmeasurements previously reported

forRAS(GarciaBecerraetal.,2010c).

MixtureswithAOT

The RAS, FORSUD, and CV365 extracts were then mixed with the commercial

surfactant,sodiumdioctylsulfosuccinate(AOT),ataratioof40%wasteextractand

60%AOTbyvolume.Aswillbeseeninthefollowingsectionforinterfacialtension,

these ratios were chosen based on the mixing ratios required to achieve low

interfacial tensions with hexane and toluene. The concentration versus surface

tensionforthesemixedsystemsareshowninFigure2‐2.

y=‐8.531ln(x)+23.873R²=0.99936

20

25

30

35

40

45

50

0.01 0.1 1 10

SurfaceTension(mN/m

)


a)40%RAS

25

Figure 22 – Surface Tension vs. concentration curves for waste biobased surfactant mixtures,containing40%biobasedsurfactant(a)RAS,b)FORSUD,c)CV365),and60%AOTbyvolume

y=‐7.937ln(x)+28.574R²=0.9998

20

25

30

35

40

45

50

0.01 0.1 1 10

SurfaceTension(mN/m

)


b)40%FORSUD

y=‐6.975ln(x)+25.738R²=0.99903

20

25

30

35

40

45

50

0.01 0.1 1 10

SurfaceTension(mN/m

)


c)40%CV365

26

These figures yield results that show much closer resemblance to commercial

surfactants,with surface tensions becoming constant at an identifiable CMC. The

CMC for the 40% RAS mixture was calculated to be 0.89 gTOC/L, with a surface

tensionof24.8mN/m.The40%FORSUDmixtureyieldedaCMCof1.66gTOC/Lwith

asurfacetensionof24.6mN/m,and40%CV365hadaCMCof1.26gTOC/Lat24.1

mN/m.

InterfacialTensionandMixtureswithAOT:The ability for the waste biomass alkaline extracts to mix with the synthetic

surfactant, sodiumdioctyl sulfosuccinate (AOT), to reduce the interfacial tensions

against various oilswas explored. RAS has not been tested for this synergism in

previousstudies. UrbanRefuseextracts,similartotheonesusedinthiswork,but

producedatalabscale,weretestedformixtureswithAOTandSDBSagainsthexane,

only. Figure 2‐3 shows the interfacial tension measurements of extract‐AOT

mixtures,atatotalsurfactantconcentrationof1gTOC/L.Theuseoftolueneasatest

oil helps asses the use of these alkaline extracts in the removal/recovery of

bituminous(heavy)oilsthathaveasimilarhydrophobicitytothatoftoluene(Kiran

etal,2009).Hexaneandhexadecane,ontheotherhand,areoilsthatarerelevantto

theextractionofaliphaticlightcrudeoils(Acostaetal,2012)

27

Figure 23 Interfacial tensions for waste biosurfactants mixed with AOT, with a constant mixtureconcentration of 1gTOC/L. Biobased surfactant% is in terms of volume (v/v). NOTE: The pHwas notcontrolled for thesemixtures.ThepHvaluesat40%bio‐surfactant forRAS,FORSUD,andCV365were10.82,7.92,and7.53respectively.Tests forRASwere laterconductedat40%solutionadjustedtopH7,andtheIFTagainsttoluenewassimilar(0.18mN/m).

0

1

2

3

4

0 20 40 60 80 100

InterfacialTension(mN/m

)

%Biobasedsurfactant

a)RASandAOTHexane

Toluene

Hexadecane

0

1

2

3

4

0 20 40 60 80 100


)

%Biobasedsurfactant

b)FORSUDandAOT

HexaneTolueneHexadecane

0

1

2

3

4

0 20 40 60 80 100


)

%Biobasedsurfactant

c)CV365andAOT

Hexane

Toluene

Hexadecane

28

The stock solutions of RAS, FORSUD, and CV365 were diluted to 1gTOC/L using

deionized water. The IFTs were measured against Toluene, Hexane, and

Hexadecane.TheleftsideofFigure2‐3representspureAOTat1gTOC/L,andthefar

right represents pure waste bio‐based surfactant, RAS, FORSUD, or CV365, at

1gTOC/L.Asexpected,theIFTsforpureAOTarelessthanthatofthepureRASand

UR surfactants. For Toluene, Hexane, and Hexadecane, the IFTs for pure AOT at

1gTOC/Lwere1.65, 2.97, and2.22mN/m respectively.RAS at 1gTOC/Lhad IFTsof

12.8,12.2,and11.5mN/m,FORSUDhad16.4,17.1,and18.9mN/m,andCV365had

17.3,22.0,and19.9mN/m,respectively.

Figure 2‐3 shows how the IFT of the RAS, FORSUD, and CV365were affected by

mixingwithAOT. MixingwithAOTdecreased IFTs significantly in comparison to

thebio‐basedsurfactants(alkalineextracts)ontheirown.Atcertaincompositions,

themixturereachedIFTsagainsttolueneandhexanethatwerelowerthanforpure

AOT. Thiseffectwasnotasevidentforhexadecane,buttheIFTswerestillgreatly

reducedfromthatofthepureRASandURsurfactants.ThelowestIFTobservedfor

RASmixtureswas0.18±0.017mN/magainsttoluene,at40%RAS.FORSUDreached

a low IFT of 0.60 0.01mN/m against toluene, and 0.75 0.096mN/m against

hexane.CV365reachedthelowestIFTofthewastebiomassextractswithavalueof

0.13±0.028mN/magainsthexane.RASandUrbanRefusemixtureswithAOTat1g/L

consistently converge to a low IFT at mixtures of approximately 40% bio‐based

surfactantbyvolume.

29

TheIFToftheash‐freeFORSUDwasalsomeasuredinmixtureswithAOTinvarious

proportions to determine if the removal ofminerals had any effect on interfacial

activity.TheIFTswereonlymeasuredagainsthexane.Thesolutionthatcontained

40%ash‐freeFORSUD,at1gTOC/L,achievedanIFTof2.28mN/m.Thisismorethan

doublethelowIFTfortheoriginalFORSUDmixtureagainsthexane,whichreached

alowIFTof0.75mN/m.

The interfacial activity of the 40% FORSUD mixture was also investigated as a

functionoftotalsurfactantconcentration.Figure2‐4showstheinterfacialtension

againsttoluene,hexane,andhexadecanefortotalconcentrationsofupto10gTOC/L.

TheIFTagainsthexadecanewas2.2mN/mat1gTOC/L,anddecreasedto0.42mN/m

ataround8gTOC/L. TheIFTagainsttoluenedecreasedfrom0.60mN/mtoalowof

0.15mN/m. The most significant change occurred for hexane. The IFT was

1.1mN/mat1gTOC/L,anddecreasedto0.019mN/mat10gTOC/L,whichisintheultra

lowIFTrange(<0.1mN/m).

30

Figure24IFTof40%FORSUD,60%AOTatincreasedtotalmixtureconcentrations

RemovalofBitumenfromcontaminatedsand:ThegoalforthesetestswastodetermineifthelowIFTsforthebio‐basedsurfactant

andAOTmixturescouldresultineffectiveremovalofbitumenfromsand.Toluene

wasusedasasolventtoaidinoilrecoverybyreducingtheviscosityofthebitumen

coating thesand. Theamountof tolueneaddedandagitationtimewerevaried to

providecleaningconditionsofdifferentdifficulties. Thesetestswerealsodoneon

soilthatwascontaminatedandthenagedforalongerperiodoftime(12months)to

determineiftheformulationscouldstillbeeffectiveinremovingoilfromagedsand

samples.

Testswerefirstdoneonsoilaged1month,withlowlevelsofsolventtocomparethe

performanceof40%RAS(1gTOC/L)andwater. Thesetestsusedlessthan20µLof

toluene (toluene to bitumen ratio of 0.4 by w/w), which proved to be difficult

0.01

0.1

1

1 10


)


40%FORSUD

Hexane

Toluene

Hexadecane

31

cleaningconditionsforbothwaterandsurfactantmixtures.When10µLoftoluene

was used, the 40% RASAOTmixture recovered 35.6 0.07% of the oil, and the

water provided no measurable oil recovery. Although this was not excellent

recovery, these results indicated the potential for bio‐based surfactant and AOT

mixtures, with much lower IFTs than water, to be advantageous in oil recovery

underchallengingextractionconditions.Additionaltestswerethenconductedwith

increasedamountstolueneandagitationtimes.Figure2‐5showsoilrecoverytests

performedusingRAS‐AOT,FORSUD‐AOT,andAOTfordifferentperiodsoftimeand

differenttolueneamounts.Thesetestswereperformedonsoilthatwasagedfor1

month.Theadditionof20µLoftoluenerepresentsasolventtobitumenratio(T:B)

of0.4,and60µLoftoluenerepresentsaT:Bratioof1.2.

32

Figure25–OilremovaltestsforRASAOT,FORSUDAOT,andAOTat1g/L.a)20µLoftolueneused(T:B0.4,b)60µLtolueneused(T:B1.2).Soilaged1month

0102030405060708090100

0 10 20 30 40 50 60 70

OilRem

oval(%)

MixingTime(Seconds)

A)20uLToluene(T:B0.4)

Water

40%RAS

40%FORSUD

AOT

0102030405060708090100

0 10 20 30 40 50 60 70

OilRem

oval(%)

MixingTime(Seconds)

b)60uLToluene(T:B1.2)

Water

40%RAS

40%FORSUD

AOT

33

At20µLoftolueneand10secondsofmixing,thereisasignificantadvantageforthe

surfactantmixtures,achieving65‐75%oilremoval,overwater,with15.1%removal.

This trend continues through to 60 seconds, as the cleaning ability for the

surfactantsappearstoplateauataround85%oilremoval,andwaterreaches62.3%.

The1g/LRAS‐AOTmixtureappears tocleanslightlybetter thantheFORSUD‐AOT

mixtureandAOTat30seconds,andat60secondstheRASandFORSUDmixtures

bothperformbetter thanpureAOT (P<0.05). Similar testswere conductedusing

60µL of toluene, providing a similar trend butwith amuch higher oil removal of

about 98% for all surfactant formulations,with no significant difference between

them, and a closer recovery performance ofwater, but still noticeably inferior oil

removalatlowmixingtimes(P<0.05).

