Environmental Chemistry for a Sustainable World Green Materials for Energy, Products and DepollutionEric LichtfouseJan SchwarzbauerDidier Robert EditorsGreen Materials for Energy, Productsand DepollutionEnvironmental Chemistry for a Sustainable WorldVolume 3For further volumes:http://www.springer.com/series/11480Series EditorsEric LichtfouseJan SchwarzbauerInstitute of Geology and Geochemistry of Petroleum and Coal,RWTH Aachen University, Aachen, GermanyDidier RobertUniversit e de Lorraine, Saint-Avold, FranceUMR, Agrocologie, Dijon, FranceICPEES, eEric Lichtfouse Jan Schwarzbauer Didier RobertEditorsGreen Materials for Energy,Products and Depollution1 3ISSN 2213-7114 ISSN 2213-7122 (electronic)ISBN 978-94-007-6835-2 ISBN 978-94-007-6836-9 (eBook)DOI 10.1007/978-94-007-6836-9Springer Dordrecht Heidelberg New York LondonLibrary of Congress Control Number: 2013945557 Springer Science+Business Media Dordrecht 2013This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part ofthe material is concerned, specically the rights of translation, reprinting, reuse of illustrations, recitation,broadcasting, reproduction on microlms or in any other physical way, and transmission or informationstorage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodologynow known or hereafter developed. Exempted from this legal reservation are brief excerpts in connectionwith reviews or scholarly analysis or material supplied specically for the purpose of being enteredand executed on a computer system, for exclusive use by the purchaser of the work. Duplication ofthis publication or parts thereof is permitted only under the provisions of the Copyright Law of thePublishers location, in its current version, and permission for use must always be obtained fromSpringer.Permissions for use may be obtained through RightsLink at the Copyright Clearance Center. Violationsare liable to prosecution under the respective Copyright Law.The use of general descriptive names, registered names, trademarks, service marks, etc. in this publicationdoes not imply, even in the absence of a specic statement, that such names are exempt from the relevantprotective laws and regulations and therefore free for general use.Whiletheadviceandinformationinthisbookarebelievedtobetrueandaccurateat thedateofpublication, neither the authors nor the editors nor the publisher can accept any legal responsibility forany errors or omissions that may be made. The publisher makes no warranty, express or implied, withrespect to the material contained herein.Printed on acid-free paperSpringer is part of Springer Science+Business Media (www.springer.com)EditorsEric LichtfouseDijon, FranceDidier RobertSaint-Avold, FranceJan SchwarzbauerInstitute of Geology and Geochemistryof Petroleum and CoalRWTH Aachen UniversityAachen, GermanyUMR, AgrocologieICPEESUniversit e de Lorraine ePrefaceThere are always owers for those who want tosee them.Henri MatisseNatureisthebest greenandenvironmental chemist. Most biological reactionsoccur at room temperature and thus save energy, whereas many synthetic reactionsneed high temperature. Most biological reactions are quantitative and thus do notproduce undesired by-products, whereas synthetic reactions are rarely quantitativeand thus generate substantial amounts of undesired, sometimes toxic, by-products.Most biological reactions occur in water, a safe solvent, whereas many syntheticreactions are still often performed in hazardous solvents. Most biological productsare useful, recyclable andrecycled, whereas manysynthetic products are notdegradable and end up polluting the environment for centuries. Living organismspossess the best catalysts called enzymes, which should be awarded the Nobel Prizein chemistry if Nobel Prizes were given to natural substances, whereas syntheticcatalysts almost never reach the natural efciency and often contain toxic metals.Biological reactions use renewable products that are usually available in the verynear surroundings of the living organism, whereas human chemistry often use non-renewablesubstancesextractedfromdeepearthoresthenshippedthousandsofmiles, thereby wasting time, money and energy. In short, despite several centuriesof advanced scientic progress, human chemistry has still a lot to learn from naturechemistry. Therefore the positive side of actual society issues or seeing owersas suggested by Henri Matisse is that there is a huge progress margin for theimagination of green chemists.Thisbookpresentsthefollowingkeyexamplesof greenmaterialsandsaferemediation: Biofuel from microalgae is a very promising fuel because microalgae is rapidlyrenewable, doesnot competewithfoodproductionanddoesnot needhugesurface area for production (Fig. 1, Chap. 1).vvi PrefaceFig. 1 Biofuel production from microalgae is fast and does not compete with food productionfrom agricultural crops. Source: Goncalves et al. Biodiesel from microalgal oil extraction, Chap. 1 Water and soil cleaning can be performed with unprecedented speed andefciency by novel techniques of electrochemistry and photocatalysis that re-move toxic metals and gas, pharmaceuticals, pathogens, chlorinated polycyclicaromatic hydrocarbons (PAH) and other pollutants (Chaps. 24, 10). Chapter 8presents cheap natural materials such as rice husk, wheat straw and y ash toremove toxic metals. Chapter 7 shows how surfactants can be used to decreasepesticide toxicity. Foodsecuritycanbeimprovedbyawidenumberofbioindicatorsincludingplants, animals and microbes, which betray the presence of pollutants in air, waterand soil (Chap. 5). Sustainable and safe clothes can be designed using natural dyes and antimicro-bials, an old practice that is regaining interest in a fossil-free society (Chap. 6).Thanks for readingEric Lichtfouse, Jan Schwarzbauer and Didier RobertFounders of thejournal Environmental ChemistryLetters andof theEuropeanAssociation of Environmental ChemistryE-mail: Eric.Lichtfouse@dijon.inra.fr, Jan.Schwarzbauer@emr.rwth-aachen.de,Didier.Robert@univ-lorraine.frOther Publications by the EditorsBooksScientic Writing for Impact Factor Journalshttp://fr.slideshare.net/lichtfouse/scientic-writing-for-impact-factor-journalshttps://www.novapublishers.com/catalog/product info.php?products id=42211Environmental Chemistryhttp://www.springer.com/book/978-3-540-22860-8Organic Contaminants inRiverine andGroundwater Systems. Aspects of theAnthropogenic Contributionhttp://www.springer.com/book/978-3-540-31169-0Sustainable AgricultureVol. 1: http://www.springer.com/book/978-90-481-2665-1Vol. 2: http://www.springer.com/book/978-94-007-0393-3R ediger pour etre publi e!http://www.springer.com/book/978-2-8178-0288-6JournalsEnvironmental Chemistry Lettershttp://www.springer.com/10311Agronomy for Sustainable Developmenthttp://www.springer.com/13593viiviii Other Publications by the EditorsBook SeriesEnvironmental Chemistry for a Sustainable WorldISSN: 2213-7114http://www.springer.com/series/11480Sustainable Agriculture Reviewshttp://www.springer.com/series/8380Contents1 Biodiesel from Microalgal Oil Extraction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1Ana L. Goncalves, Jos e C.M. Pires, and Manuel Sim oes2 Electrochemistry and Water Pollution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Subramanyan Vasudevan and Mehmet A. Oturan3 Heterogeneous Photocatalysis for PharmaceuticalWastewater Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69Devagi Kanakaraju, Beverley D. Glass,and Michael Oelgem oller4 Water Depollution Using Ferrites Photocatalysts . . . . . . . . . . . . . . . . . . . . . . . 135Virender K. Sharma, Chun He, Ruey-an Doong,and Dionysios D. Dionysiou5 Bioindicators of Toxic Metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151Slavka Stankovic and Ana R. Stankovic6 Natural Dyes and Antimicrobials for Textiles . . . . . . . . . . . . . . . . . . . . . . . . . . . 229Masoud B. Kasiri and Siyamak Safapour7 Surfactants in Agriculture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287Mariano J.L. Castro, Carlos Ojeda,and Alicia Fern andez Cirelli8 Cheap Materials to Clean Heavy Metal Polluted Waters . . . . . . . . . . . . . . 335Pei-Sin Keng, Siew-Ling Lee, Sie-Tiong Ha, Yung-Tse Hung,and Siew-Teng Ong9 Water Quality Monitoring by Aquatic Bryophytes . . . . . . . . . . . . . . . . . . . . . 415Gana Gecheva and Lilyana Yurukovaixx Contents10 Halogenated PAH Contamination in Urban Soils. . . . . . . . . . . . . . . . . . . . . . . 449Takeshi Ohura, Teru Yamamoto, Kazuo Higashino,and Yuko SasakiIndex. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467ContributorsMariano J.L. Castro Facultad de Ciencias Veterinarias, Centro de EstudiosTransdisciplinariosdel Agua(CETA-INBA-CONICET), UniversidaddeBuenosAires, Ciudad de Buenos Aires, ArgentinaAliciaFern andezCirelli Facultad de Ciencias Veterinarias, Centro de EstudiosTransdisciplinariosdel Agua(CETA-INBA-CONICET), UniversidaddeBuenosAires, Ciudad de Buenos Aires, ArgentinaDionysiosD. Dionysiou EnvironmentalEngineeringandScienceProgram, 705Engineering Research Center, University of Cincinnati, Cincinnati, OH, USARuey-an Doong Department of Biomedical Engineering and EnvironmentalSciences, National Tsing Hua University, Hsinchu, TaiwanGana Gecheva Faculty of Biology, University of Plovdiv, Plovdiv, BulgariaBeverleyD. Glass School of PharmacyandMolecular Sciences, JamesCookUniversity, Townsville, QLD, AustraliaAna L. Goncalves Faculdade de Engenharia, LEPAE, Departamento deEngenharia Qumica, Universidade do Porto, Porto, PortugalSie-TiongHa Facultyof Science, Universiti TunkuAbdul Rahman, Kampar,Perak, MalaysiaChun He School of Environmental Science and Engineering, Sun Yat-senUniversity, Guangzhou, ChinaKazuo Higashino The Tokyo Metropolitan Research Institute for EnvironmentalProtection, Koto-ku Tokyo, JapanYung-Tse Hung Department of Civil and Environmental Engineering, ClevelandState University, Cleveland, OH, USADevagiKanakaraju School of Pharmacy and Molecular Sciences, James CookUniversity, Townsville, QLD, Australiaxixii ContributorsMasoud B. Kasiri Faculty of Applied Arts, Tabriz Islamic Art University, Tabriz,IranPei-SinKeng Department of Pharmaceutical Chemistry, International MedicalUniversity, Kuala Lumpur, MalaysiaSiew-LingLee IbnuSinaInstituteforFundamentalScienceStudies, UniversitiTeknologi Malaysia, Skudai, Johor, MalaysiaMichael Oelgem oller School of Pharmacy and Molecular Sciences, James CookUniversity, Townsville, QLD, AustraliaTakeshi Ohura Faculty of Agriculture, Department of Environmental Bioscience,Meijo University, Nagoya, JapanCarlosOjeda FacultaddeCienciasVeterinarias, CentrodeEstudiosTransdis-ciplinarios del Agua (CETA-INBA-CONICET), Universidadde Buenos Aires,Ciudad de Buenos Aires, ArgentinaSiew-TengOng FacultyofScience, UniversitiTunkuAbdulRahman, Kampar,Perak, MalaysiaMehmet A. Oturan Laboratoire G eomat eriaux et Environnement (LGE),Universit e Paris-Est, EA 4508, UPEMLV, 77454 Marne-la-Vall ee, FranceJos e C.M. Pires Faculdade de Engenharia, LEPAE, Departamento de EngenhariaQumica, Universidade do Porto, Porto, PortugalSiyamak Safapour Faculty of Carpet, Tabriz Islamic Art University, Tabriz, IranYuko Sasaki The Tokyo Metropolitan Research Institute for EnvironmentalProtection, Koto-ku Tokyo, JapanVirenderK. Sharma Center of Ferrate Excellence and Chemistry Department,Florida Institute of Technology, Melbourne, FL, USAManuel Sim oes Faculdade de Engenharia, LEPAE, Departamento de EngenhariaQumica, Universidade do Porto, Porto, PortugalSlavka Stankovic Faculty of Technology and Metallurgy, University of Belgrade,Belgrade, SerbiaAna R. Stankovic Faculty of Technology and Metallurgy, University of Belgrade,Belgrade, SerbiaSubramanyanVasudevan ElectroinorganicChemicals Division, CSIR- CentralElectrochemical Research Institute, Karaikudi, IndiaTeruYamamoto The Tokyo Metropolitan Research Institute for EnvironmentalProtection, Koto-ku Tokyo, JapanLilyanaYurukova Institute of Biodiversity and Ecosystem Research, BulgarianAcademy of Sciences, Soa, BulgariaChapter 1Biodiesel from Microalgal Oil ExtractionAna L. Goncalves, Jos e C.M. Pires, and Manuel Sim oesAbstract The rapid development of the modern society has resulted in an increaseddemandfor energy, andconsequentlyanincreaseduseof fossil fuel reserves.Burningfossil fuels is nowadays oneof themainthreats totheenvironment,especially due to the accumulation of greenhouse gases in the atmosphere, whichare responsible for global warming. Furthermore, the continuous use of this non-renewable source of energy will lead to an energy crisis because fossil fuels areof limited availability. In response to this energy and environmental crisis, it is ofextreme importance to search for different energy supplies that are renewable andmore environmentally friendly. Microalgae are a promising sustainable resource thatcan reduce the dependence on fossil fuel. Biodiesel production through microalgaeisactuallyhighlystudied. Itincludesseveral steps, suchascellcultivationandharvesting, oil extraction and biodiesel synthesis. Although several attempts havebeen made to improve biodiesel yields from microalgae, further studies are requiredto optimize production conditions and to reduce production costs.This chapter reviews recent developments on oil extraction for biodiesel pro-duction. Two different processes are distinguished: (i) an indirect route, in whichmicroalgal oil is recoveredinanappropriatesolvent andthenconvertedintobiodiesel through transesterication; and (ii) a direct route, in which the productionof biodiesel is performed directly from the harvested biomass. Both routes, directand indirect, should be preceded by cell wall disruption because this step facilitatesthe access of solvents to microalgal oil. The most advantageous disruption methodsfor lipidextractionareenzymaticandpulsedelectricelddisruptionbecauseenzymes present higher selectivity towards cell walls. In addition pulsed electriceld requires less energy than other disruption methods.A.L. Goncalves J.C.M. Pires () M. Sim oesLEPAE, Departamento de Engenharia Qumica, Universidade do Porto,Rua Dr. Roberto Frias, Porto 4200-465, Portugale-mail: jcpires@fe.up.ptE. Lichtfouse et al. (eds.), Green Materials for Energy, Products and Depollution,Environmental Chemistry for a Sustainable World 3, DOI 10.1007/978-94-007-6836-91, Springer ScienceBusiness Media Dordrecht 201312 A.L. Goncalves et al.For theindirect route, it ispossibletousethreedifferent typesof solventsto recover microalgal oil. Although extraction with supercritical uids has higherextraction efciencies and is safer for the environment, costs are very high. The useof ionic liquids is also safer for the environment, but their cost is also very high. Analternative is the use of organic solvents such as n-hexane because it is less harmfuland has a higher selectivity for neutral oil fractions than other organic solvents. Weconclude that the direct route, which involves production of biodiesel directly fromthe microalgal biomass, is more efcient. Indeed, the application of the direct routeto the microalga Schizochytrium limacinum resulted in a biodiesel yield of 72.8 %,while the indirect route, in the same conditions, has resulted in a biodiesel yield of63.7 %.Keywords Biofuel Algae Microalgae Oil extraction Liquid Transesterication Chlorella vulgaris Schizochytrium Limacinum Cellwall disruption Pulse electric eld Enzymatic disruptionContents1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2 Lipid Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41.2.1 Cell Disruption Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71.2.2 Lipid Extraction Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111.3 Biodiesel Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151.3.1 Transesterication Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231.1 IntroductionThedepletionof fossil fuelsreservesandtheeffect of exhaust gasemissionsonglobal climatechangehavestimulatedthesearchforsustainablesourcesofenergy that are carbon neutral or renewable. As an alternative energy source, muchattention has been paid to biodiesel production from vegetable oil crops, such aspalm, rapeseed and soybean, and animal fats (Demirbas 2011; Ranjan et al. 2010).However, biodiesel production yields from oil crops and animal fats do not achievethe current demand for transport fuels (Chisti 2007; Demirbas 2011). Furthermore,producingbiodiesel fromvegetablecropsistimeconsumingandrequiresgreatareas of arable land that would compete with the one used in food crops, leadingto starvation in developing countries (Costa and de Morais 2011; Demirbas 2011;Demirbas and Demirbas 2011).Avoiding the competition between energy and food production, attentions arenow focused on evaluating the potential of microalgae as oil source for biodieselproduction. Microalgae are eukaryotic photosynthetic microorganisms that can befound in aquatic or terrestrial ecosystems (Fig. 1.1). They present several advantages1 Biodiesel from Microalgal Oil Extraction 3Fig. 1.1 Microalgae: (a) microscopic photograph of the microalgaChlorellavulgaris; (b) mi-croscopic