Applied Microbiology and Biotechnology (2019) 103:8825–8838
https://doi.org/10.1007/s00253-019-10128-2
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Complementing urea hydrolysis and nitrate reduction for improved
microbially induced calcium carbonate precipitation
Xuejiao Zhu 1,2 & Jianyun Wang 3 & Nele De Belie 1 & Nico Boon 2
Received: 5 June 2019 / Revised: 30 August 2019 / Accepted: 8 September 2019 / Published online: 21 October 2019
# Springer-Verlag GmbH Germany, part of Springer Nature 2019
Abstract
Microbial-induced CaCO3 precipitation has been widely applied in bacterial-based self-healing concrete. However, the limited
biogenetic CaCO3 production by bacteria after they were introduced into the incompatible concrete matrix is a major challenge of
this technology. In the present study, the potential of combining two metabolic pathways, urea hydrolysis and nitrate reduction,
simultaneously in one bacteria strain for improving the bacterial CaCO3 yield has been investigated. One bacterial strain,
Ralstonia eutropha H16, which has the highest Ca2+ tolerance and is capable of performing both urea hydrolysis and nitrate
reduction in combined media was selected among three bacterial candidates based on the enzymatic examinations. Results
showed that H16 does not need oxygen for urea hydrolysis and urease activity was determined primarily by cell concentration.
However, the additional urea in the combined medium slowed down the nitrate reduction rate to 7 days until full NO3−
decomposition. Moreover, the nitrate reduction of H16 was significantly restricted by an increased Ca2+ ion concentration in
the media. Nevertheless, the overall CaCO3 precipitation yield can be improved by 20 to 30% after optimization through the
combination of two metabolic pathways. The highest total CaCO3 precipitation yield achieved in an orthogonal experiment was
14 g/L. It can be concluded that Ralstonia eutropha H16 is a suitable bacterium for simultaneous activation of urea hydrolysis and
nitrate reduction for improving the CaCO3 precipitation and it can be studied later, on activation of multiple metabolic pathways
in bacteria-based self-healing concrete.
Keywords Ralstonia eutropha H16 . Urea hydrolysis . Nitrate reduction . CaCO3 precipitation . Self-healing concrete
Introduction
During last decade, microbially induced calcium carbonate
precipitation (MICP) has been developed as an eco-friendly
technique which has been proposed in various constructional
and environmental engineering fields, such as industrial
wastewater treatment, soil reinforcement, CO2 sequestration,
and restoration of building materials (Cheng et al. 2013; De
* Nico Boon
nico.boon@ugent.be
1
Magnel Laboratory for Concrete Research, Department of Structural
Engineering, Faculty of Engineering and Architecture, Ghent
University, Tech Lane Ghent Science Park, Campus A,
Technologiepark Zwijnaarde 60, 9052 Ghent, Belgium
2
Center for Microbial Ecology and Technology (CMET), Ghent
University, Coupure Links 653, Building A-9000, Ghent, Belgium
3
Department of Civil Engineering, Xi’an Jiaotong University,
Yanxiang Road 99, Xi’an 710029, China
Muynck et al. 2008; DeJong et al. 2006; Jiménez-López et al.
2007). Particularly, MICP-based self-healing concrete is one
of the very promising applications developed so far, in which
concrete cracks are able to be closed autonomously through
bacterial mineral producing processes (Jonkers et al. 2010;
Van Tittelboom and De Belie 2013; Wang et al. 2012;
Wiktor and Jonkers 2011). In this approach, typically, mineral
producing bacteria and nutrients is mixed in fresh concrete
before casting. When a crack is formed, bacteria inside the
crack are activated upon exposure to water and oxygen, and
thus produce a biogenic precipitation layer that heals the crack
(Jonkers et al. 2010). However, many studies so far have
shown limitations about this technology, for example, the bacterial metabolic activity which induces calcium carbonate precipitation can be significantly inhibited after introduction of
bacteria into the concrete (Basaran 2013; Bravo da Silva 2015;
Jonkers and Schlangen 2007). The reasons causing bacterial
activity loss are mainly related to the unfavorable concrete
environment, such as (1) high alkalinity, (2) limited oxygen
and moisture, and (3) limited living space. What’s more, up to
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now, most of the MICP processes applied in self-healing concrete are based on a single bacterial metabolic pathway, such
as oxidation of organic carbon (Jonkers 2007; Jonkers and
Schlangen 2008), urea hydrolysis (Wang et al. 2017), nitrate
reduction, and sulfate reduction (Dong et al. 2016; Seifan et al.
2016; Siddique and Chahal 2011). The possibility of combining multiple metabolic pathways for MICP enhancement has
not been studied yet. Therefore, in this study, investigation
will be carried out to explore the chance of improving
CaCO3 production through the combination of multiple metabolic pathways.
So far, the mostly used bacterial metabolic pathways applied in self-healing concrete are urea hydrolysis. Alkalitolerant ureolytic strains which produce considerable amount
of urease, such as Sporosarcina pasteurii (previously named
Bacillus pasteurii), Bacillus sphaericus, and Bacillus
megaterium, are often used. In this process, 1 mol of urea
firstly decomposes into 1 mol of ammonia and 1 mol of carbamate which on further hydrolysis, leads to an accumulation
of ammonia and carbonic acid. These products subsequently
equilibrate in water and form bicarbonate, 2 mol of ammonium, and hydroxide ions which results in increase of pH and
ultimately shifts the bicarbonate equilibrium, resulting in carbonate ions formation. In most of the studies, urea hydrolysis
presents several advantages such as a simple hydrolysis mechanism, rapid reaction speed, and high CaCO3 yield under controlled laboratory conditions. However, the negative influence
of concrete mentioned above imparts huge effects on bacteria
growth and urease activity during the healing process, and the
viability and performance of these bacteria in situ need to be
optimized. Therefore, the present study will examine the influence of pH, initial cell number, oxygen concentration, and
cell damage on urease activity performance, in order to provide a clearer understanding of ureolytic activity inside real
concrete environment.
