Bacterial indicator reduction in dairy manure using hybrid zero-valent iron (h-ZVI) system


Novel and efficient animal wastewater treatment technologies of bacteria reduction are needed for preventing disease outbreak in animal herds and safeguarding environmental health. Zero-valent iron (ZVI) has been used to treat bacteria contaminated water for the past decades, but its passivation issue has been a major challenge. In this study, batch tests were performed to evaluate the effect of a hybrid zero-valent iron (h-ZVI) or a mixed ZVI/Fe3O4 media system on reduction of Escherichia coli (E. coli) levels. The h-ZVI media was created through a wet chemical process that uses nitrate to oxidize ZVI in the presence of externally added Fe2+ (aq.). Transforming ZVI into a h-ZVI system could overcome the passivation of ZVI and increase the reactivity of the media. The results demonstrated that E. coli cells in the bulk phase were removed rapidly by h-ZVI media. Majority of E. coli was attached (or adsorbed) to the surface of h-ZVI media within a few minutes, which suggested that adsorption was the dominant mechanism for bacterial removal in the initial phase. This adsorption was confirmed by fluorescence microscopy with CTC-DAPI double staining and transmission electron microscopy (TEM). Increasing contact time steadily inactivated E. coli; all cells were inactivated after 120 min of contact. The TEM results indicated that h-ZVI inactivated E. coli by causing direct damage on bacterial cell membrane. The results of this study strongly suggest that h-ZVI treatment can be used in water treatment industry where bacterial contamination is concerned.

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Colony forming unit


Tetrazolium salt 5-cyano-2,3-ditolyltetrazolium chloride



DDI water:

Deoxygenated and deionized water

E. coli :

Escherichia coli


Hybrid zero-valent iron




Optical density


Revolutions per minute


Transmission electron microscopy


Tryptic soy broth/Agar




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This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors, but was supported by Texas A&M Agrilife and administered by the Department of Bio and Agricultural engineering at Texas A&M University.

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Correspondence to Zong Liu.

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Han, S., Huang, Y. & Liu, Z. Bacterial indicator reduction in dairy manure using hybrid zero-valent iron (h-ZVI) system. Environ Sci Pollut Res 26, 10790–10799 (2019).

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  • Hybrid zero-valent iron (h-ZVI)
  • Pathogen indicator reduction
  • Manure water treatment
  • Bacterial adsorption
  • Bacterial inactivation
  • Bacterial removal mechanism