Multiscale destabilization of anaerobic digestion by chloramphenicol: Divergence between methanogen detectability and...

Antibiotic residues in livestock waste streams can affect anaerobic digestion (AD), yet their functional impact on microbial viability and metabolic pathways remains unclear. This study evaluated concentration-dependent effects of chloramphenicol (CAP) by integrating process kinetics, cellular integrity, dissolved-phase responses, and functional gene profiles. Methane yield was maintained at ≤ 50 mg/L CAP but declined sharply at higher concentrations, reaching near-complete inhibition at 1,000 mg/L. Estimated inhibition thresholds were derived as IC₃₀ = 285 mg/L, IC₆₀ = 535 mg/L, and IC₉₀ = 852 mg/L from the fitted concentration-response relationship. Severe inhibition coincided with residual organic acid accumulation, pH decline, and enrichment of propionate and butyrate fractions with undetectable acetate. Flow cytometry revealed a marked CAP-dependent decline in intact biomass, with live-cell (P2) counts decreasing from 4.8 × 10⁶ cells/mL in the control to 1.0 × 10⁵ cells/mL at 1,000 mg/L, accompanied by increased forward- (FSC-A) and side-scatter (SSC-A) area indicative of structural stress. Fluorescence excitation-emission matrix (FEEM) analysis showed concentration-dependent enrichment of soluble microbial products (SMPs) fluorescence, and inoculum-only incubation confirmed biomass-associated solubilization under CAP exposure. Although methanogens remained numerically detectable (76.0% vs. 81.1%), key genes related to cofactor synthesis and electron transfer (comD, frhB, fwdA/fwdC, mcr) declined substantially at 1,000 mg/L. These convergent signals were consistent with multiscale destabilization. Given that microbial activity was not directly measured, the observed discrepancy between methanogen detectability and methane recovery should be interpreted as indicative of a potential functional imbalance rather than definitive evidence of functional decoupling.