From an environmental point of view, urea hydrolysis has
the disadvantage of producing toxic by-product ammonia.
Therefore, another bacterial metabolic pathway, nitrate reduction, has been suggested as a promising alternative technology
for MICP in self-healing concrete (Erşan et al. 2015a) (Erşan
et al. 2015b). The nitrate reduction–driven MICP does not
produce any toxic by-products and it can also lead to the
production of NO2− which is known as a corrosion inhibitor
(Erşan et al. 2016). Typically, nitrate reduction occurs in anoxic environments where the concentration of dissolved and
freely available oxygen is depleted. In these cases, nitrate
(NO3−) or nitrite (NO2−) can be used as a substitute terminal
electron acceptor instead of oxygen and in the meantime, the
process generates CO32− and HCO3− ions which contribute to
the production of CaCO3 precipitation.
From a biological point of view, urea hydrolysis and nitrate
reduction are two essential procedures involved in N transformation from natural environments. Urea hydrolysis is easy to
Appl Microbiol Biotechnol (2019) 103:8825–8838
be triggered by microorganisms which possess urease activity
(UA); the intracellular or extracellular urease enzyme will
catalyze the hydrolysis of urea into ammonia and carbonate
ions, followed by calcium carbonate precipitation as shown in
Eqs. 1 and 2. While nitrate reduction occurs in facultative
anaerobic bacteria as part of the cell respiration for
replenishing energy consumption, it happens mostly in elevated pH and anoxic conditions by denitrifying species, by utilizing the nitrate reductase (NR). Nitrate (NO3−) is reduced
and ultimately produces molecular nitrogen (N2) through a
series of intermediate gaseous nitrogen oxide products. The
reactions can be seen in Eq. 3–7. Theoretically, these two
biological processes can be activated together in a single bacterial strain since different enzymatic patterns are utilized during the reaction. The combination of both processes may allow to increase the generation of biogenetic calcium carbonate
precipitation. Therefore, in present study, the goal has been set
to stimulate both processes in one bacterial strain in order to
investigate the possibility of improving CaCO3 precipitation
yield using limited cell numbers.
Urease
COðNH2 Þ þ 2H2 O → 2NH4 þ CO3 2−
ð1Þ
CO3 2− þ Ca2þ →CaCO3
−
2HCOO þ 2NO3
−
þ 2H
þ
ð2Þ
→ 2CO2 þ 2H 2 O þ 2NO2
HCOO− þ 2NO2 − þ 3H þ →CO2 þ 2NO þ 2H 2 O
−
þ
−
ð3Þ
ð4Þ
HCOO þ 2NO þ H →CO2 þ N2 O þ H2 O
ð5Þ
HCOO− þ N2 O þ Hþ →CO2 þ N2 þ H2 O
ð6Þ
2þ
Ca
þ CO2 þ H2 O→CaCO3 þ 2H
þ
ð7Þ
Materials and methods
Bacteria cultivation and growth condition
Three different bacterial species: Bacillus sphaericus LMG
22257 (Wang et al. 2017), Ralstonia eutropha H16
(Cupriavidus necator ATCC 17699 (Kuchta et al. 2007)),
and Diaphorobacter nitroreducens M5 (DSM 15985 (Erşan
et al. 2015a)), named LMG, H16, and M5, were chosen as
bacterial candidates for MICP evaluation in this study. Strain
LMG is an urease-positive strain from our previously study
(Wang et al. 2017) Strain M5 has been found efficient in
nitrate reduction and was studied also for nitrate reducing
CaCO3 precipitating in our previous work (Erşan et al.
2015a), while H16 is a denitrifying strain which has been
reported positive for poly-hydroxyalkanoates (PHA) and
poly-β-hydroxybutyrate (PHB) accumulation (Kuchta et al.
2007; Pohlmann et al. 2000). The growth medium consisting
of yeast extract and urea (UYE media) was used for cell
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Appl Microbiol Biotechnol (2019) 103:8825–8838
cultivation. The yeast extract solution (20 g/L) was first
autoclaved for 20 min at 120 °C. The urea solution was filter
sterilized using a 0.2-μm Millipore filter (Millipore, USA) and
then added into yeast solution, until the final concentration
was 20 g/L. The pH of the medium was adjusted to 7.0 before
use. For the following experiments, the cells were harvested
by centrifuging at 7000 rpm for 5 min (Sorvall RC 6+,
Thermo Scientific™) and collected pellets were resuspended in a sterile saline solution (NaCl, 8.5 g/L) before
inoculation into test bottles. The initial cell density of the
culture was determined by using flow cytometry, and given
as cells/mL throughout the study. After 24 h, when the OD600
value reached between 1.0 and 1.2, the concentration of the
bacteria in the suspension was around 109 cells/mL.
