Periodic Reporting for period 1 - DMC-ReaDy (Dormant Microbial Communities Reactivation Dynamics in Lake Sediments)
Reporting period: 2021-04-01 to 2023-03-31
In aquatic sediments microorganisms are gradually buried by newly deposited layers and cut off from their main energy source, organic matter from overlying water or adjacent land. Yet, large populations of microorganisms are found in buried sediments, living in a state of strongly reduced metabolic activity. The evolutionary advantages of survival in this dormancy-like state remain unclear and may be related to rare events where buried microorganisms are returned to energy-replete conditions after centuries or millennia of starvation. The aim of this project was to observe if and how fast different sediment-inhabiting microbes are able to reactivate their metabolism and resume growth, and to determine the physiological changes and molecular mechanisms associated with the process.
To initiate reactivation of starved microorganisms in a natural setting, deep layers of lake sediment were transplanted to energy-rich surface sediments in diffusion chambers allowing nutrients to enter the chamber while preventing invasion of other microbes. During the six months of experiment no change in cell numbers, size or shape, and gene transcription were detected indicating no return of buried microbes to surface layer activity levels. This result contradicts previous findings from experiments performed in more artificial conditions where the detected reactivation might have stemmed from growth of contaminating surface organisms. It is thus a valuable contribution to the debate about microbial ‘dormancy’ in aquatic sediments and limits and nature of microbial survival in general.
Apart from the new insights into microbial ‘dormancy’ in sediments, the project also generated valuable data on changes of microbial metabolism with the progression of burial: from sediment surface to deeper layers and knowledge on metabolic capabilities of many abundant yet uncultured groups of sediment bacteria. This new knowledge further helped to understand how lake trophic status shapes the composition of microbial communities in the deposited sediments.
To initiate reactivation of starved microorganisms in a natural setting, deep layers of lake sediment were transplanted to energy-rich surface sediments in diffusion chambers allowing nutrients to enter the chamber while preventing invasion of other microbes. During the six months of experiment no change in cell numbers, size or shape, and gene transcription were detected indicating no return of buried microbes to surface layer activity levels. This result contradicts previous findings from experiments performed in more artificial conditions where the detected reactivation might have stemmed from growth of contaminating surface organisms. It is thus a valuable contribution to the debate about microbial ‘dormancy’ in aquatic sediments and limits and nature of microbial survival in general.
Apart from the new insights into microbial ‘dormancy’ in sediments, the project also generated valuable data on changes of microbial metabolism with the progression of burial: from sediment surface to deeper layers and knowledge on metabolic capabilities of many abundant yet uncultured groups of sediment bacteria. This new knowledge further helped to understand how lake trophic status shapes the composition of microbial communities in the deposited sediments.
An experimental set-up that allows incubation of deep sediment in the surface sediment layers of the same lake under near natural conditions was established. Avoiding oxygen exposure, deep sediments (1.20 m buried for ~ 2000 years) were placed in chambers where a membrane separates deep sediment from surface sediment. The membrane allows for diffusion of dissolved nutrients and prevents an exchange of microbial cells. The chambers were placed in surface layers of sediment cores from the same lake and the cores were placed in a flow-through aquarium with bottom water from the same lake. With the aquarium being placed in a dark climate-controlled room, the incubation conditions are as close to the ones at lake bottom as possible. Samples for analysis of gene expression and microscopic analysis have been taken from different depths of a fresh sediment core, to track the transition of microorganisms from active to a ‘dormant’ state, and from different time points of a half-a-year-long incubation of deep sediments in diffusion chambers.
During the project the RNA extraction protocol was optimized and the yields of RNA increased significantly, which allowed for a successful analysis of genes transcribed by the sediment-inhabiting microorganisms. Based on the obtained information, active microorganisms and metabolic pathways they use to generate energy and obtain carbon could be identified for the upper sediment layers. In the deeper sediment layers, the DNA of near-surface microorganisms was still present but only very few of them were active. The transplantation of sediment to the upper layers described above did not induce any change in activity of microorganisms buried deep in the sediment. This means that the possibility of reactivation of long-buried starved sediment microorganisms remains unconfirmed and the cells might be in an irreversible inactive state, slowly approaching cell death. With different types of inactive cellular states being wide-spread in the microbial world, the results are also relevant for other areas of microbiology and environmental sciences. Comparing different types of reversible and irreversible inactive states will help to better define the rather broad term of ‘microbial dormancy’.
