Advances in Biochemical Engineering/Biotechnology 151 Series Editor: T. Scheper Georg M. Guebitz Alexander Bauer Guenther Bochmann Andreas Gronauer Stefan Weiss Editors Biogas Science and Technology 151 Advances in Biochemical Engineering/Biotechnology Series editor T. Scheper, Hannover, Germany Editorial Board S. Belkin, Jerusalem, Israel P.M. Doran, Hawthorn, Australia I. Endo, Saitama, Japan M.B. Gu, Seoul, Korea W.-S. Hu, Minneapolis, MN, USA B. Mattiasson, Lund, Sweden J. Nielsen, Göteborg, Sweden H. Seitz, Potsdam, Germany G.N. Stephanopoulos, Cambridge, MA, USA R. Ulber, Kaiserslautern, Germany A.-P. Zeng, Hamburg, Germany J.-J. Zhong, Shanghai, China W. Zhou, Shanghai, China Aims and Scope This book series reviews current trends in modern biotechnology and biochemical engineering. Its aim is to cover all aspects of these interdisciplinary disciplines, whereknowledge,methodsandexpertisearerequiredfromchemistry,biochemistry, microbiology, molecular biology, chemical engineering and computer science. Volumes are organized topically and provide a comprehensive discussion of developments in the field over the past 3–5 years. The series also discusses new discoveries and applications. Special volumes are dedicated to selected topics which focus on new biotechnological products and new processes for their synthesis and purification. In general, volumes are edited by well-known guest editors. The series editor and publisher will, however, always be pleased to receive suggestions and supplemen- tary information. Manuscripts are accepted in English. In references, Advances in Biochemical Engineering/Biotechnology is abbreviated as Adv. Biochem. Engin./Biotechnol. and cited as a journal. More information about this series at http://www.springer.com/series/10 Georg M. Guebitz Alexander Bauer (cid:129) Guenther Bochmann Andreas Gronauer (cid:129) Stefan Weiss Editors Biogas Science and Technology With contributions by (cid:1) (cid:1) T.M. Callaghan C. Carliell-Marquet G. Collins (cid:1) é (cid:1) (cid:1) V. Dollhofer C.D. Dub G. Esposito F.G. Fermoso á (cid:1) ö (cid:1) (cid:1) K. Fliegerov B. Fr schle L. Frunzo T. Gehring fi (cid:1) (cid:1) (cid:1) G. Wyn Grif th G.M. Guebitz G. Guibaud S.R. Guiot (cid:1) (cid:1) (cid:1) M. Heiermann K. Koch P. Kosse M. Lebuhn ü (cid:1) ä (cid:1) (cid:1) ö M. L bken U. Messelh usser B. Munk M. Pl chl (cid:1) (cid:1) S.M. Podmirseg S.K.M.R. Rittmann B.H. Svensson (cid:1) (cid:1) ß E.D. van Hullebusch J.P.M. Vink S. Wei M. Wichern 123 Editors Georg M.Guebitz Andreas Gronauer Institute of Environmental Biotechnology Institute of Agricultural Engineering BOKU Vienna—University of Natural BOKU Vienna—University of Natural ResourcesandLife Sciences ResourcesandLife Sciences Tulln Tulln Austria Austria Alexander Bauer StefanWeiss Institute of Agricultural Engineering Institute of Environmental Biotechnology BOKU Vienna—University of Natural BOKU Vienna—University of Natural ResourcesandLife Sciences ResourcesandLife Sciences Tulln Tulln Austria Austria Guenther Bochmann Institute of Environmental Biotechnology BOKU Vienna—University of Natural ResourcesandLife Sciences Tulln Austria ISSN 0724-6145 ISSN 1616-8542 (electronic) Advances in Biochemical Engineering/Biotechnology ISBN978-3-319-21992-9 ISBN978-3-319-21993-6 (eBook) DOI 10.1007/978-3-319-21993-6 LibraryofCongressControlNumber:2015945326 SpringerChamHeidelbergNewYorkDordrechtLondon ©SpringerInternationalPublishingSwitzerland2015 Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpart of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission orinformationstorageandretrieval,electronicadaptation,computersoftware,orbysimilarordissimilar methodologynowknownorhereafterdeveloped. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publicationdoesnotimply,evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfrom therelevantprotectivelawsandregulationsandthereforefreeforgeneraluse. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authorsortheeditorsgiveawarranty,expressorimplied,withrespecttothematerialcontainedhereinor foranyerrorsoromissionsthatmayhavebeenmade. Printedonacid-freepaper SpringerInternationalPublishingAGSwitzerlandispartofSpringerScience+BusinessMedia (www.springer.com) Contents Microbiology and Molecular Biology Tools for Biogas Process Analysis, Diagnosis and Control. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Michael Lebuhn, Stefan Weiß, Bernhard Munk and Georg M. Guebitz Anaerobic Fungi and Their Potential for Biogas Production . . . . . . . . 41 Veronika Dollhofer, Sabine Marie Podmirseg, Tony Martin Callaghan, Gareth Wyn Griffith and Kateřina Fliegerová Hygiene and Sanitation in Biogas Plants. . . . . . . . . . . . . . . . . . . . . . . 63 Bianca Fröschle, Monika Heiermann, Michael Lebuhn, Ute Messelhäusser and Matthias Plöchl Direct Interspecies Electron Transfer in Anaerobic Digestion: A Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Charles-David Dubé and Serge R. Guiot A Critical Assessment of Microbiological Biogas to Biomethane Upgrading Systems. . . . . . . . . . . . . . . . . . . . . . . . . . . 117 Simon K.-M.R. Rittmann Influent Fractionation for Modeling Continuous Anaerobic Digestion Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 Manfred Lübken, Pascal Kosse, Konrad Koch, Tito Gehring and Marc Wichern Fate of Trace Metals in Anaerobic Digestion. . . . . . . . . . . . . . . . . . . . 171 F.G. Fermoso, E.D. van Hullebusch, G. Guibaud, G. Collins, B.H. Svensson, C. Carliell-Marquet, J.P.M. Vink, G. Esposito and L. Frunzo Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 v Microbiology and Molecular Biology Tools for Biogas Process Analysis, Diagnosis and Control Michael Lebuhn, Stefan Weiß, Bernhard Munk and Georg M. Guebitz Abstract Many biotechnological processes such as biogas production or defined biotransformations are carried out by microorganisms or tightly cooperating microbial communities. Process breakdown is the maximum credible accident for the operator. Any time savings that can be provided by suitable early-warning systems and allow for specific countermeasures are of great value. Process distur- bance, frequently due to nutritional shortcomings, malfunction or operational def- icits, is evidenced conventionally by process chemistry parameters. However, knowledge on systems microbiology and its function has essentially increased in the last two decades, and molecular biology tools, most of which are directed against nucleic acids, have been developed to analyze and diagnose the process. Some of these systems have been shown to indicate changes of the process status considerably earlier than the conventionally applied process chemistry parameters. Thisisreasonablebecausethetriggeringcatalystisdetermined,activitychangesof themicrobesthatperformthereaction.Thesemolecularbiologytoolshavethusthe potential to add to and improve the established process diagnosis system. This chapter is dealing with the actual state of the art of biogas process analysis in practice, and introduces molecular biology tools that have been shown to be of particular value in complementing the current systems of process monitoring and diagnosis, with emphasis on nucleic acid targeted molecular biology systems. (cid:1) (cid:1) Keywords Biogas process parameters Molecular biology tools Quantitative (cid:1) (cid:1) (cid:1) Real-TimePCR Nextgenerationsequencing Meta-omics Fluorescence-insitu (cid:1) hybridization Metabolic quotient M.Lebuhn(&)(cid:1)B.Munk DepartmentforQualityAssuranceandAnalytics,BavarianStateResearchCenterfor Agriculture(LfL),LangePoint6,85354Freising,Germany S.Weiß AustrianCentreofIndustrialBiotechnology(ACIB),Petersgasse14,8010Graz,Austria G.M.Guebitz InstituteofEnvironmentalBiotechnology,BOKUVienna—UniversityofNaturalResources andLifeSciences,KonradLorenzStr.20,A-3430Tulln,Austria e-mail:[email protected] ©SpringerInternationalPublishingSwitzerland2015 1 G.M.Guebitzetal.