Urease activity
Urease is the key enzyme that dominates the urea hydrolysis
process, and it catalyzes the hydrolysis of urea to ammonia
and CO2; thus, urease activity (amount of urea that can be
decomposed per time unit) is the most important parameter
that indicates the efficiency of ureolytic bacterial-induced
CaCO3 precipitation. In this study, two types of methods were
used for urease activity measurement, depending on the specific situation: the conductivity measurement and the IC measurement. Conductivity measurement is applied when urease
activity is determined in pure urea solution, and the amount of
urea decomposed is measured by the conductivity of the urea
solution. During urea hydrolysis, 1 mol of urea transfers into
2 mol of NH4+ and 1 mol of CO32−, the production of ionic
species from non-ionic substrates causing an overall increase
of conductivity of the solution. In this study, the conductivity
of the urea solution was recorded by a Consort C3010 sensor.
The relationship between urea decomposition and conductivity can be determined by the method described in (Whiffin
2004). The rate of conductivity increase (ms min−1) was converted to urea hydrolysis rate (mM urea hydrolysed) according
to the specific formula at 10, 20, and 28 °C shown in
Eqs.(8)–(10). It is important to note that by using this method
no other ions including Ca2+ should be present in the solution,
since these ions will affect the overall conductivity and make
it difficult to relate conductivity with the amount of urea
decomposed (Wang et al. 2017).
At 10°C : urea decomposed ðmMÞ
¼ conductivity ms cm−1 11:19
ð8Þ
At 20°C : urea decomposed ðmMÞ
¼ conductivity ms cm−1 11:08
ð9Þ
At 28°C : urea decomposed ðmMÞ
¼ conductivity ms cm−1 10:54
ð10Þ
In the presence of Ca2+ ions and other cations, the urease
activity was measured by testing the ammonium concentration
in the media through compact ion chromatography (IC)
(Metrohm 761).
Influence of initial cell density and oxygen concentration
on urease activity
Strains LMG 22257 and H16 were selected for the subsequent
experiments. Both strains were first cultured in UYE media
(20 g/L yeast extract and 20 g/L urea) for 24 h until the OD
reached 1, then a series of initial bacterial cell concentrations
(109, 108, and 107 cells/mL) was obtained through serial dilution and finally settled by flow cytometry (Accuri C6). Before
inoculation, PYREX® serum bottles (Corning, USA) with
rubber stoppers and metal caps containing 100 mL pure urea
solution (20 g/L urea) were prepared. The volume of the penicillin bottle was 125 mL and hence, the volume of the headspace was 25 mL. They were then subjected to Argon (Ar) gas
flushing for different cycles in order to achieve different oxygen concentrations in the headspace. In this experiment, oxygen contents of 1%, 3%, 8%, and 13% were used. Finally, 10,
1, and 0.1 mL of the initially grown bacteria suspension
(109 cells/mL) were added into the serum bottle through syringe injection, making the urea solution a total volume of
100 mL, resulting in a final bacterial concentration of 108,
107, and 106 cells/mL. The incubations were carried out at
28 °C on 120 rpm shaker for 24 h. After each 2 h, 5 mL of
sample was taken from each bottle for conductivity monitoring for a total of 10 h. A schematic illustration of the experiment setup can be found in Fig. 1. Experiments were carried
out in triplicates and the mean values with standard deviations
are plotted.
A two-step cultivation for urease activity examination
In order to investigate the restoration of urease activity after
the bacteria were applied into concrete, a two-step cultivation
process including some further subculture steps by simulating
concrete environment was conducted. Bacterial cells of both
strains LMG 22257 and H16 were firstly grown in UYE media for 24 h until cell concentration reached 109 cells/mL, then
they were removed from the medium by centrifugation and resuspended immediately into four kinds of new medium in
order to examine the urease activity restoration. These media
are (1) identical UYE media, (2) pure urea solution, (3) UYE
media at pH 13 (pH was adjusted by NaOH solution made in
PBS buffer). In addition, (4) cells after inactivation were
added to a new identical UYE medium. Inactivation was carried out by using the method described by Işik, and cells were
first resuspended in 1% formaldehyde for 4 h and then dried in
an oven at 70 °C for 24 h (Işik 2008). Hence, the cells were
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Fig. 1 A schematic illustration of
the urease activity experiment
setup
considered as non-living and metabolically inactivated. The
urease activity was determined immediately after transform
into new medium and again after 24 h of incubation.
Cultivation was conducted in 125-mL serum bottles in anoxic
conditions (oxygen concentration 1%); throughout the experiment, 5 mL of samples from each bottle was collected every
2 h and urease activity was monitored until 56 h by testing the
conductivity as mentioned above.
Nitrate reduction ability
Three kinds of different media were prepared for nitrate reduction examination of both strains LMG 22257 and H16.
Carbon source type and concentration are essential elements
that determine the nitrate reduction activity. In this study, yeast
extract (YD media), media containing more abundant carbon
supply (NB media), and media with minimal nutrients (M9
media) were used for nitrate reduction examination in order to
compare the efficiency in different nutrient situations, components are shown in Table 1, NO3− concentration were kept the
same among three medium (600 mg/L). A total of 125-mL
serum bottles containing 100 mL of each medium with
approximately 0% oxygen concentration in the headspace
were used. Ten milliliters of the initially grown bacteria
suspension (109 cells/mL) was added into the serum bottle, making the final bacteria concentration in the bottle
108 cells/ml. Samples were taken at each day for NO3−
and NO2− measurement by using compact ion chromatography (IC) (Metrohm 761). In the meantime, total gas
pressure in each bottle was recorded by using a UMSTensio meter (Infield 7). All experiments were carried
out in triplicate and the nitrate reduction performance
was monitored for a total of 14 days.
