Zhu, Xinyu

Anaerob udrådning (AD) er en mikrobiel proces, hvor organiske materialer nedbrydes til biogas (CH4 og CO2). AD foregår i mange naturligt anaerobe miljøer og er en essentiel del af det globale kulstof-kredsløb. I konstruerede AD systemer, dvs. biogas-reaktorer, øges den metanogene aktivitet ved at anvende empirisk baserede operationelle forhold for at accelerere produktionen af metan til energiformål. I Danmark udgør biogas produceret ved AD en betragtelig del af den vedvarende energiproduktion, og denne andel forventes at øge yderligere. Således vil en mere effektiv drift af biogasanlæggene gavne Danmarks bæredygtige udvikling betydeligt. Da AD afhænger af kompleks mikrobiel aktivitet, vil en dybere indsigt i de mikrobielle konsortier og deres aktivitet udgøre fundamental viden om proceskontrol og optimering.I AD er metabolismerne overvejende termodynamisk begrænset og den obligatoriske syntrofi er et essentielt led. Derfor er størstedelen af mikrobiotaen i AD processen ikke karakteriseret via kultiverings-baserede metoder. Udviklingen af mere tilgængelige sekventerings-teknologier (dvs. next generation sequencing, NGS) og bioinformatik-baserede metoder har gjort det muligt for forskere at udføre in-situ analyser på ikke-karakteriserede mikrobielle samfund. Anvendelsen af NGS-teknologien har vist sig at være et effektivt redskab til at afsløre den mikrobielle økologi i den anaerobe udrådningsproces. De detaljerede mekanismer er dog fortsat langt fra alle belyst, grundet den komplicerede proces.Dette Ph.D.-projekt fokuserer på omfattende at undersøge de mikrobielle samfund involveret i AD, med det mål at kunne optimere processen og belyse de fundamentale metabolismer. Specifikt blev 16s amplicon sekventering, i forbindelse med procesoptimering, benyttet til at identificere, analysere og løse de operationelle udfordringer under opstart af en termofil UASB (up-flow anaerobic sludge blanket) reaktor. For at belyse de fundamentale metabolismer blev genom-centrisk metagenetik benyttet til at karakterisere metanogene mikrobielle samfund under nedbrydning af veldefinerede substrater. Dette Ph.D.-projekt bidrager med viden til forståelsen af den mikrobielle økologi i AD-processen ved at foreslå og karakterisere en ny Candidatus art, som er allestedsnærværende i AD-systemer.Opstarten af UASB reaktorer blev undersøgt i laboratorie-skala reaktorer inokuleret med mesofile granulater. Efter at have øget temperaturen fra mesofil til termofil blev det observeret at flygtige fedtsyrer (VFAs) og alkoholer var de dominerende nedbrydningsprodukter. Metanproduktionen påbegyndtes dog først efter yderligere bicarbonat blev tilsat, som ekstern pH-kontrol. Dynamikken i sammensætningen af de mikrobielle samfund i granulaterne ifm. temperaturskiftet indikerede, at hovedparten af de mesofile mikrober ikke kunne overleve de termofile forhold. De blev erstattet af termofile mikrober, som først indfandt sig i UASB reaktorens væskefase for derefter at blive indkapslet i granulatstrukturerne. Væksten hos disse bakterier genetablerede hurtigt hydrolysen, acidogenesen og acetogenesen i reaktoren. De termofile metanogener voksede derimod meget langsommere, hvilket resulterede i alvorlig ubalance i processen (dvs. akkumulering af VFA’er og alkoholer). Således kunne det slås fast, at væksten af de termofile metanogener var ”flaskehalsen” ifm. temperaturskiftet. For at løse dette blev det foreslået at benytte bioaugmentation, dvs. tilsætning af exogene mikrober, for at accelerere tilpasningen af det mikrobielle samfund. Den bedste strategi til at udføre bioaugmentation var injektion af axeniske metanogene kulturer. Denne praksis øgede den termofile metanproduktion signikant med 40 % sammenlignet med kontrollen. Den øgede metanproduktion bidrog til udviklingen af exogene Methanothermobacter thermautotrophicus, samt dennes syntrofiske partnerorganismer, i den granulære struktur. De positive effekter ved bioaugmentation blev bibeholdt grundet tilbageholdelsen af granulaterne i UASB-reaktoren.For at undersøge den fundamentale mikrobielle metabolisme og økologi, blev metanogene mikrobielle samfund beriget i en laboratorie-skala kontinuert omrørt tank-reaktor (CSTR) med syntetisk fødemateriale. Gennem eksperimentet blev substraterne gradvist forsimplet (hhv. polysakkarider, monosakkarider, kortkædede fedtsyrer, acetat) for at efterligne de fire dele af AD-processen. Gennem den kontinuere drift blev sammensætningen af det mikrobielle samfund væsentligt simplere, da de mikrober, der ikke metaboliserede de specifikke komponenter, blev vasket ud. Det samlede mikrobielle samfund bestod af kun 35 metagenome assembles genomer (MAGs; 31 bakterielle og 4 archaea). Tilstedeværelsen af disse MAGs varierede betragteligt i de mikrobielle samfund afhængigt af fødematerialet. De skift der blev observeret i de mikrobielle samfunds sammensætning indikerer, at disse MAGs har