photograph of the microalga Pseudokirchneriella subcapitata; (c) microalgal culturinginsmallasks;(d)microalgalculturinginhorizontaltubularphotobioreactors;(e)microalgalculturing in raceway ponds; and (f) harvesting of microalgae through sedimentation. (a), (b), (c),and (f) were obtained from our research group; (d) from http://badger.uvm.edu; and (e) from http://www.abc.net.auover oil crops, including: (i) higher oil contents; (ii) higher growth and biomassproductionrates; (iii) shorter maturityrates; and(iv) requirefar lesslanddueto higher oil productivities (Chisti 2007; Mercer and Armenta 2011). As well asmicroalgae, there are other photosynthetic microorganisms with potential interest.These prokaryotic microorganisms, known as cyanobacteria, behave similarly tomicroalgae and present the same advantages. Despite the referred advantages, the4 A.L. Goncalves et al.production of biodiesel from microalgae is not economically viable. Technolog-ical improvementsshouldbeperformedtoreducetheassociatedcosts, includ-ing: (i) improvement of photosynthetic efciency by the study of photobioreactordesign; (ii) reduction of water and carbon dioxide losses in microalgal cultures;(iii)improvement ofenergybalanceforwaterpumping, CO2transfer, biomassharvesting, oil extraction and biodiesel synthesis; and (iv) use of ue gas as CO2source. This review focuses on the oil extraction and biodiesel synthesis, presentingthe research advances in the associated processes.1.2 Lipid RecoveryAfter cell cultivation, the downstream process towards the production of biodieselincludes: (i) harvesting of microalgae; (ii) drying or dewatering; (iii) cell disruptionand oil extraction; and (iv) transesterication reaction (Amaro et al. 2011; Brennanand Owende 2010), as it is possible to see in Fig. 1.2.Oilextractionfrombiomassrequiresaspecicsolvent withgreat afnitytomicroalgal oil. Extraction procedures involving organic solvents, supercritical uidsandionicliquids arethemost commonappliedmethods torecover oil frommicroalgae (Amaro et al. 2011; Mercer and Armenta 2011; Taher et al. 2011; Kimet al. 2012). Although these procedures can be applied directly to the dewateredbiomass, it is reported that their efciency is very low because microalgae presentcell walls that block the access of solvents to cytosol (Cravotto et al. 2008; Lee et al.2010), the cell compartment where the majority of microalgal lipids accumulate(Chen et al. 2009). To overcome the low efciencies associated to the application ofsolvent extraction methods, some authors have reported the application of cell walldisruption methods, such as (i) enzymatic disruption; (ii) pulsed electric eld; (iii)ultrasound and microwave; and (iv) expeller pressing (Amaro et al. 2011; Cravottoet al. 2008; Lee et al. 2010; Mercer and Armenta 2011; Taher et al. 2011). Table 1.1Fig. 1.2 Stepsinvolvedintheproductionofbiodiesel frommicroalgal biomass. PEFpulsedelectric eld1 Biodiesel from Microalgal Oil Extraction 5Table1.1EffectivenessofsomeoilextractionmethodsappliedinfattyacidrecoveryfromdifferentmicroalgaeExtractionmethodMicroorganismTotalfattyacidsa%(wFA/wDW)ReferenceOrganicsolvent(n-hexane)Crypthecodiniumcohnii4.8Cravottoetal.(2008)Ultrasonic-assistedOrganicsolvent(n-hexane)Crypthecodiniumcohnii25.9Microwave-assistedOrganicsolvent(n-hexane)Crypthecodiniumcohnii17.8Organicsolvent(ethanol:n-hexane1:1v/v)Scenedesmusdimorphus6.3Shenetal.(2009)Ultrasonic-assistedOrganicsolvent(ethanol:n-hexane1:1v/v)Scenedesmusdimorphus21.0FrenchpressOrganicsolvent(ethanol:n-hexane1:1v/v)Scenedesmusdimorphus21.2Organicsolvent(ethanol:n-hexane1:1v/v)Chlorellaprotothecoides5.6Ultrasonic-assistedOrganicsolvent(ethanol:n-hexane1:1v/v)Chlorellaprotothecoides10.7FrenchpressOrganicsolvent(ethanol:n-hexane1:1v/v)Chlorellaprotothecoides14.9Organicsolvent(chloroform:methanol,3:1v/v)Scenedesmussp.2.0Ranjanetal.(2010)Ultrasonic-assistedBlighandDyer(chloroform:methanol,3:1v/v)Scenedesmussp.6.0Organicsolvent(n-hexane)Scenedesmussp.0.6Organicsolvent(chloroform:methanol,1:1v/v)Botryococcussp.7.9Leeetal.(2010)Microwave-assistedOrganicsolvent(chloroform:methanol,1:1v/v)Botryococcussp.28.6Organicsolvent(chloroform:methanol,1:1v/v)Chlorellavulgaris4.9Microwave-assistedOrganicsolvent(chloroform:methanol,1:1v/v)Chlorellavulgaris9.9Organicsolvent(chloroform:methanol,1:1v/v)Scenedesmussp.1.9Microwave-assistedOrganicsolvent(chloroform:methanol,1:1v/v)Scenedesmussp.10.4Organicsolvent(n-hexane;10h)Scenedesmusobliquus46.9Balasubramanianetal.(2011)Microwave-assistedOrganicsolvent(n-hexane,0.5h)Scenedesmusobliquus77.1Organicsolvent(water:methanol:chloroform,0.8:2:1v/v/v;18h)Marinemicroheterotroph25.8Lewisetal.(2000)Organicsolvent(chloroform:methanol:water,1:2:0.8v/v/v;18h)Marinemicroheterotroph35.0Organicsolvent(n-hexane)Spirulinamaxima4.1GouveiaandOliveira(2009)(continued)6 A.L. Goncalves et al.Table1.1(continued)ExtractionmethodMicroorganismTotalfattyacidsa%(wFA/wDW)ReferenceOrganicsolvent(n-hexane)Chlorellavulgaris5.1Organicsolvent(n-hexane)Scenedesmusobliquus17.7Organicsolvent(n-hexane)Dunaliellatertiolecta16.7Organicsolvent(n-hexane)Nannochloropsissp.28.7Organicsolvent(n-hexane)Neochlorisoleoabundans29.0Organicsolvent(ethanol,96%v/v)Phaeodactylumtricornutum6.4Fajardoetal.(2007)scCO2(70.0MPa;55C;10kg.h

1;1.4h)Nannochloropsissp.23.0Andrichetal.(2005)Organicsolvent(n-hexane;8h)Spirulinaplatensis7.8Andrichetal.(2006)scCO2(70.0MPa;55C;10kg.h

1;0.75h)Spirulinaplatensis7.7Organicsolvent(chloroform:methanol,2:1v/v)Crypthecodiniumcohnii19.9Coutoetal.(2010)scCO2(20.0MPa;40C;0.6kg.h

1;3h)Crypthecodiniumcohnii6.9scCO2(25.0MPa;40C;0.6kg.h

1;3h)Crypthecodiniumcohnii6.3scCO2(30.0MPa;40C;0.6kg.h

1;3h)Crypthecodiniumcohnii5.7scCO2(20.0MPa;50C;0.6kg.h

1;3h)Crypthecodiniumcohnii5.6scCO2(25.0MPa;50C;0.6kg.h

1;3h)Crypthecodiniumcohnii7.1scCO2(30.0MPa;50C;0.6kg.h

1;3h)Crypthecodiniumcohnii8.6Organicsolvent(n-hexane;5.5h)Chlorococcumsp.3.2Halimetal.(2011)scCO2(30.0MPa;60C;18.5kg.h

1;1.3h)Chlorococcumsp.5.8scCO2(30.0MPa;80C;18.5kg.h

1;1.3h)Chlorococcumsp.4.8scCO2(35.0MPa;70C;6kg.h

1;4.5h)Nannochloropsisgranulata2.8Bjornssonetal.(2012)IL([Emim]MeSO4)andmethanolDunaliellasp.8.6Youngetal.(2010)IL([Emim]MeSO4)andmethanolChlorellasp.38.0Organicsolvent(chloroform:methanol,2:1v/v)Chlorellavulgaris11.1Kimetal.(2012)IL([Bmim]CF3SO3)andmethanolChlorellavulgaris19.0IL([Bmim]MeSO4)andmethanolChlorellavulgaris17.4aTotalfattyacidsrecoveredarerepresentedasafraction(in%)ofoilweightinthebiomassdryweight1 Biodiesel from Microalgal Oil Extraction 7Table 1.2 Advantages and disadvantages of the cell wall disruption methods and oil extractionprocedures applied to microalgaeProcedure Advantages DisadvantagesCell walldisruptionmethodsEnzymaticdisruptionHigher degradationselectivityEnzymes are very expensiveRequirement of less energythan mechanical methodsPulsed electriceldRequirement of less time andenergy than other appliedmethodsDifculties in operating atlarge scaleHigh operational andmaintenance costsUltrasound andMicrowaveHigher efciencies andreduced extraction timesModerate to high energeticcostsIncreased yieldsExpeller pressing Simple method High power consumptionand maintenance costsUseful for large scaleapplicationsLipidextractionmethodsOrganic solventextractionSimple and inexpensivemethodThe majority of organicsolvents are toxic,harmful and non-reusableTime-consumingSupercriticaluidextractionSupercritical uids arenon-toxic and presenthigher mass transfer ratesHigh energetic andmaintenance costsRecovery of fatty acids iseasierDifculties in scale-upRequirement of less timethan organic solventextractionIonic liquidmediatedextractionIonic liquids are non-toxicand non-volatile andpresent higher solvatationcapacitiesIonic liquids are expensiveIonic liquids can be producedspecically, according totheir applicationshows the most applied extraction methods and the achieved mass percentages ofrecovered oil and Table 1.2 summarizes the main advantages and disadvantages ofthe presented cell disruption methods and oil extraction procedures.1.2.1 Cell Disruption MethodsCell disruption methods aimto disintegrate cell walls to allowthe release of intracel-lular components into an adequate solvent. The methods used in cell wall disruptioncan be classied into mechanical, where cell wall destruction is non-specic, and8 A.L. Goncalves et al.non-mechanical, wheremethodsaremorespecic. Mechanicalmethodsincludebead mill, French press, ultrasound, microwaves and high pressure homogenizer,whereas non-mechanical methods comprise the physical methods thermolysis, de-compression