Simultaneous activation of urea hydrolysis
and nitrate reduction
In this part of experiment, the combination media (UBD media) for simultaneous activation of urea hydrolysis and nitrate
reduction were made by adding 10 g/L urea into NO3− containing NB media. In the experiment, 125-mL serum bottles
with rubber stoppers and metal caps containing 100 mL UBD
media were used. The head space of the bottles was firstly
flushed with Argon (Ar) gas for several cycles in order to
remove the oxygen as much as possible (final oxygen concentration around 0 to 2%). Then the bottles were inoculated with
10 mL of bacteria inoculation (109 cells/mL) to a final concentration of 108 cells/mL. The bottles were then kept in a
28 °C room for 7 days and samples were taken each day for
urea decomposition and nitrate reduction measurements. The
nitrate reduction performance was determined by testing the
concentration of NO3− and NO2−, recorded by using compact
ion chromatography (IC) (Metrohm 761), while the urea decomposition was tested by recording N-NH4+ and Ca2+
Table 1
Composition of three media for denitrification
Compounds (g/L)
NBD media
Peptone
Beef extract
NaCl
Yeast
Na-formate
Methanol
KNO3
Na3PO4
Initial pH
10.0
3.0
5.0
YD media
M9 media
10.0
1.0
1.0
7.0
7.0
6.0
4.0
2.0
0.0105
7.0
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concentrations by using cation ion chromatography (761
Compact IC, Metrohm with 837 Eluent Degasser). All the
experiments were performed in triplicate. Control samples
by inoculating bacteria in pure NB media and urea solution
(10 g/L) were also conducted as a base to compare with the
performance in combined media. Due to the fact that certain
amount of Ca2+ ions in the media can interfere with the regulation of bacterial metabolic activities, both strains were inoculated in UBD media with and without Ca ions (10 g/L),
separately, in order to investigate the effect of Ca2+ on two
metabolic pathways and for further regulating the simultaneous activation procedure.
Total bacterial calcium carbonate precipitation
In order to achieve the maximum overall CaCO3 production,
the media components for combination were adjusted before
applying in the concrete. In this study, a L16(44) orthogonal
table designed by SPSS was employed and 16 representative
experiments were carried out in order to investigate the feasibility of the proposed process and optimize the media components. Four factors chosen in this experiment are KNO3, urea,
CaCl2 concentration, and bacterial initial biomass, as they are
major components in the media and necessary precursors for
both reactions. The experiment design can be seen in Table 2;
other factors such as nutrient concentration, oxygen concentration, and pH were kept the same among different groups
(NB media, 18 g/L; O2, 2%; and pH 7.0). The final CaCO3
and relative CaCO3 (g: CaCO3/g: Ca) yield were calculated
based on the recorded concentration of Ca2+; a mean value of
three replicate samples with a standard deviation < 2% was
used as the final Ca2+ concentration. According to the results,
the range analysis and analysis of variance (ANOVA) were
carried out to determine the optimal level within the observed
range and the predominance of each factor.
The range analysis is usually used to determine the most
sensitive factor that influences the target index. In this study,
the range analysis was employed to discriminate the comparative significance of each factor, which was defined as the
Table 2 Orthogonal experiment design (units: conc.: g/L, initial
biomass: cells/mL)
Levels
Factors
KNO3 conc.
1
2
3
4
1
2
5
10
Urea conc.
5
10
20
30
CaCl2 conc.
5
15
30
50
Initial biomass
6
10
107
108
109
difference between the maximum and minimum value of Iji,
noted as Rj:
n o
n o
R j ¼ max Iji –min Iji
ð11Þ
A larger Rj means a greater importance of the factor (Zhao
et al. 2013). I ji was the average value of each experimental
factor at the same level i in the orthogonal experiments, which
was used to determine the optimal level and the optimal combination of factors. I ji could be expressed as:
Iji ¼ I ji =4 j
ð12Þ
where the Arabic numerals i (i = 1,2,3,4) were the level number and j represents a certain factor. Iji was the sum of the
targeting indexes of all levels in each factor j; 4j refers to the
total 4 levels of each corresponding factor.
Results
Urease activity
The three bacterial candidates LMG, H16, and M5 are all
calcium carbonate–producing species based on our previous
results, among which LMG is a widely used urease-positive
strain which produces calcium carbonate through urea hydrolysis (Wang et al. 2017), while the other two strains H16 and
M5 are denitrifying strains producing CaCO3 precipitation
through nitrate reduction (Erşan et al. 2015a; Erşan et al.
2016). However, it is not known whether several metabolic
pathways can be activated simultaneously by one strain
through combining and adjusting media components. So firstly, the urease activity of all the three strains in anoxic conditions has been tested, and as it shown in Fig. 2, the urea
decomposition rate of strain H16 showed similar developing
trend as strain LMG. Almost all the urea has been completely
decomposed after 8 h for both strains LMG and H16, and no
further decomposition is noticed for the next 6 h until the end
of the experiment, which suggests that the denitrifying strain
H16 can also produce urease in anoxic conditions, while no
urea reduction was detected in M5 cultured media, which
indicates that strain M5 is not urease positive.
