Environmental Research 208 (2022) 112738

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Environmental Research
journal homepage: www.elsevier.com/locate/envres

A quantitative, high-throughput urease activity assay for comparison and

rapid screening of ureolytic bacteria
Ming-Juan Cui a, b, 1, Aloysius Teng c, 1, Jian Chu b, **, Bin Cao b, c, *
a
College of Civil Engineering, Fuzhou University, Fuzhou, 350108, Fujian, China
b
School of Civil and Environmental Engineering, Nanyang Technological University, 50 Nanyang Ave, Singapore, 639798, Singapore
c
Singapore Centre for Environmental Life Sciences Engineering, Interdisciplinary Graduate Programme, Graduate College, Nanyang Technological University, 60
Nanyang Dr, Singapore, 637551, Singapore

A R T I C L E I N F O A B S T R A C T

Keywords: Urease is a dinickel enzyme commonly found in numerous organisms that catalyses the hydrolysis of urea into
Urease ammonia and carbon dioxide. The microbially induced carbonate precipitation (MICP) process mediated by
Urease-producing bacteria urease-producing bacteria (UPB) can be used for many applications including, environmental bioremediation,
MICP
soil improvement, healing of cracks in concrete, and sealing of rock joints. Despite the importance of urease and
Biomineralization
UPB in various applications, a quantitative, high-throughput assay for the comparison of urease activity in UPB
and rapid screening of UPB from diverse environments is lacking. Herein, we reported a quantitative, 96-well
plate assay for urease activity based on the Christensen’s urea agar test. Using this assay, we compared urease
activity of six bacterial strains (E. coli BL21, P. putida KT2440, P. aeruginosa PAO1, S. oneidensis MR-1, S. pasteurii
DSM 33, and B. megaterium DSM 319) and showed that S. pasteurii DSM 33 exhibited the highest urease activity.
We then applied this assay to quantify the inhibitory effect of calcium on urease activity of S. pasteurii DSM 33.
No significant inhibition was observed in the presence of calcium at concentrations below 10 mM, while the
urease activity decreased rapidly at higher concentrations. At a concentration higher than 200 mM, calcium
completely inhibited urease activity under the tested conditions. We further applied this assay to screen for
highly active UPB from a wastewater enrichment and identified a strain of S. pasteurii exhibiting a substantially
higher urease activity than DSM 33. Taken together, we established a 96-well plate-based quantitative, high-
throughput urease activity assay that can be used for comparison and rapid screening of UPB. As UPB and
urease activity are of interest to environmental, civil, and medical researchers and practitioners, we envisage
wide applications of the assay reported in this study.

1. Introduction
Urease (urea amidohydrolase; EC 3.5.1.5) is a dinickel enzyme that
catalyses the hydrolysis of urea to ammonia and carbon dioxide. It is the
first identified metalloenzyme commonly found in bacteria, fungi, and
plants (Mobley and Hausinger, 1989; Mobley et al., 1995; Krajewska and
Ureases, 2009). Urease-producing bacteria (UPB), i.e., ureolytic bacte­
ria, are capable of inducing precipitation of calcite through the release of
carbonate ions and the increase in pH. Microbially induced calcite
precipitation (MICP) enabled by UPB has been harnessed for many ap­
plications, including soil improvement (Ivanov and Chu, 2008; van
Paassen et al., 2010), healing of cracks in concrete (Jonkers et al., 2010;
Mian and Qian, 2016), and sealing of rock joints underground (Wu et al.,
2019). In particular, UPB-mediated MICP provides an efficient and
cost-effective biomineralization approach for environmental applica­
tions. For example, MICP can be used to immobilize subsurface con­
taminants such as toxic radionuclides (Fujita et al., 2008; Lauchnor
et al., 2013) and heavy metals (Cuaxinque-Flores et al., 2020; Fang et al.,
2021; Yong et al., 2021), seal fluid leakages from underground pipes
(Phillips et al., 2016), and treat hypersaline water generated during the
production of shale oil and gas (Lei et al., 2021). In addition, MICP can
also be used to mitigate the impact of climate change as it can enhance
the capture and sequestration of atmospheric carbon dioxide into un­
derground geological formations (Mitchell et al., 2010; Okyay et al.,