(eds.),BiogasScienceandTechnology, AdvancesinBiochemicalEngineering/Biotechnology151, DOI10.1007/978-3-319-21993-6_1 2 M.Lebuhnetal. Abbreviations BB Bead-beating BMP Biological/biochemical methane potential BLAST Basic local alignment search tool Bp Base pair(s) cDNA Complementary DNA (transcribed from RNA species) CLSM Confocal laser scanning microscopy COD Chemical oxygen demand DGGE Denaturing-gradient gel electrophoresis DNA Deoxyribonucleic acid FISH Fluorescence in situ hybridization LCB Lignocellulosic biomass LM Light microscopy MQ Metabolic quotient mRNA Messenger RNA NA Nucleic acid(s) NGS Next generation sequencing OLR Organic loading rate PC(o)A Principal coordinate/Principal component analysis PCR Polymerase chain reaction PSM Process simulation model qPCR Quantitative Real-Time PCR rDNA Ribosomal deoxyribonucleic acid RNA Ribonucleic acid rRNA Ribosomal ribonucleic acid RT Reverse transcription SCFA Short-chain fatty acid(s) or also VFA SEM Scanning electron microscopy SMA Specific methanogenic activity TEM Transmission electron microscopy TGGE Temperature-gradient gel electrophoresis TVA/TIC Total volatile acids/total inorganic carbon VFA Volatile fatty acids VOA Volatile organic acids Contents 1 Introduction.......................................................................................................................... 3 2 Physico-ChemicalandBiochemicalProcessParameters.................................................... 4 2.1 GasProduction............................................................................................................ 5 2.2 ProcessIntermediates,SCFA,TotalandVolatileSolids,andSpecific Determinants............................................................................................................... 5 2.3 EarlyWarning—TheTVA/TICRatio........................................................................ 7 2.4 Nutrients,ToxicandDisturbingAgents..................................................................... 7 MicrobiologyandMolecularBiologyTools… 3 2.5 BiologicalMethanePotential,andActivity,ToxicityandSupplementationTests... 8 2.6 EnzymeTestsandApplications................................................................................. 9 2.7 ClassicalMicrobiologyApproaches........................................................................... 11 3 MolecularBiologyApproaches........................................................................................... 12 3.1 PCRBasedApproachesandNucleicAcidSequencing............................................ 12 3.2 MicroscopyBasedDetectionofMicroorganisms:Specificand Non-specificImaging.................................................................................................. 25 References.................................................................................................................................. 29 1 Introduction Biogasproductionbyanaerobicdigestionoforganicmatterisabio-technologywith verylongtraditionforsome2,000–3,000years.Itwasappliedinitiallyforsanitation purposesandonlylateradditionallyforenergyproduction.Theissuesanitationwith itsbeneficialeffectsforthesocietyispresentedwithinthisbookinChap.3.Allofthe processstepsareperformedinafoodchainbydifferentmicroorganisms,governedby processengineeringinasuitabletechnicalenvironment.Someofthesemicrobeshave to cooperate extremely efficiently in syntrophic dependency in order to be able to thriveandproliferateattheminimumlimitofpossibleenergygain[1,2]. Methanogenic archaea, and among these particularly the acetoclastic Methanosaetaceae,appeartobemostsensitiveinbiogasprocessestostressfactors such as short retention times, high ammonia, oxygen and short-chain fatty acid (SCFA)concentration, lack ofcertaintraceelementsandincreasedtemperature [3, 4].Duetotheirrelativelylowapparentmaximumturnovernumber(K )foracetate m and long doubling times [5], the acetoclastic methanogens are disfavored at short retention times and increasing acetate concentration in the fermenter [6]. They are increasingly washed out if their proliferation cannot compensate out-dilution. This effect is even pronounced at additional stress conditions, favoring the activity and growth of syntrophic associations with hydrogenotrophic methanogens to the det- riment of active Methanosaetaceae and acetoclastic activity [3, 4, 7]. It is incor- porated as a central point in the bioindicator concept of process diagnosis [4] (see also Sects. 