Identifying lignocellulose-degrading strategies in denitrifying microbial communities using meta-omics approaches

Abstract

Microorganisms are everywhere, and millions of species are estimated to exist; however, only a small fraction have been isolated and in-depth characterized. For this reason, many habitats remain largely unexplored and may hold untapped potential in the discovery of new enzymes and pathways to curb or counteract many of the environmental challenges we face today, including the everincreasing emission of greenhouse gases, increases in global temperature, and the loss of biodiversity worldwide. Culture-independent methods, such as the meta-omics, have over the last decades revolutionized microbial ecology and contributed massively to new understanding of the microbial “dark matter”. In this thesis I have explored defined habitats that are rich in nitrate and lignocellulose. This combination is of interest to me because lignocellulose degradation under denitrifying conditions is to date not well studied and may hold potential for new discoveries. Lignocellulose degradation has been widely studied under aerobic conditions, showcasing an important role for oxidative enzymes in the degradation of both lignin and (hemi)cellulose. It has also been well-studied under anaerobic conditions where microorganisms utilize sophisticated enzyme systems for coordinated and efficient breakdown. However, under denitrification, questions remain to be answered regarding the degrading strategies used by the microorganisms, and even a potential utilization of oxidative enzymes. Using meta-omics, and in particular metagenomics and metaproteomics, I aim to provide in this PhD thesis a deeper understanding of how denitrifying microorganisms enzymatically degrade lignocellulose in various anaerobic environments. Firstly, we studied a microbial community originating from a woodchip bioreactor in Denmark, and the microbial community’s potential, as well as active degradation strategies for lignocellulose turnover. This study explores the activity of carbohydrate-active enzymes (CAZymes) in every metagenome- assembled genome (MAG) and further reconstruct the metabolic potentials and active pathways to obtain a comprehensive view of the functional dynamics of the community. The results suggested a broad degradation of lignocellulose subfractions, even including expressed oxidative enzymes whose functionality is puzzling under strict anaerobic conditions. Secondly, we have analyzed the microbial communities present in multiple nearby lakes in Frogn and Ski municipalities. These lakes are large catchment areas for agriculture run-offs and are designated with a high eutrophic status. Fertilizer (rich in nitrate and phosphorous) leach into the lakes and together with falling leaves and branches in the riparian zone (close to the shore), this constructs a niche habitat with high nitrate and lignocellulose levels. Again, using metagenomics and metaproteomics, in combination with gas analyzes, we have analyzed the microbes’ potential as well as active degradation strategies for lignocellulose turnover. Here, we have focused on identifying a core microbiome present in all the eutrophic lakes and particularly investigated their metabolic potential and active pathways. A broader microbial community analysis identified key lignocellulose degraders expressing CAZymes, here also including expressed oxidative enzymes, raising questions about “dark oxygen” production in these systems. Finally, we have also developed a novel bioinformatic workflow for metagenomics with integrated workflows for metatranscriptomics and metaproteomics within the Galaxy suit of tools. In connection with this, we also developed interactive tools for the visualization of meta-omics stats and annotations in the new software app ViMO, providing advanced analysis in a user-friendly environment. Overall, this PhD work has produced a catalog of more than a thousand MAGs found in lakes and woodchip bioreactors, many of which are lignocellulosedegrading denitrifiers. This may serve as a large resource for future work. Isolation attempts of these may characterize their denitrifying phenotype, their expression of enzymes in mono- or co-culture with respect to anoxic (denitrifying), microoxic, and oxic conditions, as well as investigate their expression and utilization of oxidative enzymes in anoxia.

Mikroorganismer finnes overalt, og man regner med at det finnes millioner av arter, men bare en liten brøkdel av dem har blitt isolert og grundig karakterisert. Derfor er mange habitater fortsatt i stor grad uutforsket, og de kan ha et uutnyttet potensial når det gjelder å finne nye enzymer og veier som kan dempe eller motvirke mange av miljøutfordringene vi står overfor i dag, blant annet de stadig økende utslippene av klimagasser, den globale temperaturøkningen og tapet av biologisk mangfold verden over. Kulturuavhengige metoder, som meta-omikk, har i løpet av de siste tiårene revolusjonert mikrobiell økologi og bidratt massivt til ny forståelse av den mikrobielle “mørke materien”. I denne avhandlingen har jeg utforsket definerte habitater som er rike på nitrat og lignocellulose. Denne kombinasjonen synes jeg er interessant fordi nedbrytning av lignocellulose under denitrifiserende forhold til dags dato er lite studert og innehar potensiale for nye oppdagelser. Nedbrytning av lignocellulose har blitt grundig studert under aerobe forhold, noe som viser at oksidative enzymer spiller en viktig rolle i nedbrytningen av både lignin og (hemi)cellulose. Det er også relativt godt studert under anaerobe forhold, der mikroorganismene bruker sofistikerte enzymsystemer for koordinert og effektiv nedbrytning. Under denitrifikasjon gjenstår det imidlertid å finne svar på hvilke nedbrytningsstrategier mikroorganismene bruker, og om de eventuelt benytter seg av oksidative enzymer.