Microorganisms of anaerobic digestion and their potential for process enhancement Author Mag. Biol. Andreas Walter Dissertation submitted to the Faculty of Biology of the Leopold-Franzens Universität Innsbruck in partial fulfilment of the requirements for the degree Doctor of Philosophy (PhD). Innsbruck, November 2017 Advisors Univ. Prof. Dr. Heribert Insam Institute of Microbiology, Universität Innsbruck Prof. Dr. Dieter Bryniok Group of Bioprocess Technology, Frauenhofer IGB Stuttgart Group of Biotechnology, Fachhochschule Hamm-Lippstadt PD Ingrid Franke-Whittle, PhD, MSc Institute of Microbiology, Universität Innsbruck Cover design & layout Andreas Walter Cover illustrations Andreas Walter Ingrid-Franke Whittle Marina Fernández-Delgado Juárez DNA: https://abm-website-assets.s3.amazonaws.com/forensicmag.com/s3fs- public/styles/content_image/public/featured_image/2017/06/shutterstock_13 4698571.jpg?itok=4Muqxq7v This work was carried out at the Institute of Microbiology, Leopold- Franzens Universität Innsbruck . It includes results from several projects that were founded by alpS GmbH in cooperation with Müller Abfallprojekte GmbH, the Tiroler Wissenschaftsfonds (TWF), the Fonds für Wissenschafliche Forschung (FWF) and the Aktion D. Swarovski KG. “The experience reminds me of a favorite saying: Most of the yield from research efforts comes from the coal that is mined while looking for diamonds.” Paul D. Boyer Table of Contents I. Background.………………………………………….......................…1 II. Proving stability and degradation efficiency in bench scale biomethane potential tests …………………................................................................13 Publication 1 - Biomethane potential of industrial paper wastes and investigation of the methanogenic communities involved….…....19 Publication 2 - Methane yields and methanogenic community changes during co-fermentation of cattle slurry with empty fruit bunches of oil palm…………………...........................…………..49 Publication 3 - Investigation into the effect of high concentrations of volatile fatty acids in anaerobic digestion on methanogenic communities………………………………………………........63 III. Monitoring of physicochemical parameters and community screenings in big scale, commercial AD plants…...…..…....………………....….91 Publication 4 - Searching for links in the biotic characteristics and abiotic parameters of nine different biogas plants………............101 Publication 5 - Biotic and abiotic dynamics of a high solid-state anaerobic digestion box-type container system………....…........133 Publication 6 - Investigation into the effect of high concentrations of volatile fatty acids in anaerobic digestion on methanogenic communities……..………………………………...……….…163 Publication 7 - Microbiome in anaerobic digestion of sewage sludge with and without co-substrates………………...………………193 IV. References…………………………………………………......…..213 V. Acknowledgement………………………………………………...219 VI. Curriculum Vitae ………………………….………....…..................221 Abstract Zusammenfassung Die Produktion von Biogas, besetzt im Angesicht anderer regenerativer Energiequellen wie Wind, Sonne und Wasser, eine kleine, aber nichts destotrotz bedeutende Nische, da neben der nachhaltigen Erzeugung von Energie gleichzeitig organische Abfallströme bedient werden können. Biogas entsteht in einem anaeroben Vergärungsprozess, in welchem organische Reststoffe durch ein Konsortium von Mikroorganismen in hauptsächlich Methan (CH 4) und Kohlendioxid (CO 2) umgewandelt werden. Die Interaktion, die in diesem Prozess zwischen Bakterien, Archaeen und ihrer Umwelt ablaufen sind so komplex und vielfältig, dass der Gesamtprozess immer noch nicht vollständig entschlüsselt werden konnte. Im Zuge dieser PhD-Arbeit wurde versucht, einige Wissenslücken zu schließen, um schlussendlich die Effizienz dieses komplexen Prozesses ein Stück steigern zu können. Laborversuche wurden durchgeführt, um das Biomethanpotential verschiedenster Substrate zu testen, die Überwachung des Abbauprozesses zu verbessern und Änderungen der mikrobiellen Gemeinschaft zu verstehen. Daneben wurden auch bestehende Großanlagen einem Monitoring unterzogen, um Fluktuationen und Verschiebungen in der bakteriellen und methanogenen Gemeinschaft zu überwachen und Zusammenhänge zwischen biotischen und abiotischen Umweltfaktoren aufzudecken. Kombinationsverfahren wie Milchsäureproduktion und nachgeschaltete Biomethanisierung erwiesen sich als geeigneter, hocheffizienter Prozess zu Bioabfallbehandlung, Rückstände aus der Palmölproduktion erwiesen sich als geeignete Substrate für anaerobe Vergärung und ein neuartiges solid-state- Verfahren, das aerobe und anaerobe Phasen kombinierte, konnte als hocheffizientes Verfahren für lignocellulosereiche Substrate bestätigt werden. Ergebnisse zeigten einerseits