Large body of work during the project was dedicated to the preparation for the transcriptome analysis. In order to attribute gene transcripts to specific microorganisms, genomes of these microorganisms had to be reconstructed from bulk DNA data. The reconstruction of these genomes enabled further analyses of the microbial community. For example, assessing what metabolic pathways of methane production are the most wide-spread ones in lake sediments. As DNA data was stemming from multiple Swiss lakes, the reconstructed genomes together with previously acquired geochemical data allowed for analysis of lake trophic status effects on the composition and metabolic capabilities of the sediment microbial communities. This coupling between the trophic status of a lake and microbial metabolic capacity has implications for the carbon cycling in lake sediments, an essential part of the global cycle of carbon sequestration and remineralization.
The results of the research have been presented at the “4th International Workshop on microbial life under extreme energy limitation” and at the D-A-CH Geobiology symposium 2022. A manuscript on methane cycling in lake sediments based on generated data is currently being prepared for submission. Two further manuscripts are being written: 1) On impact of lake trophic status on metabolic capabilities of microorganisms and 2) the lack of reactivation of deeply buried microorganisms upon sediment transplantation. Further collaborative studies using the generated data are ongoing and will likely lead to further publications.
During the project the RNA extraction protocol was optimized and the yields of RNA increased significantly, which allowed for a successful analysis of genes transcribed by the sediment-inhabiting microorganisms. Based on the obtained information, active microorganisms and metabolic pathways they use to generate energy and obtain carbon could be identified for the upper sediment layers. In the deeper sediment layers, the DNA of near-surface microorganisms was still present but only very few of them were active. The transplantation of sediment to the upper layers described above did not induce any change in activity of microorganisms buried deep in the sediment. This means that the possibility of reactivation of long-buried starved sediment microorganisms remains unconfirmed and the cells might be in an irreversible inactive state, slowly approaching cell death. With different types of inactive cellular states being wide-spread in the microbial world, the results are also relevant for other areas of microbiology and environmental sciences. Comparing different types of reversible and irreversible inactive states will help to better define the rather broad term of ‘microbial dormancy’.
Large body of work during the project was dedicated to the preparation for the transcriptome analysis. In order to attribute gene transcripts to specific microorganisms, genomes of these microorganisms had to be reconstructed from bulk DNA data. The reconstruction of these genomes enabled further analyses of the microbial community. For example, assessing what metabolic pathways of methane production are the most wide-spread ones in lake sediments. As DNA data was stemming from multiple Swiss lakes, the reconstructed genomes together with previously acquired geochemical data allowed for analysis of lake trophic status effects on the composition and metabolic capabilities of the sediment microbial communities. This coupling between the trophic status of a lake and microbial metabolic capacity has implications for the carbon cycling in lake sediments, an essential part of the global cycle of carbon sequestration and remineralization.
The results of the research have been presented at the “4th International Workshop on microbial life under extreme energy limitation” and at the D-A-CH Geobiology symposium 2022. A manuscript on methane cycling in lake sediments based on generated data is currently being prepared for submission. Two further manuscripts are being written: 1) On impact of lake trophic status on metabolic capabilities of microorganisms and 2) the lack of reactivation of deeply buried microorganisms upon sediment transplantation. Further collaborative studies using the generated data are ongoing and will likely lead to further publications.
The project presents first transcriptional data resolved down to individual microbial populations from a reactivation attempt of deeply buried sediment bacteria. Unlike in previous studies, the reactivation has been attempted in conditions close to natural, which might explain the results contradicting previous observations of increased microbial activity. It is an important addition to the exploration of inactive states of microorganisms and their reversibility. I also hope that, once published, the RNA extraction, sequencing, and analysis procedures will inspire the scientific community to apply these methods in fresh-water and marine sediments. The analysis of the reconstructed genomes alone provided new knowledge on metabolisms of common yet uncultured freshwater sediment microorganisms. For example, I could explore which pathways of methane generation dominate among lake sediment microorganisms. The results were surprising when compared to predictions made based on geochemical data and thermodynamic calculations. Exploration of this contradiction led to a new hypothesis on the nature of wide-spread interactions between methane producing and fermenting microorganisms. I further identified certain microbial groups that are indicative of an oligotrophic lake water during the time of sediment deposition and who persist deeper sediment layers even after the lake turned eutrophic. This can be a useful tool for reconstructing past changes of trophic status of lakes.