Effect of initial cell density and oxygen concentration
on urease activity
The urea decomposition of strains LMG and H16 at different
initial cell densities and different initial oxygen concentrations is
shown in Fig. 3. It can be seen that for both strains, the urea
decomposition rate under different oxygen concentrations (from
1 to 13%) follows always a similar increasing trend, regardless
of the initial cell concentration. Especially when the cells are
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on the urea hydrolysis rate. Furthermore, it is worth to notify
that cell concentration had a significant influence on urea hydrolysis rate. When cell density reaches the highest value of
108 cells/mL (Fig. 3 a(1), b(1)), the urea decomposed increased
to 20 g/L within 10 h, which means a completely hydrolyzation
was achieved, for both H16 and BS. However, only 25% of the
urea was completed decomposed within 10 h when cell concentrations equaled 107 and 106 cells/mL.
Urease activity in a two-step cultivation procedure
Fig. 2 Urea decomposition of three bacterial candidates in anoxic
condition (O2 concentration 2%)
present in a concentration of 107 and 106 cells/mL, lines overlap
with each other completely (Fig. 3 a(2–3), b(2–3)), which indicates that urea decomposition rate kept the same regardless of
different oxygen concentrations. The oxygen concentration thus
has only marginal influence on urease activity. In some previous
studies, it has been shown that aeration or increase of oxygen
flow will increase the final CaCO3 yield (Seifan et al. 2017;
Zhang et al. 2016), which is due to oxygen can accelerate cell
growth and thus finally increase the biogenetic precipitation
yield. However, in this study, we found that in the condition
of constant initial cell number, oxygen has no significant effects
The urease activity restoration after different treatments is
shown in Fig. 4, for the first 24 h, both strains LMG and
H16 were grown until exponential phase in UYE media before
re-inoculation. Despite the same concentration of cells being
added to different media, a different recovery in urease activity was observed depending on the subculture medium. For
the LMG cells re-inoculated in the identical UYE media, immediately after transfer they kept up growth still stationary
phase. Within 8 h, the OD600 increased to around 1.2 and no
further increase was detected, at the same growing conditions.
At the same time, the highest NH4-N concentrations were
achieved (35 mg/L) in the culture within 8 h, which indicates
a full recovery of urease in a second UYE media (Fig. 4 (a(1–
2)). For the LMG cells transferred to pure urea solution, similar variation of enzyme activity was detected. Cells grew up
Fig. 3 Urea decomposition of strains LMG (a1–3) and H16 (b1–3) under different initial cell densities (1–3, 108; 107; 106 cells/mL) and initial oxygen
concentrations (1–13%)
Appl Microbiol Biotechnol (2019) 103:8825–8838
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Fig. 4 Growth curve and urease activity of strains LMG (a1–2) and H16 (b1–2) in a two-step cultivation. The second activation was performed in the
identical UYE media (circles); pure urea solution (squares); UYE media at pH 13 (triangles); cells after inactivation in the same UYE media (diamonds)
very slowly until stationary phase and a relatively slower
NH4-N production speed was detected compared with cells
in UYE media, which indicates that a small amount of biomass increase can still be expected in pure urea solution (although without any other nutrient) and urease activity can be
regained in such condition. Interestingly, although no sustainable growth was detected when LMG cells were transferred to
UYE media under pH 13, a high amount of NH4-N generation
was still achieved, similar to the samples in pure urea solution.
This indicated that high pH in the concrete may restrict the
sustainable growth of bacteria cells. However, the enzyme
activity is still able to be restored, in a certain level of biomass,
and can be recovered to an active state when cells are again
subjected to urea solution. Similar enzyme activity variation
happened in H16 strain (Fig. 4 (b(1–2)). LMG and H16, subjected to inactivation before being inoculated to the second
media, showed an overall loss of activity, neither bacterial
growth nor urea decomposition was detected throughout the
subculture phase, which indicated that urease activity can be
restored in low active cells, but not in completely dead cells.
Nitrate reduction performance
Nitrate reduction performance in different media
The nitrate reduction performance of both bacteria strains was
greatly influenced by the components in the medium. As
shown in Fig. 5 b(1), strain H16 was able to reduce NO3−
from an initial concentration of 600 mg/L to a final concentration of 60 mg/L within 4 days when it was inoculated in NB
media. At the same time, a NO2− accumulation in the media
from 0 to 80 mg/L was detected in the bottle, with a short
increase of total gas pressure up to around 37 hPa on the first
day of the measurement (Fig. 5 b(2)). However, when the
strain was inoculated in YD media and M9 media, it took
7 days and 14 days, respectively, for the complete reduction
of NO3−, and also a relatively feebler gas pressure increase
was detected (around 28 hPa and 21 hPa, respectively). For
bacterial strain LMG, similar situation was discovered (Fig. 5
a(1,2)). NO3− concentration was reduced to 1/10 of the initial
concentration on the 4th day when cells were inoculated in the
NB media, and on 7th and 14th day, respectively, when cells
were inoculated in YD and M9 media. This indicated that
bacterial strain LMG is also nitrate reduction positive when
it is inoculated in proper media and growing condition. The
nitrate reduction rate of LMG in NB media was more than 2
times higher than in YD media and 3 times higher than in M9
media. It appears that, at the same NO 3− concentration
(600 mg/L), when more abundant carbon sources appear in
the media, the nitrate reduction happens faster. The benefits of
a nutrient-sufficient environment, likely offset additional energy costs of NO3−, transport into the cell. Overall, both strains
showed similar nitrate reduction activity development when
they were inoculated in NB, YD, and M9 media in anoxic
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Fig. 5 Denitrification ability of strains LMG (a1) and H16 (b1) in different media with the total gas pressure (a(2) and b(2))
condition. The obtained NO3− reduction activity in NB media
was quite promising and could be used as reference for further
concrete applications.