Testswerealsocarriedoutwith40%RAS,FORSUD,andCV365mixtureswithAOT,

as well as pure AOT, for soil that was aged for a longer period of time after

contamination.Figure2‐6showstheresultsforsoilaged12months.Theseresults

show that thebitumenbecomesmoredifficult to removewith increasedaging, as

60µL (T:B of 1.2) was required for meaningful cleaning results. However, the

surfactantmixturecontinuestorecovertheoilconsiderablybetterthanwater.The

surfactantmixturesshowedsimilarremovalcapabilitiestoeachother,witharange

of92‐95%recovery.

34

Figure 26 – Soil aged 12 months, solution mixed for 3 minutes. 60µL of toluene used (T:B 1.2).Surfactantmixturesconcentrationsof1g/L

2.4.2 DiscussionSurfaceTension:

TheshapeoftheRASgraphinFigure2‐1indicatesbehaviourthatissimilartothat

expectedwithconventionalsurfactants.Thesurfacetensionoftheextractremains

constant beyond a certain concentration, indicating the formation ofmicelles and

thepresenceofacriticalmicelleconcentration(CMC). TheCMCof1.34gTOC/Lfor

thisparticularRASextractionwasconsistentwiththatreportedbyGarcia‐Becerra

etal.(2010c),whichisencouragingconsideringthepotentialforthecompositionof

theRAStovaryfromyeartoyear,aswellaswithintheyearfromseasontoseason.

Thesurfacetensionwasclosertotheextractionthattookplaceduringthesimilar

timeofyear(springof2008,ST=37.2mN/m).

0102030405060708090100

Water 40%RAS 40%FORSUD 40%CV365 AOT

OilRem

oval(%)

Soilaged1year(60uLtoluene)

35

ThesurfacetensionversusconcentrationgraphsshowninFigure2‐1fortheUrban

Refuse extracts, FORSUD and CV365 showed signs of surface activity as

demonstrated by the reduction of surface tension with increased concentrations.

However, the surface tension did not become constant, and CMC could not be

readilyidentified,aswouldbeexpectedforaconventionalsurfactant.

The surface tension for RAS on its ownwas comparable to that of a commercial

surfactant,sodiumdodecylbenzenesulfonate(SDBS),witharangeof30‐35mN/m.

However, RAS has amuch higher CMC compared to the 25mg/L CMC of SDBS in

salinesolution(Rosen,2004).TheCMCoftheAOTusedfortheseexperimentswas

0.77gTOC/L,andasurfacetensionof26mN/m.RASalsocompareswelltootherbio‐

based surfactants, such as sodium lignosulfonates, reported to havemuch higher

CMCs of 5‐10g/L, and surface tension of 42‐45mN/m (Askvik, Are Gundersen,

Sjoblom, Merta, & Stenius, 1999). The dynamic surface tensions achieved with

FORSUDandCV365morecloselyresembledthevaluesreportedforhumicmaterial,

with CMC of 8g/L and surface tension of 48mN/m (Guetzloff & Rice, 1994). As

mentionedintheresultssection,previousworkhasreportedlowersurfacetensions

and CMCs for UR extracts (Montoneri et al., 2010). The surface tension

measurements in those testswere taken for200minutes, allowingmore time for

theURcomponentstoadsorbtothesurfaceandreachequilibrium.Theprocedure

formeasuringsurfacetensioninthisworkinvolvedmeasuringtheURextractsfor

only10minutes,andmaynothaveallowedformaximumsurfaceadsorption.

36

When theUR samplesweremixedwith the synthetic surfactant,AOT, the surface

tensionisothermshowedtheclassicalbreakpointusedtodeterminetheCMCofthe

mixture.WhenRASwasmixedwithAOT,theCMCofthemixturewasslightlylower

than that of RAS alone, and the surface tension after the CMCwas lowered to 25

mN/m.Thegraphsofthethreewastebio‐basedsurfactantsshowedsimilarsurface

tensions, and similar rangesofCMCs. TheFORSUDmixturehad thehighestCMC,

then CV365, and RAS had the lowest. When the RAS, FORSUD, and CV365were

mixedwithAOT,asshowninFigure2‐2,thesurfacetensionsbecamecomparableto

thoseofconventionalmicrobiallyproducedbiosurfacants(Lin,1996).

The loweredsurfacetensionsof thewastebio‐basedsurfactantswhenmixedwith

AOT may suggest some synergism. The CMC for RAS‐AOT was lower than RAS

alone,whiletheFORSUDandCV365didn’tapproachaCMCbeforebeingmixedwith

AOT.However,thiscouldalsobeanindicationofAOTbeingthedominantfactor,as

theCMCandSTscloselyresembletheresultsachievedforAOTalone.

The CMC of surfactant mixtures has been modeled using a combination of the

pseudo phase separation model and regular solution theory by Rubingh (1979),

suchthat:

and

37

Where thecmci is theCMC foreach individual surfactant, cmc* is theCMC for the

mixture,αi is themolar fractionofeachsurfactant in thebulkphase,andxi is the

molarfractioninthemicelle.Theβparameterrepresentsthenatureofinteractions

between the two surfactants, and is the key parameter of interest. Themodel of

RubinghwasusedtodescribeandfittheCMCobtainedfortheRAS‐AOTmixturein

Figure2‐2. Aβvalueof‐0.216wascalculatedfortheRAS‐AOTsurfactantmixture

(Refer to the Appendix for calculations). The negative value is an indication of

synergistic interactions between RAS and AOT, and the formation of a mixed

micelle. Thisβ value is comparable to other anionic‐anionic surfactantmixtures,

and is relatively smallwhencompared toβ values foranionic‐nonionic surfactant

mixtures,whichrangebetween‐1.6and‐4(Rosen,2004;Joshietal.,2005).

InterfacialTensionofmixtureswithAOT:ThepotentialsynergiesbetweenURextractsandAOTwerepreviouslyobservedby

Montonerietal.(2010),buttheextractsevaluatedinthatworkwereproducedata

labscaleandwere testedathigherconcentrations (10‐13g/L). Thesamplesused

for the tests in this paper were produced in pilot plant facilities, suggesting the

potential scalability forproducing the formulationsdescribed in thiswork. Itwas

interesting to see the same type of interaction take place with RAS, which is a

differenttypeofwastebiomass,andwasextractedusingadifferenttypeofalkaline

extraction method. The IFTs against Hexane for CV365 decreased below that of

38

hexadecane and toluene as it approached a low value at a composition of 40%

CV365. The RAS‐AOT mixtures achieved the lowest IFT with toluene, which

suggeststhattheCV365mixturehasahigheraffinityforhexane.

Urban Refuse waste surfactants have been mixed with the anionic commercial

surfactant,sodiumdodecylbenzenesulfonate(SDBS),inpreviouswork(Montoneri

etal.,2010).SDBSwasabletolowertheIFTmixturebelowthatoftheURextract

surfactants, but did not lower the IFT to levels below that of pure SDBS. The

synergistic effect was not quite as strong as with AOT. Urban refuse extracts in

mixture with AOT from these previous tests were able to achieve IFTs within a

rangeof0.26‐0.30mN/magainsthexane(Montonerietal.,2010).

The IFTs for RAS, FORSUD, and CV365 when mixed with AOT were generally

decreased by one order ofmagnitude. This was especially the case for RAS and

toluene,wheretheIFTcameclosetoreachingultra‐lowIFTs(<0.1mN/m),whichis

an indication that the mixture would possibly be useful for environmental

remediationorenhancedoilrecovery.Thisdrasticdifferencewasalsoobservedfor

bio‐surfactant/syntheticsurfactantmixturescontainingRhamnolipidbio‐surfactant

and anionic alkyl propoxylated sulfate surfactants. However, with a better

performingbio‐surfactantandtheuseofoptimalsalinitylevels,ultralowIFTvalues

of 0.035 mN/m and 0.033 mN/m against toluene and hexane were obtained

(Nguyenetal.,2008).TheRASandURextractmixturesdidnotreachultralowIFTs

atatotalconcentrationof1gTOC/L.

39

However,when40%FORSUDmixturewas increased to10gTOC/L, aspresented in

Figure 2‐4, an ultra low interfacial tension of 0.019mN/m was achieved against

hexane. This is lower than the Rhamnolipidmixtures prepared by Nguyen et al.

(2008).ItisalsomuchlowerthantheIFTof0.26mN/machievedbyFORSUD‐AOT

mixtures at 10g/L in previous work by Montoneri et al. (2010), which uses a

FORSUDextractproducedfromaslightlydifferentextractionprocessperformedat

a lab scale. However, caution must be taken in comparing the concentrations

directly. The concentration of 10g/L reported by Monteri et al. (2010) was

measured in termsofmassofdriedextractsolids,whichwerere‐dispersed in the

aqueoussolution.ThismaynotdirectlytranslatetothemassunitsofTotalOrganic

Carbon(gTOC)reportedinthiswork,asTOConlyaccountsforaportionofthetotal

solid mass. Nonetheless, the differences in IFT performance could suggest

differences in the chemicalmake up of the UR extracts produced in a pilot plant

facility(versuslabscale),andpotentialadvantagesusingthescaledupprocess. It

has been suggested that changes in the chemical nature of UR extracts can have

opposite effects on surface and interfacial tension, where a more hydrophilic

substancecanyieldlowerIFT,buthigherST(Montonerietal.,2010).

An IFTcomparisonwasdonewithFORSUDandash‐freeFORSUD,againsthexane.

ThehigherIFToftheash‐freemixturewithAOTdemonstratedthatfurthermineral

(ash) separationmay be detrimental to the IFT. This could be beneficial from a

processingpointofview,aslessprocessingstepscanresultinabetterperforming

surfactant.Onemajordifferencebetweenthetwosamplesisthecalciumcontent,as

40

showninelementalanalysisinTable2‐2.TheunfilteredFORSUDcontainsover10

timesmore calcium in proportion to the amount of organic carbonpresent. This

excessinCa2+ionscouldplayaroleinneutralizinganyanionicgroupspresentinthe

solution,makingthesurfactantmorehydrophobic,whichwouldreducetheIFTwith

hydrophobicoils. TheCa2+content, inrelationtotheorganiccarboncontent,was

highest for CV365, which could be an explanation for that surfactant mixture

reachingthelowestIFTwithhexane,atatotalsurfactantconcentrationof1gTOC/L.

