Hypothesis

Under combined thermal (+2°C) and acidification (−0.2 pH) stress, low‑abundance bacterial taxa that increase extracellular polysaccharide (EPS) secretion create a localized alkaline microenvironment at the coral‑calcifying interface, thereby sustaining internal pH and calcification rates despite external OA.

Mechanistic Basis

• Rare bacteria respond functionally before taxonomic shifts (1), producing EPS that binds protons and reduces H⁺ diffusion to the calcifying epithelium.
• This EPS layer works synergistically with the coral's own pH‑upregulation mechanisms (5), extending the capacity to sustain aragonite saturation internally.
• The effect is independent of trophic plasticity but can enhance heterotrophic feeding efficiency by stabilizing mucus matrices (2).

Predictions

1. Corals inoculated with EPS‑producing isolates will show higher intracellular pH (measured with pH‑sensitive dyes) and calcification than controls under +2°C/−0.2 pH.
2. Targeted suppression of EPS production (e.g., via specific enzyme inhibitors or antisense oligonucleotides) will abolish the pH‑buffering advantage, leading to calcification rates comparable to aposymbiotic, stressed corals.
3. The magnitude of EPS‑mediated pH elevation will correlate with the relative abundance of the low‑abundance taxa identified in baseline microbiome surveys.

Experimental Approach

• Microbiome manipulation: Isolate candidate EPS‑producing bacteria from resilient Acropora and Porites colonies; generate EPS‑deficient mutants via CRISPRi of polysaccharide synthase genes.
• Inoculation: Apply suspensions to nubbins in flow‑through mesocosms; include antibiotic‑treated controls to reduce native low‑abundance taxa.
• Stress exposure: Maintain tanks at +2°C and −0.2 pH for 4 weeks.
• Measurements:
  • Intracellular pH at the calcifying epithelium using pH‑sensitive fluorescent probes (e.g., SNARF‑1) imaged by confocal microscopy.
  • Calcification rates via buoyant weight and alkalinity anomaly techniques.
  • EPS concentration in mucus layers quantified by phenol‑sulfuric acid assay.
  • 16S rRNA amplicon sequencing to track taxon abundance and functional gene expression (metatranscriptomics).

Potential Outcomes and Implications

• If hypothesis holds: Demonstrates a microbiome‑driven chemical refugium that complements biological refugia (7), revealing a new lever for assisted evolution (probiotic EPS‑enhancers).
• If hypothesis falsified: Suggests that observed functional shifts in rare taxa are epiphenomenal, redirecting focus to host‑centric pH regulation or symbiont‑mediated mechanisms.

This framework directly links the early functional response of low‑abundance microbiota (1) to the long‑term carbonate budget under combined stressors (4, 6), offering a testable, mechanistic bridge between microbiome dynamics and reef persistence.

We don't expect EPS to be the sole factor, but we anticipate a detectable effect. It's important to control for variations in mucus thickness when interpreting pH measurements. We're planning to run parallel assays with fluorescently labeled EPS to visualize its spatial distribution.