Sungeun Lee

A critical step in developing new biosensing devices is to ensure biofilm adaptability to electrochemical conditions and responsiveness to varying environmental factors. In this study, we investigated the biofilm enrichment from French vertical flow treatment wetland (VFTW) sludge in air-cathode MFCs with varying external resistances (100 and 1000 Ω). Electrochemical Impedance Spectroscopy, Environmental Scanning Electron Microscope, and metagenomic analysis showed that the 100 Ω condition promoted higher current output, lower anode resistance, thicker biofilm formation, and stronger enrichment of the Geobacteraceae. In contrast, 1000 Ω condition resulted lower current output and a reduced abundance of electroactive bacteria. This was aligned with the limited biofilm coverage and pseudocapacitive behavior, which could offer advantages for long-term biosensor operation by controlling electrode fouling and enabling continuous electron transfer. Interestingly, spatial biofilm heterogeneity along the anode depth promoted distinct microbial community compositions. The internal biofilm layers presented an increase in the relative expressions of c-type cytochrome omcB, omcE, omcM, which could potentially regulate its extracellular electron transfer (EET). These findings demonstrate the high adaptability of VFTWs-enriched electroactive biofilms and provide valuable insight for designing strategies to influence biofilm dynamics and stable signal output, fostering the application of MFCs for clogging indication in French VFTWs.

Les virus du sol sont capables d'influencer la structure de la communauté microbienne et le fonctionnement de l'écosystème en affectant l'abondance des cellules hôtes par lyse et par leurs caractéristiques à transférer des gènes entre les hôtes. Bien que notre compréhension sur la diversité et la fonction virales s’améliore, la connaissance des interactions hôte-virus dans le sol reste limitée. Pour mieux comprendre les interactions hôte-virus, un gradient du sol à long terme manipulé par le pH dans lequel la communauté microbienne change à travers, a été étudié. Les principaux objectifs de cette thèse ont consisté à (1) déterminer l'influence de la structure de la communauté microbienne et du pH du soil sur les virus par séquençage d’ADN haut-débit (Chapitre II), (2) déterminer l’infectivité des populations virales à partir de niches de sol co-localisées et non co-localisées avec son hôte grâce à une approche de l’essai de plaque combinée à un séquençage hybride (Chapitre III), (3) identifier les populations virales infectant des groupes fonctionnels microbiens spécifiques du sol, en particulier les méthanotrophes (Chapitre IV) et les nitrifiants (Chapitre V ), à l’aide d’une sonde isotopique stable à l'ADN. Nos premiers résultats ont montré que la structure de la communauté virale change selon le pH du sol, ce qui souligne que la communauté virale est étroitement liée aux populations hôtes. L’analyse de CRISPR systèmes a révélé des interactions virus-hôte dynamiques, avec le nombre et la taille des CRISPR systèmes distincts selon le soil à pH contrasté. L’analyse taxonomique de cette CRISPR systèmes suggère que les virus jouent un rôle essentiel dans la composition et de la fonction de la communauté procaryote du sol. Les processus co-évolutionnaires entre l'hôte (le système de restriction-modification et le CRISPR-Cas système) et les populations virales co-localisées (la mutation d’une séquence espaceur « spacer » et la méthyltransférase codée par le virus) fournissent des preuves de l'adaptation locale et que les interactions virus-hôte jouent un rôle important dans la susceptibilité d'un hôte à l'infection et par conséquent la régulation des populations bactériennes du sol. L'ADN-SIP-métagénomique ciblant des groupes fonctionnels microbiens spécifiques a permis l’analyse des populations hôte-virus individuelles. Le suivi du flux de carbone à travers les populations procaryotes et virales a révélé des interactions actives entre les virus et les hôtes méthanotrophes et nitrifiants, et les préférences de niches de pH du sol. Notre étude a montré une preuve de transfert horizontal de gènes et des gènes métaboliques auxiliaires codés par le virus, indiquant que les virus contribuent de manière significative aux cycles biogéochimiques dans le sol, tels que le carbone (les gènes qui codent pour les familles GH, peptidases et la sous-unité C de méthane monooxygénase particulaire), et l'azote (les gènes qui codent pour la nitrogénase et le cytochrome cd1-nitrite réductase). Dans l’ensemble, ces résultats ont montré que les virus du sol sont des régulateurs importants des communautés microbiennes par la lyse spécifique de l’hôte et des interactions dynamiques virus-hôte.

