Resource Partitioning and Co-occurrence of Size-Fractionated Microbiomes in the Delaware and Chesapeake Bays

🧠 Background & Motivation

Microbial communities in aquatic ecosystems are structured into free-living (FL) and particle-attached (PA) fractions, representing distinct ecological niches with different access to substrates, nutrients, and microenvironments. These size-fractionated communities play complementary roles in carbon cycling, nutrient transformation, and ecosystem functioning, yet their community assembly, metabolic strategies, and interaction networks remain poorly resolved, particularly under dynamic environmental conditions.

The Chesapeake and Delaware Bays, two geographically proximate but environmentally distinct estuaries along the U.S. Atlantic coast, differ in salinity gradients, particle load, light availability, and anthropogenic influence. These contrasts provide a natural framework to investigate how resource partitioning, substrate uptake, and microbial interactions shape FL and PA communities across seasons.

In this project, I applied read-based metagenomic and metatranscriptomic analyses across spring, summer, and fall to quantify taxonomic composition, metabolic potential, activity, co-occurrence patterns, and functional redundancy in FL and PA microbial communities.

🎯 Research Questions & Objectives

• How do free-living and particle-attached microbiomes differ in taxonomic and functional diversity?
• Do FL and PA communities exhibit distinct co-occurrence and interaction networks?
• Does substrate uptake and resource partitioning drive niche differentiation between size fractions?
• How does functional redundancy vary between FL and PA communities and across seasons?
• Are active (RNA-based) interaction networks different from total (DNA-based) networks?

👨‍🔬 My Role

This project is part of my primary postdoctoral research program.

• Designed the conceptual framework linking size fractionation, resource partitioning, co-occurrence, and functional redundancy
• Performed read-based metagenomic and metatranscriptomic analyses across seasonal datasets
• Quantified substrate uptake pathways and metabolic specialization
• Constructed co-occurrence networks for FL and PA communities
• Developed a read-based functional redundancy matrix adapted from MAG-based frameworks
• Integrated total (DNA) and active (RNA) community analyses
• Conducted differential expression analysis and MTX modeling
• Led data interpretation and visualization

🧩 Challenges & Solutions

Challenge 1: Distinguishing true differential expression from differences driven by gene copy number variation
Solution: Implemented MTX modeling and DNA:RNA normalization approaches to decouple transcriptional activity from genomic abundance.

Challenge 2: Lack of an established functional redundancy framework for read-based datasets
Solution: Transformed and adapted MAG-based functional redundancy matrices for read-level data, enabling pathway-level FRed quantification without relying solely on genome bins.

Challenge 3: Inconsistent taxonomic lineage and trait assignments across profiling tools
Solution: Built a GTDB-Tk–based reference framework to harmonize outputs from HUMAnN3, Kaiju, mOTUs, and MetaPhlAn, integrating Pfam- and KO-based functional annotations.

Challenge 4: Inferring robust microbial interaction networks from compositional data
Solution: Applied composition-aware network methods and cross-validated co-occurrence patterns across seasons, size fractions, and DNA/RNA datasets.

🛠 Methods & Tools

*Data & Sequencing

• Shotgun metagenomics
• Metatranscriptomics
• Read mapping and expression quantification
• Functional redundancy (FRed) modeling
• MTX modeling
• Co-occurrence and network analysis

*Bioinformatics & Network Analysis

*Taxonomy & Function

• Kaiju
• mOTUs
• HUMAnN3
• eggNOG-mapper
• dbCAN
• DIAMOND

*Mapping & Quantification

• Bowtie2
• SAMtools

*Network Analysis

• SparCC
• SPIEC-EASI
• igraph (R)

*Languages & Workflow

• Python
• Bash
• R
• Snakemake
• SLURM (HPC scheduling)

📄 Publications

• Resource partitioning and co-occurrence shape functional redundancy of size-fractionated microbiomes in the Delaware and Chesapeake Bays
  Manuscript in preparation.

🎤 Conferences & Talks

• Metabolic flexibility, secondary metabolism, and seasonal dynamics in particle-associated and free-living microorganisms in the Chesapeake and Delaware Bays
  Jojy John, Mir A. Ahmed, Barbara J. Campbell
  ASM Microbe 2025, Los Angeles, USA

🧑‍🔬 Collaborator / Reference

Dr. Barbara J. Campbell
Dean’s Distinguished Professor
Department of Biological Sciences, Clemson University
Email: bcampb7@clemson.edu