Investigating the activity of indigenous microbial communities from Italian depleted gas reservoirs and their possible impact on underground hydrogen storage.

H2 produced from renewable energies will play a central role in both greenhouse gas reduction and decarbonization by 2050. Nonetheless, to improve H2 diffusion and utilization as a fuel, large storage capacity systems are needed. Underground storage of natural gas in depleted reservoirs, aquifers and salt caverns is a well-established technology. However, new challenges arise when it comes to storing hydrogen due to the occurrence and activity of indigenous microbial populations in deep geological formations. In a previous study, four Italian natural gas reservoirs were characterized both from a hydro-chemical and microbiological point of view, and predictive functional analyses were carried out with the perspective of underground hydrogen storage (UHS). In the present work, formation waters from the same reservoirs were used as inoculant during batch cultivation tests to characterize microbial activity and its effects on different gas mixtures. Results evidence a predominant acidogenic/acetogenic activity, whilst methanogenic and sulfate reducing activity were only marginal for all tested inoculants. Furthermore, the microbial activation of tested samples is strongly influenced by nutrient availability. Obtained results were fitted and screened in a computational model which would allow deep insights in the study of microbial activity in the context of UHS.

Biogeochemical characterization of four depleted gas reservoirs for conversion into underground hydrogen storage.

Depleted gas reservoirs are a valuable option for underground hydrogen storage (UHS). However, different classes of microorganisms, which are capable of using free H2 as a reducing agent for their metabolism, inhabit deep underground formations and can potentially affect the storage. This study integrates metagenomics based on Illumina-NGS sequencing of bacterial and archaeal 16S rRNA and dsrB and mcrA functional genes to unveil the composition and the variability of indigenous microbial populations of four Italian depleted reservoirs. The obtained mcrA sequences allow us to implement the existing taxonomic database for mcrA gene sequences with newly classified sequences obtained from the Italian gas reservoirs. Moreover, the KEGG and COG predictive functional annotation was used to highlight the metabolic pathways potentially associated with hydrogenotrophic metabolisms. The analyses revealed the specificity of each reservoir microbial community, and taxonomic and functional data highlighted the presence of an enriched number of taxa, whose activity depends on both reservoir hydrochemical composition and nutrient availability, of potential relevance in the context of UHS. This study is the very first to address the profiling of the microbial population and allowed us to perform a preliminary assessment of UHS feasibility in Italy.

Perspective on the use of methanogens in lithium recovery from brines.

Methanogenic archaea stand out as multipurpose biocatalysts for different applications in wide-ranging industrial sectors due to their crucial role in the methane (CH4) cycle and ubiquity in natural environments. The increasing demand for raw materials required by the manufacturing sector (i.e., metals-, concrete-, chemicals-, plastic- and lubricants-based industries) represents a milestone for the global economy and one of the main sources of CO2 emissions. Recovery of critical raw materials (CRMs) from byproducts generated along their supply chain, rather than massive mining operations for mineral extraction and metal smelting, represents a sustainable choice. Demand for lithium (Li), included among CRMs in 2023, grew by 17.1% in the last decades, mostly due to its application in rechargeable lithium-ion batteries. In addition to mineral deposits, the natural resources of Li comprise water, ranging from low Li concentrations (seawater and freshwater) to higher ones (salt lakes and artificial brines). Brines from water desalination can be high in Li content which can be recovered. However, biological brine treatment is not a popular methodology. The methanogenic community has already demonstrated its ability to recover several CRMs which are not essential to their metabolism. Here, we attempt to interconnect the well-established biomethanation process with Li recovery from brines, by analyzing the methanogenic species which may be suitable to grow in brine-like environments and the corresponding mechanism of recovery. Moreover, key factors which should be considered to establish the techno-economic feasibility of this process are here discussed.

Phytophthora zoospores: From perception of environmental signals to inoculum formation on the host-root surface.

