This study probed the effect of the water potential (Ψ) on anaerobic methane production and a microbial consortium. The Ψ level of the investigated anaerobic digester (n = 20) was in the range from −0.10 to −2.09 MPa with a mean value of −1.23 MPa, and the Ψ level of the anaerobic digester was significantly correlated with the SCOD, TKN, NH⁴⁺-N, alkalinity, salinity (S_PS), NH₄⁺, Na⁺, K⁺, Cl⁻, NO₃⁻, and PO₄³⁻ (p < 0.001). The maximum methane production rate (R_m) of the Control (−0.40 MPa) was 8.11 mL day⁻¹ and decreased to 1.70 mL day⁻¹ at −3.91 MPa (K5), and the lag growth phase time (λ) was delayed to 35.96 and 25.34 days at −2.85 MPa (K4) and −3.91 MPa (K5), respectively. The ultimate methane potential (B_u) was 0.264 Nm³ kg⁻¹-VS_added for the Control, and when Ψ was adjusted, B_u increased to 0.278 Nm³ kg⁻¹-VS_added at −1.49 MPa (K3) but decreased to 0.203 and 0.172 Nm³ kg⁻¹-VS_added at −2.85 MPa (K4) and −3.91 MPa (K5), respectively. Therefore, the methane yield was inhibited due to the decrease in Ψ, and the methane yield is predicted to be inhibited from about −1.65 MPa. In the genus-level taxonomic classification of the microbial community, the relative abundance of Methanosarcina decreased significantly to 36.76% at −3.91 MPa (K5) compared to 58.15% for the Control; however, the relative abundance of Methanoculleus significantly increased to 35.16% at −3.91 MPa (K5) compared to 14.85% for the Control.

This study investigated the relationship between water potential (Ψ) and the cation-induced inhibition of methane production in anaerobic digesters. The Ψ around methanogens was manipulated using polyethylene glycol (PEG) in a batch anaerobic reactor, ranging from −0.92 to −5.10 MPa. The ultimate methane potential (B_u) decreased significantly from 0.293 to 0.002 Nm³ kg⁻¹-VS_added as Ψ decreased. When Ψ lowered from −0.92 MPa to −1.48 MPa, the community distribution of acetoclastic Methanosarcina decreased from 59.62% to 40.44%, while those of hydrogenotrophic Methanoculleus and Methanobacterium increased from 17.70% and 1.30% to 36.30% and 18.07%, respectively. These results mirrored changes observed in methanogenic communities affected by cation inhibition with KCl. Our findings strongly indicate that the inhibitory effect of cations on methane production may stem more from the water stress induced by cations than from their direct toxic effects. This study highlights the importance of considering Ψ dynamics in understanding cation-mediated inhibition in anaerobic digesters, providing insights into optimizing microbial processes for enhanced methane production from organic substrates.