SIGMA - SIngle-cell Genomics and Metabolic modeling for tailored microbial consortiA design

Project Details

Principal Investigator Laura Treu

Type of Grant CoG

Current position at UNIPD Associate Professor

Project acronym SIGMA

Project title       SIngle-cell Genomics and Metabolic modeling for tailored microbial consortiA design

Host Department       Department of Biology

Research cost     € 175.000,00

Start date of the project      01/02/2024

Duration of the project 30 months

People

Research plan

WP1. Single-cell technology applied to AD and BU communities T1.1 Single or few cells will be captured in gel droplets, forming microreactors to support growth of (co)isolated cells. To analyze single-cell sequencing data, a bioinformatics toolbox will be developed using genome-centric metagenomics strategies (T1.2) to reconstruct draft genomes by pooling sequences from cells of the same species. Genome-centric metatranscriptomics will integrate DNA/RNA-seq and bioinformatics to infer microbial functions and activity (T1.3). D&M: D1.1 Report on a microfluidic droplet method for anaerobic microorganisms; D1.2 Multi-omics characterization plan (genomics, transcriptomics); M1 Release of new pipelines to analyze target species in communities.

WP2. Recognition of phage-host interaction DNA from samples in WP1 and WP3 (T2.1) will be used to study interactions between phages, microbiomes, and free-living phages. Integrated prophages and free viruses will be analyzed to define infection mechanisms and microbial defenses (e.g., CRISPR-Cas). Phage-driven community dynamics will be characterized (T2.2). Environmental conditions inducing phages targeting unwanted bacteria will be selected to shape communities (T2.3). D&M: D2.1 Global map of microbial–viral interactions; D2.2 Phages and conditions for microbiota control; M2 Strategy to control microbial–phage dynamics in AD microbiomes.

WP3. Reconstruction of community exchange networks Gas composition, pressure, and carbon sources will be experimentally tested (T3.1). Growth rate, gases, and VFAs will be monitored by GC, flow cytometry, and microscopy. Microbiome evolution will be tracked by time-series single-cell genomics and transcriptomics to validate syntrophic interactions (T3.2). Flux balance analysis and condition-specific genome-scale metabolic models will be optimized for primary CO₂ consumers (T3.3). D&M: D3.1 Report on selected strains and syntrophic consortia; D3.2 Metabolic models integrating metabolomics and transcriptomics; M3 Strategy to obtain optimized microbial consortia.

WP4. Innovation outreach, dissemination, and exploitation. Results will be disseminated through high-impact journals (T4.1), international conferences (T4.2), and outreach to young researchers via social media and congresses (T4.3). D&M: Three high-impact publications (e.g., Environmental Science & Technology, Microbiome, The ISME Journal); D4.2 Conference dissemination; M4 Report on communication activities.

International Conferences Contributes

• 19th International Symposium on Microbial Ecology (ISME19), August 18th-23th 2024, Cape Town, South Africa. INTERNATIONAL CONGRESS, POSTER: De Bernardini N, Giangeri G, Savio F, Ling M, Fraulini S, Orellana E, Ji M, Campanaro S, Treu L. Genetic variability in a carbon dioxide reducing microbial community: a comparison between metagenomics and single-cell sequencing.

• 19th International Symposium on Microbial Ecology (ISME19), August 18th-23th 2024, Cape Town, South Africa. INTERNATIONAL CONGRESS, POSTER: Orellana E., De Bernandini N., Zampieri G., Serna R., Jalali F., Campanaro S., Treu L. Syntrophy in anaerobic microbial communities: amino acids as key players in CO2 methanation.

• EMBO | EMBL Symposium. New approaches and concepts in microbiology, June 24th-27th 2025, Heidelberg, Germany. INTERNATIONAL SYMPOSIUM, POSTER: De Bernardini N., Savio F., Orellana E., Campanaro S., Treu L. The hidden influence of phage-host interactions on carbon dioxide methanation 

• 5th International conference on biogas microbiology (ICBM-5), May 26th-29th 2025, Galway, Ireland. INTERNATIONAL CONGRESS, SELECTED ORAL PRESENTATION: Savio F., De Bernardini N., Orellana E., Ji M., Campanaro S., Treu L. Integrating single-cell DNA sequencing and metagenomics to uncover overlooked genetic features in a CO₂-fixing microbiome for biogas upgrading. 