* Corresponding author. School of Civil and Environmental Engineering, Nanyang Technological University, 50 Nanyang Ave, Singapore, 639798, Singapore.
** Corresponding author.
E-mail addresses: cjchu@ntu.edu.sg (J. Chu), bincao@ntu.edu.sg (B. Cao).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.envres.2022.112738
Received 30 November 2021; Received in revised form 12 January 2022; Accepted 12 January 2022
Available online 15 January 2022
0013-9351/© 2022 Elsevier Inc. All rights reserved.
M.-J. Cui et al.
2016) and lessen the impact of thermal stress on coral reefs (Biscéré
et al., 2018).
Because of the ubiquitous presence and wide applications of UPB,
several methods have been developed to assess urease activity (Taba­
tabai, 1994; Tabatabai et al., 2002). For example, Stuart’s urea broth can
be used to screen for the presence of ureolytic activity in environmental
and clinical samples. This assay is based on the colour change of the
phenol red indicator caused by alkalinisation of the media during urea
hydrolysis (Brink, 2010). It is often used to qualitatively differentiate
organisms with high urease activity such as Proteus from other genera
within the Enterobacteriaceae. The Christensen’s urea agar test is another
method for screening UPB by observing a similar colour change of the
phenol red indicator in the agar. Compared with the Stuart’s urea broth,
the Christensen’s urea agar is supplemented with peptone and glucose so
that it can be used to screen a higher diversity of bacteria, including
those that are unable to use ammonia as the sole nitrogen source.
Furthermore, the reduced buffer capacity of the Christensen’s urea agar
enables more sensitive detection of ammonia, allowing the screening of
UPB that fail to produce sufficient ammonia to be detected in Stuart’s
urea broth (Brink, 2010). Both the Stuart’s and Christensen’s
biochemical tests for urease activity are qualitative in nature. Urease
activity can be quantified by measuring the release of ammonia using
the Nessler method (Krug et al., 1979). However, this method requires
the use of toxic mercuric salts, and the preparation of reagents is
time-consuming. Another method quantifies ammonia using the Ber­
thelot reagent (Cussac et al., 1992; van Vliet et al., 2001), which requires
sonication and incubation for a prolonged period of time. Although
several high-throughput methods for the screening and assay of urease
activity have been proposed, they either require long periods of incu­
bation (Onal Okyay and Frigi Rodrigues, 2013), the use of heavy inoc­
ulum (Qadri et al., 1984a, 1984b) or consumption of large volumes of
reagents (Cordero et al., 2019).
The objective of this study was to develop a quantitative, high-
throughput urease activity assay for rapid assessment of bacterial ure­
olytic potential. Specifically, we established a quantitative, 96-well plate
assay based on the Christensen’s urea agar test. To demonstrate the
2
Environmental Research 208 (2022) 112738
validity of our proposed assay, we quantitatively compared urease ac­
tivity of selected bacteria, including two well-known ureolytic bacteria,
Sporosarcina pasteurii and Bacillus megaterium. We then applied this assay
to quantify the inhibitory effect of calcium on urease activity of
S. pasteurii. Finally, we demonstrated the feasibility of using this assay to
screen for highly active ureolytic bacteria from environmental samples.
2. Materials and methods
2.1. Bacterial strains and culture conditions
Bacterial strains used in this study were Escherichia coli BL21, Pseu­
domonas putida KT2440, Pseudomonas aeruginosa PAO1, Shewanella
oneidensis MR-1, S. pasteurii DSM 33, and B. megaterium DSM 319.
S. pasteurii DSM 33 and B. megaterium DSM 319 were purchased from the
German Collection of Microorganisms and Cell Cultures GmbH, while
the other strains were obtained from our current laboratory stocks. All
strains were maintained in 25% glycerol stock stored at − 80 ◦ C and
grown in their respective liquid or agar media when required.
B. megaterium DSM 319 was grown in Luria-Bertani (LB) broth supple­
mented with 0.1 mM NiCl2 while S. pasteurii DSM 33 was grown in an
ammonium-yeast extract medium containing 20 g/L yeast extract, 15 g/
L ammonium chloride and 0.1 mM NiCl2. The ammonium-yeast extract
medium was then adjusted to a pH of 9.25 using NaOH. All the other
strains were grown in LB Miller broth at either 30 or 37 ◦ C.
2.2. 96-well plate-based urease activity assay
A 96-well plate-based urease activity assay was developed using the
Christensen’s urea agar (Fig. 1). The Christensen’s urea agar comprised
1 g/L of peptone, 1 g/L of dextrose, 5 g/L of sodium chloride, 2 g/L of
potassium phosphate, 20 g/L of urea, 0.012 g/L of phenol red, and 5 g/L
of agar. Bacterial cells were grown in 24-well plates and then harvested
by centrifugation and resuspended in PBS buffer. 200 μL of the cell
suspension was introduced into each well of the 96-well plate followed
by pelleting the cells by centrifugation and removal of supernatant. Cells
Fig. 1. Schematic illustration of the 96-well plate-
based urease activity assay. Tested bacteria were
grown in 24-well plates before the cells were pelleted
and washed with PBS. Then, 200 μL of cell suspension
was transferred into each well of the 96-well plate.
After the cells have been pelleted to the bottom, 200
μL of the Christensen’s urea agar was added into each
well. A layer of sterile cotton gauze moistened with
hydrochloric acid was used to cover the plate to
prevent cross-well contamination by ammonia gas
released by urease-active bacteria. The absorbance at
560 nm was measured for each well to quantify ure­
ase activity.
M.-J. Cui et al.