and osmotic shock, the chemical methods, where antibiotics, chelatingagents, solvents and detergents are applied, and the enzymatic methods, which uselytic enzymes (Geciova et al. 2002; Chisti and Moo-Young 1986).Enzymatic DisruptionEnzymes can be applied in oil extraction from microalgae, as they can mediate thehydrolysis of cell walls, enabling the release of their content into an appropriatesolvent. Application of lytic enzymes with little volumes of organic solvent canimprove oil recovery yields, as well as extraction times (Mercer and Armenta 2011).Forcellwalldegradation, cellulasesarethemostappliedenzymes(MercerandArmenta 2011; Sander and Murthy 2009). Although enzymatic extraction has notyet been applied to microalgae, it has been successfully used in oil extraction fromJatropha curcas L. seeds (Shah et al. 2004). Three phase partitioning (TPP) methodand an enzyme-assisted TPP (EATPP) method were applied to recoveroil fromthese seeds. The TPP consisted in the addition of three solvents to the seeds, inorder to form a three phase system. The applied solvents were water, ammoniumsulphate, and t-butanol. The three phases were separated by centrifugation and thephase containing the recovered oil was the one containingt-butanol, which waseliminated through evaporation. TPP and EATPP were performed at different pHconditions: 4.0, 7.0, and 9.0. Higher oil yields were obtained at pH 9.0: (i) 32.0 %(wt.) for TPP; and (ii) 36.8 % (wt.) for EATPP (Shah et al. 2004).Despite being expensive, enzymes offer several advantages over other cell walldisruption methods. They present higher degradation selectivity than mechanicaldisruption methods. Furthermore, microalgal cell walls are more recalcitrant thanthe ones of other microorganisms, being very resistant to degradation. Thus, theuse of mechanical disruption methods requires higher energy amounts (Sander andMurthy 2009).Pulsed Electric FieldThe pulsed electric eld (PEF) technology seems to be a potential alternative for oilextraction from microalgae (Taher et al. 2011). This technique applies brief pulsesof a strong electric eld to cells, which induces non-thermal permeabilization ofmembranes(Guderjan et al. 2005; Taher et al. 2011). In determined conditions,PEF can also cause signicant damage in microalgal cell walls (main barrier for oilextraction in most microalgae), membrane and it can led to complete disruption ofcells into fragments. PEF is a relatively new method that has not yet been appliedto extract microalgal oil. However, evidence of high extraction efciencies in plantproducts, such as maize, olives (Guderjan et al. 2005) andBrassicanapus cells1 Biodiesel from Microalgal Oil Extraction 9(Guderjan et al. 2007), suggests that this can be a suitable method to improve thepermeabilization of microalgal membranes and efciently extract their oil (Mercerand Armenta 2011). Guderjan et al. (2005) used PEF to induce stress in plant cellsand thus recover functional food ingredients, such as phytosterols and polyphenols.The authors applied 120 pulses with eld strength of 0.6 and 7.3 kV.cm-1to maizeand they added a small amount of n-hexane to perform the oil extraction. On theother hand, olives were treated using the following conditions: 30 pulses with eldstrength of 0.7 kV.cm-1and 100 pulses with eld strength of 1.3 kV.cm-1. Afterapplication of PEF the membranes were completely disintegrated and the oil contentwas recovered by centrifugation. Oil recovery obtained for dried maize was 23.5 and23.9 % (wt.) for pulses with 0.6 and 7.3 kV.cm-1, respectively. These results wereobtained for maize treated with PEF and n-hexane and further drying for 24 h, at38 C. Application of electric pulses with eld strength of 0.6 kV.cm-1, followedby n-hexane addition, an incubation period of 24 h and drying for 24 h at 38 C,resulted in an oil yield of 43.7 %, which means that incubation of the mixture withthe organic solvent allows higher oil recovery. Regarding olives, the application ofPEF with strength of 0.7 and 1.3 kV.cm-1followed by centrifugation resulted inan oil yield of 6.5 and 7.4 goil per gmash. Guderjan et al. (2007) applied 120 pulseswith a eld strength of 7.0 kV.cm-1and a duration of 30ps to hulled rapeseed,followed by drying at 50 C for 10 h and an extraction step with n-hexane. Withthese extraction procedures, the authors obtained an oil yield of 32 % (wt.), against23 % obtained without PEF application.PEF requires less time and energy than other applied methods (Guderjan et al.2005) and its use as a pre-treatment for organic solvent extraction requires far lessorganic solvents (usually presenting high toxicity) than the conventional organicsolvent extraction methods, which reduces the energy needs in the extraction process(Guderjan et al. 2007).Ultrasound and MicrowaveAnother method that can be used to promote cell wall disruption of microalgal cellsis the applicationof ultrasounds and microwaves. In ultrasonic-assisted method,microalgal oil canberecoveredbycavitation. Thisphenomenonoccurs whenvapour bubbles of the liquid are formed when the pressure is lower than its vapourpressure. Asthesebubblesgrowwhenpressureisnegativeandcompressunderpositivepressure, aviolent collapseofthebubblesispromoted. Whenbubblescollapse near cell walls, damage can occur, leading to the release of cell contents(MercerandArmenta2011; Taheret al. 2011). Applicationofthisultrasound-assistedmethodtomicroalgal biomass canimprove extractionefciencies byreducingextractiontimes andincreasingoil recoveryyields. Theexperimentsperformed by Cravotto et al. (2008) with the microalgaCrypthecodiniumcohniishowed that cell disruption using ultrasounds increased oil extraction yields from4.8 %, when applying Soxhlet extraction with n-hexane, to 25.9 % (wt.). Shen et al.(2009) used the microalgae Scenedesmus dimorphus and Chlorella protothecoides10 A.L. Goncalves et al.toevaluatetheeffectofsonicationbeforesolventextractionusingamixtureofethanol:hexane in a ratio of 1:1 (v/v). Application of ultrasound-assisted disruptionincreased the oil yields from 6.3 % to 21.0 % (wt.) for S. dimorphus and from 5.6 %to 10.7 % (wt.) for C. protothecoides. Additionally, Ranjan et al. (2010) comparedoil extraction yields from Scenedesmus sp. using the following methods: (i) Blighand Dyers method (1959), organic solvent extraction using a solvent mixture ofchloroform, methanol and water, where solvents were applied in a ratio of 3:1:0(v/v); (ii) ultrasound-assisted extraction followed by the Bligh and Dyers method,using the same mixture of chloroform and methanol. Results obtained with theseexperimentsshowedanincreaseinoilyieldsfrom2.0%to6.0%(wt.), whenapplying method (i) and (ii), respectively. One possible reason for this increase inoil recovery is that when both methods are applied, oil extraction is a result of theinteraction between two phenomena: oil diffusion across the cell wall and disruptionof the cell wall with release of its contents to the solvent (Ranjan et al. 2010).Microwave-assisted method is supported by the principle that microwavesdirectly affect polar solvents and materials. Even when they are applied to driedcells, traceamountsofmoistureareaffected: temperatureincreasesduetomi-crowaves, moisture is evaporated, and pressure in the cells increases, leading to adamage or rupture of the cell wall followed by the release of its contents. Microwavetheory and the extraction principle are described in detail by Mandal et al. (2007).The use of microwaves followed by organic solvent extraction using small amountsof solvent contributes to an efcient and inexpensive extraction procedure, whichdoes not requireprevious biomass dehydration. Cravottoet al. (2008) appliedorganic solvent extraction with n-hexane and microwave-assisted solvent extraction(using the same solvent) to the microalga C. cohnii, achieving oil recovery yieldsof 4.8 % and 17.8 % (wt.), respectively. Furthermore, Lee et al. (2010) used theBlighandDyersmethod(1959)withamixtureofchloroform:methanol intheratioof1:1(v/v)precededbytheapplicationofamicrowavestreatmenttothemicroalgaeBotryococcus sp., Chlorellavulgaris, andScenedesmus sp. With thismicrowave-assisted method, the oil extraction yields obtained for these organismswere28.6, 9.9, and10.4%(wt.), respectively, against the7.9, 4.9, and1.9%(wt.) obtained for the control method Bligh and Dyers method (1959; Lee et al.2010). Recently, Balasubramanian et al. (2011) promoted cell wall disruption ofScenedesmus obliquususingthemicrowave-assistedmethodandcomparedtheachieved