3 and “Microbial Guilds, Bioindicators and Transcriptional Profiling”). The second bottleneck is the thermodynamically difficult hydrogen, formate or electron-releasingconversionofshortchainfattyacids(SCFAs),alcoholsandother intermediatesofthebiogasprocess.Mostofthesereactionsareendergonicatstandard conditions but can be realized by syntrophic associations involving product-scavenging methanogens [8, 9]. Methanogenic archaea are able to remove thereactionproductsbyconvertingthemfinallytobiogas,predominantlyCH and 4 CO ,whichsegregatesfromthefermentersludgetothegasheadspaceandisfurther 2 withdrawn by gas utilization. Syntrophic bacteria or anaerobic fungi partners of methanogensaredifficulttocultivateandtostudywithouttheirproduct-consuming associate.Moderncharacterizationistypicallyinitiatedbygenomeormetagenome analysis,possiblyleadingtoinsightsaboutspecialrequirementsthatallowcultivation ofpureisolatesandstudyingtheirspecialphysiologicalperformances[10–12]. 4 M.Lebuhnetal. Athirdrecognizedbottleneckistheinitialrate-limitinghydrolysisofrecalcitrant substrates such as lignocellulose-rich biomass (LCB). When compared to aerobic degradation of lignocellulose, considerably less is known on the corresponding anaerobicprocessandtheorganismsinvolved.Besidesbacteria,otherorganismssuch as anaerobic fungi may be involved in efficient initial LCB attack and degradation [13].Chapter2inthisbookisdedicatedtoanaerobicfungiandrecentperceptionsof theirroleinanaerobicLCBdigestion.Forsomeofthesecellulolyticorganisms,the genomehasbeensequenced[14,15].Suchgenomeinformationisaninvaluabledata basisforprocessoptimizationandfurtherbiotechnologicalexploitation. Microbialprocessesinanaerobicdigestionaredrivenbyboth,bioticandabiotic factors. The physical and chemical environment (e.g. nutritional factors and redox status)arebasictoanddeterminethebioticactivity,thesubstrateconversionbythe microbes. Biotic measures to regulate the process (e.g. bioaugmentation) however are scarce as briefly discussed in Sect. 2.7. The most important issue in process optimization is to avoid the worst case, processdisturbanceorevenbreakdown.Thisrequiresaprocesscontrolstrategythat includes reliable process diagnosis based on meaningful analytical data. Since the activity of bioindicator microbes, organisms that are typical for certain process conditions, does react before conventionally used process chemical parameters indicate process failure, a promising approach for successful process control is to assesstheactivityofthesebioindicatorsasintegralpartofanearly-warningsystem [4]. The relevant actors, i.e. bioindicators performing the crucial biogas process steps, must hence be identified, and suitable analysis tools must be used or developed to track these key organisms and their activity quantitatively. In the following chapters, microbiology and molecular biology tools for biogas process diagnosis and control are compiled and discussed. Since several important process dynamics such as SCFA and total solid (TS) turnover as well as gas quality/quantity are the result of microbial activity, and respective wet chemistry and physico-chemical analyses are and will be indispensable part of conventional practice but have revealed limitations, recent experience with these conventional applications for agricultural single-stage biogas processes is presented in the fol- lowing Sect. (2). Molecular biology approaches have only recently emerged and may be introduced into practice after comparison or along with established physico-chemicalroutines.Some ofthese molecular tools, however, arepromising candidates to be implemented in a holistic suite of analytical tools for process diagnosis and control. 2 Physico-Chemical and Biochemical Process Parameters The spectrum of physico-chemical parameters actually employed for process diagnosis of agricultural and category 2 biowaste (untreated non-infectious to humans, animals or plants), biogas plants has originally been adopted from anaerobic sewage sludge digestion. Many of these parameters and respective