were subsequently covered by 200 μL of the Christensen’s urea agar. A
thin layer of sterile cotton gauze soaked with hydrochloric acid was used
to cover the 96-well plate to prevent cross-well contamination by
gaseous ammonia. The colour change of the agar with respect to time
was then monitored by measuring the absorbance at 560 nm using a
Tecan Infinite Pro II microplate reader.
2.3. Validation of the 96-well plate-based urease activity assay
To validate the 96-well plate-based urease activity assay, we tested
urease-positive bacteria S. pasteurii DSM 33 and B. megaterium DSM 319,
urease-negative bacteria E. coli BL21 and S. oneidensis MR-1, as well as
P. aeruginosa PAO1 and P. putida KT2440. Fresh colonies of each bac­
terial strain were picked and inoculated into their respective growth
media in 24-well plates and incubated at 30 ◦ C for approximately 5 h.
The cells for urease activity assay should be harvested at the exponential
phase. All the tested cultures reached the exponential growth phase after
5-h incubation. The cell cultures were then washed three times with 1.5
mL of phosphate-buffered saline (PBS) and spun down at 4000 rpm for 2
min. The bacterial cells were then adjusted to the same OD600 (~ 0.10)
before 200 μL of bacterial cell culture was transferred into the 96-well
plate in triplicates. The cells were then pelleted to the bottom of the
wells and 200 μL of the Christensen’s urea agar was added into each
well.
2.4. Quantification of inhibitory effect of calcium on urease activity
S. pasteurii DSM 33 was used as a model urease-positive organism to
quantify inhibition of calcium on urease activity. This organism was
chosen because of its high urease activity and wide applications in
geotechnical and environmental engineering (Ivanov and Chu, 2008;
Phillips et al., 2016; Mitchell et al., 2010). Filter-sterilized calcium
chloride stock solution was diluted with PBS and mixed with the
Christensen’s urea agar to achieve different final Ca2+ concentrations (5,
10, 25, 50, 75, 100, 200, 250, 500 mM) and then transferred into the
96-well plate. Cells from an overnight culture of S. pasteurii DSM 33 were
harvested and washed three times with PBS and the cell pellets were
then resuspended in 5 mL of PBS before 200 μL of the cell suspension was
transferred into each well. The absorbance at 560 nm was measured in
15 min intervals. Relative urease activity at various Ca2+ concentrations
was indicated by the rate of increase in OD560 from t = 15 min to t = 30
min. Model fitting of the relative activity at different calcium concen­
trations was carried out using the DoseResp function of OriginLab Pro.
The non-competitive inhibition model was used:
v = Vm (
1
)(
[S]
) of calcium allows more rapid formation of calcium carbonate crystals
1 + K[I]I KM + [S]
where v is the rate of urea hydrolysis, Vm is the maximum rate of urea
hydrolysis, [S] is the concentration of urea, [I] is the concentration of
Ca2+, KI is the inhibition constant, and KM is the Michaelis constant.
2.5. Screening for ureolytic bacteria from wastewater
Rapid screening for ureolytic bacteria from a wastewater enrichment
(Yang et al., 2020) was carried out using the 96-well plate-based urease
activity assay. Serial dilutions of an overnight culture of the enrichment
were plated onto the ammonium-yeast extract agar. After an overnight
incubation at 30 ◦ C, individual colonies with unique morphologies were
picked for the 96-well plate-based screening of urease activity. Colony
PCR was carried out to amplify the 16S rRNA gene with the primers 27F
(5′ -AGAGTTTGATCMTGGCTCAG-3′ ) and 1492R (5′ -TACGGYTACCTT
GTTACGACTT-3′ ) using Q5 High-Fidelity DNA polymerase (NEB, USA).
Clean up of the PCR products was carried out using the Wizard SV Gel
and PCR Clean-Up System (Promega, USA) before they were sent for
sequencing. Phylogenetic analysis was then carried out using the MEGA
3
Environmental Research 208 (2022) 112738
X software according to the protocol described by Hall (2013). The
phylogenetic tree was constructed using the maximum-likelihood algo­
rithm, and bootstrap values were obtained from an analysis of 1000
re-samplings of the alignments.
3. Results and discussion
3.1. Quantitative comparison of urease activity
The relative urease activity of 6 bacterial strains quantified using the
96-well plate-based urease activity assay is shown in Fig. 2. S. pasteurii
DSM 33 exhibited the fastest colour change. The rapid change of colour
indicated a high urease activity of S. pasteurii DSM 33. For E. coli BL21
and S. oneidensis MR-1, no change in OD560 was observed within 3 h,
corroborating the lack of urease activity in these two strains.
Interestingly, although B. megaterium DSM 319 is known to be
urease-positive, no apparent change in OD560 in the first 3 h was
observed. Similarly, neither P. aeruginosa PAO1 nor P. putida KT2440
showed apparent change in OD560 despite being shown capable of pro­
ducing urease. This could be attributed to the lower urease activity of
these strains than S. pasteurii DSM 33 that would require a longer re­
action time for them to release the necessary amount of ammonia to
overcome the buffer capacity of the Christensen’s urea agar. In addition,
the nutrients present in urease agar used to maintain bacteria growth
and urease activity might be another factor contributing to the lower
urease activity (Stewart, 1965).
3.2. Quantification of inhibitory effect of calcium on urease activity
By using S. pasteurii DSM 33 as a model ureolytic organism, the
inhibitory effect of calcium on urease activity was quantified using the
96-well plate-based urease activity assay. No significant difference was
