Role of biofilm on virus inactivation in limestone aquifers: implications for managed aquifer recharge

  • Amirhosein Ramazanpour EsfahaniEmail author
  • Okke Batelaan
  • John L. Hutson
  • Howard J. Fallowfield
Research article



Virus, as nano-sized microorganisms are prevalent in aquifers, which threaten groundwater quality and human health wellbeing. Virus inactivation by attachment onto the limestone surfaces is a determining factor in the transport and retention behavior of virus in carbonaceous aquifers.


In the present study, the inactivation of MS2 -as a model virus- by attachment onto the surfaces of limestone grains was investigated in a series of batch experiments under different conditions such as limestone particle size distribution (0.25–0.50, 0.5–1 and 1–2 mm), treated wastewater and RO water, temperature (4 and 22 °C), initial MS2 concentrations (103–107 PFU/mL) and static and dynamic conditions. The experimental data of MS2 inactivation was also fitted to a non-linear kinetic model with shoulder and tailing. The characteristics of biofilm on the surfaces of limestone aquifer materials were assessed using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM).


The inactivation rate of virus decreased with increasing the adsorbent diameter. Furthermore, virus inactivation was greater at room temperature (22 °C) than 4 °C, in both static and dynamic conditions. The inactivation of virus via attachment onto the limestone aquifer materials in dynamic conditions was higher than under static conditions. In addition, fitting the experimental data with a kinetic model showed that virus inactivation was high at higher temperature, smaller limestone grains and dynamic conditions. Moreover, the experiments with treated wastewater showed that in authentic aqueous media, the virus inactivation was considerably higher than in RO water, due to the presence of either monovalent or divalent cations and surface roughness created by biofilms.


Finally, in terms of managed aquifer recharge systems, the presence of biofilm increases bacteria and virus retention onto the aquifer surfaces.

Graphical abstract



Virus inactivation Biofilm Batch experiment MS2 Limestone Temperature 



Amirhosein Ramazanpour Esfahani was in receipt of an Australian Government Research and Training Program (RTP) at Flinders University. We like to thank Mr. Raj Indela (Flinders University) for laboratory support, Mr. Michael Ferraro Olympic Drilling, South Australia for provision of authentic aquifer substrates, Dr. Allan Pring (Flinders University) for providing guidance in the spectroscopic analyses. Dr. Jason Gascooke (Flinders University) for providing assistance in preparation of SEM images. Furthermore, the authors acknowledge Flinders Microscopy and Microanalysis and the expertise and supports provided by Dr. Jennifer Fendler for the imaging measurements on the Leica TCS SP5 Laser Scanning Confocal Microscope.

Compliance with ethical standards

Conflict of interest

The authors confirm that they have no real or perceived conflict of interest which might influence the results of the research. The final version of manuscript has been approved by all authors.


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Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Amirhosein Ramazanpour Esfahani
    • 1
    • 2
    Email author
  • Okke Batelaan
    • 1
    • 2
  • John L. Hutson
    • 1
  • Howard J. Fallowfield
    • 1
    • 2
  1. 1.College of Science and EngineeringFlinders UniversityAdelaideSouth Australia
  2. 2.National Centre for Groundwater Research and TrainingBedford ParkAustralia

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