Physiological Responses of Methanosarcina barkeri under Ammonia Stress at the Molecular Level: The Unignorable Lipid Reprogramming

Abstract

Acetotrophic methanogens' dysfunction in anaerobic digestion under ammonia pressure has been widely concerned. Lipids, the main cytomembrane structural biomolecules, normally play indispensable roles in guaranteeing cell functionality. However, no studies explored the effects of high ammonia on acetotrophic methanogens' lipids. Here, a high-throughput lipidomic interrogation deciphered lipid reprogramming in representative acetoclastic methanogen (Methanosarcina barkeri) upon high ammonia exposure. The results showed that high ammonia conspicuously reduced polyunsaturated lipids and longerchain lipids, while accumulating lipids with shorter chains and/or more saturation. Also, the correlation network analysis visualized some sphingolipids as the most active participant in lipid−lipid communications, implying that the ammonia-induced enrichment in these sphingolipids triggered other lipid changes. In addition, we discovered the decreased integrity, elevated permeability, depolarization, and diminished fluidity of lipid-supported membranes under ammonia restraint, verifying the noxious ramifications of lipid abnormalities. Additional analysis revealed that high ammonia destabilized the structure of extracellular polymeric substances (EPSs) capable of protecting lipids, e.g., declining α-helix/(β-sheet + random coil) and 3-turn helix ratios. Furthermore, the abiotic impairment of critical EPS bonds, including C−OH, C�O−NH−, and S−S, and the biotic downregulation of functional proteins involved in transcription, translation, and EPS building blocks' supply were unraveled under ammonia stress and implied as the crucial mechanisms for EPS reshaping.

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“…The results show 0.9–1.8 times higher fluorescence intensity of the DMP exposure group than that of the control (Figure S6B), indicating a drastically increased membrane permeability under DMP stress. Consistently, measurement of the membrane depolarization level by using lipophilic potentiometric dye diSC3(5) as a fluorescence probe showed 2.5–2.6 times higher fluorescence intensity in the DMP-treated group (Figure S6C). In addition, the SEM images show the presence of distinct membrane pores and deformations for the DMP-treated bacterial cells against the smooth and intact cell membrane of the control (Figure S6D).…”

“…The 2′,7′-dichlorofluorescein diacetate (DCFH-DA), N -phenyl-1-naphthylamine (NPN), lipophilic potentiometric dye 3,3′-dipropylthiadicarbocyanine iodide (diSC3(5)) were used to determine the intracellular ROS level, membrane permeability, and membrane depolarization. , The fluorescence intensity of the samples was measured by using a multifunctional microplate reader (Synergistic H1, BioTek, USA). Details of the measurement methods are given in the SI.…”

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“…Conversely, the trend in the PM:LB arrangement showed a variability in ROM enhancement (Figure S3). Such variations might have stemmed from the wide range of ammonia concentrations found in PM (from 3–13 gN/L). ,,− , Higher ammonia concentrations can negatively impact microbial health by compromising cell membrane function, leading to decreased integrity, elevated permeability, depolarization, and diminished fluidity . Additionally, the energy expenditure for microbes to regulate intracellular pH through the potassium pump increases due to the diffusion of free ammonia. , Despite this, methanogens have shown an ability to withstand certain levels of ammonia toxicity, with thresholds identified at 4 and 7 gN/L for acetoclastic and hydrogenotrophic methanogenesis, respectively …”

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