ThesynergismobservedwiththemixturesofAOTandalkalinewasteextractscan

be explained by the Hydrophilic‐Lipophilic Difference (HLD) framework used for

mixturesofionicsurfactants.Thesemi‐empiricalHLDexpressionis:

(Acostaetal.,2008)

Where S is the electrolyte concentration (salinity); K is a constant; EACN is the

EquivalentAlkaneCarbonNumber(e.g.Hexane=6,Toluene=1),whichrepresents

thehydrophobicityoftheoilphase;xi isthemolefractionofeachsurfactantinthe

mixture, and Cc is the characteristic curvature for each surfactant. The

characteristic curvature is a parameter that represents a surfactant’s tendency to

formmicellesorreversemicelles.ApositiveCcwillfavourtheformationofreverse

micelles, and a negative Cc will favour micelle formation. The hydrophobic (or

lipophilic)natureofAOTisreflectedbytheCcof+2.5,whichcanleadtoapositive

HLDvalue,andtendencytoformreversemicelles(Acostaetal.,2008).Mixturesof

this surfactantwithamorehydrophilic component (negativeCc) tend to shift the

41

valueofHLDofthemixturetowardszero,whichleadstolowerinterfacialtensions

(Acosta et al., 2003, 2012). Therefore, the synergisms observed in the IFTswith

mixtures indicate that the alkaline extracts of RAS and UR are more hydrophilic

(negativeCc),andhelpproduceanHLDthatapproacheszerowhenmixedwithAOT.

BitumenRemovalfromContaminatedSand:Theoil removal tests presented in Figure2‐5andFigure2‐6 show somepositive

results for the surfactant mixtures of waste bio‐surfactants and AOT. All tests

consistently produced improved oil removal in comparison to deionized water,

which is indicative of the performance benefits from lower interfacial tensions.

UndertheconditionsofFigure2‐5a(1monthofsoilaging,20µLtoluene,30seconds

ofmixing),40%RASwasabletoremovemorebitumenthan40%FORSUDandAOT.

Thiscouldbereflectiveofthefactthatthe40%RASmixturereachedthelowestIFT

against toluene. At a longer mixing time of 60 seconds, the 40% RAS and 40%

FORSUD formulations performed better than AOT on its own, suggesting the

potentialbenefitsofmixturesincomparisontopureAOT.

Increases in the toluene to bitumen ratio (T:B) resulted in improved oil removal,

which is consistent with previous work by Quraishi et al. (2012). This can be

explainedbyareductioninbitumenviscosity.Theteststhatusedahighertoluene

content of 60µL did not showmuch variation among the RAS and UR surfactant

mixtures,buttheydidshowbenefitscomparedtowatercleaning,especiallyatthe

lowestmixing time. This could prove to be useful in industrial processes,where

42

shorterresidencetimescanleadtoreducedcosts. Thebitumenwasmoredifficult

toremovefromsoilthatwasagedforayearaftercontamination,asdemonstrated

by loweroil removalofwater. However, theRASandURsurfactant formulations

stillperformedwellwiththeaidofaT:Bratioof1.2(60µLToluene).Althoughthe

oilrecoveryusinghigheraT:Bratiodoesnotvarysignificantlyamongthevarious

surfactantformulations,theredoesappeartobearelationshipbetweenthelowered

IFTs and the enhanced ability to remove heavy oil from sand in comparison to

water.

Althoughitwouldbeidealtonotrequireanysolvent,thereareindustrialprocesses

thatusesolventoilrecovery,suchassolvent‐aidedsteamassistedgravitydrainage

(SAGD) (Gates & Chakrabarty, 2008; El Naggar et al., 2010). SAGD uses thermal

energytoreducetheviscosityofheavyoiltoimproveitsrecovery,whichisasimilar

purpose to toluene used in these experiments. The RAS and UR surfactant

formulations could potentially be useful in reducing the amount solvent or steam

requiredfortheseindustrialoilrecoveryprocesses.

2.5 ConclusionAlkaline extracts from various waste biomass sources were used to produce

effectivebio‐basedsurfactants. Theextracts thatcontainedorganicmaterial from

Return Activated Sludge (RAS) and Urban Refuse (UR) solid waste were surface

active, and when combined with a hydrophobic commercial surfactant, AOT,

produced low interfacial tensions against toluene, hexane, and hexadecane.

43

Although this had been observed at higher surfactant concentrationswith similar

URextractsobtainedfromalabscaleprocess,similarbehaviourwasobservedwith

URsamplesobtainedfromapilotscaleprocess.ThealkalineRASextractshoweda

similar synergywithAOT, as demonstrated bymixtures that could be reduced to

lowIFTs(<1mN/m).Whenthetotalsurfactantconcentrationwasincreased,oneof

the UR surfactantmixtures reached ultra‐low IFTs against hexanewithout added

electrolyte. SynergismbetweenRASandAOTwasalso suggestedby thenegative

value of the mixed surfactant interaction parameter, β, which indicates the

formationofmixedmicelles.

The RAS and URwaste extracts inmixtureswith AOTmixtureswere effective in

bitumenremovalfromcontaminatedsand,possiblyaresultoflowIFTs. Thistype

ofbehaviorcanbeusefulforapplicationssuchassurfactantenhancedoilrecovery

andenvironmentalremediation.Furtherworkcanbedonetofurtheroptimizethe

surface activity of the extracts through the additionof salt, or by findingbiomass

sourceswithahigherlipidcontent.Thereisalsoanopportunitytotrymixingthese

wastebiomassextractswithdifferentsyntheticsurfactants,orotherbiosurfactants.

44

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47

CHAPTER3 Woodadhesiveformulationusingalkalineextractsfromreturn

activatedsludgeandmustardprotein

3.1 AbstractIn this work, the strength of adhesives formulated with protein‐rich alkaline

biomassextractswereoptimizedusingstrategiesthattookadvantageoftheprotein

cross‐linking capability of glutaraldehyde. The renewable biological resources

exploredwereReturnActivateSludge(RAS)andmustardprotein(MP),whichwere

both extracted using alkaline solutions. In one strategy,mixtures of extracts and

cross‐linking agent, glutaraldehyde, in solution were heat treated prior to their

application on wood, which resulted in modest adhesive strength improvements

overpreviouslyreported formulations. In thesecondstrategy, theglutaraldehyde

wasapplieddirectlyontothewoodseparatelyfromtheMPandRASsolutions(in‐

situmixing). Ahighshearstrengthof2.3MPawasreachedforMPadhesivewhen

glutaraldehydewasmixedinsituuponwoodapplication.RASadhesivesachieveda

high shear strength of 0.88MPa. These bond strengthswere achieved under low

pressure(8.6kPa)andambienttemperaturepressingconditions, ina60%relative

humidity environment, and showed improvements to previous RAS and MP

adhesive work under similar conditions. This work showed the potential for

protein‐basedadhesivesforwoodworkingapplicationsatambientconditions.

48

3.2 IntroductionTheneedforthedevelopmentofwoodadhesivesfromenvironmentallyfriendlyand

renewable resources continues to grow. Current commercialwoodadhesives are

primarilyderived frompetrochemical resourcesbecause theyare relatively cheap

andprovidesuperiorbondstrengthandwaterresistance(W.H.Wang,Li,&Zhang,

2008). However, concerns over the depletion of petroleum resources,

environmental issuesfromtheirextraction,healthissues,andincreasingcosts,are

leading to a growing need to develop products from inexpensive, renewable

resources with competitive adhesive performance. (Park et al., 2000; Lin &

Gunasekaran,2010).

Adhesives have been produced from awide variety of biological sources, such as

proteins from agricultural by‐products like bone meal, cow blood, soy, and red

onion skin, peanut hull, andwattle bark (Odozi & Agiri, 1986; Chen, 1982; Pizzi,

1978; Park et al., 2000; Lin & Gunasekaran, 2010; Kalapathy et al., 1995).

Polysaccharides have been used in the formulation of adhesives, as well as

microbially producedbiopolymermixtures from fermentationwastes. (Weimer et

al., 2003; Haag et al., 2004). Soy protein isolates have been heavily explored

because soybean protein adhesives were extensively used in the interior grade

plywoodproductionintheearly1900s.Theyhavesincebeensubstantiallyreplaced

bypetroleumbasedadhesives, suchasphenol‐formaldehyderesins,due tohigher

strengths and lower costs. However, with renewed interest in using sustainable

resources, protein modifications have been studied to enhance performance and

49

compete with synthetic adhesives (Frihart, 2005; Kalapathy et al., 1995,

Hettiarachchyetal.,1995).

There are some limitations with biopolymer based adhesives, such as moisture

sensitivity, thermal instability, and processing difficulties (Haag et al., 2006). In

order to address these issues, a variety of physical, chemical, and enzymatic

methods have been used to modify hydrophobicity, molecular weight, and

biopolymer conformation (Wang et al., 2008). Protein denaturing is a common

methodforenhancedperformanceofproteinbasedadhesives.Methodsusingheat

exposure,alkali/acid treatment,organicsolvents,andureahavebeen investigated

tobreakthestronginternalbondsofcoiledproteinmoleculestoallowforunfolding

and better exposure of polar groups for adhesion to wood (Wu & Inglet, 1974,

Lambuth,1994).Crosslinkingischemicalmodificationthatinvolvestwomolecular

components being joined together by a covalent bond to form a larger molecule

(Wong,1993).

