C1

State of the art

Influences of trophic changes of lakes on fermentation processes in the corresponding lake sediments have not been studied so far. Due to very high numbers of bacteria in sediments (10⁸-10⁹ cells per ml), their enormous diversity (up to 10 000 species) and their high functional redundancy, we assume that microbial communities in sediments are highly resilient towards changes in food supply. Moreover, bacteria are known to survive long periods of insufficient substrate supply at low numbers, thus securing a high degree of reversibility after periods of disturbance. Degradation of the epilimnetic primary production to methane and carbon dioxide in the sediment typically proceeds in a three-step process including primary fermentations, the obligately syntrophic secondary fermentations, and methanogenesis. Alternatively, primary and secondary fermentations may be coupled in a single organism which cooperates directly with methanogenic partners (two-step process). Due to the low energy supply of methanogenic communities in general (Schink and Stams 2013) and in oligotrophic lakes in particular, we hypothesize that the two-step process is typical of an oligotrophic sediment whereas the three-step process represents a system adapted to high and periodic substrate import as typical of, e.g., a eutrophic lake sediment. In the project presented here, we want to characterize the numerically dominant communities of sugar-degrading fermenting bacteria in sediments of different trophic states and to follow the transition from one trophic state to the other in chemostats at different substrate supply rates, thus examining the resilience and the reversibility of the two different fermentation modes under conditions of changing substrate supply. Beyond examining our hypothesis above, we want to identify – on the basis of a broad variety of novel syntrophically fermenting bacteria – reliable biochemical and molecular markers which allow to quantify the relative contribution of two-step versus three-step degradation processes within methanogenic microbial communities under the influence of changes in the trophic state.

Preparatory work

An obligately syntrophic glucose-oxidizing bacterium cooperating with methanogens in a two-step mode was isolated in our lab from profundal sediments of Lake Constance as the numerically dominant sugar utilizer which outnumbered conventional sugar-fermenting bacteria in the sediment by two orders of magnitude (Müller et al. 2008, 2015)(Müller et al., 2008; 2015). Further bacteria which oxidize amino acids in a similar syntrophic mode were enriched from the same source (Y. Patil, unpublished). Our lab has ample experience in the cultivation, characterization and biochemical analysis of syntrophic and other strictly anaerobic bacteria, including cultivation in chemostats, and also maintains all analytical equipment to quantify important microbial metabolites including formate and hydrogen at micromolar concentrations.

Proposed project and role within the RTG

In this project, we want to examine the hypothesis whether methane formation in an oligotrophic sediment follows a two-step strategy whereas a eutrophic system selects for a three-step process as outlined above. We will follow cultivation-dependent and molecular approaches to quantify syntrophic (two-step) and non-syntrophic (three-step) sugar-fermenting bacteria in lake sediments of different trophic status (Upper and Lower Lake Constance, Mindelsee) and under changing trophic conditions (chemostat experiments with shifts in substrate supply with sediments from Upper Lake Constance and Mindelsee). Quantitative community analysis by dilution-to-extinction cultivation in the presence and absence of hydrogen-scavenging methanogens with different sugars will elucidate the relative importance of two-step versus three-step fermenting communities. These enrichment experiments are to be accompanied by molecular biological analyses following the community development on the basis of phylogenetic (16S rRNA) or functional (hexokinase gene) markers. Numerically dominant sugar degraders are to be characterized with respect to specific enzymes (e. g., bifurcating enzymes; Herrmann et al. 2008) that may allow to develop specifically designed molecular probes to quantify populations of obligately syntrophic sugar fermenters (two-step process) in sediment material. Analysis of fatty acid pools (Montag and Schink 2016) and dissolved organic matter in sediment pore water will allow to calculate the energetic options for the two different modes of methanogenic degradation in the various sediments. Determination of enzyme activities in cell-free extracts from the same source will help to quantify the overall contribution of syntrophic key enzyme reactions (hydrogenases, formate dehydrogenases) to the overall degradation processes.

The project will cooperate closely with project A1 (paleo-limnology of sediments), A2 (bacterio- and phytoplankton), B4 (biofilms), C2 (nitrifying microbes) and C3 (carbon flow dynamics in the water column). Beyond these, we might apply carbon isotope fractionation (C3) as an analytic tool for identification of different sugar degradation strategies.