eine große Variabilität der bakteriellen Komponente der Mikrobiota, und andererseits eine große Stabilität der Archaeengemeinschaft. Die methanogenen Archaeen werden deshalb als wenig geeignet angesehen, das Prozessmonitoring frühzeitig zu unterstützten, während die bakterielle Biota durchaus Potenzial aufweist, zur Früherkennung von Prozessproblemen beizutragen. I Abstract Abstract Beside common renewable energy sources, such as solid biofuels, hydro, wind and solar power, the production of biogas is a small niche, achieving waste management and energy recovery at the same time. Biogas is produced in an anaerobic digestion process, where biodegradable material is transformed into mostly methane (CH 4) and carbon dioxide (CO 2) by a consortium of bacteria and archaea. Interactions among these microorganisms and their environment are complex and diverse, thus the anaerobic digestion process is still far away from being fully understood. The main objective of the present PhD thesis was to fill some of the related knowledge gaps, helping to finally improve the overall anaerobic digestion process efficiency. Three lab-scale experiments were conducted to review the biomethane potential (BMP) and the process stability of multifaceted substrates. High CH 4 yields of fermentation residues from a lactic-acid generating biorefining approach and of fibre-containing residues from the palm oil and paper industries, revealed a suitable potential for full-scale application. The pretreatment of complex organic matter by mechanical, chemical or biological applications was found to be a viable option for increasing the overall degradation efficiency and decreasing reactor retention time. In all bench-scale reactors, acetoclastic Methanosarcina was found to be the dominant methanogen, accompanied by a hydrogenotrophic consortium of Methanobacterium , Methanoculleus and Methanosphaera . Changes in reactor temperatures, co-substrate or pretreatment type caused minor effects on the methanogenic community composition. Twelve full-scale biogas plants were monitored three times a year to find links between biotic characteristics and abiotic process parameters. Acetoclastic Methanosarcina or Methanosaeta were found to be dominant in reactors operated under mesophilic conditions, accompanied by hydrogenotrophic consortia of Methanobacterium , Methanobrevibacter , Methanoculleus , Methanocorpusculum and Methanosphaera . High abundances of Methanothermobacter were found in reactors operated at 55 °C. Correlation analysis to determine any links between the microbial communities found, and the physico-chemical parameters investigated revealed a positive relationship between Methanosarcina and concentrations of acetate. II Abstract In solid-state AD reactors, high yields of biogas correlated positively with the abundance of this genus. High numbers of Methanocorpusculum and extensive percolation, however, were found to negatively correlate with biogas production in these fermenters. In all the different reactors, the archaeal communities were found to remain stable over time. Even short-term acidification appeared to have no significant effects on these communities. Thus, methanogen community dynamics would not seem to be an appropriate indicator regarding BMP, when the microbial community is fully established. In another project, sludges of two North Tyrolian biogas plants were monitored and sampled, when VFA levels were high, as well when VFA levels were low. Aim of this study was to determine if differences in the VFAs levels could result in variations in the indigenous methanogenic communities. The methanogenic reactor was shown to be dominated by a more diverse community, comprising Methanosarcina , Methanoculleus , Methanobacterium and Methanosaeta , while the thermophilic community was dominated by Methanothermobacter . Communities in both reactors remained stable over time. Hence, even huge changes in the VFA level in the AD digesters did not greatly alter the methanogen communities, probably due to good buffering capacities in both AD systems. Finally, seven full-scale anaerobic digesters of five different wastewater treatment plants (WWTPs) were analysed by conducting biotic and abiotic investigations: A combination of robust community fingerprinting and Illumina MiSeq sequencing revealed a core bacterial community dominated by Chloroflexi , Firmicutes Bacteroidetes and Proteobacteria , with variations in the profiles due to differences in the co-substrate feeding regime. Despite these differences, the physicochemical properties and dynamics of biomethane generation revealed a stable performance of all reactors, indicating a resilient bacterial microbiota in all full-scale reactors. Within the Archaea , Methanosaeta dominated in