Severalchemicals, suchassimpledialdehydes,haveshowntheability tocrosslink

proteins. Glutaraldehyde isoneof themostextensivelyusedcross‐linkingagents,

and it has beenused to improve themechanical properties andwater stability of

various protein basedmaterials for use in food, textile, andmedical applications

(Reddy et al., 2008; Wong, 1993). The crosslinking reaction mechanism of

glutaraldehyde is not completely understood, but the reaction results in the

connection of proteins to form a larger molecule and a more stabilized network

(Reddy et al., 2008). It has been proposed that glutaraldehyde polymerizes the

50

protein molecules through a Schiff base, whereby glutaraldehyde forms an

unsaturatedpolymerthatcross‐linkstheaminogroupsoftheproteins,asshownin

Figure3‐1. Glutaraldehydecan formpolymers in solution, inaneutralor slightly

alkalineenvironment(Wong,1993).Itisbelievedthattheaminogroupsassociated

with the amino acid, lysine, are the most reactive to aldehydes that promote

crosslinking(Lundblad,2005;Quiocho&Richards,1966). Mustardprotein,oneof

thesourcesconsideredinthiswork,isrichinlysine(Aideretal.,2012).Theuseof

glutaraldehdyehasbeenshowntoenhancetheadhesionandmoistureresistanceof

soyprotein isolate (SPI)basedadhesives, and foradhesivesbasedon thealkaline

extraction of Return Activated Sludge (Wang et al., 2007; Garcia‐Becerra et al.,

2012).

Figure31Reactionbetweenglutaraldehydeandprotein(Wong,1993)

Two biological resources were explored for adhesion optimization in this work;

ReturnActivatedSludge(RAS)andsolublemustardseedproteinisolate,whichwill

bereferredtoasmustardprotein(MP). Alkalineextractionmethodswereusedto

extractbothsources.ReturnActivatedSludgeisaby‐productofbiologicallytreated

51

municipalwastewater.Thisisanabundantsourceoforganicmaterialthatcontains

extracullar and intracellular biopolymeric substances with surface‐active

capabilities (Garcia‐Becerra et al., 2010b).Themain constituents inRAS are cells,

proteins,polysaccharides,lipids,andhumicsubstances(Frolund,Keiding,&Nielsen,

1996). Therehasbeenother adhesivework involvingheterogeneousbiopolymer

mixes (Weimer et al., 2003). Previous characterization of RAS alkaline extract

indicates that proteins and polysaccharides account for approximately 20% and

12% of the organic content, respectively (Garcia‐Becerra et al., 2010). Alkaline

extraction induces cell lysis to occur, and the liberation of organic content of the

cellsgivesthesurface‐activecharacteristicsofthefinalaqueoussolution.

The other biomass source of interest for adhesive production ismustard protein

(MP).Proteinextractedfrommustardseedhasbeenstudiedasapotentiallyuseful

source for human nutrition due to its abundance and well balanced amino acid

composition (Xu et al., 2003; Aider et al., 2012). Mustard seed ismainly used to

produce edible oil, but can contain 23‐30% protein (Alireza‐Sadeghi et al., 2006;

Newkirketal.,1997).Mustardproteinisrichinlysine,whichisimportantforcross‐

linkingwith glutaraldehyde (Aider et al., 2012;Lundblad, 2005). The isolationof

mustardproteinfromtheseedcanbecarriedoutusinganalkalineextraction.

RAS andMPwere assessed for adhesive capabilities for the first time by Garcia‐

Becerraetal.(2012).MPwasofinterestbecauseproteins,primarilysoy,havebeen

usedascommercialadhesives.RASisnotapureproteinmaterial,asitismadeup

52

ofavarietyofbiopolymers.However,itwasapparentthattheproteincomposition

withintheorganicsolidscontentplayedakeyroleinadhesiveperformance(Garcia‐

Becerraetal.,2012).Bothbiopolymersourcesshowedpotentialaswoodadhesives,

and incorporated theuseofglutaraldehydeasa cross‐linker. Glutaraldehydewas

abletointeractwithbothRASandMP.However,thehighestbondstrengthsforRAS

andMPcamefromhotpressconditions(260kPa,150°C),restrictingtheapplication

potential of these formulations to limited bonding processes such as plywood

production.

The goal of this research was to develop formulations of RAS and MP based

adhesives that could form a strong bond with wood when applied under low

pressure and ambient temperature pressing conditions. This was done through

adjustments in the RAS and MP concentrations, and cross‐linking. The idea of

encouraging the cross‐linking reaction with the use of a heat treatment on the

adhesive formulation, containing cross‐linker, before being applied to wood was

explored. Anewmethodofapplicationwasalso investigated,whereby theMPor

RAS solution was added to the wood separately from the cross‐linker,

glutaraldehyde,toallowforin‐situmixingofproteinandcross‐linker. Thegoalof

the in‐situapplicationmethodwas to formastrongcross‐linkedproteinnetwork,

whileallowingforwoodpenetration,whichisessentialforbondformation. Shear

testswereperformedtodeterminetheeffectivenessofthesetreatments.

53

3.3 MaterialsandMethods

3.3.1 MaterialsNaOHsolution(50%w/w,reagentgrade)foralkalineextractionofReturnActivate

Sludge (RAS) and glutaraldehyde solution (50% w/w, reagent grade) were

purchased from Sigma‐Aldrich (Oakville, Canada). Lyophilized mustard seed

soluble protein isolate, which will be referred to as mustard protein (MP) was

providedbyProfessorDiosady,UniversityofToronto. Maplewoodchips,cut into

2.54x10.16x0.32cmpieces,werepreparedbytheUniversityofTorontoCarpenter

Shop(UofTFacilitiesandServices,Toronto).

3.3.2 MethodsProductionofRASExtract:The procedure used for the extraction of Return Activated Sludge follows that

developed by Garcia‐Becerra et al. (2010). The aerobic return activated sludge

(RAS)wascollectedfromthemetropolitanAshrbidgesBayWastewaterTreatment

Plant (1400 population equivalent; average capacity: 725,000m3/day; sludge

retentiontime:2.5days;aerateiontime:6‐8h).TheRASsamplingandextractionfor

theseparticularstudiestookplace inMayof2011. TheRASwascollected,placed

into an ice bath, transported to the lab, and was left to settle for 2 hours. This

allowed for thebio‐solids to settle. Theclear, supernatantwater sittingon topof

the bio‐solidswas then removed. Thiswas done to reduce the amount of NaOH

required to raise the solution to the appropriate pH, andwould allow for amore

54

concentratedextract.ThepHoftheconcentratedRASwasraisedto12.6,using50%

NaOH solution, and mixed for four hours at 500rpm at room temperature. The

alkalineextract solutionwas thenstoredat4°C. Theextractwas then frozenat ‐

80°C, and lyophilized at 0.012mBar and ‐80°C, to obtain a dried powder of the

extractedRASsolids,storedatroomtemperature.

PreparationofalkalimodifiedMustardProtein:MP was produced from yellow mustard seed using a technique developed by

MarnochandDiosady(Marnoch&Diosady,2006).Theproteinwasextractedfrom

defattedyellowmustardseedatpH11,ultrafiltered,diafiltered,precipitatedatpH

5,andlyophilized.TheMPunderwentanalkalimodificationbyadjustingthepHof

a suspendedMPsolution to12,usingNaOH,and incubating it at40°C for1hour.

Thissolutionwasfrozen,lyophilized,andthenstoredatroomtemperature.

AdhesiveSamplePreparation:MPwaspreparedin15,25,and40%(allweightbasisunlessotherwisespecified).

solutionsindeionizedwater.RASwaspreparedsimilarly,butwith30%,45%,and

60%freeze‐driedsolids.TheRASandMPsolidpowderweremixedwithwaterfor

30minutes. For testswhereglutaraldehydewasadded to the formulationbefore

adhesive application, the glutaraldehyde was mixed with the solution for 30

minutes. Formulations thatwere pre‐treated before being applied towoodwere

placed inanovenat80°C(Fisher IsotempVacuumOven,Model281) in the20mL

scintillation vials the formulations were initially prepared in. All heat‐treated

55

samplescontained2gofformulation,andwereheatedbetween1and18hours.For

thetestsperformedusingthein‐situapplicationstrategy,theMPandRASsolutions

were added to one wood piece, and glutaraldehyde was added separately and

quicklymixedwiththeproteinsolutionbeforethesecondwoodpiecewaspressed

ontop.

Theadhesiveformulationswereappliedtothewoodbyplacing120mgofadhesive

toa2.54x2.54cmareaononeendofthewoodpiece(pre‐conditionedat25°Cand

60%relativehumidity(RH).Asecondpieceofwoodwasplacedontopofthearea

whereadhesivewasapplied.Thegluedpiecesweresubjectto8.6kPafor1houror

18hoursatroomtemperatureand60%RH.Thegluedpieceswereequilibratedat

roomtemperature(23‐25°C)and60%RHfor5daysbeforesheartesting.

Soakingtestswereperformedtotestmoistureresistance.Thegluedwoodsamples

weresubmergedintapwaterat23°Cfor24h. Thespecimenswerethendriedat

25°C and 60% relative humidity for 5 days before shear strength tests. This

procedure is consistent with water resistance procedure used by Wang et al.,

(2007),whichwasperformedaccordingtoASTMStandardMethodD1151‐00.

ShearStrength:The shear strength was measure from a lap shear test method, using a Sintech

Computerizedsystemfortensiletesting(Sintech20,withMTSSintechTestWorks

V2.1 Software). This equipmentmeasured the force required to break the glued

56

jointbypullingapart the twoedgesata loadingrateof1.00mm/min. Theshear

strengthatmaximumloadwasrecorded,andreportedvaluewas themeanof4‐5

specimens.

RheologicalProperties:TheviscosityofadhesiveformulationswasmeasuredusingaTAInstrumentsCarri‐

Med Rheometer (Model CSL2 500, TA Instruments Rheology Solutions Software

DataV1.2.2)usinga2cmdiameterconegeometryandvariousshearrates.

ThermalProperties:The phase transitions of cross‐linked MP was evaluated through differential

scanningcalorimetry(DSC),usingaDSCQ100(TAInstruments). Thecross‐linked

formulationswere freeze‐dried, and4‐6mgofdriedpowderwasplaced in aDSC

pan, and capped. Samples were scanned from 20 to 230°C at a heating rate of

10°C/minute.

3.4 ResultsandDiscussion

3.4.1 ResultsRheologicalPropertiesFigure 3‐2 presents the viscosity of mustard protein (MP) and RAS based

formulations, at various shear rates. The results help indicate whether or not

protein cross‐linking occurredwith addition of glutaraldehyde or heat treatment.