Combined performance of urea hydrolysis and nitrate
reduction
Urea decomposition and nitrate reduction in the media
without Ca2+
Figure 6 shows the combined performance of urea decomposition and nitrate reduction of strains LMG (a) and H16 (b) in
different media (without Ca2+). No significant difference was
discovered for the two processes when the bacteria were inoculated in the combined UBD media compared with the separate media. As it shown in Fig. 6 a(1), in the test period of
7 days, the NO3− concentration in UBD and NB media both
reduced from 600 to around 30 mg/L in a similar decreasing
trend, the reduction rate increased at the beginning and the end
of the test and kept stable (around 20 mg/d) in the middle.
There is also an increasing NO2− concentration detected in the
media, the highest concentration detected was 50 mg/L on the
6th day. This indicates that bacterial strain LMG is able to
convert NO3− to NO2− when no Ca2+ presence in the media
and the extra urea supplemented will not affect nitrate reduction. In the examination of urea decomposition (Fig. 6 a(2)),
NH4-N concentration increased rapidly from 0 to 8.8 g/L on
the first day and then kept going up until the highest value of
10.2 g/L, this indicate the full decomposition of urea in the
solution. Interestingly, the urea decomposition in UBD media
again showed a similar increasing trend with it in the pure urea
solution, the highest NH4-N concentration was also detected
on the second day in pure urea solution, this revealed the fact
that once there is presence of urea in the solution, bacterial
urease can be activated and nitrate reduction can still happen
at the same time. These two processes can be activated simultaneously in one medium and will not affect each other. For
bacterial strain H16, similar situation was discovered (Fig. 6
b(1, 2)). However, it is worthwhile to notice that a more rapid
nitrate reduction rate of H16 was detected compared with
LMG; thus, it may indicate that strain H16 is able to work
more efficiently in the anoxic condition compared with strain
LMG.
Urea decomposition and nitrate reduction by H16
in the media with Ca2+
From the above evaluation, it can be seen that both strains
showed nearly equal performance for these two metabolic
pathways when they were inoculated in combined media.
However, due to the reason that strain H16 showed superior
biomass growth than LMG in the anoxic condition, and it
exhibited slightly higher nitrate reduction efficiency in the
simultaneous activation procedure afterwards (see Fig. 6), it
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Appl Microbiol Biotechnol (2019) 103:8825–8838
Fig. 6 Nitrate reduction and urea decomposition of strains LMG (a) and H16 (b) in combined media without Ca2+ supplement
was selected for further investigation in the combined media
with Ca2+ ions for CaCO3 yield optimization. Overall, Ca2+
ions in the media showed huge inhibition effects on bacterial
nitrate reduction activity, but not on the urea decomposition
process. As shown in Fig. 7a, when bacteria were inoculated
in pure urea solution with the presence of Ca2+ (concentration
10 g/L), urea decomposition can still be detected within a
short period and the highest NH4-N concentration (9.7 g/L)
occurred on the first day of the test. However, a significant
decrease of nitrate reduction rate was found when bacteria
were inoculated in NB media with Ca2+; only 100 mg/L of
NO3− has been reduced after 7 days, with an extremely low
amount of NO2− accumulation (Fig. 7b). Moreover, when
bacteria were inoculated in combined UBD media with
Ca2+, the inhibition showed again, no decrease of nitrate concentration was detected in the media on the first 4 days.
Nevertheless, it goes down again rapidly after the 5th day
while the NH4-N concentration in the solution reached steady
state (Fig. 7d). The data of dissolved Ca2+ concentration in
different media also showed that more Ca2+ was deposited
when there is urea decomposition happening in the media
(Fig. 7c). This indicates that the amount of CaCO3 production
by strain H16 from nitrate reduction is relatively slower and
less compared with urea hydrolysis.
Bacterial urea hydrolysis and nitrate reduction
under different Ca2+ concentrations
Ca2+ is essential for the biogenetic formation of CaCO3.
Nevertheless, in the present study, the Ca2+ concentration
showed dramatic effect on the nitrate reduction process.
As the results in Fig. 8 reveal, the urea decomposition of
H16 displayed a clear rising-to-stationary pattern, which,
noticeable, was not markedly impacted by the addition of
Ca2+ ions in the media. However, the bacterial nitrate
reduction (NR) was severely restrained in response to
the high Ca2+ concentration. Therefore, the enzyme activities of LMG and H16 challenged by a series of Ca2+
dosages, from 0 to 10 g/L in the UBD media, were further
studied. As can be seen from Fig. 8, a nearly linear decrease of UA with the increase of Ca2+ concentration was
detected over the whole urea hydrolysis period. It can be
presumed that the high concentration of Ca2+ at some
point can affect the level of cell growth and division, thus
further affects the urease amounts and conclusively the
enzyme activities. More interestingly, a non-linear relationship between Ca2+ concentration and NR was discovered throughout the nitrate reduction process in both
strains. Specifically, nearly 80% of nitrate (around
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Appl Microbiol Biotechnol (2019) 103:8825–8838
Fig. 7 Nitrate reduction and urea decomposition of strain H16 in media with Ca2+ supplement (a in pure urea solution, b in NB media, c, d in UBD
media)
500 mg/L) was reduced when Ca2+ concentration was
around 0.5 g/L in the media; however, the value dropped
dramatically to around 20 to 30% for both strains when
Ca2+ concentration increased to 1 g/L, and steadily declined at even higher Ca2+ concentration. This indicates
that Ca2+ ions had severe inhibition effects on bacterial
nitrate reduction activity, but not on the urea decomposition process.
