Elsevier

Environmental Pollution

Volume 247, April 2019, Pages 1100-1109
Environmental Pollution

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

https://doi.org/10.1016/j.envpol.2019.01.005Get rights and content

Highlights

  • FA enhanced inhibition effect of FT on E.coli survival in river water.

  • K+, Fe3+ and NaFeEDTA further promoted E.coli survival, but others were opposite.

  • Increased reductase activities were beneficial for E.coli survival under FT.

  • Bacterial aggregation was beneficial for E.coli survival under FT.

Abstract

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.

Introduction

The microorganism survival patterns under subzero conditions were investigated (Ray et al., 1998; Chintalapati et al., 2004; Siamak et al., 2015), but many known microorganisms in nature will not stay below freezing point forever. In some cases, they may experience freeze-thaw (FT) cycles. Abnormal climates caused by solar activities and global warming have induced more frequent and longer FT phenomenon with seasonal changes and during the course of a day at middle and high latitudes (Hughes et al., 2017; Groffman et al., 2015). Actually, pathogenic bacteria in such regions would experience higher FT stress, which will have influences on their survival. Hence, more attentions should be paid to researches on pathogenic bacterial survival subjected to FT stress because their survival is related with infection changes caused by pathogenic bacterial during seasonal changes.

Many studies have demonstrated mesophilic bacterial survival patterns treated by different FT frequencies, freezing time and temperatures (Sorensen et al., 2018; Asadishad et al., 2014; Pesaro et al., 2003), but little information about the changes of cell activities and structures. Indeed, osmotic pressure was widely considered as pivot factor and measured (Mao et al., 2003; Gunnarsdóttir et al., 2012), but it was still not deep and comprehensive enough to explain the survival patterns and resistance mechanisms of mesophilic bacteria under FT stress. The consideration being relevant with ions in natural environment should be not only osmotic pressure, but also the ion types and contents. Several researches reported that ion specie and electrical property had significant influences on bacterial activities at normal atmospheric temperatures (Reczek and Chandel, 2018; Fu et al., 2017). Besides, bacterial survival also related with alterations in enzymatic system (Churgin et al., 2005; Nguyen et al., 2015; Shokrollahzade et al., 2015) and surface structure (Marine et al., 2018). Then the most common protection enzymes, such as catalase, superoxide dismutase and so on as well as cellular surface characters must be vital factors when mesophilic bacteria are stressed by FT. Williams et al. (2009) and Yanyan et al. also proved that bacterial morphology influenced the survival responses when stressed by oxidative matter, extreme salinity and temperature in nature, so morphologic changes of mesophilic bacteria under FT stress should be observed too. Yusuke et al. (2004) indicated that abundant organic matter content of soil was beneficial to recover to regular state of bacterial cell function from FT stress and thus improved the survival of bacteria. Similarly, there is abundant dissolved organic matter in natural water. But how the coexistence of dissolved organic matter and ions which accords with mainly natural water contents influence mesophilic bacterial survival is not clear enough yet.

To test above hypothesizes, Escherichia coli, the most common gram-negative pathogenic bacteria which could cause bloodstream infection in all population and countries of the whole world (Chazan et al., 2009; Freeman et al., 2009), was studied in this work. Fulvic acid, widely found in water bodies, was selected as the typical dissolved organic matter for the following researches. The effects of fulvic acid and coexisting fulvic acid and various ions (four types of cations and three types of anions) on Escherichia coli survival patterns in natural water treated by FT stress were investigated, respectively, meanwhile the Escherichia coli mechanisms resisting FT stress based on experimental results of antioxidant enzyme, cell surface hydrophobicity and scanning electron microscope at various sampling time points were discussed.

Section snippets

Experimental water sample

The water sample was collected from the upper reaches of Songhua River (SR, 126.5264°E,43.8075°N), then filtered through 0.45 microporous filter membrane in lab in order to remove aquatic organisms and suspended particulate matter in water. Filtered sample, namely background water, was preserved in polythene plastic bottle with airtight and light-free at 4 °C and was applied for the determinations of water properties and component content, as well as the following experiments.

The water

Bacterial survival in FA systems

The bacterial survival quantities in SR and FA systems are demonstrated with sampling time increasing in Fig. 1. There were no significant differences in bacterial survival numbers with sampling time increasing in SR and FA systems among UFT groups, and their survival quantities at the last sampling time were slightly lower than 108 CFU/mL (decreasing less than 10 times). Although there was no relationship between the bacterial survival quantities and FA concentrations in both UFT and FT

Conclusion

FT significantly reduced E.coli survival in every system, and the survival continuously decreased with sampling time increasing. Generally, survival were depressed by FA under FT stress, and the coexistences of FA and K+,Fe3+ or NaFeEDTA separately enhanced the survival when E.coli cells were subjected to repeated FT cycles in water. However, the presence of FA and Ca2+ or anions separately acted completely opposite roles in E.coli survival. The survival strategies of E.coli under repeated FT

Acknowledgments

I would like to extend my deep gratitude to all those who have offered cordial and selfless support in writing this thesis.

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