Periodic Reporting for period 4 - VOLCANO (Microbiology of extremely acidic terrestrial volcanic ecosystems)
Periodo di rendicontazione: 2020-07-01 al 2020-12-31
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. The microbial collective of volcanic ecosystems has a major impact on geochemical cycles and greenhouse gas emissions. However, especially the unique microbial communities shaped by high temperatures and extreme acidic conditions with their exceptional properties have hardly been explored and not been exploited to date. The identity, diversity, ecophysiological properties and interactions of the microorganisms present in these volcanic ecosystems are in urgent need of investigation. The aim of this project is to obtain a fundamental understanding of the microbial ecology of extremely acid terrestrial mud volcanoes with special emphasis on the microbial diversity, cellular structure, metabolic potential, and the molecular response to changing environmental conditions of microorganisms involved in the key elemental cycles of sulfur, methane, hydrogen and nitrogen.
A. The metagenomic approach involved samples from the Solfatara, Pantelleria Island and Vulcano Island (all Italy). Emissions and nutrient profiles at the sites were analyzed and the extracted DNA was sequenced using next generation sequencing technology. The metagenome data were analyzed using ‘in house’ pipelines. The Solfatara metagenome was dominated by representatives of the genera Acidithiobacillus, Thermoplasma, Picrophilus, Ferroplasma, and Sulfolobus. The metagenomic analysis of the Pantelleria Island soil showed a high relative abundance of a novel methanogen, a Methanocella sp. Several metagenome assembled genomes (MAGs) were analyzed in detail to resolve the metabolic capacity.
B. 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. We showed that methanethiol and in addition, hydrogen sulfide were consumed by strain SolV resulting in increased biomass concentration.
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 was purified and this enzyme is a high potential candidate in biotechnological applications. We also demonstrated that the methanotroph Methylacidimicrobium tartarophylax strain 4AC could also grow on hydrogen and carbon dioxide but only under oxygen limited conditions using an extremely oxygen-sensitive hydrogenase.
The presence of a third pmo operon (encoding a monooxygenase with an unknown function) prompted us to examine as alternative substrates for growth. Using batch and methanol limited continuous cultures, we showed that strain SolV was able to oxidize the short-chain alkanes ethane and propane and use them as a substrate for growth. In addition, growth was possible on natural gas. Full transcriptomes (RNA-seq) of cells growing on short-chain alkanes were investigated.
The membrane protein complexes of strain SolV were resolved using complexome profiling. We were able to identify 296 unambiguous proteins including the important protein complexes in methane oxidation pathway (pMMO, MDH, putative membrane-bound FDH), carbon fixation (RuBisCO), and the electron transport chain (Complexes I to V).
D. Several new isolates of bacteria consuming methane, hydrogen and carbon monoxide were obtained using the samples from Pantelleria Island soil as inoculum. These included autotrophic hydrogen-consuming sulfate-reducers, H2-consuming (aerobic conditions) Kyrpidia sp., methane consuming Methylacidimicrobium and Methylocaldum-like species and a novel H2-consuming autrotroph that produces and excretes high concentrations of amino acids. All isolates were characterized in detail using physiological experiments and genome sequencing.
E. Knowledge on N-cycling processes in low pH ecosystems is limiting, despite the presence of both oxidized and reduced nitrogen species at μM to mM concentrations. As a proof of principle we have obtained an novel isolate, Nitrosacidococcus tergens, capable to oxidize ammonia even at pH 2.5. Molecular analysis of the Pantelleria ecosystems showed the presence of close relatives.
F. Expanding the world and use of verrucomicrobial methanotrophs.
We studied the production of methanol on a combination hydrogen and methane under rare earth element (REE) limitation and showed the potential of conversion of methane (or biogas) into liquid biofuel (methanol) using acidophilic methanotrophs. 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. Furthermore, we purified methanol dehydrogenases from strain SolV with europium or lanthanum in its active site.
F. Microbial interactions coupling the different nutrient cycles. The different bacteria and archaea living in the hostile volcanic ecosystems will have to compete for limiting resources or to cooperate for the removal of toxic end products. As part of possible interaction between methane and ammonia-oxidizing microorganisms we investigated the nitrosative stress handling in M. fumariolicum SolV.
B. 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. We showed that methanethiol and in addition, hydrogen sulfide were consumed by strain SolV resulting in increased biomass concentration.
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 was purified and this enzyme is a high potential candidate in biotechnological applications. We also demonstrated that the methanotroph Methylacidimicrobium tartarophylax strain 4AC could also grow on hydrogen and carbon dioxide but only under oxygen limited conditions using an extremely oxygen-sensitive hydrogenase.
The presence of a third pmo operon (encoding a monooxygenase with an unknown function) prompted us to examine as alternative substrates for growth. Using batch and methanol limited continuous cultures, we showed that strain SolV was able to oxidize the short-chain alkanes ethane and propane and use them as a substrate for growth. In addition, growth was possible on natural gas. Full transcriptomes (RNA-seq) of cells growing on short-chain alkanes were investigated.
The membrane protein complexes of strain SolV were resolved using complexome profiling. We were able to identify 296 unambiguous proteins including the important protein complexes in methane oxidation pathway (pMMO, MDH, putative membrane-bound FDH), carbon fixation (RuBisCO), and the electron transport chain (Complexes I to V).
D. Several new isolates of bacteria consuming methane, hydrogen and carbon monoxide were obtained using the samples from Pantelleria Island soil as inoculum. These included autotrophic hydrogen-consuming sulfate-reducers, H2-consuming (aerobic conditions) Kyrpidia sp., methane consuming Methylacidimicrobium and Methylocaldum-like species and a novel H2-consuming autrotroph that produces and excretes high concentrations of amino acids. All isolates were characterized in detail using physiological experiments and genome sequencing.
E. Knowledge on N-cycling processes in low pH ecosystems is limiting, despite the presence of both oxidized and reduced nitrogen species at μM to mM concentrations. As a proof of principle we have obtained an novel isolate, Nitrosacidococcus tergens, capable to oxidize ammonia even at pH 2.5. Molecular analysis of the Pantelleria ecosystems showed the presence of close relatives.
F. Expanding the world and use of verrucomicrobial methanotrophs.
We studied the production of methanol on a combination hydrogen and methane under rare earth element (REE) limitation and showed the potential of conversion of methane (or biogas) into liquid biofuel (methanol) using acidophilic methanotrophs. 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. Furthermore, we purified methanol dehydrogenases from strain SolV with europium or lanthanum in its active site.
F. Microbial interactions coupling the different nutrient cycles. The different bacteria and archaea living in the hostile volcanic ecosystems will have to compete for limiting resources or to cooperate for the removal of toxic end products. As part of possible interaction between methane and ammonia-oxidizing microorganisms we investigated the nitrosative stress handling in M. fumariolicum SolV.
Our research on verrucomicrobial methanotrophs isolated from volcanic ecosystems has been influential and innovative in the field of microbial ecology and novel biochemistry. Nowadays many research groups in the world include the verrucomicrobial methanotrophs in their biodiversity surveys. The VOLCANO project pioneered the field of chemolithoautrophy in acidic terrestrial volcanic ecosystems: (a) in studying the biodiversity of geochemically relevant microorganisms, (b) in applying new techniques to address microbiological problems, (c) in looking for innovative environmental biotechnology, (d) in its multidisciplinary nature spanning from the molecule to the climate of the earth. Scientific innovation were born where different disciplines engaged in partnership. The project made discoveries of presently unknown microbial metabolisms. Apart from the methodological innovation, the project stimulated 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.