Fig. 8 Nitrate reduction and
urease activity of strains LMG
and H16 after 7 days at different
Ca2+ dosage. (decomposed urea
concentration (circular),
decomposed NO3− concentration
(triangular))
Total bacterial calcium carbonate
precipitation yield
According to the Rj values shown in Table 3 of each factor, the
factors with larger Rj value having a bigger impact on the final
experimental results, CaCl2 conc. (5.75) > initial biomass
(5.60) > KNO3 conc. (3.14) > urea conc. (0.39). The Rj value
of the CaCl2 conc. was the largest one among all the ranges,
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Appl Microbiol Biotechnol (2019) 103:8825–8838
Table 3
Orthogonal experimental analysis of the total bacterial calcium carbonate precipitation yield
KNO3 conc. (g/L)
Urea conc. (g/L)
CaCl2 conc. (g/L)
Initial biomass
(cells/mL)
1
2
3
4
1
10
5
10
10
5
20
30
15
15
15
50
107
109
106
106
5
6
7
8
9
10
11
12
13
14
15
16
Ij1
Ij2
Ij3
Ij4
Rj
2
10
5
1
1
2
1
5
2
5
10
2
7.34
4.21
6.43
6.27
3.14
20
20
30
20
5
30
5
30
5
10
10
10
7.54
5.54
6.00
6.98
0.39
5
30
5
50
5
15
30
30
50
50
5
30
2.46
5.79
8.22
7.78
5.75
109
107
107
108
106
108
108
109
107
109
108
106
4.09
4.72
6.76
9.69
5.60
CaCO3 yield (g/L)
Relative CaCO3 yield
(g/g: Ca)
5.09
8.87
3.25
5.83
0.94
1.64
0.60
0.32
4.07
7.38
2.00
9.30
0.80
5.92
8.84
14.17
4.36
11.63
2.97
2.46
2.26
0.68
1.11
0.51
0.44
1.09
0.81
1.31
0.24
0.64
1.65
0.22
Values in italic are the maximum CaCO3 yield (14.17) and the highest relative CaCO3 yield (2.26) obtained in this study
implying that the CaCl2 concentration was the most significant factor influencing the CaCO3 precipitation by the means
of simultaneous activation of both metabolic pathways.
Considering the previous results, it can be deduced that although urea hydrolysis can remain in steady state regardless
of the Ca2+ concentration in the media, the nitrate reduction
showed strictly inhibited results due to high amount of Ca2+ in
the reaction system. Therefore, the CaCl2 concentration
can affect the final experimental CaCO3 yield by affecting mostly the nitrate reduction process. Besides, considering that the bacterial initial biomass was the second
most important parameter affecting the precipitation
yield, it is always a priority to apply as high amount of
biomass as possible (until 109 cells/mL) to obtain high
amount of CaCO3 precipitation. However, when talking
about in-field application in concrete, it still needs further investigation of the effect that excess biomass will
bring to the concrete. Overall, the maximum CaCO 3
yield obtained in this study is 14.17 g/L by applying
1 g/L KNO3, 30 g/L urea, and 30 g/L CaCl2 with initial
bacterial biomass 109 cells/mL in the UBD media, while
the highest relative CaCO 3 yield was 2.26 g/g Ca,
achieved in the media consisting of 2 g/L KNO3, 20 g/
L urea, and 5 g/L CaCl2 with initial bacterial biomass
109 cells/mL.
Discussion
Interactions of urea hydrolysis and nitrate reduction
during combination
Urea hydrolysis and nitrate reduction are two essential
steps involv ed in the nitrogen cycle in nature.
Interactions of these two processes in marine and soil
systems balance the inorganic nitrogen concentration in
the form of NO3−, NO2−, and NH4+ through multiple bacterial enzyme activities. Urease activity (UA) provokes
the hydrolysis of urea into NH3 and CO2 and it has been
found existing in a wide range of microorganisms and
plants (Ciurli et al. 1996; Mobley et al. 1995). Nitrate
reduction takes place during the microbial oxidation of
organic matter by use of NO3− as an electron acceptor
during the cellular respiration; the denitrifying microorganism species express nitrate reductase (NR) specifically
for NO3− reduction (Lomas 2004). In the present study, in
the condition of no calcium ions supplement, it was found
that for both strains LMG and H16, the addition of urea to
NB (denitrifying) media resulted in a slight decrease in
measured NR, as shown in Fig. 6 a(1), nitrate uptake ratio
displayed a clear drop of around 5% compared with control samples (cultured in pure denitrifying media)
8836
throughout the test period. The results were consistent
with the examination carried out by Lund, who for the
diatom Skeletonema costatum also found that the presence
of urea caused a NO3− uptake rate decrease by around
12% (Lund 1987). Other studies with enriched seawater cultures also showed that urea inhibits NO3− uptake (Grant et al.