observed in the relative activity of urease in the presence of calcium at
concentrations below 10 mM, while the relative activity decreased
rapidly at higher calcium concentrations (Fig. 3 and Figure S1). The
inhibition of calcium on urease activity followed the non-competitive
inhibition model (R2 = 0.99).
Calcite precipitation induced by UPB has many applications such as
improving the strength of soil, healing concrete cracks and plugging of
rock joints (Ivanov and Chu, 2008; Jonkers et al., 2010; Wu et al., 2019).
One key factor influencing the efficiency of the mineralization process
and strength of the improved soil is the amount of calcium carbonate
produced in each treatment which in turn relies on the calcium content
present in the cementation solution used for MICP (Krajewska, 2018;
Tang et al., 2020). Several studies have shown that a high concentration
responsible for the strengthening of soil (Qabany et al., 2012; Ng et al.,
2014). However, excessive levels of calcium in the cementation solution
will inhibit the urease activity and is detrimental to MICP (Nemati and
Voordouw, 2003; Nemati et al., 2005; Lai et al., 2021). Therefore, one
key factor to improve the efficiency of any MICP based methods is to
obtain a urease enzyme that can tolerate a higher calcium concentration.
Tools that can quantify calcium tolerance of UPB in a high-throughput
manner would facilitate the search for highly tolerant bacterial
strains. The 96-well plate-based urease activity assay developed in this
study was demonstrated to have the capability of quantifying the
inhibitory effect of calcium on urease activity in UPB. The high R2 value
of the fitting curve obtained shows a good fit between the experimental
OD560 absorbance values with a non-competitive inhibition model. The
assay developed here may also be employed to quantify the inhibitory
effects of other chemicals or metals on urease (Shaw, 1954).
3.3. Screening for ureolytic bacteria from wastewater
The 96-well plate-based urease activity assay was employed to
screen for bacteria with high urease activity from a previously reported
M.-J. Cui et al.
Fig. 2. Christensen’s urea agar assay of common laboratory strains. a. Colour change of Christensen’s urea agar for the laboratory strains tested from 0 to 3 h. b.
OD560 readings of the Christensen’s urea agar with respect to time. c. Relative urease activity of the tested bacterial strains with respect to S. pasteurii DSM 33. (For
interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
(Yang et al., 2020) wastewater enrichment. Four strains A, B, C, and D
were isolated from the enriched culture based on distinct colony mor­
phologies. Urease activity of the 4 isolates and the model urease-positive
bacterium S. pasteurii DSM 33 was compared using the 96-well
plate-based assay. Intriguingly, strain B exhibited higher rate of in­
crease in OD560 than all the other strains, including S. pasteurii DSM 33
(Fig. 4 and Figure S2). Both strains A and C showed a slightly higher rate
of increase in OD560 than S. pasteurii DSM 33 while no increase in OD560
for strain D was observed (Fig. 4 and Figure S2). These results suggested
that among all the tested strains, strain B has the highest urease activity.
Phylogenetic analyses suggested that strains A and B showed a high
degree of sequence homology to S. pasteurii. Strain C is most closely
related to S. pasteurii BNCC 337394, while strain D is closely related to
the uncultured Sporosarcina sp. clone FACULANA16. Detailed analysis of
the full genome of strains C and D is required to better understand their
phylogenetic ancestry due to the low bootstrap value obtained. Strains
A, B and C are likely to be aerobic, urease-positive and Gram-positive
bacteria from the genus of Sporosarcina or Bacillus (Claus et al., 2006).
Interestingly, among the three strains analysed, strain B showed the
4
Environmental Research 208 (2022) 112738
Fig. 3. The inhibitory effect of calcium on urease
activity in S. pasteurii DSM 33 quantified using the 96-
well plate-based assay. a. OD560 in the presence of
CaCl2. b. Relative urease activity at different calcium
concentrations. Relative urease activity was deter­
mined as the rate of colour change (OD560) of the
Christensen’s urea agar from t = 15 min to t = 30
min. Fitting curve was generated by fitting experi­
mental data to the non-competitive inhibition model
(R2 = 0.99). (For interpretation of the references to
colour in this figure legend, the reader is referred to
the Web version of this article.)
highest 16S rRNA gene similarity with S. pasteurii although the urease
activity assay showed that strain B had a much higher urease activity
than S. pasteurii DSM 33. The difference in the ureolytic potential of
different bacterial strains from the same species could be attributed to
strain-specific differences in their urease-encoding genes (Hammes
et al., 2003). Further genomic and transcriptomic analyses are required
in the future to understand strain-specific differences in urease activity.
4. Conclusions
In this paper, we reported a 96-well plate-based quantitative, high-
throughput urease activity assay. Using this assay, we quantitatively
compared urease activity of six bacterial strains (E. coli BL21, P. putida
KT2440, P. aeruginosa PAO1, S. oneidensis MR-1, S. pasteurii DSM 33, and
B. megaterium DSM 319) and showed that S. pasteurii DSM 33 exhibited
the highest urease activity, while urease activity in E. coli BL21 and
S. oneidensis MR-1 was undetectable. We then applied this assay to
quantify the inhibitory effect of calcium on urease activity of S. pasteurii
DSM 33. No significant inhibition was observed in the presence of
M.-J. Cui et al. Environmental Research 208 (2022) 112738

