Austrian Science Fund project F₄₂₀

Because size does matter: F₄₂₀ tail length in methanogens

Start of the project

November 2023

Prinicipal Investigator

Andreas O. Wagner

Research context

Cofactors are indispensable in many enzymatically catalyzed processes. In biogas processes, the coenzyme F420 is involved in methanogenesis and inevitable for methane generation. This remarkable cofactor is equipped with a polyglutamic-tail that varies in length. The project is designed to study F420 tail length in methanogens.

Hypotheses

F420 tail length spectra vary along with functionality, taxonomy and/or physiological status of methanogenic pure or mixed cultures.

• Tail length profiles vary between different (groups of) methanogens under optimal growth conditions.
• The electron donor affects F420 tail length profiles and therefore tail length provides information on the dominant catabolic pathway in a methanogenic culture.
• Incubation temperature exhibits an effect on tail length expression in methanogenic pure cultures.
• A switch in substrate and/or loading rate during AD can be traced back by the evaluation of F420 tail length profiles in a mixed methanogenic community.

Objectives

Elucidation of the role of F420 tail length in methanogenic pure and mixed culture systems:

1. Systematic analyses of cofactor tail length in various meso- and thermophilic, acetoclastic, hydrogeno- and methylotrophic methanogenic pure cultures as a function of growth phase, electron donors as well as incubation temperature (meso-/thermophilic).
2. Applying RNA based analyses intends to gain an insight into the regulation of F420 tail length expression in methanogenic pure cultures with regard to different environmental conditions.
3. Investigation of F420 tail length profiles from methanogenic mixed cultures with regard to varying environmental conditions.

Methods

A mixture of analytical, classical microbiological and highly modern approaches will be applied including anaerobic cultivation, gas chromatography, high performance liquid chromatography, AmpliconSeq, differential-RNA-sequencing with primary transcript enrichment, dPCR, including the respective data analyses and processing techniques.

Originality

F420 exhibits an essential role in electron transfer during one-carbon-reactions in methanogenesis. Although this process is well studied, little is known about of F420tail-length profiles in methanogens. Latest research elucidated the genes involved in the formation of F420-n. However, in situ analyses of methanogenic organisms regarding F420 tail-length are largely missing. Hence, this is a microbiologically orientated approach to systematically investigate the cofactor tail length in methanogenic pure-, then co- and subsequently mixed cultures to finally understand how F420 tail length is connected to the performance of anaerobic digestion systems.