To explore moist soils and to target host plants, phytopathogenic Phytophthora species utilize the sensory and propulsion capabilities of the biflagellate unicellular zoospores they produce. Zoospore motion and interactions with the microenvironment are of primary importance for Phytophthora physiology. These are also of critical significance for plant pathology in early infection sequential events and their regulation: the directed zoospore migration toward the host, the local aggregation and adhesion at the host penetration site. In the soil, these early events preceding the root colonization are orchestrated by guidance factors, released from the soil particles in water films, or emitted within microbiota and by host plants. This signaling network is perceived by zoospores and results in coordinated behavior and preferential localization in the rhizosphere. Recent computational and structural studies suggest that rhizospheric ion and plant metabolite sensing is a key determinant in driving zoospore motion, orientation and aggregation. To reach their target, zoospores respond to various molecular, chemical and electrical stimuli. However, it is not yet clear how these signals are generated in local soil niches and which gene functions govern the sensing and subsequent responses of zoospores. Here we review studies on the soil, microbial and host-plant factors that drive zoospore motion, as well as the adaptations governing zoospore behavior. We propose several research directions that could be explored to characterize the role of zoospore microbial ecology in disease.

Transcriptomic and Ultrastructural Signatures of K+-Induced Aggregation in Phytophthora parasitica Zoospores.

Most pathogenic oomycetes of the genus Phytophthora spread in water films as flagellated zoospores. Zoospores perceive and produce signals attracting other zoospores, resulting in autoaggregation in vitro or biofilm formation on plant surface. The mechanisms underlying intercellular communication and consequent attraction, adhesion and aggregation are largely unknown. In Phytophthora parasitica, the perception of a K+ gradient induces coordinated motion and aggregation. To define cellular and molecular events associated with oomycete aggregation, we combined transcriptomic and ultrastructural analyses. Results indicate involvement of electroception in K+ sensing. They establish that the transcriptome repertoire required for swimming and aggregation is already fully functional at zoospore release. At the time points analyzed, aggregates are mainly constituted of zoospores. They produce vesicular and fibrillary material discharged at cell-to-cell contacts. Consistently, the signature of transcriptome dynamics during transition to aggregates is an upregulation of genes potentially related to vesicular trafficking. Moreover, transcriptomic and functional analyses show a strong enhancement of carbonic anhydrase activity, indicating that pH homeostasis may contribute to aggregation by acting on both zoospore movement and adhesion. This study poses the molecular and cellular bases of aggregative behavior within oomycetes and expands the current knowledge of ion perception-mediated dissemination of propagules in the rhizosphere.

Hydrogen-Fueled Microbial Pathways in Biogas Upgrading Systems Revealed by Genome-Centric Metagenomics.

Biogas upgrading via carbon dioxide hydrogenation is an emerging technology for electrofuel production. The biomethanation efficiency is strongly dependent on a balanced microbial consortium, whose high- resolution characterization along with their functional potential and interactions are pivotal for process optimization. The present work is the first genome-centric metagenomic study on mesophilic and thermophilic biogas upgrading reactors aiming to define the metabolic profile of more than 200 uncultivated microbes involved in hydrogen assisted methanogenesis. The outcomes from predictive functional analyses were correlated with microbial abundance variations to clarify the effect of process parameters on the community. The operational temperature significantly influenced the microbial richness of the reactors, while the H2 addition distinctively alternated the abundance of the taxa. Two different Methanoculleus species (one mesophilic and one thermophilic) were identified as the main responsible ones for methane metabolism. Finally, it was demonstrated that the addition of H2 exerted a selective pressure on the concerted or syntrophic interactions of specific microbes functionally related to carbon fixation, propionate and butanoate metabolisms. Novel bacteria were identified as candidate syntrophic acetate oxidizers (e.g., Tepidanaerobacter sp. DTU063), while the addition of H2 favored the proliferation of potential homoacetogens (e.g., Clostridia sp. DTU183). Population genomes encoding genes of Wood-Ljungdahl pathway were mainly thermophilic, while propionate degraders were mostly identified at mesophilic conditions. Finally, putative syntrophic interactions were identified between microbes that have either versatile metabolic abilities or are obligate/facultative syntrophs.

Optimization of hydrogen dispersion in thermophilic up-flow reactors for ex situ biogas upgrading.

