Water quality and microbial load: a double-threshold identification procedure intended for space applications

Stefano Amalfitano, Caterina Levantesi, Laurent Garrelly, Donatella Giacosa, Francesca Bersani, Simona Rossetti

Preprint posted on 26 July 2018

Article now published in Frontiers in Microbiology at

Monitoring microbiological water quality in space: a culture-independent approach.

Selected by Daphne Ng

Categories: microbiology


Water is essential for the survival of every living thing, astronauts included. Space exploration necessitates the development of microbiological methods for in-flight water monitoring to ensure a safe water supply on long missions or in confined spaces such as the International Space Station (ISS). Culture-independent microbiological methods are preferred as many bacteria are viable but not culturable (VBNC). Hence, they are often underestimated by conventional culture-dependent methods, which rely on visible microbial growth, such as heterotrophic plate counts.


Key findings

In this study, the microbial loads in various water samples (chlorinated tap water, unchlorinated tap water, ground water, river water and waste water) were evaluated using heterotrophic plate counts (HPC) and several culture-independent techniques (ATP measurements, quantitative real-time PCR and flow cytometry). New water quality standards based on a double-threshold (warning and alarm) peak microbial load identification procedure were proposed to alert astronauts to possible microbial contamination in water.

To validate the findings of the culture-independent techniques, HPC was performed. However, HPC was significantly influenced by growth conditions, incubation time and initial microbial load. Further differences in culturability may arise due to factors such as microgravity as well as varying cosmic radiation levels. In addition, astronauts may be supplied with water from several space agencies. Hence, heterotrophic plate counts may not be reliable and reproducible, unless confirmed with other culture-independent techniques.

All culture-independent techniques in the study showed significant differences in microbial loads between the different water samples. Measurements of cellular ATP content were able to consistently distinguish between the water samples based on microbial contamination levels. The biggest difference between warning and alarm thresholds was also observed. However, microbial cells contain varying amounts of ATP. As such, ATP measurements may not be able to accurately represent variations in microbial community structure. Microbial load evaluations based on ATP-metry may be strengthened by measuring other cell specific parameters such as total cell counts and cell size.

The abundance of 16S rRNA as measured by quantitative real-time PCR (qPCR) was significantly different among the water types. 16S rRNA gene copies correlated with microbial loads assessed by other parameters. Water with high and low microbial loads could be differentiated based on measurements of 16S rRNA by qPCR. Although several hours may be required for the sampling and extraction of nucleic acids, qPCR may be a feasible method for water quality monitoring through the quantification of genes and species of interest.

There was significant correlation between the findings of flow cytometry and microbial loads as assessed by HPC, ATP-metry and qPCR. However, cell aggregates and debris in the water may influence the accuracy of flow cytometry. Therefore, further calibration and validation of the performance of flow cytometry is required before they can be used for space applications.


What I like about this preprint

Routine culture-based microbiological water quality monitoring methodologies may not accurately reflect the microorganisms present as many microbes are in the VBNC state. As observed from this study, microbial growth is also affected by environmental factors such as gravity and background radiation. Hence, microbial contamination of water may escape detection using these methods. The study highlights the importance of developing alternative techniques and standards to detect microbial contamination events. As on-board water treatment processes in spacecraft may fail, accurate monitoring of microbiological water quality in space is vital. The last thing astronauts want to experience while in space is falling sick after drinking contaminated water!


Future directions and questions for the author

  1. Using the methods in the study, the authors should consider measuring other microbial indicators of water quality such as fecal indicator bacteria to see if these can be quantified accurately.


  1. Can pathogens be detected using these methods?

Tags: water quality

Posted on: 27 August 2018


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Author's response

Stefano Amalfitano shared

I agree that the next step should be to work on water pathogens. qPCR is already applicable for direct pathogen detection in space waters. Flow cytometry is also a promising technology but it would require further technological advances in view of possible automated analyses.

However, microbiological threats could be too many to be routinely identified in space. The described procedure will hopefully help to select a limited subset of samples (e.g., those above the identified thresholds) on which to work for pathogen detection.

One more important point is that the analyses should be performed on-site directly (i.e., onboard the space craft) and it will take years to develop, make fly, and test those instruments that are conventionally used for water analysis in Earth-based applications.

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