Terry J. McGenity Kenneth N. Timmis Balbina Nogales Editors Hydrocarbon and Lipid Microbiology Protocols Biochemical Methods Springer Protocols Handbooks More information about this series at http://www.springer.com/series/8623 Terry J. McGenity Kenneth N. Timmis Balbina Nogales (cid:129) (cid:129) Editors Hydrocarbon and Lipid Microbiology Protocols Biochemical Methods Scientific Advisory Board JackGilbert,IanHead,MandyJoye,VictordeLorenzo, JanRoelofvanderMeer,ColinMurrell,JoshNeufeld, RogerPrince,JuanLuisRamos,WilfredRo¨ling, HeinzWilkes,MichailYakimov Editors TerryJ.McGenity KennethN.Timmis SchoolofBiologicalSciences InstituteofMicrobiology UniversityofEssex TechnicalUniversityBraunschweig Colchester,Essex,UK Braunschweig,Germany BalbinaNogales DepartmentofBiology UniversityoftheBalearicIslands andMediterraneanInstituteforAdvanced Studies(IMEDEA,UIB-CSIC) PalmadeMallorca,Spain ISSN1949-2448 ISSN1949-2456 (electronic) SpringerProtocolsHandbooks ISBN978-3-662-49135-5 ISBN978-3-662-49137-9 (eBook) DOI10.1007/978-3-662-49137-9 LibraryofCongressControlNumber:2016938230 #Springer-VerlagBerlinHeidelberg2016 Thisworkis subjectto copyright.Allrightsarereservedbythe Publisher,whetherthe wholeorpartofthe materialis concerned,specificallytherightsoftranslation,reprinting,reuseofillustrations,recitation,broadcasting,reproductionon microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computersoftware,orbysimilarordissimilarmethodologynowknownorhereafterdeveloped. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply, evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulationsand thereforefreeforgeneraluse. Thepublisher,theauthorsandtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedtobe trueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty,expressor implied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeenmade. Printedonacid-freepaper ThisSpringerimprintispublishedbySpringerNature TheregisteredcompanyisSpringer-VerlagGmbHBerlinHeidelberg Preface to Hydrocarbon and Lipid Microbiology Protocols1 Allactivecellularsystemsrequirewaterastheprincipalmediumandsolventfortheirmetabolicand ecophysiological activities. Hydrophobic compounds and structures, which tend to exclude water, althoughprovidinginteraliaexcellentsourcesofenergyandameansofbiologicalcompartmental- ization, present problems of cellular handling, poor bioavailability and, in some cases, toxicity. Microbesbothsynthesizeandexploitavastrangeofhydrophobicorganics,whichincludesbiogenic lipids, oils and volatile compounds, geochemically transformed organics of biological origin (i.e. petroleum and other fossil hydrocarbons) and manufactured industrial organics. The underlying interactionsbetweenmicrobesandhydrophobiccompoundshavemajorconsequencesnotonlyfor thelifestylesofthemicrobesinvolvedbutalsoforbiogeochemistry,climatechange,environmental pollution, human health and a range of biotechnological applications. The significance of this “greasymicrobiology”isreflectedinboththescaleandbreadthofresearchonthevariousaspects ofthetopic.Despitethis,therewas,asfarasweknow,notreatiseavailablethatcoversthesubject. In an attempt to capture the essence of greasy microbiology, the Handbook of Hydrocarbon and Lipid Microbiology (http://www.springer.com/life+sciences/microbiology/book/978-3-540- 77584-3) was published by Springer in 2010 (Timmis 2010). This five-volume handbook is, we believe, unique and of considerable service to the community and its research endeavours, as evidencedbythelargenumberofchapterdownloads.Volume5ofthe handbook,unlikevolumes 1–4whichsummarizecurrentknowledgeonhydrocarbonmicrobiology,consistsofacollectionof experimentalprotocolsandappendicespertinenttoresearchonthetopic. Asecondeditionofthehandbookisnowinpreparationandadecisionwastakentosplitoffthe methods section and publish it separately as part of the Springer Protocols program (http://www. springerprotocols.com/).Themulti-volumeworkHydrocarbonandLipidMicrobiologyProtocols, whilerootedinVolume5oftheHandbook,hasevolvedsignificantly, intermsofrange oftopics, conceptual structure and protocol format. Research methods, as well as instrumentation and strategic approaches to problems and analyses, are evolving at an unprecedented pace, which can be bewildering for newcomers to the field and to experienced researchers desiring to take new approaches to problems. In attempting to be comprehensive – a one-stop source of protocols for researchingreasymicrobiology–theprotocolvolumesinevitablycontainbothsubject-specificand more generic protocols, including sampling in the field, chemical analyses, detection of specific functionalgroupsofmicroorganismsandcommunitycomposition,isolationandcultivationofsuch organisms, biochemical analyses and activity measurements, ultrastructure and imaging methods, geneticandgenomicanalyses,systemsandsyntheticbiologytoolusage,diverseapplications,and 1AdaptedinpartfromthePrefacetoHandbookofHydrocarbonandLipidMicrobiology. v vi PrefacetoHydrocarbonandLipidMicrobiologyProtocols theexploitationofbioinformatic,statisticalandmodellingtools.Thus,whiletheworkisaimedat researchers working on the microbiologyof