results with organic solvent extraction with n-hexane. Disruption using mi-crowaves was performed for 30 min, while organic solvent extraction was performedby 10 h. Oil recovery yields obtained with solvent extraction and the microwave-assistedmethodwere46.9%and77.1%(wt.), respectively(Balasubramanianet al. 2011).Both methods improve signicantly oil extraction from microalgae, presentinghigher efciency, reduced extraction times and increased yields, as well as moderatecosts and negligible added toxicity.1 Biodiesel from Microalgal Oil Extraction 11Expeller PressingPressing techniques lie on the principle that when microalgal cells are submitted tohigh pressures, they start to crush, releasing their contents to an adequate solvent.As the methods described before, pressing techniques can be advantageous whenusingas apre-treatment for organicsolvent extraction. Apre-treatment usingFrench press was applied by Shen et al. (2009) to the microalgaeS. dimorphusandC. protothecoides. After pressing microalgae, the oil was recovered using asolvent system containing ethanol and n-hexane in a 1:1 (v/v) ratio. Comparingextracted oil yields with those obtained without pre-treatment, oil content achievedfor S. dimorphus raised from 6.3 % to 21.2 % (wt.), while for C. protothecoides itraised from 5.6 % to 14.9 % (wt.).Although this method is very simple and has reduced equipment costs, it presentssome disadvantages when compared to other cell disruption methods, such as highpower consumption and maintenance costs.1.2.2 Lipid Extraction MethodsExtraction of microalgal oil can be performed directed to the harvested biomassor in addition to a cell wall disruption method. The second methodology generallypresents higher lipid recoveries, as cell contents are released to the solvent applied.Intherecoveryofmicroalgal oil, it isveryimportant tochooseanappropriatesolvent because this choice can improve lipid recovery yields and reduce processcosts. Additionally, the majority of solvents applied are harmful and toxic, meaningthat the selection of the solvents used should take into account their impact in theenvironment and in public health. The extraction procedures commonly applied toextract microalgal oil include the use of organic solvents, supercritical uids andionic liquids (Mercer and Armenta 2011; Kim et al. 2012).Organic Solvent ExtractionThe use of organic solvents to extract microalgal oil is the most applied extractionmethod. The main organic solvents applied include hexane, cyclohexane, benzene,ethanol, acetone, and chloroform (Brennan and Owende 2010; Mercer and Armenta2011; Grima et al. 2003). These solvents have shown to be quite effective in oilextractionfrommicroalgae. Agoodsolvent for oil extractionmaypresent thefollowingcharacteristics: (i)tobeinsolubleinwater; (ii)tohavehighafnityfor oil, i.e. non-polar, to increase its permeability to cell membrane and also tosolubilise the target compounds; (iii) to have a low boiling point to facilitate itsremoval after extraction; (iv) to have a considerably different density from that ofwater. Furthermore, the organic solvent applied should be inexpensive, non-toxicand reusable (Mercer and Armenta 2011).12 A.L. Goncalves et al.Several studieshavereportedtheuseof achloroform, methanol andwatermixture, known as the Bligh and Dyers method (1959), to extract microalgal oil(Mercer and Armenta 2011). Lewis et al. (2000) studied the effect of applying thesolvents chloroform, methanol and water in different sequences and proportions onan oil-producing strain of a marine microheterotroph, Thraustochytrid ACEM 6063.The authors used the following sequences and ratios: (i) water:methanol:chloroform(0.8:2:1, v/v/v); (ii) chloroform:methanol:water (1:2:0.8, v/v/v); (iii) chloro-form:methanol:water (1:4:0.8, v/v/v). Total fatty acids extracted with these threesolvent systems were 258.5, 350.0, and 326.5 mg.g-1dry weight, respectively. With thisstudy, the authors concluded that changing solvent sequence can have signicanteffectsonextractionyieldsandthat increasingtheproportionof methanol didnot signicantlyaffect theextractionefciency. Later, Leeet al. (2010)usedamixture of chloroform and methanol (1:1, v/v) to extract oil from the organismsBotryococcus sp., C. vulgaris, and Scenedesmus sp. Extraction yields obtained withthismethodwere12.0, 24.9, and18.8mg.g-1dry weightfor Botryococcussp., C.vulgaris, and Scenedesmus sp., respectively.Another common organic solvent applied to extract microalgal oil is n-hexane.Gouveia andOliveira (2009) usedn-hexane todetermine oil contents of themicroalgae Spirulina maxima, C. vulgaris, S. obliquus, Dunaliella tertiolecta,Nannochloropsis sp., andNeochlorisoleoabundans. Oil yields obtained with thissolvent rangedbetween4.1%(wt.) fromS. maximaand29.0%(wt.) fromN. oleoabundans (Gouveia and Oliveira 2009). Shen et al. (2009) applied a solventsystem composed by a mixture of ethanol:n-hexane (1:1, v/v) to the microalgaeS. dimorphus and C. protothecoides. Oil contents obtained with this method were6.3 % and 5.6 % (wt.) for S. dimorphus and C. protothecoides, respectively (Shenet al. 2009).Ranjan et al. (2010) compared oil extraction from theScenedesmus sp. usingtwo organic solvent methods: Soxhlet extraction with n-hexane, and the Bligh andDyers (1959), using a chloroform and methanol mixture in a ratio of 3:1 (v/v). Theachieved oil contents were 0.6 % (wt.) for extraction with n-hexane and 2.0 % (wt.)for extraction with the Bligh and Dyers method, showing that the last method ismost efcient. This can be explained by the non-polar character of n-hexane, whichresults in a lower selectivity of microalgal oil, mainly composed by unsaturated fattyacids, toward n-hexane. On the other hand, chloroform has a polar nature, whichallows a higher selectivity of microalgal oil toward this organic solvent (Ranjanet al. 2010).Fajardo et al. (2007) used ethanol as an organic solvent for oil extraction fromthe microalga Phaeodactylum tricornutum. The authors applied an ethanol solution(96 % v/v) to freeze dried biomass, obtaining a oil yield of 6.4 % (wt.) (Fajardoet al. 2007).Although n-hexane may be less efcient than chloroform, it is less toxic and ithas an apparently higher selectivity for neutral oil fractions (Amaro et al. 2011).The application of this organic solvent coupled with an efcient cell wall disruptionmethod could be a promising alternative to avoid the harmfulness of chloroform.1 Biodiesel from Microalgal Oil Extraction 13Supercritical Fluid ExtractionAn alternative to the use of volatile and toxic organic solvents in microalgal oilextraction is the application of supercritical uids as solvents (Amaro et al. 2011;Halim et al. 2011; Mercer and Armenta 2011). Supercritical uids are compoundsthat behave both as a liquid or a gas when exposed to temperatures and pressuresabove their critical values. The most used supercritical uid for oil extraction isCO2(scCO2) because it presents low critical temperature (31.1 C) and pressure(72.9 atm) (Mercer and Armenta 2011).The scCO2extraction presents several advantages over the traditional organicsolvent extraction procedures, such as: (i) tuneable solvating power; (ii) low toxicityand ammability; (iii) favourable mass transfer rates; and (iv) production of solventfreeextractsbecauseat roomtemperatureCO2behavesasagas(Amaroet al.2011; Crampon et al. 2011; Halim et al. 2011; Macas-S anchez et al. 2007). Themaindisadvantageistheassociatedcost, whichismainlyduetotherequiredinfrastructure and operational conditions (Halim et al. 2011).Efcienciesof oil extractionusingscCO2dependonthefollowingfactors:(i) pressure; (ii) temperature; (iii) CO2owrate; and (iv) extraction time.Furthermore, the use of modiers or co-solvents, such as ethanol can be adjustedto optimize extractions. When ethanol is applied as a co-solvent, polarity of CO2increases and its viscosity is altered, increasing the uid solvating power. In theseconditions, lower temperature and pressure are required, improving the extractionefciency. Another limiting factor of scCO2 extraction is the level of moisture inthe sample. High moisture content can reduce contact time between the solvent andbiomass, making difcult the diffusion of CO2into the sample and the diffusionof oil out of the cell, because microalgal biomass tends to gain a thick consistency(Halim et al. 2011).Studies performed by Mendes et al. (1995) showed that application of scCO2with a gas ow rate of 21.4 kg.h-1at 35.0 MPa and 55 C toC. vulgaris cellsresultedinanoil yieldof 13.3%(wt.). Applicationof