Fig. 4. a. Urease activity assay of the isolates A, B, C and D from a wastewater enrichment. DSM 33 and E. coli BL21 were included for comparison. b. Relative urease
activity of the isolates with reference to S. pasteurii DSM 33 and E. coli BL21. c. Phylogenetic analysis of the isolates A, B, C and D from the wastewater enrichment.
The phylogenetic tree was constructed based on the 16S rRNA sequencing results obtained from the unique colonies and other closely related bacteria, including
those that has not been cultured before. Bootstrap values obtained from 1000 re-samplings of the data are represented on the nodes.

calcium at concentrations below 10 mM, while the relative activity of
urease decreased rapidly at higher concentrations. At a concentration
higher than 200 mM, calcium completely inhibited urease activity in
S. pasteurii DSM 33 under the tested conditions. We further applied our
proposed assay to screen for highly active UPB from a wastewater
enrichment and identified a strain of S. pasteurii (strain B) exhibiting a
substantially higher urease activity than DSM 33. Taken together, we
established a 96-well plate-based quantitative, high-throughput urease
activity assay that can be used for comparison and rapid screening of
ureolytic bacteria. As ureolytic bacteria and urease activity are of in­
terest to environmental, civil, and medical researchers and practitioners,
we envisage wide applications of the assay reported in this study.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This research was supported by Grant No MOE2015-T2-2-142
provided by the Ministry of Education (MOE), Singapore, the Centre for
Urban Solutions, Nanyang Technological University, Singapore, and the
National Research Foundation and MOE Singapore, under its Research
Centre of Excellence Programme, Singapore Centre for Environmental
Life Sciences Engineering (SCELSE) (M4330005.C70 to B.C.), Nanyang
Technological University, Singapore.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.envres.2022.112738.
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