Nitrobacter strain NHB1 is a nitrite-oxidizing bacterium previously demonstrated to form a consortium capable of nitrification under acidic conditions when cocultivated with a neutrophilic ammonia-oxidizing bacterium. Here, we characterize the growth of isolated NHB1 under different pH and nitrite (NO2−) concentrations, as well as its influence on the activity of obligately acidophilic soil ammonia-oxidizing archaea (AOA) isolated from acidic soils when grown in coculture. NHB1 is acidotolerant with optimal growth at pH 6.0 (range: 5.0–7.5) at an initial NO2− concentration of 500 μM. However, at lower NO2− concentrations, closer to those found in soil, its pH optimum decreases to 5.0 and with detectable growth extended to pH 3.5. In coculture, NHB1 enhances the growth of the acidophilic AOA Nitrosotalea devaniterrae Nd1 and Nitrosotalea sinensis Nd2, which are highly sensitive to NO2-derived compounds and typically oxidize only ~200 to 300 μM ammonia (NH3) when grown in batch cultures as isolates. However, in coculture with NHB1, both strains oxidized up to ~3 mM NH3, limited only by the buffering capacity of the medium, and their pH range was also extended downward by ~0.5 units. NHB1 also possesses a cyanase, enabling reciprocal cross-feeding through cyanate-derived NH3 production while utilizing AOA-derived NO2−. These findings suggest that NO2− removal is essential for ammonia oxidizer growth in acidic soils and emphasize the importance of considering substrate and metabolic product concentrations when characterizing ecophysiology. Genome analysis reveals that NHB1 is distinct from validated species, and we propose the name “Nitrobacter laanbroekii.”

Bacteriophages (phages) can infect a range of hosts in highly diverse soil bacterial communities. However, selection of host communities across ecological gradients and co-evolutionary processes may influence both the distribution of phages and the susceptibility of individual hosts through virus interactions and local adaptation within distinct ecological niches. Metagenomic-based analyses have revealed that soil pH selects for distinct populations and community structures for both phage and hosts at local and global scales. However, whether contrasting soil pH represents a selective barrier for phages capable of infecting an individual host is unknown. To examine the influence of pH on individual host-virus interactions, two closely related Bacillus strains were isolated and characterized from pH 7.5 soil associated with a long-term contiguous pH gradient (pH 4.5 to 7.5). Phages infecting each strain were subsequently enriched from soils across the pH gradient (pH 4.5, 5.5, 6.5 and 7.5), enumerated using a plaque assay, and characterized via metagenomic analysis. Phages infecting each strain were cultivated from all soils but their community composition and abundance varied with pH. Phage populations infecting each of the two strains were distinct despite the close relatedness of the two hosts, indicating relatively narrow host ranges for each virus. These results suggest that while phage community structures vary substantially across an ecological gradient, soil pH alone does not represent a barrier for the distribution of phages capable of infecting an individual host.

Soil virus communities are diverse and dynamic but contributions to specific processes, such as nitrification, are largely uncharacterised. Chemolithoautotrophic nitrifiers perform this essential component of the nitrogen cycle and are established model groups for linking phylogeny, evolution and ecophysiology due to limited taxonomic and functional diversity. Ammonia-oxidising bacteria (AOB) dominate the first step of ammonia oxidation at high supply rates, with ammonia-oxidising archaea (AOA) and complete ammonia-oxidising Nitrospira (comammox) often active at lower supply rates or when AOB are inactive, and nitrite-oxidising bacteria (NOB) completing canonical nitrification. Here, the diversity and genome content of dsDNA viruses infecting different nitrifier groups were characterised after in situ enrichment via differential host inhibition, a selective approach that alleviates competition for non-inhibited populations to determine relative activity. Microcosms were incubated with urea to stimulate nitrification and amended with 1-octyne or 3,4-dimethylpyrazole phosphate (AOB inhibited), acetylene (all ammonia oxidisers inhibited), or no inhibitor (AOB stimulated), and virus-targeted metagenomes characterised using databases of host genomes, reference (pro)viruses and hallmark genes. Increases in the relative abundance of nitrifier host groups were consistent with predicted inhibition profiles and concomitant with increases in the relative abundance of their viruses, represented by 200 viral operational taxonomic units. These included 61 high-quality/complete virus genomes 35-173 kb in length and possessing minimal similarity to validated families. Most AOA viruses were placed within a unique lineage and viromes were enriched in AOA multicopper oxidase genes. These findings demonstrate that focussed incubation studies facilitate characterisation of host-virus interactions associated with specific functional processes.