organicsolvents, suchasacetoneandn-hexane, resultedinanoil yieldof16.8%and18.5%(wt.),respectively (Mendes et al. 1995). Andrich et al. (2005) applied different extractionconditions using scCO2and also organic solvent extraction with n-hexane to themicroalga Nannochloropsis sp. Extraction procedures allowed the achievement of250 mg.g-1dry weight (23.0 %) using scCO2 (gas ow rate of 10 kg.h-1, 70.0 MPa,and 55 C) and 120 mg.g-1dry weight(12.0 %) using n-hexane at both 52 C androomtemperature(Andrichet al. 2005; Cramponet al. 2011). Later, Andrichet al. (2006) used Spirulina platensis to verify the extraction efciency of scCO2technique using four different pressures (25.0, 40.0, 55, and 70.0 MPa) and twodifferent temperatures (40and55C), withagas owrateof 10kg.h-1. Inaddition to this method, the authors also tested solvent extraction with n-hexane.Results showed that after 45 min, the amount of oil extracted reached its maximum(78.2 mg.g-1dry weight) for extraction performed at 55 C and 70.0 MPa. The sameamountofextractedoilwasobtainedafter2.5handafter3.5h, forextraction14 A.L. Goncalves et al.performed at 55 C and 40.0 MPa and at 40 C and 40.0 MPa, respectively. Forsolvent extraction with n-hexane, the highest oil recovery (77.7 mg.g-1dry weight) wasachieved after 8 h of reaction (Andrich et al. 2006). Couto et al. (2010) performedsupercritical uid extraction from the microalga C. cohnii at temperatures of 40 and50 C and pressures of 20.0, 25.0 and 30.0 MPa. Gas ow rate was 0.6 kg.h-1andextractiontimewas3h. Optimumextractionconditionswerefoundtobe30.0MPaand50 C(8.6%), againstthe19.9%(wt.)achievedbyapplicationof BlighandDyers method(1959). Withthis work, it was possibletostatethatatthehighestpressures(25.0and30.0MPa)theextractionyieldincreaseswiththetemperature, whileat thelowest pressure(20.0MPa), anincreaseintemperatureleadstoadecreasedyield. Temperatureinuenceontheextractionefciency results from the combination of the following antagonic thermodynamiceffects: (i) at constant pressure, an increase in temperature leads to a decrease inthe density of the supercritical uid and thus its solvatation capacity; (ii) vapourpressure of solutes increase with the temperature, resulting in an high solubilityinthesupercritical uid(Coutoet al. 2010). UsingscCO2toextract oil fromthe microalgaChlorococcum sp., Halim et al. (2011) achieved an oil recovery of58 mg.g-1dry weight (5.8 %) at a ow rate of 18.5 kg h-1, a pressure of 30.0 MPaand a temperature of 60 C, during 80 min. By increasing temperature to 80 C,oil yield was 48 mg.g-1dry weight (4.8 %). The authors also applied organic solventextraction (using n-hexane) obtaining a oil yield of 32 mg.g-1dry weight (3.2 %) after areaction time of 5.5 h (Halim et al. 2011). Bjornsson et al. (2012) used the microalgaNannochloropsis granulata to study the effect of pressure, time and temperature inoil extraction yields. Firstly, the authors evaluated the effect of pressure (35, 45,and 55 MPa; 50 C; 6 kg h-1; 3 h), concluding that no signicant differences inoil yields were obtained by varying this process variable. Later, different extractiontimes were applied (3, 4.5, and 6 h), maintaining pressure, temperature and gasowrateconstant (35MPa; 50 C; 6kg.h-1). Theincreaseofextractiontimeresultedindifferencesinoil yieldthat rangedfrom8.67mg.g-1dry weight(over180min)to15.56mg.g-1dry weight(over270min)andto16.91mg.g-1dry weight(over 360 min). However, the differences in yields were not statistically signicant.Finally, scCO2extraction was performed by keeping pressure, gas ow rate, andtime constant (35 MPa, 6 kg.h-1, and 4.5 h), and by varying temperature (50, 70, and90 C). The increase of extraction temperature resulted in a statistically signicantincrease in oil yield from 15.56 mg.g-1dry weighat 50 C to 28.45 mg.g-1dry weightat 70Candto25.75mg.g-1dry weightat 90C. Althoughoil yielddecreasedbyincreasingtemperaturefrom70to90 C, thisdecreasewasnot statisticallysignicant (Bjornsson et al. 2012).Application of scCO2 to extract microalgal oil is very attractive, as it is a greentechnology and it allows a complete characterization of the extracted oil and theresulting biofuel. However, it still needs to be improved, because of its high cost-effectiveness and high energy-consuming drying step required before supercriticaluid extraction (Crampon et al. 2011).1 Biodiesel from Microalgal Oil Extraction 15Ionic Liquid Mediated ExtractionIonic liquids (ILs) have been reported as an attractive alternative for volatile andtoxicorganicsolventsbecauseof their non-volatilecharacter, thermal stability,and high solvatation capacity (Kim et al. 2012; Lateef et al. 2009). ILs are saltsof relativelylargeasymmetricorganiccationscoupledwithsmaller organicorinorganic anions. These organic salts can be liquid at room temperatures or lowmelting point solids (Cd >Cr >Zn. Apart from the EMF, other parametricconditions like metal concentration, electrode material and afnity existing betweenthe pollutant inuence the deposition. The main advantage of the electrodepositionisthedepositedmetalscanbeeasilyrecycledbyelectrometallurgical process.The disadvantage of the process is the surface of the cathode is modied duringelectrodeposition and it needs additional process control.Dechlorination of organic toxicants can occur not only by anodically but also ca-thodically, proved using graphite/carbon as electrode material. It was concluded thatthe dehalogenation results in a decreased toxicity and an increased biodegradability,52 S. Vasudevan and M.A. Oturanthus enabling further biological treatment. It is concluded that, energy consumptionand conversion rates are such that a technically and economically viable method forthe detoxication of waste waters can be developed.2.3.8 Aerogel and Electrochemistry for CleaningContaminated WaterThe method called capacitive deionization (CDI), consumes only 1632 Wh/gal. InCDI, contaminated water ows in a serpentine manner down stack of electrochemi-cal cells. Attached to each side of the electrodes is a sheet of carbon aerogel, a highlyporous material that has very large surface area. The aerogel is made by dipping athin carbon sheet in a mixture of resorcinol and formaldehyde and then carbonizingat around 1,000C in a vacuum or inert atmosphere. A prototype system has reducedsalt concentration 100 mg/L5.0 g L-1),thereby requiring the consumption of excessive amounts of expensive reactants thatmakes the treatment less cost affordable.Electrochemistry constitutes one of the clean and effective ways toproduce in situ hydroxyl radical (OH), a highly strong oxidizing agent oforganic matter in waters. Due to its very high standard oxidation power(E(OH/H2O) =2.80 V/SHE), this radical species is able to react non-selectivelywith organic or organometallic pollutants yielding dehydrogenated or hydroxylatedderivatives, which can be in turn completely mineralized, i.e., converted into CO2,water and inorganic ions.Recently, the electrochemical advanced oxidation processes (EAOPs) have re-ceived great attention by their environmental safety and compatibility (operating atmild conditions), versatility, high efciency and amenability of automation (Brillaset al. 2009).Anodic OxidationAnodic oxidation constitutes a direct way to generate hydroxyl radical by electro-chemistry. This technique does not use any chemicals, hydroxyl radicals are formed54 S. Vasudevan and M.A. Oturanby oxidation of water on high O2evolution overvoltage anodes (Pera-Titus et al.2004; Martnez-Huitle and Brillas 2008; Oturan 2000a). Firstly Pt and PbO2 anodeswere investigated and on the obtained experimental results (Marselli et al. 2003)proposed the following mechanism for the oxidation of organics with concomitantoxygen evolution, assuming that both formation of OH and oxygen evolution takeplace on such anodes from the discharge of water (Reaction 2.36). FormedOHare heterogeneous since they are adsorbed on anode material (M) and react withorganic matter (R) leading to its mineralization, i.e. transformation of R to CO2 andH2O (Reaction 2.37), when R does not contain heteroatom.MH2O M(-OH) He-(2.36)M(-OH) R MmCO2nH2O (2.37)Recently a new and powerful anode material, the boron-doped diamond (BDD)thin lm anode appeared as an emerging anode material. This anode possesses verygood properties for the electrochemical treatment of wastewaters contaminated byorganicpollutants. Ithasagreatchemicalandelectrochemicalstability, awideelectrochemical working range. It has been shown that this new anode has an O2overvoltage