57

Figure3‐2showstheviscosityofMPandRASsolutionswithincreasingamountsof

glutaraldehyde, and solutions containing glutaraldehyde that were heat‐treated.

TheMPformulationsmeasuredforviscositycontained7.5%mustardprotein.

Figure 32 – Viscosity versus shear rate curves for MP and RAS solutions that were treated withglutaraldehyde (GL) and heat treatment (HT). GL1 indicates 1%wt. glutaraldehyde. a)MP solutionsweremadewith7.5%MustardProtein,andb)RASwith45%solidRAScontent.

0

50

100

150

200

250

300

350

400

0.1 1 10 100

Viscosity(Pa.s)

ShearRate(1/s)

a)MustardProteinAdhesives

MP7.5GL1

MP7.5GL2

MP7.5GL0.5HT

0102030405060708090100

0.1 1 10 100 1000

Viscosity(Pa.s)

ShearRate(1/s)

b)RASAdhesives

RAS45

RAS45GL1

RAS45GL2

RAS45GL6

RAS45GL1HT

58

At low shear rates, the viscosity of MP formulations increased by one order of

magnitude as the glutaraldehyde composition doubled from 1% to 2%. It is

apparentthatheattreatmentincreasedtheviscosityofthesolutioncontaining0.5%

glutaraldehyde. MPformulationswithnoglutaraldehdyeand0.5%glutaraldehyde

(noheat treatment) resembled the consistencyofwater, andwere too fluid tobe

accuratelymeasured by the rheometer. Although therewere no values obtained,

this in itselfwas an indication that viscosity increasedwith the addition ofmore

glutaraldehydeandheattreatment.

Althoughhigherconcentrationswereused foradhesivestrength tests (15%,25%,

45%), the addition of glutaraldehyde to these formulations formed a solid‐like

materialthatcouldnotbeappliedtothegeometryoftherheometer.However,this

anecdotaldescriptionisconsistentwiththeideathattheadditionofglutaraldehyde

increasedtheviscosityoftheMPsolutions.

TheRASformulationresultspresented inFigure3‐2balsoshowhigherviscosities

with glutaraldehyde addition andheat treatment. This trend ismore apparent at

low shear rates. However, the viscosity was not affected until a much higher

amountofglutaraldehyde,6%,wasaddedtotheformulation.

Theseresults indicatethepossibilityofproteincross‐linkingtakingplacewiththe

addition of glutaraldehyde, and the ability for heat treatment to encourage the

cross‐linking reaction with the presence of glutaraldehyde. The relationship of

59

increasedviscositywithcross‐linking is consistentwithpreviousworkonprotein

cross‐linking (Haag et al., 2006,Wang et al., 2007, Garcia‐Becerra et al., 2012,

Yamadaetal.,2000).

EffectsofpreheattreatmentonMPadhesive:Theeffectofheat‐treatingtheMPbasedadhesiveformulations,withthepresenceof

the cross‐linker, glutaraldehyde, on adhesive strength is presented in Figure 3‐3.

ThesetestswereconductedwithaconstantMPandglutaraldehydeconcentrationof

15%and0.5%,respectively.Theformulationsweretreatedinanovenat80°Cfor

differentperiodsoftime,beforebeingcooledandappliedonwoodsamples.These

adhesivetestswerepreparedbypressingthegluedwoodsamplesunder8.6kPaof

pressure for 18 hours. The extended press periodwas used to encouragewood

penetration.

Figure33ShearstrengthofMPadhesivewith15%MPand0.5%GLandpreheattreatmentforvariousperiodsof time. HT1means1hour,HT2is2hours,etc. Sampleswerepressedfor18hoursunderadeadweight,forapressureof8.6kPa

0

1

2

3

HT1 HT2 HT5 HT18

ShearStrength(MPa)

60

The results inFigure3‐3 indicate that exposing these formulations toheatwitha

glutaraldehyde can be advantageous to adhesive strength. When the adhesive

formulation of 15%MP, 0.5% GL was not exposed to heat, a bond could not be

maintained. Whenexposedtoheat,therewasanobservablechangeinthetexture

towardsamoregel‐likeconsistency.Theamountoftimeexposedtoheatappearsto

improvethestrength.Thelowestshearstrengthof0.42MPawasobtainedwiththe

formulation exposed toheat for the shortest periodof time. Thehighest average

shear strength reached for these tests was 1.10MPa, and this was for the

formulation exposed to heat for the longest period of time, 18 hours. The shear

strength for 15%MP samples that was heated for 18 hours was not statistically

greaterthantheformulationheattreatedfor5hours. Howeverthe18hourand5

hour tests had stronger shear strengths than the formulations treated for 1 hour

and2hours(P<0.05).

MustardProteinConcentration,GlutaraldehydeAddition,andInsituMixing:Increases inMP and glutaraldehyde concentrations led to significant increases in

theviscosityofadhesive, to thepoint that it formedasolidmaterial. Therefore,a

suitable adhesive could not be formulated with higher MP and glutaraldehyde

concentrationsbecausetheycouldnotpenetratethewoodsurfacetoformabond.

61

Inordertotakeadvantageofthepotentiallystrongbondsofthecross‐linkedMP,a

newapplicationstrategywasdevelopedwherebythemustardproteinsolutionand

glutaraldehydewereappliedontothewoodseparatelyforin‐situmixing,resulting

inin‐situcross‐linking.Theshearstrengthresultsfromin‐situmixingwithvarying

MPandglutaraldehydeconcentrationsarepresentedinFigure3‐4.

Figure 34 – Shear strength for MP adhesives containing 15%, 25%, and 40% (labeledMP15, MP25,MP40),withglutarldehyde (GL)mixed insitu. Sampleswerepressed for1hourunderadeadweightwithapressureof8.6kPa.

Figure3‐4showstheshearstrengthresultsofMPadhesiveformulationscontaining

15%,25%,and40%mustardprotein. These solutionsweremixedwithdifferent

amountsofglutaraldehyde,rangingfrom1.2‐7.2µL,whichasapercentofthe120mg

appliedtoeachpieceofwoodtranslatesto1‐6%GL.BoththeMPconcentrationand

GLcontentappearedtoaffecttheadhesivestrength.

At15%MP,theadhesionstrengthimprovedastheGLcontentincreasedfrom1%to

4%,whereahighstrengthof0.84MPawasachieved.Thestrengthdidnotimprove

0

1

2

3

GL1% GL2% GL4% GL5% GL6%

ShearStrenght(MPa)

MP15

MP25

MP40

62

astheGLcontentincreasesbeyond4%(P<0.05).Thiswasasimilartrendfor25%

and40%MPformulations,butwithhighershearstrengths.At4%glutaraldehyde,

25% MP formulation achieved a strength of 1.71MPa, and 40% MP reached

2.31MPa.

Asoaking testwasperformedon thebestperforming formulation,MP40GL4%, to

testforwaterresistance. Theaveragebondstrengthaftersoakinganddryingwas

slightly less than the dry sample at 2.07MPa, but this result was not statistically

lower when considering the variance in the results (P>0.05). Therefore, the MP

formulationwasabletomaintainmostofitsbondstrengthaftersoaking.

Oneissuewiththisin‐situapplicationmethodisthatitcanbesensitivetoexecution

andtiming. Theproteinsolutioncangetsoakedinthewoodif it is leftsittingtoo

longbeforetheglutaraldehydeisadded, leavingnothingforglutaraldehydetomix

andformabondwith.Also,theglutaraldehydemustbeproperlymixedbeforethe

secondpiece ofwood is pressedon top. However, if leftmixing for too long, the

solution can quickly cross‐link and harden,which can prevent penetration to the

secondpieceofwood.

RASExtractAdhesion:Although RAS is made up of multiple constituents, previous work indicated that

RAS’s adhesive capabilitywas correlated to protein content (Garcia‐Becerra et al,

2012). Therefore,theconcepts learnedfromtheMPmodificationsweretestedon

63

RAS to examine if this would translate to enhanced adhesive performance. The

shear strength results are presented in Figure 3‐5. The graph shows RAS

concentrationsof45%and60%,intermsofweightpercentageoffreezedriedRAS

powder. Themodifications to the samples included glutaraldehyde addition, pre‐

heattreatment,andin‐situmixingwithglutaraldehyde.RASconcentrationsof30%

werealsotested,butdidnotyieldmeasureableadhesivestrengths.

Figure35–Shear testresults forRAS formulations. The formulationsunderwentpreheat treatment(HT),additionofglutaraldehyde(GL),orweremixed insituwithglutaraldehyde(IM). Sampleswerepresseddownfor18hoursunder8.6kPa.

Themodifications involvingpre‐heat treatment, glutaraldehyde content, or in situ

applicationmethod,wereperformedonadhesiveformulationscontaining45%RAS.

Formulationscontaining30%RASweretestedforglutaraldehydeaddition(1%GL),

and pre‐heat treatmentwith 1% GL for 18 hours. The pre‐heat treated samples

weretheonlyonestomaintainabond,butthebondswerenotstrongenoughtobe

0

0.2

0.4

0.6

0.8

1

1.2

ShearStrength(MPa)

64

measurewithinthelimitsofthesheartestingequipment(0.25MPawasthelowest

measurablestrengthrecordedbytheequipmentforthesetests).

RAS45%formulationshowedashearstrengthof0.63MPa.RAS45%with2%GL

reached a strength of 0.76MPa, andwas the only formulationwith the sameRAS

concentration that showed a statistical improvement to the RAS 45% solution

(P<0.05).IncreasedRASconcentrationalsoresultedinastatisticalimprovementto

RAS45. The formulation containing 60%RAS (RAS60)with 6%GLmixed in situ

showed the highest average strength of the RAS adhesive tests, with 0.88MPa

(58.9kg).

RASadhesiveswerenotabletomaintainabondaftersoaking.

ThermalPropertiesDSC can determine thermal transition points ofmaterials through the amount of

energyreleasedorabsorbedbyasampleataparticulartemperature.Thethermal

transition properties of the MP adhesives can be detected, and variations in the

structureduetocross‐linkingwiththeadditionofglutaraldehydecanbereflected

by changes in the thermal behaviour. The temperatures and heat flow associated

withthetransitionwererecordedbyDSC.Figure3‐6showsaDSCthermogramfor

MP protein that has no been modified, MP that has been cross‐linked with

glutaraldehyde,andMPtreatedwithglutaraldehydeandpre‐heattreatment.