specifikke funktionelle roller i AD-fødekæden, og at deres roller ikke altid defineres fysiologisk gennem de 4 AD-skridt. Derudover var de omfattende nedbrydningsmønstre rekonstruerede fra den funktionelle annotation af MAG. En ny model for nedbrydning af glukose er desuden foreslået, baseret på syntrofisk aktivitet mellem Clostridiaceae sp. og Methanoculleus thermophilus. I denne model produceres ikke acetat som et mellemprodukt.Den genom-centriske metagenetic afslører adskillige MAGs, som ikke taxonomisk kunne relateres til karakteriserede arter. Et MAG ekstraheret fra sammenblanding af 8 AD metagenomer var specielt interessant grundet dens udbredelse i AD-systemer og dens høje tilstedeværelse under specifikke forhold. Fra den funktionelle annotation og genekspressions-profil kan det bekræftes at dette MAG udfører hydrogenotrofisk metanogenese i AD-systemet. Dette genom er tilstede i 40 forskellige prøver, både fra laboratorie- og fuldskala AD-reaktorer. Prøverne viser desuden, at tilstedeværelsen af dette MAG øges under termofile forhold med relativt korte hydrauliske opholdstider. Rekonstruktionen af fylogenien, baseret på 400 bevarede gener, og på 16S rRNA gener viste begge, at dette MAG er tæt relateret til Methanoculleus bourgensis MS2T. Den gennemsnitlige overensstemmelse mellem nukleotiderne i M. bourgensis MS2T og det valgte MAG er kun 89%, hvilket er for lavt til at tilskrive dette MAG på arts-niveau. Vi foreslår derfor dette MAG som en ny Candidatus art i slægten Methanoculleus. Grundet de metaboliske egenskaber er den tildelt navnet Candidatus Methanoculleus thermohydrogenotrophicum, sp. nov.Alt i alt demonstrerede resultaterne fra nærværende Ph.D.-studium forskellige anvendelser af NGS-teknologien i AD-systemer. Den opnåede indsigt i sammensætningen af de mikrobielle samfund og dynamikkerne blev direkte benyttet til at løse tekniske udfordringer i driften af AD-reaktorer. Dybere indsigt i den mikrobielle metabolisme og økologi har udvidet den nuværende forståelse af AD-processen væsentligt. Den opnåede viden udgør værdifulde forudsætninger for fremtidig AD proceskontrol og -optimering.Anaerobic digestion (AD) is a microbial mediated process where organic compounds are degraded to biogas (CH4 and CO2). AD occurs in many natural anoxic environments and is an essential step for global carbon circle. Engineered AD systems, i.e. biogas reactors, enhance methanogenic activity by applying empirical operational conditions, in order to accelerate the methane production for energetic purposes. In Denmark, biogas produced from AD has a considerable share in renewable energy with the expectation to expand. Thus, the more effective operation of biogas plants will significantly benefit Denmark’s sustainable development. As AD relies on complex microbial activity, a more comprehensive understanding of the AD microbial consortia and their activity provides the fundamental knowledge for process control and optimization.In AD, the microbial metabolisms are mostly thermodynamically constrained and the obligatory syntrophy is an essential intermediary step. Thus, the majority of AD microbiota remains uncharacterized since in the past it was mainly investigated using cultivated-based methods. The advent of more powerful sequencing technology (i.e. next generation sequencing, NGS) and newly developed bioinformatic methods enable researchers to perform in-situ analyses on uncharacterized microbial communities. The applications of NGS technology were proved to be effective tools to reveal AD microbial ecology. However, the detailed mechanisms of microbial activity are still far from fully elucidated due to the intricacy of AD process.This Ph.D. project relied on comprehensive investigations of microbial communities in order to optimize the AD process and elucidate the fundamental metabolisms. Specifically, in the case of process optimization, 16S rRNA amplicon sequencing was used to identify, analyse and solve the operational challenges during the start-up of thermophilic up-flow anaerobic sludge blanket (UASB) reactors. To elucidate the microbial metabolisms, genome-centric metagenomics was applied to characterize methanogenic communities degrading a set of defined substrates. In addition, the Ph.D. study also expands the understanding of AD microbial ecology by proposing and characterizing a novel Candidatus species ubiquitously present in AD systems.The start-up of thermophilic UASB reactors was investigated in lab-scale reactors inoculated with mesophilic granules. After increasing the operational temperature from mesophilic to thermophilic, volatile fatty acids (VFAs) and alcohols were found as the main digestion products. Methane production, on the other hand, only initiated after bicarbonate addition as external pH control. The dynamicity of microbial community composition in the granules during the temperature shift suggested