much higher than that of conventional anodes such as Pt, PbO2, dopedSnO2, and IrO2(Canizares et al. 2004). Thus the BDD anode is able to producelarger amount ofOH physisorbed on anode surface, BDD(OH), from Reaction(2.36) that are more labile and reactive compared other anode materials, leadingto a rapid and efcient destruction of organic pollutants. The effectiveness of theBDDanodewasshownfortheoxidationofawiderangeofpollutants(Ozcanet al. 2008; Canizares et al. 2008) with almost mineralization of treated solutions.These studies were highlighted that the current efciency is inuenced by appliedcurrent density and initial pollutant concentration, low organic concentration andhigh applied current density favoring the mineralization degree.Electro-Fenton ProcessThe secondEAOPs is the electro-Fentonprocess inwhichhydroxyl radicalsaregeneratedindirectlyviaFentonsreagent (mixtureofH2O2andiron(II))inhomogeneous medium. H2O2 is electrogenerated in-situ by 2-electron reduction ofdissolved O2 in acidic medium (Reaction 2.38) in presence of a catalytic amountof ferrous ions (Oturan et al. 2000). The most widely applied cathode materialsfor the effective H2O2 are based on carbonaceous materials (carbon felt, reticulatedvitreous carbon (RVC), carbon sponges, carbon nanotubes (NT), or graphite).O22H2 e-H2O2(2.38)Compared to other chemical oxidant, H2O2 is a weak oxidant, but its oxidationpower is enhanced in presence of Fe2 ions (Fenton 1894) via Fentons reaction:2 Electrochemistry and Water Pollution 55Fe3+Fe2+2e-2e-e-OH-H+H2OH2O21/2 O21/2 O2 + 2H+O2 + 2H+-OHFig. 2.7 Schematic representation of the electrocatalytic production of hydroxyl radicals by theelectro-Fenton process (Oturan et al. 2000)H2O2Fe2Fe3OH--OH (2.39)Thisenhancement isattributedtotheformationof highlypowerful oxidanthydroxyl radical viaReaction(2.39). Fe3formedbythisreactionisreducedelectrochemicallyat thecathodetoregenerateferrousironinordertocatalyseFentons reaction:Fe3e-Fe2(2.40)The simultaneous generation of H2O2and regeneration of ferrous iron at thecathode allow continuous and catalytic formation of hydroxyl radicals that react onorganic pollutants by hydrogen atom abstraction or addition reactions:Organic pollutants -OH oxidation intermediates (2.41a)Intermediates -OH CO2H2O inorganic ions (2.41b)When the anode is Pt, the O2 needed for the Reaction (2.38) to produce H2O2isformedontheanodebyoxidationof water (Oturanet al. 2001). Thus, theelectro-Fentonprocessconstitutesanoverallcatalyticsystem. Bothcatalyticallycycle taking place during this system for the continuous regeneration of Fe2 andH2O2 in order to produce hydroxyl radicals via Fenton reaction are schematized inFig. 2.7.56 S. Vasudevan and M.A. Oturan0481216200 100 200 300 400 500 600TOC/ppmTime/mnFig. 2.8 Effect of pH and medium on TOC removal efciency during degradation of 0.2 mMmethyl parathion (insecticide) aqueous solution by electro-Fenton process at I =150 mA. pH:1 (); 3 () and 4 () in H2SO4 medium; pH: 1 (); 3 (-) and 4 () in HClO4 medium (Diagneet al. 2007). TOC total organic carbonEffect of Operating Parameter on the Process EfciencyThe electro-Fenton process is governed by a number of operating parameters, themost important being solution pH, applied current, catalyst concentration, medium(supporting electrolyte) and initial organic content.Effect of Solution pH and MediumThe solution pH is one of key parameter in electro-Fenton process. Now the optimalvalue of this parameter is well known. The value of pH 3 was suggested rstly (Sunand Pignatello 1993) and conrmed thereafter by other groups (Diagne et al. 2007).This value is valid for the Fenton process and all related process including electro-Fenton process (Fig. 2.8). However electro-Fenton process can be carried out moreor less effectively in the range of 2.53.5. It is suggested that iron ions (catalyst)are lost from the solution by precipitation for pH>4. For pHFenton(67.8%) >UV/H2O2(40.6%) >UV(14.2%). Onthebasisof operating costs, the Fenton process was the most cost-effective in comparisonwith the other four AOPs (Table 3.9), Dai et al. (2012). As shown in Table 3.9,UV/TiO2 and UV can be considered as costly approaches for wastewater treatment.However the authors reported that this statement can be challenged on the groundsthat the cost of photocatalytic treatment can be reduced by applying sunlight as theirradiation source.The removal efciency by UV-A (350400 nm) and solar irradiation provided bya solar simulator on CBZ was investigated along with another API, IBP, Achilleoset al. (2010b). Greater degradation was attained with UV-A irradiation than witharticial solar radiation for both APIs. Degradation of CBZ in pure water was foundto be more dependent on changes in the loading of Degussa P25 TiO2 compared toIBP. Conversion of 74 %CBZ was achieved after 120 min of reaction with 100 mg/L120 D. Kanakaraju et al.Table3.8HighlightsofheterogeneousphotocatalyticstudiesonantiepilepticsCompound(s)WatermatrixExperimentalfeaturesAnalyticalmethodsFindingsReferenceCarbamazepineDeionizedwaterMediumpressureHglamp(30W)HPLCComparisonofUV/TiO2withUV,UV/H2O2andUV/FentonshowedtheUV/TiO2yielded70.4%degradationwhichwasslightlylowerthanUV/Fentonat86.9%degradation.Allprocessesfollowedpseudo-rstorderkineticsDaietal.(2012)Initialconcentration:4.2pMTiO2loading:0.52.0g/LTemperature:25CpH:6.5HospitalwastewaterAnnularslurryphotoreactorUV-Ablacklight(8W)HPLC,CODPhotodegradationathighconcentrationofCBZinsynthetichospitalwastewaterwasttedtotheL-HkineticsmodelChongandJin(2012)Initialconcentration:5,000pg/LTiO2AnataseparticlesMilli-QwaterUV-Alamp:2.17mW/cm2HPLC,IC,TOCUV-AandUV-CirradiationefcienttodegradeCBZImetal.(2012)UV-Clamp:3.56mW/cm2Initialconcentration:0.021mMTiO2loading:0.5g/LpH:5.4CarbamazepinemixturesCarbamazepine(andibuprofen)Milli-QwaterandwastewaterfromWWTPSolarsimulator(1,000WPhillipXelamp)andUV-Alamp(9WRadiumlamp)UV-vis(CBZ:284nm),DOCDegradationunderUV-AirradiationinpurewaterwassensitivetoTiO2P25loading.SolarandUV-AphotocatalysisappearstobeefcientonCBZdegradationAchilleosetal.(2010b)Initialconcentration:520mg/LTiO2loading:503,000mg/LTypeofTiO2:DegussaP25,HombikatUV100,Aldrich,3 Heterogeneous Photocatalysis for Pharmaceutical Wastewater Treatment 121TronoxAK-1,TronoxTRHP-2,TronoxTRH2O2concentration:0.071.4mMpH:310Carbamazepine(Clobricacid,iomeprol)Milli-QwaterSolarsimulatorXeshortarclamp(1,000W)(cut-off296nm)UVvis,HPLC,DOCDegradationofCBZdependentontheconcentrationofNOMasitgreatlyinuencesitskineticsDollandFrimmel(2005a)Initialconcentration:15.5mg/LTiO2loading(P25andHombikatUV100):500mg/LCarbamazepine(clobricacid,iomeprol)UltrapurewaterSolarsimulatorXeshort-arclamp(1,000W)HPLC,DOC,ICP-AES,HPLC/TQMSPseudo-rstorderdegradationrateofCBZforTiO2P25andUV100were4.710

3and0.1310

3s

1DollandFrimmel(2005b)Initialconcentration:4.3mg/LTiO2andHombikatUV100loading:0.1g/LTemperature:202CCarbamazepine(Clobricacid,iomeprol,iopromide)Milli-QwaterSolarsimulatorXeshortarclamp(1,000W)HPLC,DOC,ICP-AES,HPLC/ESMS,DOCDegradationofCBZwasfasterwithTiO2P25duetohigheradsorptionDollandFrimmel(2004)Initialconcentration:1.0and4.3mg/LTiO2P25loading:0.011,000mg/LTemperature:202CHombikatUV100loading:0.11,000mg/L122 D. Kanakaraju et al.Table 3.9 Comparison ofoperating costs of advancedoxidation processes (AOP)studied (Data taken from Daiet al. (2012))Process Treatment cost (US$/kg)UV 210.4UV/H2O242.6Fenton 5.4UV/Fenton 26.7UV/TiO270.2P25 under UV-A irradiation, but it decreased to 35 % upon solar irradiation, whichwas attributed to completely different reactor set-ups. Wastewater samples spikedwith 10 mg/L CBZ had a detrimental effect on DOC removal, due to the presenceof scavengers of HOradicals and naturally occurring DOC in the raw wastewatersample. Nevertheless, thestudyprovedthat UV-Aandsolarirradiationcanbeapplied to CBZ removal (and also for IBP). Direct photolysis with a UV-A lampproduced negligible degradation and DOC removal for CBZ.The results from another study conrmed that direct photolysis supplied by aUV-C irradiation source is not effective for CBZ elimination, Im et al. (2012). Animportant contribution of the study is that UV-C irradiation increased the removalefciency compared to that of UV-A irradiation employed on the 0.021 mM CBZin the presence of 0.5 g/L TiO2 P25. Addition of radical scavengers decreased theremoval rate, implying that HOhas a crucial role to play in the photocatalytic degra-dation of this compound. Addition of oxygen also enhanced the mineralization.A TiO2 nanober was evaluated as a pre-treatment option and for