65

Figure36DSCthermogramofunmodifiedMustardSeedProteinIsolate(MP)andMPcrosslinkedbywith2%glutaraldehydew/w(MPGL2),andwith0.5%glutaraldehydeand18hoursofheattreatmentat80°C(MPGL0.5HT18)

TheDSC thermograms indicate thepossibilityof structuralvariations takingplace

due to cross‐linking. The key thermal transition points are represented by the

peaks,whichwerepresumablyduetothethermaldenaturingofthenativeprotein

structure.Eachsampleshowskeythermal transitionbehaviourat89‐95°C,andat

around 200°C, that could represent glass transition (Tg) or melting temperature

(Tm).UnmodifiedMPshowstwoseparatepeaksthroughoutthehighertemperature

transition points between 198 and 210°C, whereas the cross‐linked proteins

showedonepeak,apossibleindicationofconformationalchangestakingplace.The

denaturingenthalpy,representedbytheareaunderthepeak,appearstobehigher

fortheproteinstreatedwithglutaraldehydeandheattreatment(withthepresence

of 0.5% glutaraldehyde). The lower heat flow peaks indicate possible

conformational changes due to cross‐linking, resulting in new structures that

requiremore thermal energy for denaturation. These results are consistentwith

66

previousworkusingDSCanalysis todetectcross‐linkingofproteins(Wang etal.,

2007,Lanttoetal.,2007).

3.4.2 DiscussionHeattreatment:

ThefirstmodificationtestedontheMPadhesiveswasthepre‐heattreatmentwith

thepresenceof theglutaraldehyde(GL)proteincross‐linker. The ideaof thepre‐

heat treatment of adhesive formulations was to encourage the cross‐linking

reaction, andpossibly simulate any other curing that occurs during heat pressing

usedfortheproductionofplywood.Thisisanassumptionbasedonthebeneficial

effectsofheatpressingonbondstrength forbothRASandMPadhesives (Garcia‐

Becerra,2012).Thishelpedimprovethestrengthoftheadhesivewithoutrequiring

theenergyintensiveprocessofheatpressing.

It is possible that heat encouraged the cross‐linking reaction. There is also the

possibility of some thermally aideduncoilingof theprotein structure, denaturing,

thatcouldhelptheproteinsflowbetterwithinthesolution,butitisverydifficultto

knowif this ishappening.Theheat‐treated formulation formedagel‐likenetwork

thatcouldindicatefurthercross‐linkingthatdidnotoccurwithoutheattreatment.

This anecdotal observation is consistent with the viscosity results from heat

treatment. Thisformationofasolid‐likematerialhasbeenobservedtobearesult

ofcross‐linkinginsoyproteinadhesiveworkaswell(Wangetal.,2007).

67

Also,theDSCthermogramspresentedFigure3‐6showthattheheat‐treatedprotein

absorbedmoreenergy, incomparisontounmodifiedmustardprotein. Thehigher

denaturing enthalpy is a possible indication of larger molecules being formed

through cross‐linking, and intertwining of those largermolecules to form amore

stable protein network. These findings are consistent with enthalpy changes

observedfromDSCanalysisinotherproteincross‐linkingwork(Wangetal.,2007,

Lanttoetal.,2007).

These tests have indicated that both the glutaraldehyde and heat treatment

contributedtoimprovedadhesiveperformance. Adhesiveformulationscontaining

15%MPand0.5%Glutaraldehydethatwerenotheattreatedwerenotabletohold

the bond to the wood in humid ambient conditions (60% RH). Also, a 15%MP

formulationwith no glutaraldehyde that underwent pre‐heat treatment could not

maintainabond,anddidnotshowanyvisiblephysicalchangesinthetexture.

Althoughthemainobjectivewastoimprovetheadhesivestrengthbycross‐linking

of the proteins before being applied to the wood, too much cross‐linking can be

detrimental.Excesscross‐linkingcanleadtotheformationofasolid‐likematerial,

whichwould inhibit theability topenetratethewood. If theproteinsbecometoo

large,thentheycannotinterlockwiththewood.Thispenetrationandinterlocking

withthewoodiskeytoastrongbond(Wangetal.,2007).Itisveryimportantfor

theproteinstobeabletohavepolargroupsexposedtoformhydrogenbondswith

thewoodstructure.Thiscanbedifficulttoachievewithlarger,cross‐linkproteins.

68

Afterbondingwith thewood,abondstrengthcanbeestablished, inpart, through

thereformationofhydrogenbondsbetweenproteinchains(Frihart,2005).

Fortunately,thepre‐heattreatedsolutionsof15%MPand0.5%GLprovidedagood

balance of mechanical strength enhancement while still allowing for wood

penetration. However, when further cross‐linking was encouraged by increasing

glutaraladehydecontentinthe15%MPsolutions,theformulationsformedastrong

gel‐likenetworkthatcouldnotpenetratethewoodandformabond.Theissuealso

occurredforformulationscontaining25%MP,asexpected. Excessglutaraldehyde

has also been shown to be detrimental to the adhesive strength of soy protein

adhesiveformulations(Wangetal.,2007).

InSituCrosslinkerMixing:A newmethod of applicationwas explored to overcome the limitations of excess

cross‐linkingpriortowoodapplication. Thein‐situmixingofproteinsolutionand

glutaraldehyde allowed for improvedwood penetration, and cross‐linking to take

place between the wood pieces, forming a strong bond. Using this application

method, strongbondswere formedwithmuchhigherprotein and glutaraldehyde

concentrations.

TheMP concentration had a strong impact on the adhesive strength. This could

simply be due to an increased abundance of interactions that could take place,

allowing more opportunity for hydrogen bonding to the wood, and within the

69

formulation.Theadditionofglutaraldehydealsocontributedtoenhancedadhesion

strengthforthein‐situmethodofapplication.

With the in‐situmixing procedure, theMP solutionwas given a chance to spread

andpenetrate intotheporousstructureof thewoodinterfacebeforecross‐linking

tookplace.Aftertheglutaraldehydewasintroduced,thewoodpieceswerequickly

pressedontoeachother, togiveachanceforMPandglutaraldehydetopenetrate

thewoodbefore reacting, and create amechanical interlockwith thewood. This

allowedfortheformationofastrongproteinnetworkbetweenthewoodpieces.An

understanding of the cross‐linking kinetics would be useful, as it could help

understandhowquicklythebondcanform,andpotentiallyposeanadvantageover

the setting times of synthetic adhesives. The viscosity and DSC results are an

indicationofthecross‐linkingthatcantakeplacebetweenthewoodpieceswiththe

additionofglutaraldehyde. Itisalsopossiblethatglutaraldehydepromotedcross‐

linkingofproteinwiththecellwallpolymersofthewood,acapabilitythathasbeen

suggestedbyXiaoetal.(2010).

Thebond strengthappeared to reachamaximumat4%GL, insteadof improving

with increased GL content. It is possible that the glutaraldehydewas present in

excess,andthereforedidnotcontinuetocontributetocross‐linkingwhenmoreGL

added. Insomecases, thebondstrengthevendecreasedwith increasedGLabove

4%.Asmentioned,glutaraldehydehasbeenshowntopromotecross‐linkingofcell

wall polymers of wood (Xiao et al., 2010). Perhaps the excess glutaraldehyde

70

encouraged cross‐linking between wood cells, forming a stronger wood network

thatismoredifficulttopenetrate.

ThemaximumstrengthofMPbasedadhesives(2.31MPa)wasstillnotquiteashigh

as the 4.40MPa achieved for commercial white glue, produced with polyvinyl

acetate(PVAc),pressedundersimilarconditions.However,theMPbasedadhesives

may have an advantage over PVAc when it comes to thermal stability. The DSC

analysisformustardproteinindicatesaglasstransitiontemperature(Tg)ofaround

90°C,whereasaTgof40°ChasbeenreportedforthePVAcpolymer(Crispimetal.,

1999).PerhapsMPbasedadhesivescanbeadvantageousinmaintainingabondin

hightemperatureenvironments.

AlthoughtheMPadhesivemaynotbeasstrongascommercialwhiteglue,thisnew

methodofapplicationhasresultedin improvementsfrompreviousadhesivework

withMPunder similarpressing conditions. WorkbyGarcia‐Becerra et al. (2012)

reportedastrengthofapproximately1MPaforMPadhesive,pressedunderadead

weight in ambient conditions. It is also important to note that those tests were

pressed under favourable pressing conditions of a higher pressure (47kPa) and

lower relative humidity curing environment of 30%RH. In thiswork, a bond of

more than double the strength was achieved under 8.6kPa in a 60% RH curing

environment.

71

The MP adhesives also demonstrated good water resistance. After soaking, the

bondstrengthoftheformulationcontaining40%MPand4%GLmixedin‐situwas

notstatisticallydifferentformthedrystrength.Themoistureresistancesuggestsa

potential performance advantage over soy protein based adhesives, which have

shownreducedadhesivestrength,andevencompletebondfailure,withsoakingor

exposure tohighhumidity environments (Hettiarachchyet al., 1995;Kalapathyet

al.,1995;Zhongetal.,2001)

RASAdhesion:

The modifications to RAS based adhesives did not result in the same kind of

adhesives enhancements as for MP, but improvements were still realized. The

viscosity measurements in Figure 3‐2 did show increased viscosities for

formulationswithglutaraldehyde,andforthosepre‐heattreatedwiththepresence

ofglutaraldehyde.Thissuggeststheabilityfortheglutaraldehydetoreactwiththe

biopolymers inRAStoencouragesomecross‐linking,andfortheheatexposureto

enhance this reaction. However, this did not necessarily translate to adhesive

strength,which is consistentwith the findings in previousRAS adhesivework by

Garcia‐Becerra et al. (2012), suggesting an unclear relationship between viscosity

and adhesive strength. The concentration of the extract appeared to have the

strongesteffectonadhesivestrength.

