Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - VOLCANO (Microbiology of extremely acidic terrestrial volcanic ecosystems)

Teaser

The acronym of this project refers to the little island of Vulcano in the Mediterranean Sea which was believed to be the chimney of the forge of Vulcan - the blacksmith of the Roman gods. We aim to increase the fundamental understanding of the microbial ecology of extremely...

Summary

The acronym of this project refers to the little island of Vulcano in the Mediterranean Sea which was believed to be the chimney of the forge of Vulcan - the blacksmith of the Roman gods. We aim to increase the fundamental understanding of the microbial ecology of extremely acid terrestrial mud volcanos with special emphasis on the elemental cycles of sulfur, methane and nitrogen.
In a metagenomic approach we have isolated DNA from Solfatara samples (Italy), sequenced it using NGS technologies and analyzed the data using ‘in house’ pipelines. In 2017 we organized a sampling campaign to Pantelleria Island (Italy) in cooperation with scientist from the Istituto Nazionale di Geofisica e Vulcanologia and the University of Palermo (Italy). Samples were taken to analyze nutrient profiles and emissions, to extract DNA/RNA for metagenomics and to start specific enrichments. We also obtained samples from Volcano Island for metagenomic studies.
The biogenic sulfuric acid is responsible for extremely acidic sulfur-rich volcanic environments like mud pots. The genome of our methanotrophic Solfatara isolate strain SolV harbors a gene encoding a putative methanethiol oxidase (MTO. We showed that methanethiol and hydrogen sulfide were consumed and in a spin-off project we were able to fully characterize a bacterial MTO and to demonstrate its presence in humans. Mutations in the human MTO results in extra-oral halitosis. From Pantelleria Island samples we successfully isolated autotrophic hydrogen-consuming sulfate-reducers.
Volcanic hydrogen is a potential energy source for methanotrophs. We showed that strain SolV can grow as a real ‘Knallgas’ bacterium, without methane. We purified the oxygen tolerant Group 1h/5 hydrogenase since this enzyme is a high potential candidate in biotechnological applications.
Using batch and methanol limited continuous cultures, we showed that strain SolV was able to oxidize ethane and propane. In addition, growth was possible on natural gas.
The membrane protein complexes of strain SolV were resolved. We were able to identify 296 unambiguous proteins including the important protein complexes in methane oxidation, carbon fixation, and the electron transport chain.
The metagenomic analysis of the Pantelleria Island sediments showed a high relative abundance of a novel methanogen, a Methanocella sp. Attempts to enrich for this methanogen resulted in enrichment of thermoacidophilic acetogens. We also started H2-consuming enrichments under aerobic conditions.
We have obtained an active enrichment showing stable performance in the oxidation of ammonium at low pH values. Metagenome sequencing resulted in a complete genome of the nitrifier involved which most likely represents a new genus. Molecular analysis of the Pantelleria ecosystems showed the presence of close relatives.
The production of methanol was studied using a combination of hydrogen and methane under rare earth element (REE) limitation. This research may show the potential of conversion of methane/ biogas) to liquid biofuel. As part of this we also study the uptake of REEs and the role of different REE’s in the methanol dehydrogenase of strain SolV. In cooperation with Lena Daumann (Ludwig-Maximilians-Universität München) we have developed a new fluorometric method to follow the uptake of REE’s. Furthermore, we purified methanol dehydrogenases from strain SolV with europium or lanthanum in its active site. Samples from the Pantelleria sampling campaign were used to start enrichments on methane and we were able to isolate a new representative of the genus Methylacidimicrobium.