While the complexity of prokaryotic communities in soil is relatively well understood, we are currently ignorant of the role of viruses in influencing their ecology. Viruses infect every living organism and affect rates of biogeochemical processes by killing active cells via cell lysis, or augmenting function through the transfer of auxiliary metabolic genes. The microbially-mediated process of nitrification is central to nitrogen losses and emissions of the greenhouse gas nitrous oxide, contributing to global warming and stratospheric ozone depletion, and viruses may impact this central step of the global N cycle. However, challenges remain in identifying active interactions with specific host or functional groups and virus populations within structurally complex and diverse soil environments. To address this, discrete active interactions between individual hosts and viruses in soil microcosms were examined using the transfer of assimilated carbon from autotrophic prokaryotes to viruses. Microcosms were established with amendments of urea to fuel nitrification and 13C carbon dioxide, followed by DNA stable-isotope probing (SIP) combined with metagenomic analyses. Hybrid analysis of GC mol% fractionation and 13C-DNA-SIP resulted in the identification of active ammonia oxidizing archaea (AOA) hosts and viruses only. To enable characterization of viruses actively infecting all active ammonia- and nitrite oxidizing bacteria (AOB, NOB), a second approach was used where incubations were performed using filtration of virus-like particles to increase the recovery of virus metagenomes. This included differential inhibition of specific nitrifier groups to alleviate competition and potentially increase the abundance of viruses infecting non-inhibited groups. Applications of DMPP or octyne were added to preferentially inhibit AOB and encourage growth of AOA, or acetylene to inhibit all ammonia oxidizing activity as a negative control. This approach dramatically increased the recovery of high-quality virus contigs, with 225 contigs associated with those infecting AOA, AOB or NOB including 69 complete or near-complete genomes. Viruses of ammonia-oxidizers contained auxiliary metabolic genes involved in central metabolic pathways, and analysis of viral hallmark genes revealed that they were distinct from previously cultivated viruses. Results demonstrate that virus infection of nitrifiers and propagation of viruses is a dynamic process during soil nitrification, providing insights into potential impacts of viruses on a specific functional process in soil.

Ammonia-oxidising archaea (AOA) are a ubiquitous component of microbial communities and dominate the first stage of nitrification in some soils. While we are beginning to understand soil virus dynamics, we have no knowledge of the composition or activity of those infecting nitrifiers or their potential to influence processes. This study aimed to characterise viruses having infected autotrophic AOA in two nitrifying soils of contrasting pH by following transfer of assimilated CO₂-derived ¹³C from host to virus via DNA stable-isotope probing and metagenomic analysis. Incorporation of ¹³C into low GC mol% AOA and virus genomes increased DNA buoyant density in CsCl gradients but resulted in co-migration with dominant non-enriched high GC mol% genomes, reducing sequencing depth and contig assembly. We therefore developed a hybrid approach where AOA and virus genomes were assembled from low buoyant density DNA with subsequent mapping of ¹³C isotopically enriched high buoyant density DNA reads to identify activity of AOA. Metagenome-assembled genomes were different between the two soils and represented a broad diversity of active populations. Sixty-four AOA-infecting viral operational taxonomic units (vOTUs) were identified with no clear relatedness to previously characterised prokaryote viruses. These vOTUs were also distinct between soils, with 42% enriched in ¹³C derived from hosts. The majority were predicted as capable of lysogeny and auxiliary metabolic genes included an AOA-specific multicopper oxidase suggesting infection may augment copper uptake essential for central metabolic functioning. These findings indicate virus infection of AOA may be a frequent process during nitrification with potential to influence host physiology and activity.

Crop harvest followed by a fallow period can act as a disturbance on soil microbial communities. Cropping systems intended to improve alpha-diversity of communities may also confer increased compositional stability during succeeding growing seasons. Over a single growing season in a long-term (18 year) agricultural field experiment incorporating conventional (CON), conservation (CA), organic (ORG) and integrated (INT) cropping systems, temporal changes in prokaryote, fungal and arbuscular mycorrhizal fungi (AMF) communities were investigated overwinter, during crop growth and at harvest. While certain prokaryote phyla were influenced by cropping system (e.g. Acidobacteria), the community as a whole was primarily driven by temporal changes over the growing season as distinct overwinter and crop-associated communities, with the same trend observed regardless of cropping system. Species-rich prokaryote communities were most stable over the growing season. Cropping system exerted a greater effect on fungal communities, with alpha-diversity highest and temporal changes most stable under CA. CON was particularly detrimental for alpha-diversity in AMF communities, with AMF alpha-diversity and stability improved under all other cropping systems. Practices that promoted alpha-diversity tended to also increase the similarity and temporal stability of soil fungal (and AMF) communities during a growing season, while prokaryote communities were largely insensitive to management.

Significance: The impact of soil viruses on prokaryotic hosts and their functional processes is largely unknown. While metagenomic sequencing of soil microbial communities enables identification of linkages between viruses and hosts, this does not necessarily identify contemporary interactions. To enable a detailed analysis of active virus–host interactions between individual populations, we focused on the critical biogeochemical process of methane (CH₄) oxidation and followed the transfer of carbon from hosts to their associated viruses in situ. Analysis of¹³C-enriched metagenomic DNA demonstrated that CH₄-derived carbon is transferred into viral biomass via both primary and secondary utilizers of CH₄and suggests viral predation is an important mechanism for releasing CH₄-derived organic carbon into the soil matrix.