This study evaluates the efficiency of four novel up-flow reactors for ex situ biogas upgrading converting externally provided CO2 and H2 to CH4, via hydrogenotrophic methanogenesis. The gases were injected through stainless steel diffusers combined with alumina ceramic sponge or through alumina ceramic membranes. Pore size, input gas loading and gas recirculation flow rate were modulated to optimize gas-liquid mass transfer, and thus methanation efficiency. Results showed that larger pore size diffusion devices achieved the best kinetics and output-gas quality converting all the injected H2 and CO2, up to 3.6L/LREACTOR·d H2 loading rate. Specifically, reactors' CH4 content increased from 23 to 96% and the CH4 yield reached 0.25LCH4/LH2. High throughput 16S rRNA gene sequencing revealed predominance of bacteria belonging to Anaerobaculum genus and to uncultured order MBA08. Additionally, the massive increase of hydrogenotrophic methanogens, such as Methanothermobacter thermautotrophicus, and syntrophic bacteria demonstrates the selection-effect of H2 on community composition.

In-situ biogas upgrading in thermophilic granular UASB reactor: key factors affecting the hydrogen mass transfer rate.

Biological biogas upgrading coupling CO2 with external H2 to form biomethane opens new avenues for sustainable biofuel production. For developing this technology, efficient H2 to liquid transfer is fundamental. This study proposes an innovative setup for in-situ biogas upgrading converting the CO2 in the biogas into CH4, via hydrogenotrophic methanogenesis. The setup consisted of a granular reactor connected to a separate chamber, where H2 was injected. Different packing materials (rashig rings and alumina ceramic sponge) were tested to increase gas-liquid mass transfer. This aspect was optimized by liquid and gas recirculation and chamber configuration. It was shown that by distributing H2 through a metallic diffuser followed by ceramic sponge in a separate chamber, having a volume of 25% of the reactor, and by applying a mild gas recirculation, CO2 content in the biogas dropped from 42 to 10% and the final biogas was upgraded from 58 to 82% CH4 content.

Deeper insight into the structure of the anaerobic digestion microbial community; the biogas microbiome database is expanded with 157 new genomes.

This research aimed to better characterize the biogas microbiome by means of high throughput metagenomic sequencing and to elucidate the core microbial consortium existing in biogas reactors independently from the operational conditions. Assembly of shotgun reads followed by an established binning strategy resulted in the highest, up to now, extraction of microbial genomes involved in biogas producing systems. From the 236 extracted genome bins, it was remarkably found that the vast majority of them could only be characterized at high taxonomic levels. This result confirms that the biogas microbiome is comprised by a consortium of unknown species. A comparative analysis between the genome bins of the current study and those extracted from a previous metagenomic assembly demonstrated a similar phylogenetic distribution of the main taxa. Finally, this analysis led to the identification of a subset of common microbes that could be considered as the core essential group in biogas production.

Biogas Upgrading via Hydrogenotrophic Methanogenesis in Two-Stage Continuous Stirred Tank Reactors at Mesophilic and Thermophilic Conditions.

This study proposes an innovative setup composed by two stage reactors to achieve biogas upgrading coupling the CO2 in the biogas with external H2 and subsequent conversion into CH4 by hydrogenotrophic methanogenesis. In this configuration, the biogas produced in the first reactor was transferred to the second one, where H2 was injected. This configuration was tested at both mesophilic and thermophilic conditions. After H2 addition, the produced biogas was upgraded to average CH4 content of 89% in the mesophilic reactor and 85% in the thermophilic. At thermophilic conditions, a higher efficiency of CH4 production and CO2 conversion was recorded. The consequent increase of pH did not inhibit the process indicating adaptation of microorganisms to higher pH levels. The effects of H2 on the microbial community were studied using high-throughput Illumina random sequences and full-length 16S rRNA genes extracted from the total sequences. The relative abundance of archaeal community markedly increased upon H2 addition with Methanoculleus as dominant genus. The increase of hydrogenotrophic methanogens and syntrophic Desulfovibrio and the decrease of aceticlastic methanogens indicate a H2-mediated shift toward the hydrogenotrophic pathway enhancing biogas upgrading. Moreover, Thermoanaerobacteraceae were likely involved in syntrophic acetate oxidation with hydrogenotrophic methanogens in absence of aceticlastic methanogenesis.