Case study

Quantification of Urease Activity via HighThroughput Conductivity Measurements

Tools:
CardioExcyte 96

Frédéric M. Lapierre,featured by Nanion Technologies
Frédéric Maurice Lapierre, Munich University

five minutes is monitored and the slope of the conductivityof Applied Sciences. His focus is thesignal correlates with the urease activity [3]. As thedevelopment of a bioprocess for microbial

determination of enzymatic activity is a time dependent

induced calcite precipitation (MICP)-relevant

parameter, it inherently takes several minutes for the data

microorganisms.

to be aquired. Measuring one sample after another withconventional methods can consequently be considered astime consuming.
To our best knowledge, no commercial high-throughput
conductivity measurement is available for such enzyme
assays. Here, the CardioExcyte 96 (Nanion Technologies)

Microbial urease catalyzes the hydrolysis of urea toammonium and carbonate, which results in an increaseof the environmental pH value. Addition of calcium ionsthen leads to calcium carbonate (= calcite) precipitation.
Microbial Induced Calcite Precipitation (MICP) is successfully
applied for, e.g., restoration of construction materials,
ground improvement or inhibition of weed growth. Urease
activity is considered a key factor for MICP performance.
Multiple studies use different techniques to adjust the urease
activity of a bacterial culture, or isolate new ureolytic
microorganisms. Consequently, quantification of urease
activity plays an important role for MICP research andapplication [1].
The conductivity assay for urease activity determination, firstpublished by Chin and Kroontje [2] and later established for
MICP-relevant microorganisms by Whiffin [3], is widely used in
MICP research. The conductivity assay is easy to perform; anurease-positive sample is added to an urea solution, resulting
in the formation of ions, increasing the overall conductivity ofthe solution. In general, the conductivity change over

Nanion's CardioExcyte 96 platform
The conductance is continuously and automaticallymonitored as a measure of urease activity. The
CardioExcyte 96 is an automated device, recordingfrom 96 wells at a time.

was used for parallelized urease activity determination,and this system proved to overcome the hurdles met withconventional measurement setups.
Nanion´s impedance-based cell monitoring technology
CardioExcyte 96 was originally developed to investigate
electrophysiology, contractility and viability recordings ofadherent cells. In general, reading the impedance of planar

“The CardioExcyte has dramatically improved our ability
to determine the urease activity. The ease-of-use and highthroughput allows for a drastic time saving when measuring large
numbers of samples.”Robert Huber, Prof. Dr., Munich University of Applied Sciences

gold-film electrodes that are used as growth substrate fortraditionally with conductivity probes (InLab 751, Mettler

adherent cells reveals changes in electrode coverage orToledo, Giessen) in a stirred beaker containing 24 ml of 1.1 M

cell behavior.

urea solution as described in Lapierre et al. [1].

Here, the CardioExcyte 96 was used to measure theA conversion of conductivity change over time (unit: µS/conductance of solution which is containing urease-positivecm/s) to urease activity (unit: U/ml) is known from previous

bacteria. In principle, an urease-positive sample is added

findings. Urease activity is defined as the amount ofto an urea solution, resulting in the formation of ions. In thissubstrate hydrolysed in 1 minute. 10 µl of each dilution of thestudy, a culture of urease-positive bacterium Sporosarcina

prepared urease-positive culture is added to each well of thepasteurii, the most used microorganism for MICP in published

CardioExcyte 96 plates. Afterwards, 190 µl of 1.1M urea solution

research, was prepared by overnight shake flask cultivationis added to each well. The plate was shaken in double orbital

at 30 °C and 250 rpm in chemically defined medium

mode for 30 seconds using a microplate reader (SpectraMax

as described by Lapierre et al. [4]. A dilution row was

iD3, Molecular Devices, United States). Afterwards, thepipetted (100%, 85%, 70%, …, 0%) using culture medium.

conductance of each well is measured using the CardioExcyte

For comparison, 1 ml of each dilution was measured

96 over the course of 5 minutes.

BTime [s]Urease activity [U/ml]

Conductance [µS]

Conductivity change (probe) [µS/cm/s]

A

Conductance change (CardioExcyte 96)[µS/s]

CardioExcyte 96 recordings. A Conductance signals for a dilution row of an urease-positive culture.
More urease-positive culture results in steeper curves. Only average values are shown (N =3, N = 1 for
100% due to pipetting errors). B Conductance change per second measured with the CardioExcyte
96 (X-axis) and InLab 751 (Y-axis) correlate nicely (R² = 0.9967). Only average values are shown(N =3, N = 1 for 100% due to pipetting errors). The relationship Urease activity [U/mL] = 21.097 ×Conductance change (microplate) [µS/s] was determined. The error bars depict SD.

Addition of urease-positive culture to an urea solution

Taken together, the CardioExcyte 96 allows for automatedresults in a linear conductance signal over the period of 300

enzyme activity measurements, enabling fast bioprocessseconds (Figure 1A). The conductance change measured

development. Overall, using MICP for soil stabilization reducesin the microplate wells correlates nicely with measurements

the environmental impact of ground improvement, since

executed in a standard conductivity probe setup (Figure 1B).

conventional ground improvement techniques often have a

Consequently, high-throughput small-scale urease activity

high impact on the environment.

measurements can be performed using the CardioExcyte 96.
High-throughput screening of conductance kinetics for
enzyme activity determination allows for drastic time savings
when measuring a large number of samples compared to thestandard method using probes, e.g. when screening for new
urease positive strains.
Another advantage of the CardioExcyte 96 system comparedto the standard method is that the required sample volume ismuch lower (20 µL instead of 1 ml).

Microbial Induced Calcite Precipitation (MICP) is successfully applied for restoration of construction materials.
Sand-derived Biocement (Photo: Alexander Bayerhoff) and, right, scanning electron microscope photo revealing
microbially precipitated calcium carbonate. The holes in the lime structure indicate bacteria (Photo: Constanze
Eulenkamp).

“My experience with the Nanion team was 100% positive, bringing
a new technology like CardioExcyte 96 on board in our lab andachieving my research goals in a very short time was striking tome.”
Frédéric Maurice Lapierre, Munich University of Applied Sciences

Contact Information

References

Frédéric Maurice Lapierre

1.

Lapierre et al. (2022), Front. Bioeng. Biotechnol. 10:936759

2.

Chin and Kroontje (1962), J. Agric. Food Chem. 10, 347–348

3.

Whiffin (2004), PhD Thesis at Murdoch University

4.

Lapierre et al. (2020), Sci. Rep. 10, 22448

M.Eng., Research Associate
Hochschule München, University of Applied Sciences,Lothstrasse 6480335 München, Germany

Robert Huber, Prof. Dr.,
Fakultät für Wirtschaftsingenieurwesen, HochschuleMünchen, University of Applied Sciences,Lothstrasse 6480335 München, Germany
www.wi.hm.edu/02_fakultaet/personen/professorinnen/huber.de.html

Acknowledgments
We thank Frédéric and Robert for sharing their viewson the importance of microbial Induced Calcite
Precipitation (MICP) and restoration of construction
materials. We are grateful for your valuable insights
and wonderful collaboration.

Nanion Technologies GmbH I Ganghoferstraße 70A, 80339 Munich, Germany+49 89 2190 95-0 I info@nanion.de I www.nanion.de