Effects of fulvic acid and fulvic ions on Escherichia coli survival in river under repeated freeze-thaw cycles

The effects of fulvic acid (FA) and ions on mesophilic pathogenic bacteria survival under freeze-thaw (FT) stress in natural water and its resistant mechanisms are rarely understood. Therefore, survival patterns of Escherichia coli in river water added with various concentrations of FA or FA-ion under FT stress were studied in this work. Meanwhile, cell surface hydrophobicity (CSH), unit activities of superoxide dismutase (SOD) and catalase (CAT) were determined and Escherichia coli morphologies were observed to explore the bacterial resistant mechanisms against FT stress. The results demonstrated that FT cycles significantly reduced bacterial quantities as sampling time, i.e. freeze-thaw cycle time increased. And the biggest reducing rate was observed after the first FT cycle in every system. Ttd values, time needed to reach detection limit under FT stress decreased under FT stress as FA was added into water, while the changes of ttd values were quite complicated when FA and various ions existed together. Generally, the ttd values of FA-cation systems exceeded that of FA system except FA-Ca²⁺ systems, but it was opposite for FA-anion systems. CSH was heightened after FT cycles and reached peak value at last sampling time in every system. Mechanical constraint from extracellular ice crystals and high CSH induced bacterial aggregation, which protect inner cells of aggregation from extracellular ice crystals. And the unit activities of SOD were significantly higher than those of CAT. Unit activities of SOD and CAT in large part of tested systems increased with sampling time under FT stress, which reduced reactive oxygen species produced from repeated FT cycles. Thus, these could improve the resistance of Escherichia coli to freeze-thaw stress and promote their survival. This work explored the survival pattern and strategy of Escherichia coli in natural water under FT stress.