Work performed

A. Nutrient profiles and meta-omics of the mud pot volcano ecosystems. DNA was isolated from mudpot and soil samples obtained from the Solfatara (Naples, Italy). DNA isolation protocols had to be optimized for the acidic ecosystems sampled. The DNA was sequenced using next generation sequencing technologies. The metagenome data were analysed using ‘in house’ pipelines. Species representing the genera Acidithiobacillus, Thermoplasma, Picrophilus, Ferroplasma, and Sulfolobus were the most dominant ones. In May 2017 we organized a sampling campaign to Pantelleria Island (Italy) in cooperation with scientist from the Istituto Nazionale di Geofisica e Vulcanologia (INGV) and the University of Palermo (Italy) (Seminar Op den Camp 2017). This Favara Grande is located in the middle of the island and is characterized by high geothermal fluxes composing of mainly CO2, but also CH4, CO, H2 and NH3. Although there is no volcanic activity, there is a high flux of geothermal gasses, the pH is low and the temperature is high. Samples to analyze nutrient profiles and emissions related to the elemental cycles of carbon, hydrogen, nitrogen and sulfur were collected. In addition samples were taken for DNA/RNA extraction and metagenome analyses. These analyses were performed on core samples obtained from the FAV2 site to detect the presence of novel genomes and to study the microbial community. The soil core was divided in three subsamples of 0-5 cm (top layer), 5-10 cm and 10-15 cm. DNA was extracted with two different methods (CTAB and Fast DNA Spin kit for soil), sequenced with Illumina MiSeq and analyzed. A total of 52,825,705 reads and 103 bins were obtained from the sequencing, with 30 bins showing >90% completeness. We also obtained samples from Volcano Island and aim to study the microbial composition of this site by metagenomic approaches.
B. Sulfur cycling in terrestrial mud volcanos. Microbial cycling of organic sulfur compounds especially dimethyl sulfide and methanethiol plays a vital role in the processes of global warming, acid precipitation, and the global sulfur cycle. Volcanoes are significant contributors to the sulfur budget. The biogenic sulfuric acid is responsible for extremely acidic sulfur-rich environments like mud pots. The genome of strain SolV, isolated from the Solfatara, was shown to harbor a gene encoding a putative methanethiol oxidase (MTO) which catalyzes the oxidation of methanethiol to formaldehyde, hydrogen sulfide and hydrogen peroxide. We showed that methanethiol and in addition, hydrogen sulfide were consumed by strain SolV resulting in increase of biomass concentration. In a spin-off project we were able to fully characterize a bacterial MTO and to demonstrate its presence in humans. Mutations in the human MTO results in extra-oral halitosis (Eyice et al. 2017; Pol et al. 2018).
The samples from Pantelleria Island were successfully used to isolate autotrophic hydrogen-consuming sulfate-reducers. Active enrichments were used to isolate pure cultures through serial dilutions and the strains are currently characterized.
C. Carbon and hydrogen cycling in terrestrial mud volcanos. In volcanic ecosystems hydrogen is present as a potential energy source for methanotrophs. The full genome of M. fumariolicum SolV revealed the presence of two hydrogen uptake hydrogenases genes, encoding an oxygen-sensitive (hup-type) and an oxygen-insensitive enzyme (hhy-type). Using growth experiments (batch and continuous cultures) together with transcriptome and kinetics analyses, we showed that strain SolV can grow as a real ‘Knallgas’ bacterium on hydrogen/carbon dioxide, without addition of methane. Expression of the two hydrogenases was analyzed. This research, published in ISME Journal (Mohammedi et al. 2017) is the first study that shows autotrophic growth on hydrogen and carbon dioxide by methanotrophs. The high oxygen tolerance of the Group 1h/5 hydrogenase in strain SolV makes this enzyme a high potential cand

Final results

Our research on verrucomicrobial methanotrophs isolated from volcanic ecosystems has been influential and innovative in the field of microbial ecology and novel biochemistry. Before one of our key publication in 2007, many microbiologists still considered that only alpha and gamma-proteobacterial methanotrophs existed. Nowadays many research groups in the world include the verrucomicrobial methanotrophs in their biodiversity surveys.
Although in the last decades chemistry has witnessed a flourishing field of mechanically interlocked structures, including catenanes, these catenanes are rare in complex biological systems. The description of the unique catenane structure of the CS2 hydrolase from the Solfatara archaeon Acidianus sp. A1-3 has resulted in the initiation of chemical oriented research into application of this catenanes
The VOLCANO project further pioneers the field of chemolithoautrophy in acidic terrestrial volcanic ecosystems: (a) in studying the biodiversity of these geochemically relevant microorganisms, (b) with the application of new techniques (meta-omics, cultivation) to address microbiological problems, (c) with potential for innovative environmental biotechnology, (d) in its multidisciplinary nature spanning from the molecule to the climate of the earth. Scientific innovation is born where different disciplines engage in partnership. Based on our large experience, the project will make future discoveries of presently unknown microbial metabolisms possible. It will also enable the detailed investigation of interesting newly isolated chemolithoautotrophs. Apart from the methodological innovation, the project will stimulate innovation in many different fields of science. The volcanic thermoacidophilic, chemosynthetic microbial communities were most probably also inhabiting hydrothermal sites in early history of the Earth, and likely, they could be good analogues of possible life forms that could develop on early Mars in similar conditions.