72

There was some indication on the ability for the pre‐heat treatment to aid in

bondingability,whichwasnotreflectedinthesheartestresults.Thereweretests

prepared for formulations containing 30% RAS extract were prepared with no

glutaraldehyde,1%GL,and1%GLwithheattreatment.Theformulationwithheat

treatment was the only one that maintained a bond after five days of curing.

Althoughbondwasnotstrongenoughtobemeasuredwithinthelimitsoftheshear

testingequipment, thiscouldbean indicationof improvedmoistureresistance,as

thebondwasmaintainedinahumid,60%RHenvironment.

Glutaraldehydeadditiondidnothavethesameimpactontheadhesivestrengthasit

did forMP. With such a heterogeneousmixture of biopolymers inRAS, it can be

expected that glutaraldehyde would not react the same as it would for a pure

proteinsolutionsuchasMP.Theproteincontentonlymakesupabout23%ofthe

organiccontentintheRAS,ascharacterizedbyGarcia‐Becceraetal.(2010),which

was the reason for trying suchahighconcentrationof60%RAS. Inaddition, the

proteins in RAS are not well characterized, and may not contain the free amino

groups,suchaslysine,thatarefavourableinglutaraldehydeaidedcross‐linking.

ImportantimprovementswerestillmadeincomparisontopreviousworkwithRAS

basedadhesives.PreviousRASextractformulationscouldonlyformabondundera

hot press (0.26MPa, 150°C). No bond was formed when pressed at room

temperatureundera lowerpressureof47kPa (Garcia‐Becceraet al., 2012). With

the modifications in this work, reasonably strong bonds were achieved when

73

pressedundera lowerpressure (8.6kPa)at room temperature, and thebondwas

maintainedinahigherhumidityenvironment(60%RH).

Crosslinkingagent:

Glutaraldehyde is one of themost extensively used protein cross‐linkers, and has

shown to be effective in thiswork, especiallywithmustard protein. However, it

wouldbe interesting toexplore theeffectsofalternativecross‐linkingagentswith

similarstructurestoglutaraldehyde,suchassimpledialdehydesthatonlydiffer in

the carbon chain lengthbetween aldehyde groups. For example,malondialdehyde

and adipaldehyde are simple dialdehydes known to form protein cross‐links.

Glutaraldehyde has a three carbon chain between aldehydes to form the

COH(CH2)3COHstructurethatcanbeseeninFigure3‐1,whereasMalondialdehyde

hasonlyoneCH2group,andadipaldehydehasfour(Wong,1993).Itispossiblethat

variations in the chain length can have an impact on cross‐linking and bond

strength.

Aconcernwiththeuseofglutaraldehydeistoxicity.Althoughitiscommonlyused

asadisinfectantatlevelsofupto2%insolution,itsvaporscanbetoxicatlowlevels

(legalexposurelimitof0.2ppm),sotheliquidformmustbehandledwithcareina

well ventilated environment (California Department of Public Health, 1995).

Therefore,alongwiththebenefitstowoodworkingapplications,theabilitytoform

a bond under low temperature conditions when incorporating glutaraldehyde is

veryimportantfortoxicityreasons. Apotentialalternativecross‐linkertoaddress

74

this issue is genepin. Genipin is a naturally occurring substance that has been

shown to cross‐link proteins. It has beenused in biomedical andpharmaceutical

applications,andhasbeenexploredasanalternativetoglutaraldehydeduetolower

toxicityandincreasedbiocompatibility(Muzzarelli,2009).

3.5 ConclusionModifications to mustard protein (MP) and Return Activate Sludge (RAS) extract

adhesive formulations led to improvedadhesivecapabilities.Theuseof thecross‐

linkingagent,glutarldehyde,andheattreatment,weremodificationsmadetoinduce

conformationalandstructuralchangestobenefitadhesiveperformance.Thein‐situ

methodofapplyingtheadhesivesolutionandcross‐linkerhadthemostsignificant

impact on the adhesive strength of MP formulations. This application method

allowedforwoodpenetration,andthe formationofastrongcross‐linkednetwork

betweenwoodpieces.

TheeffectsofthesemodificationswerenotaspronouncedforRASbasedadhesives.

However, with increased concentrations, bonds could be formed with RAS

formulations under low pressure and ambient temperature pressing conditions,

which cannotbe said forpreviousRASadhesivework. Theability to formbonds

underlowpressureandambienttemperaturesexpandstheapplicabilityofMPand

RAS based adhesives to uses such as cabinetry,musical instrument building, arts

andcrafts,andothergeneralinteriorwoodassembly.

75

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Frihart,C.R.(2005).Woodadhesionandadhesives.InR.M.Rowell(Ed),Handbook

ofwoodchemistryandwoodcomposites(pp.15‐273).BocaRaton,FL:CRCPress,Inc.

GarciaBecerra,F.Y.,Acosta,E.J.,Allen,D.G.(2010).Alkalineextractionof

wastewateractivatedsludgebiosolids. Bioresour.Technol.,101(18),6983‐6991.

Garcia‐Becerra,F.Y.,Acosta,E.J.,Allen,D.G.(2012).Woodadhesivesonalkaline

extractsfromwastewaterbiosolidsandmustardprotein.J.Am.OilChem.Soc.,89(7).1315‐1323.

Haag,A.P.(2006).Mechanicalpropertiesofbacterialexopolymeric adhesivesandtheircommercialdevelopment.InA.M.Smith,&J.A.Callow

(Eds.),Biologicaladhesives(pp.1‐19).Berlin:Springer.Haag,A.P.,Maier,R.M.,Combie,J.,&Geesey,G.G.(2004).Bacteriallyderived

biopolymersaswoodadhesives.Int.J.Adhes.Adhes.,24(6),495‐502.Hettiarachchy,N.S.,Kalapathy,U.,&Myers,D.J.(1995).Alkali‐modifiedsoyprotein

withimprovedadhesivesandhydrophobicproperties.J.Am.OilChem.Soc.,72(12),1461‐1464.

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Kalapathy,U.,Hettiarachchy,N.S.,Myers,D.,&Hanna,M.A.(1995).Modificationof

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CHAPTER4 ConclusionandRecommendations

4.1 ConclusionIt was very encouraging to see the hydrophilic RAS and UR extracts show

synergisms in mixtures with AOT, a relatively hydrophobic surfactant. A small

amount of AOT was required to reduce the IFTs by an order of magnitude in

comparisontothewasteextractsurfactantsalone.FurtheradditionofAOTresulted

inoptimalmixturesreachinglowIFTs(<1mN/m)thatwerewellbelowthatofAOT

alone, and approached levels that are relevant to industrial applications.

Furthermore,ultralowIFTs(<0.1mN/m)werereachforamixtureofaURalkaline

extractandAOTwithincreasedconcentrationsandnoadditionofsalt. Thisultra‐

low IFT was reached against hexane, indicating relevance to heavy crude oil

extractionprocesses. The surface tension andCMC results forRAS‐AOTmixtures

alsohadimplicationsforsynergismsbetweenthewastealkalineextractsandAOT.

This was reflected by a negative interaction parameter, β, for mixed surfactants,

indicating surfactant interactions and the formation of mixed micelles. The

synergismwithAOTwas very encouraging for the potential applicability of these

waste biomass extracts as surfactants. Mixtures of RAS and UR alkaline extracts

withAOTwerethenshowntobeeffective inbitumenremoval fromcontaminated

sand.

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These results have very positive implications for waste sources to be used to

produceeffectivesurfactants. RASandURareabundantsourcesof surface‐active

organicmatterthatcanbeextractedusingverysimplealkalineextractionmethods,

buttheseextractsmaynotperformcompetitivelyontheirown.Withtheabilityto

interactinasynergisticmannerwithanothersurfactant,thewasteextractsbecome

an important component in a commercially competitive surfactant formulation.

Thiscanalsoencouragefurtherexplorationofdifferentwastebiomasssourcesfor

useassurfactants.

Theworkonformulationsofadhesives fromalkalineextractsofRASandmustard

protein(MP)revealedthattheimportantfactorsencouragingbondstrengthunder

low pressure, ambient pressing conditions were solids content, cross‐linking of

proteins,andtheabilitytoachievewoodpenetration.FortheMPbasedadhesives,

the in‐situmixingmethodallowedforgoodwoodpenetrationtobeachievedwith

theuseofhigherproteinandcross‐linkercontents,resultingingreatimprovements

tobond strength.The applicationmethodhelped achieve a bond strengthof over

doublethatachieved inpreviousworkwithMPundersimilarpressingconditions.

ImprovementsforRASadhesiveswereprimarilymadethroughtheincreaseinRAS

solidscontent. TheRASadhesivebondstrengthswerenotashighas formustard

protein,butimportantimprovementsweremadeonpreviouswork,wherenobond

couldbeformedforRASundersimilarpressingconditions.

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The success of the in‐situ cross‐linking method was very encouraging for new

applications of protein based adhesives. The primary commercial application for

protein based adhesives has been interior grade plywood production, which

requireshotpressing. Thisworkshowedanewway to take fulladvantageof the

cross‐linking reaction tohelpmaximizewoodpenetrationandbond strength, and

couldpotentiallybeeffectiveforotherproteins.Thestrongbondstrengthachieved

under low pressure and temperature pressing conditions shows potential for

expanding the applicability of protein based adhesives to new interior wood

workingapplicationsthatdonotrequirehotpressing,suchascabinetryandgeneral

artsandcrafts.