Deliverables and Milestones 

The Deliverables and Milestones reported here are provided as a summary, more detailed information may be made available upon request (PI, laura.treu @ unipd.it), subject to data sensitivity and publication constraints.

WP1

• D1.1: Report on a microfluidic droplet method for anaerobic microorganisms (Month 4)

The development of this method focused on establishing a microfluidic single-cell approach to investigate anaerobic microbiota using single permeable capsules (SPCs) as picoliter micro-reactors. SPCs are generated with the ONYX instrument under nitrogen-protected handling and loaded in a low-occupancy Poisson regime (target λ≈0.1) based on impedance cell counts (Bactobox) and the effective cell concentration. The workflow enables controlled enzymatic lysis followed by MDA-based whole genome amplification (WGA) and genome barcoding. Although capsule production and theoretical occupancy were reproducible, the fraction of WGA-positive SPCs (SYTO9 readout) was lower than expected, indicating downstream bottlenecks (taxon-dependent lysis), with archaea likely under-recovered. Next steps optimize lysis while preserving SPC integrity and implement enrichment/sorting strategies to increase recovery of informative SPCs.

• D1.2: Multi-omics characterization plan (Month 8)

Bioinformatic approaches combining single-cell genomics, metagenomics, and transcriptomics have been developed to characterize microbial communities. Initial proof-of-concept work with a mock community (E. coli and L. lactis strains) demonstrated successful strain-level genome reconstruction and metabolic modeling from single-cell data, despite low genome coverage (6% average completeness). Application to a biogas upgrading community generated 706 single amplified genomes (SAGs) with 11.9% mean completeness. While individual SAGs showed limited coverage, combining multiple SAGs from the same species enabled near-complete genome reconstruction. Methanothermobacter thermautotrophicus was identified as the dominant with the highest transcriptional activity on hydrogenotrophic methanogenesis. Single-cell sequencing revealed 73 SAGs from species undetected by metagenomics, demonstrating its value for comprehensive microbiome profiling beyond traditional approaches. A bioinformatics toolbox for analyzing single-cell DNA datasets was developed and is available upon request on the Github repository (https://github.com/MetabioinfomicsLab).

• M1: Strategy to analyze target species at community-level (Month 8)

A bioinformatic toolbox for single-cell sequencing data has been developed, with tailored strategies enabling the reconstruction of SAGs and their comparison with corresponding MAGs.

WP2

• D2.1: Global map of the microbial-viral interactions (Month 12)

Microbial-viral interactions have been investigated by exposing experimental communities to different stress factors (UV, Mitomycin C, CuCl₂, and H₂O₂). The H₂O₂ treatment caused community collapse and was excluded. Using combined single-cell and metagenomic sequencing, we reconstructed 30 high-quality microbial genomes and 476 viral genomes (grouped into 80 species-level clusters). The viral populations included integrated proviruses (16), temperate phages (36%), and lytic phages (64%). We identified 42 virus-host associations: 14 confirmed computational predictions and 28 were novel discoveries detected exclusively through single-cell sequencing, demonstrating the power of this approach for mapping microbial-viral interactions.

• D2.2: Phages-mediated condition for microbiota composition  (Month 16)

Among the recovered 80 viral operational taxonomic units (vOTUs), 49 were linked to their putative hosts spanning 15 bacterial classes. Comparative coverage analyses under different stress conditions revealed effective phage induction, with several vOTUs increasing in abundance and specific phage–host interactions emerging under UV and Mitomycin C treatments. Despite these dynamics, most hosts and integrated proviruses remained stable or dormant, and overall community structure and methanogenic performance were preserved. These findings expand our current understanding of BU viral diversity, induction dynamics, and phage–host relationships.

• M2: Strategy to control microbial-phages dynamics (Month 18)

A strategy for single-cell virome-host dynamics control has been developed, integrating viral induction assays with scDNA-seq and metagenomics.

WP3

• D3.1: Report on selected strains and syntrophic consortia (Month 25)

ongoing

• D3.2: Report on condition-specific metabolic model  (Month 28)

ongoing

• M3: Strategy to control microbial-phages dynamics (Month 28)

ongoing

WP4

• D4.1: Manuscript publication (Month 28)

ongoing

• D4.2: Dissemination in Conferences  (Month 28)

Please refer to the above section "International Conferences Contributes".

• M4: Report on dissemination and communication activities (Month 28)

ongoing