that the majority of the mesophilic microbes could not tolerate the thermophilic conditions. Moreover, it was demonstrated that the fermentative thermophiles first evolved in the liquid phase of UASB reactor and then were encapsulated in the granular structure of the sludge. The growth of these bacteria rapidly restored the hydrolysis, acidogenesis and acetogenesis in the reactor. On the contrary, the thermophilic methanogens grew much slower than fermentative bacteria leading to severe process imbalance (i.e. accumulation of VFAs and alcohols). Thus, the evolvement of thermophilic methanogens was recognized as the biological ‘bottleneck’ during the temperature transition. To overcome the identified obstacle, bioaugmentation, i.e. provision of exogenous microbes, was proposed to accelerate the microbial community adaptation. The best strategy found to perform bioaugmentation was the injection of axenic methanogenic cultures. This practice significantly increased the thermophilic methane production rate by 40% compared with the control reactor (i.e. without bioaugmentation). The enhancement of methane production was attributed to the evolvement of exogenous Methanothermobacter thermautotrophicus and the concomitant growth of its syntrophic partners in the granular structure. The positive effects brought by bioaugmentation were persistent in UASB reactor due to the retention of the microbes in the granular sludge. For the investigation of the basic microbial metabolism and ecology, methanogenic microbial communities were enriched in a lab-scale continuous stirred-tank reactor (CSTR) fed with synthetic feedstocks. In the experiment, the substrates used were stepwise simplified (i.e. polysaccharide, monosaccharide, short chain fatty acids, acetate) to mimic the four steps of AD process. During the continuous operation, the microbial community was substantially simplified, because the microbes that could not metabolize the specific compounds were washed out. The overall microbial community consisted of only 35 metagenome assembled genomes (MAGs) (31 bacterial and 4 archaeal). The abundance of these MAGs dramatically varied in the communities adapted to different substrates. The shifts in microbial community composition indicate that MAGs have specific functional roles in AD food chain and their roles cannot always be physiologically defined in accordance with 4 AD steps. Moreover, the explicit degradation pathways were reconstructed from the functional annotation of MAGs. It is notable that, a novel glucose degradation model was proposed with the syntrophic activity of Clostridiaceae sp. and Methanoculleus thermophilus. In this model, acetate is not produced as intermediate compound. The genome-centric metagenomics reveals a considerable number of MAGs that could not be taxonomically assigned to characterized species. A MAG extracted from co-assembly of 8 AD metagenomes was especially emphasized due to its ubiquity in AD system and its high abundance under specific conditions. From the functional annotation and gene expression profile, it is confirmed that this MAG performs hydrogenotrophic methanogenesis in AD system and is found dominant from the reactors where H2 was added. This genome is present in 40 different samples from both full-scale and lab-scale AD reactors. The MAG was found in higher abundance during thermophilic reactor operations with relatively short hydraulic retention times. The phylogenetic assignment was based on 400 conserved genes and on 16S rRNA genes. The two methods concordantly showed that this MAG is closely related to Methanoculleus bourgensis MS2T. However, the average nucleotide identity between M. bourgensis MS2T and the selected MAG was only 89%, which is too low similarity to assign the MAG at the species level. Thus, we propose a novel Candidatus species inside the Methanoculleus genus. According to the metabolic traits, it is named as Candidatus Methanoculleus thermohydrogenotrophicum, sp. nov.Overall, the results from this Ph.D. study bring new knowledge on the AD process based on NGS technology. Practically, the gained information regarding microbial community composition and dynamicity was directly used to solve technical challenges in AD operations. Fundamentally, deeper insights into the microbial metabolisms and ecology substantially expanded the current understanding of AD. The revealed knowledge provides pivotal prerequisites for future AD process control and optimization. <br/

Online Research Database In Technology

Deciphering the microbial ecology in bio- gas reactors for optimizing the anaerobic digestion process

http://orbit.dtu.dk/ws/files/142938103/Thesis_online_version_Xinyu_Zhu.pdf

Deciphering the microbial ecology in bio- gas reactors for optimizing the anaerobic digestion process

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Online Research Database In Technology