1967; McCarthy 1972). One other study on interactions between urea and NR (Mathew 1981) concluded that NR in ten
chlorophyte species was constitutively expressed during
growth on urea. Similar to the present results, Mathew
(1981) found that nitrogen-sufficient growth on urea led to
significantly decreased, but still detectable NR. However,
the additional NO3− showed no dramatic impact on urea uptake rates and UA (Fig. 6 a(2)), The present data suggest that
(1) without the present of Ca2+ in the media, UA and NR can
be activated simultaneously in strains LMG and H16, (2) urea
would decrease NO3− uptake and NR in NO3− grown cultures,
and (3) NO3− would not decrease urea uptake and UA in ureagrown cultures. Corresponding with our previous results on
urease activity, oxygen was not necessarily needed during
bacterial urea hydrolysis; however, it is needed for bacterial
growth and spores germination, this might be a bottleneck
problem for the aerobic bacteria-based self-healing system.
Thus, it is necessary to maintain and, moreover, improve the
cell viability and enzyme activity in oxygen limited conditions
inside deeper crack zones. Accordingly, a more oxygen tolerant pathway, such as nitrate reduction, is highly recommended
to be introduced into the system, a schematic illustration can
be seen in Fig. 9. Therefore, present study offers a possible
strategy to improve the CaCO3 precipitation yield by activating both pathways, urea hydrolysis, and nitrate reduction simultaneously in one bacterial strain, through adjusting and
combining the media components corresponded for MICP.
Fig. 9 Schematic illustration for
summary of urea hydrolysis and
nitrate reduction
Appl Microbiol Biotechnol (2019) 103:8825–8838
The effect of Ca2+ concentration on bacterial urea
hydrolysis and nitrate reduction
Ca2+ ions concentration had profound effect on microbialinduced CaCO3 precipitation activity. On the one hand,
Ca2+is an essential component for CaCO3 generation, on the
other hand, Ca2+ ions influence the bacterial ureolytic activity
as demonstrated in this study. Excess amount of Ca2+ is toxic to
bacteria since they only need limited amount to regulate their
life activities. This explanation is also supported by the observation discovered by De Muynck in 2008, in which a calcium
dosage of 17 g Ca2+ cm−2 was found to induce the highest
consolidation for limestone prisms with no improvement at
higher concentrations through urea hydrolysis–based MICP
(De Muynck et al. 2010). It can be demonstrated that, first of
all, due to the detrimental effects of Ca2+ concentration on the
activity of the microorganisms, not all urea could be hydrolyzed by a given amount of cells. Secondly, higher concentrations of calcium ions result in a higher saturation state of the
system. As a result, a nucleation around the negatively charged
cell wall occurs, obstructs the nutrients and urea uptake, and
further decreases the UA performance. On the other hand, it is
noticeable from Fig. 8 that the potential of nitrate reduction was
suppressed even more than the UA performance. This apparent
uncoupling of Ca2+ concentration and NR has also been observed before in soil environment (Hyun and Lee 2004;
Lützow et al. 2006), the respiration rate and nitrate reduction
Bsystem^ become much lower in alkaline conditions and in the
presence of excess Ca2+. The inhibition of respiration and NR
in the presence of Ca2+ might be explained by the organicmineral interactions. Bivalent cations like Ca2+ or Al3+ acting
as cation bridges between minerals and dissolved organic matters (DOM) exist in the system. This keeps the organic-mineral
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Appl Microbiol Biotechnol (2019) 103:8825–8838
complexes more flocculated and condensed (Baldock and
Skjemstad 2000; Blanco-Canqui and Lal 2004). The excess
Ca2+ in the solution may therefore be stabilizing the organic
matter by inhibiting the microbial processing of organic C and
N by reducing the efficiency of enzymatic activity.
In this study, strains Bacillus sphaericus LMG 22257 and
Ralstonia eutropha H16 have been found capable of producing urease and nitrate reductase simultaneously in combined
media. However, there are caveats with regard to the cations
Ca2+ added in the system. In the condition without Ca2+ supplement, the additional urea in the combination media would
decrease the activity of nitrate reductase (NR) at a negligible
degree, and NO3− will not affect the urease activity (UA) and
urea decomposition. Nitrate reduction can still happen following urea decomposition when pH is elevated and oxygen
levels are low. The clear difference in UA and NR profile
has important implications for understanding each stage during biogenetic CaCO3 generation. While Ca2+ ions were included in the system, however, elevated pH combined with
excess Ca2+ ions actually suppresses nitrate reduction potential at the early stage. This inhibition effect happens because
the bivalent cations impair the microbial access to organic C
and N resources and further thwarts the nitrate reduction pathway. However, in the later period, the higher NR rates occurring after UA consumption still provide speculation that the
recovery of nitrate reduction is possible after urea hydrolysis.
To summarize, in the first stage of the process, urease activity
occurs without being affected by additional nitrate and high
Ca2+ concentration. In the second stage, after the urea hydrolysis has finished, due to an increase of pH, elimination of
oxygen, and reduction of Ca2+ in the medium, the condition
becomes favorable for a recovery of nitrate reduction. This
most probably explains why in the later period, a higher NR
rate was detected following the UA consumption. Thus, this
study verifies the possibility of improving biologically catalyzed CaCO3 precipitation yield via the combination of two
metabolic pathways, urea hydrolysis and nitrate reduction, in
single bacterial strains. The results from these experiments,
most importantly, offer a pathway for improving crack healing
efficiency in self-healing concrete.
Funding information This work is part of a research project jointly supported by the Center for Microbial Ecology and Technology (CMET) and
Magnel Laboratory for Concrete Research from Ghent University. It is
granted by the China Scholarship Council (File No. 201706140108) and
BOF co-funding from Ghent University (reference code: 01SC4918).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
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