hydrocarbons, lipids and other hydrophobic organics, much of it will be equally applicable to research in environmental microbiology and, indeed, microbiologyingeneral.This,webelieve,isasignificantstrengthofthesevolumes. We are extremely grateful to the members of our Scientific Advisory Board, who have made invaluable suggestions of topics and authors, as well as contributing protocols themselves, and to generous ad hoc advisors like Wei Huang, Manfred Auer and Lars Blank. We also express our appreciationofJuttaLindenbornofSpringerwhosteeredthisworkwithprofessionalism,patience andgoodhumour. Colchester,Essex,UK TerryJ.McGenity Braunschweig,Germany KennethN.Timmis PalmadeMallorca,Spain BalbinaNogales Reference TimmisKN(ed)(2010)Handbookofhydrocarbonandlipidmicrobiology.Springer,Berlin,Heidelberg Contents Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 HermannJ.Heipieper ProtocolsfortheIsolationandAnalysisofLipopeptidesandBioemulsifiers. . . . . . . . . 3 ThomasJ.Smyth,MichelleRudden,KonstantinaTsaousi, RogerMarchant,andIbrahimM.Banat ProtocolsfortheDetectionandChemicalCharacterisation ofMicrobialGlycolipids.. . . .. . . . .. . . . .. . . .. . . . .. . . . .. . . .. . . . .. . . . .. . . .. . 29 ThomasJ.P.Smyth,MichelleRudden,KonstantinaTsaousi, RogerMarchant,andIbrahimM.Banat IsolationofGlycoproteinBioemulsifiersProducedbyMarineBacteria. . . . . . . . . . . . . 61 TonyGutierrezandIbrahimM.Banat ProtocolsonLipidExtractionfromWetAlgalBiomass. . . . . . . . . . . . . . . . . . .. . . . . . 75 ForoughGhasemiNaghdiandPeerM.Schenk IsolationandCharacterizationofLipidDropletsfromYeast. . . . . . . . . . . . . . . . . . . . 81 KarinAthenstaedt ProtocolsforIsolationandAnalysisofPolyhydroxyalkanoates. . . . . . . . . . . . . . . . . . . 93 Mo`nicaBassas-Galia` PurificationofLipidRaftsfromBacterialMembranes. . . . . . . . . . . . . . . . . . . . . . . . . 113 CharlotteWermserandDanielLopez IsolationandAnalysisofMembranePhospholipidsasBiomarkers. . . . . . . . . . . . . . . . 123 NancyHachichoandHermannJ.Heipieper ProtocolsforPurifyingandCharacterizingIntegralMembraneAlkBEnzymes. . . . . . 133 RachelNarehoodAustin,DavidBorn,ThomasJ.Lawton, andGraceE.Hamilton ProtocolsforStructuralandFunctionalAnalysisofParticulateMethane MonooxygenasefromMethylocystisSpeciesStrainRockwell(ATCC49242). . . . . . . . . 149 SarahSirajuddinandAmyC.Rosenzweig vii viii Contents Phenotype-BasedIdentificationofKeyEnzymesforPolycyclicAromatic Hydrocarbon(PAH)MetabolismfromMycobacteriaUsingTransposon MutagenesisandaPAHSprayPlate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 Seong-JaeKim,OhgewKweon,andCarlE.Cerniglia MicrocalorimetryasaGeneralTechniquetoCharacterizeLigandBinding. . . . . . . . . 177 TinoKrell,MiriamRico-Jime´nez,Andre´sCorralLugo, Jose´ AntonioReyesDarias,A´lvaroOrtega,andAbdelaliDaddaoua About the Editors Terry J. McGenity is a Reader at the University of Essex, UK. His Ph.D., investigating the microbial ecology of ancient salt deposits (University of Leicester), was followed by postdoctoral positions at the Japan Marine Science and Technology Centre (JAMSTEC, Yoko- suka) and the Postgraduate Research Institute for Sedimentology (UniversityofReading).Hisoverarchingresearch interestistounder- stand how microbial communities function and interact to influence major biogeochemical processes. He worked as a postdoc with Ken TimmisattheUniversityofEssex,wherehewasinspiredtoinvestigate microbialinteractionswithhydrocarbonsatmultiplescales,fromcommunitiestocells,andasboth asourceoffoodandstress.Hehasbroadinterestsinmicrobialecologyanddiversity,particularly withrespecttocarboncycling(especiallythesecondmostabundantlyproducedhydrocarboninthe atmosphere, isoprene), and is driven to better understand how microbes cope with, or flourish in hypersaline,desiccatedandpoly-extremeenvironments. Kenneth N. Timmis read microbiology and obtained his Ph.D. at Bristol University, where he became fascinated with the topics of environmental microbiology and microbial pathogenesis, and their interfacepathogenecology.Heundertookpostdoctoraltrainingatthe Ruhr-University Bochum with Uli Winkler, Yale with Don Marvin, andStanfordwithStanCohen,atthelattertwoinstitutionsasaFellow oftheHelenHayWhitneyFoundation,whereheacquiredthetoolsand strategiesofgeneticapproachestoinvestigatemechanismsandcausal relationships underlying microbial activities. He was subsequently appointedHeadofanIndependentResearchGroupattheMaxPlanck InstituteforMolecularGeneticsinBerlin,thenProfessorofBiochem- istry in the University of Geneva Faculty of Medicine. Thereafter, he became Director of the Division of Microbiology at the National Research Centre for Biotechnology (GBF)/now the Helmholtz Centre for Infection Research (HZI) and Professor of Microbiology at the Technical UniversityBraunschweig.Hisgrouphasworkedformanyyears,interalia,onthebiodegradationof oil hydrocarbons, especially the genetics and regulation of toluene degradation, pioneered the genetic designand experimental evolutionof novel catabolic activities,discovered the new group of marine hydrocarbonoclastic bacteria, and conducted early genome sequencing of bacteria that ix