4.2 RecommendationsforFutureworkBiobasedSurfactants

o Mixturesandsustainability:giventheability for thewastealkalineextracts

to form synergisms with a hydrophobic synthetic surfactant, it would be

worth examining their ability to form a synergy with more hydrophobic

biosurfactants, further maintaining the theme of sustainability and

improvingtheperformanceofbio‐basedproducts

o New sludge sources: apply alkaline extraction to different industrialwaste

watersourcesthatmaybericherinproteinsandlipids,possiblyresultingin

moresurfaceactiveextracts

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o Performbitumenremoval testunderconditions thatmaybemorerelevant

toindustrialapplications

o Modeling: the CMC and surface tension data formixtureswas applied to a

simplemodeltoexploresynergisms.Furthermodelingcouldbeusedtohelp

characterize the waste extracts, and the interfacial tension behaviour of

mixtures with AOT, using frameworks such as HLD‐NAC (Hydrophilic‐

LipophilicDifference–NetAverageCurvature)

BiobasedAdhesives

o ProteinfromWasteSources:thebondstrengthofRASappearstobestrongly

correlatedtoproteincontent.Toimprovethestrengthofadhesivesbasedon

RAS, future work should explore extracting out the proteins, possibly

through acid precipitation. Also, other sludge sources that may be more

proteinrichthanRASshouldbeexplored

o Cross‐linking agents: Glutaraldehyde was effective, but other cross‐linking

agentsmaybeevenmoreeffective in forminga strongbond. Tests canbe

conducted with other dialdehydes with varying structures between the

aldhehydeendgroups.Also,alesstoxiccross‐linkingagent,suchasgenipin,

should be investigated to address health concerns associated with using

glutaraldehyde

o Other proteins: Given the success of the in‐situ mixing method, other

proteins that are commercially available and have been used for adhesive

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production,suchassoyprotein,shouldbetestedusingthisnewapplication

method

o Applications: Given the encouraging results for wood bonding, MP or RAS

basedadhesivescouldbetestedinotheradhesiveapplications,suchaspaper

orcardboardbinding

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NomenclatureAOT–SodiumDioctylSulfosuccinate(AerosolOT)

β‐Aninteractionparameterformixedsurfactantsystemsandmixedmicellization

CMC–CriticalMicelleConcentration(gTOC/L)

CV365–UrbanRefuseextractfrommixofpublicgreenparktrimmingandfood

residuesaerobicallycompostedfor365days

DSC–DifferentialScanningCalorimetry

FORSUD–UrbanRefuseextractfromanaerobicdigestateofsolidurbanwaste

GL–Glutaraldehyde

HLD–Hydrophilic‐LipophilicDifference

HT–HeatTreatment

IFT–InterfacialTension(mN/m)

MP–MustardProtein

RAS–ReturnActivatedSludge

ST–SurfaceTension(mN/m)

TOC–TotalOrganicCarbon

UR–UrbanRefuse

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APPENDIXCalculationsandDiagrams

CriticalMicelleConcentrationCalculation

InChapter2, thecriticalmicelleconcentration(CMC)wascalculatedbasedon the

typical surface tension vs. log(concentration) graph. The Gibbs adsorption

relationship was used to calculate a precise CMC for the bio‐based surfactant

formulations.Itisbasedonalinearrelationshipbetweensurfacetensionandlogof

concentration (logC), until the breaking point where surface tension remains

constant. The linearrelationshipwas firstdetermined,andtheCMCoccursat the

intersection between the linear equation and the surface tension after the CMC.

FigureA‐1demonstratesthegeneralrelationship(Rosen,2004).

FigureA1(Rosen,2004)

SampleCalculation:

The following sample calculation is for Return Activated Sludge (RAS). The

followingfigure,whichcanbeseeninFigure2‐1presentedintheChapter2results,

shows the relationship between surface tension and concentration of RAS. The

concentrationisintermsofTotalOrganicCarbon(gTOC/L).

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FigureA2

Step1:AlinearrelationshipwasestablishedbetweensurfacetensionandLog(C)untilwhat

appears to be the breaking point, inwhich the surface tension becomes constant.

Thesurfacetensionisrepresentedbythesymbol,γ.

(1)

Whichinthisexamplecameoutto:

Step2:FindtheaveragesurfacetensionbeyondtheCMC,asitappearstoremainconstant:

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Step3:Usethatsurfacetensionvaluetosolveforconcentrationintheequationobtainedin

Step1.ThisconcentrationisthemreportsastheCMC.

SolveforCMC:

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BitumenRemovalAnalysis

InChapter2,theabilityforsurfactantformulationstoremovebitumenfrom

contaminatedsandwasinvestigated.Therearetwokeycomponentstocalculating

theoilrecovery(%)forthebitumenremovalexperiments.

1)Surfactantwashing–Residualoilremoval(ROR)The contaminated sand is first washed with the surfactant formulations. The

amountofoilthatwasremovedbythesurfactant isdeterminedbycalculatingthe

amount of oil that was not removed, residual oil, in terms of absorbance. The

residualoil iscompletelyremovedbytolueneandtheabsorbancereadingistaken

for Residual Oil Removal (AROR). The absorbance reading is taken by diluting the

washingfluidexperimentinthecuvette(20µLin1.5mLoftoluene).RefertoFigure

A‐3belowforaschematicofthisprocedure.

FigureA3–Soilwashingandresidualoilreadings

2)Referenceabsorbance–Positivecontrolfor100%Recovery

A reference absorbance reading must be obtained to represent 100% bitumen

removal(AR).Thisisobtainedbywashingcontaminatedsand(5wt%bitumen)with

4mLofToluene.Thetoluenecontainingallofthebitumenisdilutedinacuvettefor

absorbancereadings.Thefollowingdiagramhelpsdemonstratethisprocess

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FigureA4–Referenceabsorbancereadingfor100%oilremoval

Calculationforoilremoval:Sincetheabsorbancereadingforoilrecoveryisbasedontheresidualoil thatwas

NOT recovered by the surfactant, the calculation for oil removal achieved by the

surfactantmustbeadjustedaccordingly:

AROR–ResidualOilRemoval

AR–Referencefor100%oilremoval

(2)

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ElementalAnalysisConcentrationsInChapter2,anelementalanalysiswasperformedandnormalizedtomassoforganiccarboninTable2‐2.Thefollowingtableshowstheelementalconcentrationsin(mg/L).TableA1

Concentration(mg/L)

Constituent RAS FORSUD FORSUDashfree

CV365

TOC 3,214.7 25,453.3 22,153.3 32,196.7TN 845.8 4847.0 3327.0 4088.0Na 2,201.0 3,590.0 2,100.0 3,480.0S 83.8 537.2 329.3 914.7K 70.9 248.0 12.2 918.0Si 52.4 522.0 460.0 2,050.0Fe 52.3 216.0 1.9 1,180.0Al 51.0 280.0 7.7 732.0Ca 31.5 554.0 24.5 3,380.0B 17.8 296.0 299.0 292.0Mg 6.5 76.8 7.7 655.0Cu 4.1 7.0 2.4 30.0Zn 2.3 17.6 0.8 65.3Ti 2.0 8.2 1.5 56.5Ba 0.4 33.0 0.3 18.6Ni 0.3 2.2 0.0 7.7Mn 0.3 9.3 0.0 87.3Cr 0.1 1.3 0.4 6.4

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CalculationofBeta(β)ParameterforRAS‐AOTMixtureAsimplephaseseparationmodel formixedmicellizationwasusedincombination

withregularsolutiontheorybyRubingh(1979).ThepaperbyHassanetal.(1995)

was used as a guide for the calculations. The calculations are based on

approximationsformolecularweightofRASmadetoconvertalloftheparameters

on a molar basis. The molecular weight of RAS was based on the chemical

characterizationbyGarcia‐Becerra(2010).

Fornon‐idealmixtures,thecmc*,theharmonicmeanoftheCMCsoftheindividual

components could be calculated using an activity coefficient (fi), representing the

degreeofinteractionbetweenspeciesinthemicelle.

(3)

Where:

(4)

αI–Bulkphasemolefractionxi‐molefractionofthecomponentinthemicelleWithβbeingthekeyparameterweareinterestedincalculating.UsingasequenceofequationsinHassanetal.(2005).

(5)

Theseequationsworkonamolarbasis,sothefirststepistoconverteverything.

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Step1:ConverttoMolarBasisConcentration:RAS:Convertmassintermsoforganiccarbon(gTOC)tototalmassofsolids(gsolids)Basedonelementalformulafororganicmatter:C4H7O2NWherecarbonmakesupfor47.5%ofthetotalmass:MolecularweightofRAS:

BasedonanalysisfromGarcie‐Becerraetal(2010):56,000Da(g/L)

ConversionFactorofgTOCtomolesofRAS:

Therefore:

CMCofRAS:CMCRAS=1.34gTOC/Lx

AOT:Molecularformula:C20H37NaO7S:Carbonmakesupfor58.8%ofthetotalsolidsMolecularWeight:444.56g/mol

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UsingsameprocessasforRAS,findtheconversionfactor:

CMCofAOT(0.77gTOC/L):

Usingtheaboveconversionfactorstoconverteverythingtoamolarbasis,themole

fractionandtheCMCofthemixturecanbedetermined:

Mixture:Themixtureusedwas40%RAS,60%AOTonaweightbasisofTotalOrganic

Carbon(TOC).

ThetotalmolesofthemixturepergramofTOCofthemixtureiscalculatedbasedon

theconversion factorsaboveand themass ratios. Thisvaluecan thenbeused to

calculatethemolarratiosandtheCMCofthemixture.

Molesofmixture:

MoleFractions:RAS(α1)=0.0065

AOT(α2)=0.9945CMCMixture:CMCMixture=2.08mol/L

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Step2:Calcualateβ Now,thateverythingisconvertedtoamolarbasis,theCMCforpureRAS,AOT,the

Mixture,andthemolar fractions,canbeappliedto thephaseseparationapproach

formixedmicellizationwith the use of an activity coefficient, calculated using an

interactionparameter,β.

Substitutingequation4intoequation5equationforeachcomponent,youget:

Where the two unknowns are the micelle mole fraction, xi, and the interaction

parameter,β. Usingasolverinexcel,themicellemolefractionofeachcomponent

wascalculated,andthenβwascalculated:

β =0.216

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ReferencesGarciaBecerra,F.Y.,Acosta,E.J.,&Allen,D.G.(2010).Alkalineextractionof

wastewateractivatedsludgebiosolids.Bioresour.Technol.,101(18),6983‐91.Hassan,P.A.,Bhagwat,S.S.,&Manohar,C.(1995).Micellizationandadsorption

behaviorofbinarysurfactantmixtures.Langmuir,11(2),470‐473.Rosen,M.J.(2004).Surfactantsandinterfacialphenomena.(3rded.,pp.379‐414).

Hoboken,N.J.:Wiley‐Interscience.Rubingh,D.N.(1979).InK.L.Mittal(Ed.)SolutionChemistryofSurfactants:Volume

1(pp.337)NewYork:PlenumPress.

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