High salinity, particularly large fluctuations in salinity, poses a significant threat to the biological treatment of high-salt industrial wastewater, while glycine betaine (GB) is commonly used for osmotic pressure balance. However, the GB metabolism mechanism and the effect of GB addition on microbial osmoadaptation responding to salt perturbations are still unclear. This study investigated the effect of GB on the stability of anaerobic process in nitrobenzene reduction under various salt perturbations and further explored the microbial osmoadaptation and metabolism mechanism. The nitrobenzene reduction was observably improved with GB addition (R+), and the microbial resilience increased due to repeated salt shocks. The generation of acetic acid showed a 46.21 % decrease in R+ compared to that without GB addition (R-) at 3 % salinity. Meanwhile, the intracellular GB concentration reached 10.76 mg L−1 to 54.76 mg L−1 with salinity increasing from 1.5 % to 6 %. 16S rRNA analysis revealed that the osmoadaptation period was shortened in R+ through the rapid osmoadaptation of salt-intolerant microorganisms (such as Propionibacteriaceae and SBR1031) and the massive enrichment of salt-tolerant microorganisms (such as Glutamicibacter). Besides, the resistance to hypersaline disturbance improved by the increasing size and complexity of the microbial community network. Moreover, the metagenomic analysis revealed that the abundance of genes involved in microbial metabolism was significantly enhanced in R+, indicating a more efficient metabolic efficiency and electrons transfer with GB addition under high-salt environments. This study provides vital insights into GB metabolism and microbial osmoadaptation responding to salt perturbations in long-term high-salt wastewater biotreatment.
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