improvementof biodegradability of 5,000pg/L CBZ in synthetic hospital wastewater, Chongand Jin (2012). This application was found to be efcient for CBZ abatement insynthetic hospital wastewater and also for COD removal. High CBZ degradationrate of 48.33pg/L min was obtained, which also resulted in 40 % COD reductionafter 4 h of treatment. The study highlighted the fact that the TiO2 based system hasa good potential as a sustainable pre-treatment system for hospital wastewater.Doll and Frimmel (2004, 2005a, b) conducted multiple photocatalytic studiesonCBZundervariousconditions, whichrevealedthat TiO2photocatalysisisapromising abatement method for CBZ degradation. When a comparison was madewith Hombikat UV100, CBZ was degraded more efciently with TiO2 P25 undersimulated solar irradiation (1,000 W), Doll and Frimmel (2004). In the subsequentstudy, photocatalytic degradationof CBZwas investigatedwithtwotypes ofTiO2photocatalysts in the presence and absence of NOM under simulated solarUVlight, Doll andFrimmel (2005a). It wasnotedthat different concentrationsof NOMhaddifferent effectsonthekineticsof CBZdegradation. Ingeneral,NOMretardedthephotocatalyticdegradationrateofCBZ. Thiswasattributedto competitive inhibition as NOM scavenged the holes. The research group alsosuggested possible photocatalytic degradation products for CBZ, Doll and Frimmel(2005b) (Scheme 3.9).3 Heterogeneous Photocatalysis for Pharmaceutical Wastewater Treatment 123NH2NH2NOOHOHONOHONNH2OON NOScheme 3.9 Photocatalytic degradation pathways of carbamazepine (CBZ), Doll and Frimmel(2005b)3.6 ConclusionA typical UV/TiO2investigation is shown in Fig. 3.7. After one or more modelcompounds have been chosen, the next step is the photocatalytic experimental set-up or the choice of the photoreactor and light source, which will have a signicanteffect ontheoutcomeofthedegradationrate, Legrini et al. (1993). Therearevarious factors that need to be taken into consideration with respect to photoreactors,namelythematerial it ismadeof, shape, geometry, radiationpathlengths, andthe cooling apparatus. Similar issues have been highlighted in a review which hascommentedonthislackofinformation, whenexplainingphotoproductsformedfrom the photocatalytic and photolytic studies, Fatta-Kassinos et al. (2011a).When considering light sources, articial light sources have been employed inmost studies. These can be grouped into monochromatic light sources such as lowpressure Hg lamps and polychromatic light source namely medium pressure Hglamps and high pressure Hg lamps. Other irradiation devices include Xe, Xe-arc,Hg and Ra lamps. Solar simulators have been commonly applied to simulate solarirradiation. LED based light sources are also emerging as an alternative light source,due to advantages which include no mercury waste, longer life time compared to Hgbased lamps and improved exibility in terms of reactor design, Landgraf (2001)andChenetal. (2007). Veryfewstudieshavemadeacomparisonbetweenthelamps spectral domain and the absorption spectrum of the reactant. Photon ux,intensity and irradiance emitted from the light source also need to be clearly statedas these values determine the rate of photons emitted, which has a direct correlationto the degradation rate. Selection of a radiation source (shape and dimension) is alsogreatly inuenced by the type of photoreactor.124 D. Kanakaraju et al.Modelcompound(s)Photocatalyticexperimentalset-upVariablesinfluence oroptimizationDegradationmonitoringMineralizationIdentificationofintermediatesand toxicityassessmentFig. 3.7 Typical steps involved in heterogeneous photocatalytic degradationReaction rates are known to be dependent on operation conditions. Optimizationof these variables thus allows for maximum degradation. Statistical approaches suchas multivariate analysis and response surface methodology have also been appliedto establish the optimal degradation rate. One of the most commonly investigatedand important factors is the TiO2 loading. It has been shown that the degradationrate is not always proportional to the catalyst load, but an optimum load needs to bedetermined. Difculty arises when comparing studies for a particular compound ofinterest. In general, optimal catalyst load is inuenced by the photoreactor designincluding its diameter and source of irradiation. Commercially available TiO2 suchas Degussa P25, Hombikat UV 100, PC 500 and Aldrich have been used in variousphotocatalytic degradation studies of pharmaceuticals. Among them, TiO2 DegussaP25 has demonstrated its superiority in removing various APIs. A concentrationbelow the optimum catalyst loading is typically chosen for further investigation,duetothefact that catalyst depositionmight occurduringtheexperiment. Forexample, 0.4 g/L TiO2 was chosen instead of 0.5 g/L which gave the best resultsfor metoprolol and propranolol degradation, Romero et al. (2011).Initial concentrations of APIs applied in photocatalytic studies tend to be higherthan the environmental level. Furthermore, in most cases single pharmaceuticalsare investigated despite the fact that real wastewaters contain complex mixtures ofpharmaceuticals. Although most studies have demonstrated efciency for API inspiked distilled water or ultrapure water, real wastewater samples are rarely used.Thepresenceofradical scavengerssuchasHCO3-, CO32-ionsandNOMinreal wastewater typically impacts on degradation efciencies. Radical scavengersor inhibitors are frequently studied to determine the role of reactive intermediates,e.g. theHOradical, O2-anionandholes inthephotocatalyticdegradation.Isopropanol, benzoquinone and iodide ions are commonly used for this purpose.Ingeneral, laboratoryscaleexperimentsaremorecommonthanpilot scaleoperationsduetoamorecontrollableenvironment. Thereisusuallynodirect3 Heterogeneous Photocatalysis for Pharmaceutical Wastewater Treatment 125correlation between the effectiveness of photodegradation in the laboratory withthat under real environmental conditions. Conducting photodegradation studies onpilot scales will provide more realistic environmental conditions such as season,latitude, and hardness of water. These are generally not encountered when workingin the laboratory. Trials using solar energy have been reported for pharmaceuticaldegradation, althoughtheresultsthusfar varybetweencompoundsof interest.Commercial and industrial scale applications of solar energy have been establishedin a few European countries, the USA and Canada.In the subsequent step, degradation monitoring of pharmaceuticals is generallycarriedout byusingHPLCdetectionor UVvisspectrophotometry. Thelatterhasbeenreportedtobeafast anduseful tool ofmonitoringalthoughit isnotthestate-of-the-art techniquefor API detection. Thekineticsof degradationinterms of percentage API removed or API disappearances versus irradiation time orconcentration change versus irradiation time (C/Co vs time) are commonly reported.The L-H model has been often applied to describe photocatalytic mineralization ofpharmaceutical degradation.Apart from determining the removal rate of APIs with photocatalytic treatment,the degree of mineralization is a critical parameter in heterogeneous photocatalysis.DOChasbeenfrequentlymeasuredasanindicationoftheformationofCO2.Inmostcases, completedegradationofthepharmaceuticaldoesnotcorresponddirectly with the mineralization rate mainly due to due to the formation of morestable compounds or transformationproducts duringthe degradation. Anon-biodegradable fraction frequently remains in the treated solution. Mineralization canbe also determined by measuring the formation of inorganic ions such as chlorine,sulphate and nitrate.Although the ultimate goal of heterogeneous photocatalysis is to achieve com-pletemineralizationof parent compounds, it is alsoimportant toevaluatetheby-products generated to ensure that there are no toxic or more persistent com-pounds produced during the treatment. This will contradict the ultimate goal of theapplication of such an AOP. However, photodegradation products are not commonlystudied. Afewpossiblereasons for this are: (i) difculties inseparatingandidentifying a large number of these transformation products formed; (ii) lack of ornon-existent analytical standards to determine the identity of these transformationproducts; and (iii) requirement for more than one analytical technique or samplepreparation technique due to their diverse physicochemical properties, Ag uera et al.(2005) and Fatta-Kassinos et al. (2011b). 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