Exogenous putrescine plays a switch-like influence on the pH stress adaptability of biofilm-based activated sludge

Abstract

Microbial community adaptability to pH stress plays a crucial role in biofilm formation. This study aims to investigate the regulatory mechanisms of exogenous putrescine on pH stress, as well as enhance understanding and application for the technical measures and molecular mechanisms of biofilm regulation. Findings demonstrated that exogenous putrescine acted as a switch-like distributor affecting microorganism pH stress, thus promoting biofilm formation under acid conditions while inhibiting it under alkaline conditions. As pH decreases, the protonation degree of putrescine increases, making putrescine more readily adsorbed. Protonated exogenous putrescine could increase cell membrane permeability, facilitating its entry into the cell. Subsequently, putrescine consumed intracellular H⁺ by enhancing the glutamate-based acid resistance strategy and the γ-aminobutyric acid metabolic pathway to reduce acid stress on cells. Furthermore, putrescine stimulated ATPase expression, allowing for better utilization of energy in H⁺ transmembrane transport and enhancing oxidative phosphorylation activity. However, putrescine protonation was limited under alkaline conditions, and the intracellular H⁺ consumption further exacerbated alkali stress and inhibits cellular metabolic activity. Exogenous putrescine promoted the proportion of fungi and acidophilic bacteria under acidic stress and alkaliphilic bacteria under alkali stress while having a limited impact on fungi in alkaline biofilms. Increasing Bdellovibrio under alkali conditions with putrescine further aggravated the biofilm decomposition. This research shed light on the unclear relationship between exogenous putrescine, environmental pH, and pH stress adaptability of biofilm. By judiciously employing putrescine, biofilm formation could be controlled to meet the needs of engineering applications with different characteristics.IMPORTANCEThe objective of this study is to unravel the regulatory mechanism by which exogenous putrescine influences biofilm pH stress adaptability and understand the role of environmental pH in this intricate process. Our findings revealed that exogenous putrescine functioned as a switch-like distributor affecting the pH stress adaptability of biofilm-based activated sludge, which promoted energy utilization for growth and reproduction processes under acidic conditions while limiting biofilm development to conserve energy under alkaline conditions. This study not only clarified the previously ambiguous relationship between exogenous putrescine, environmental pH, and biofilm pH stress adaptability but also offered fresh insights into enhancing biofilm stability within extreme environments. Through the modulation of energy utilization, exerting control over biofilm growth and achieving more effective engineering goals could be possible.

Keywords: biofilm formation; exogenous putrescine; pH stress adaptability; resistance mechanism; tolerance response.

Fig 1

Biomass and microbial activity response to exogenous putrescine. (a) The dynamic biomass density at 48 h under various putrescine concentrations. Data of the y-axis represent the difference in biofilm mass calculated by subtracting the control group biofilm mass from the treatment group under the same operating pH. (b) The life cycles of microorganisms in the biofilm at 48 h (Alive cells: Intact and Early apoptosis; Dead cells: Late apoptosis and Broken). (c)–(e) The dynamic biofilm growth curve. Acid: pH 3–4. pl: pH 5–6. Alkali: pH 8–9. Ribbons around fitting curves represent the 95% confidence intervals. Error bars represent the standard deviation of the data set. P-value is calculated using t test, significance level, P < 0.05(*), P < 0.01(**), P < 0.001(***).

Fig 2

Biofilm attachment and absorption exogenous putrescine. (a) EPS concentration consists of PN and PS at 24-h cultivation. (b) Cell membrane permeability level at 24-h cultivation. (c) Intracellular total nitrogen concentration at 4-h absorption. (d) Schematic diagram of adsorption and absorption differential mechanisms of exogenous putrescine at different pHs. Acid: pH 3–4. pl: pH 5–6. Alkali: pH 8–9. Error bars represent the standard deviation of the data set. P-value is calculated using t test, significance level, P < 0.05(*), P < 0.01(**), P < 0.001(***).

Fig 3

Exogenous putrescine decreased microorganism intracellular H⁺ concentration in biofilm. (a–c) Intracellular OH^- concentration histogram of microorganisms at 48-h cultivation (left). Average intracellular OH^- concentration signal strength of microorganisms (right). (d) Intracellular GABA concentration. (e) Glutamate metabolism capacity. (f) Intracellular GTP concentration. (g) Major pHi increasing mechanisms of microorganisms affected by exogenous putrescine at different pHs. Acid: pH 3–4. pl: pH 5–6. Alkali: pH 8–9. Error bars represent the standard deviation of the data set. P-value is calculated using t test, significance level, P < 0.05(*), P < 0.01(**), P < 0.001(***).

Fig 4

Exogenous putrescine affects microorganism oxidative phosphorylation activity. (a) Biofilm ATP and ADP dynamic equilibrium concentrations at 48-h cultivation. (b) Average oxidative phosphorylation activity of biofilms at 48-h cultivation. (c) Mechanism of exogenous putrescine effects on oxidative phosphorylation activity at different pHs. Acid: pH 3–4. pl: pH 5–6. Alkali: pH 8–9. Error bars represent the standard deviation of the data set. P-value is calculated using t test, significance level, P < 0.05(*), P < 0.01(**), P < 0.001(***).

Fig 5

Functional genes and microbial diversity analysis. (a) Effects of exogenous putrescine on genes relative abundance about putrescine oxide and active transport, glutamate metabolism AR system, proton dynamics, and oxidative phosphorylation. (b) Changes of fungus, acidophile, and alkaliphile under exogenous putrescine and pH influence. Acid: pH 3 ~ 4. Alkali: pH 8 ~ 9. Error bars represent the standard deviation of the data set. P-value is calculated by t.test, significance level P < 0.05(*), P < 0.01(**), P < 0.001(***).