Journal of Environmental Management 256 (2020) 109938

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Journal of Environmental Management

journal homepage: http://www.elsevier.com/locate/jenvman

Research article

Application of microbe-induced carbonate precipitation for copper removal

from copper-enriched waters: Challenges to future industrial application
Carla Duarte-Nass a, b, Katherina Rebolledo c, Tamara Valenzuela d, Matías Kopp b, David Jeison e,
Mariella Rivas f, Laura Azo�car g, h, Alvaro
� Torres-Aravena e, Gustavo Ciudad b, i, *
a
Doctorate in Engineering Sciences with Specialization in Bioprocesses, Universidad de La Frontera, Avenida Francisco Salazar #01145, Temuco, Chile
b
Departamento de Ingeniería Química, Universidad de La Frontera, Avenida Francisco Salazar #01145, Temuco, Chile
c
Facultad de Ingeniería, Universidad Cat�olica de Temuco, Avenida Rudecindo Ortega #02950, Temuco, Chile
d
Departamento de Ciencias Químicas, Universidad de La Frontera, Avenida Francisco Salazar #01145, Temuco, Chile
e
Escuela de Ingeniería Bioquímica, Pontificia Universidad Cat� olica de Valparaíso, Avenida Brasil #2085, Valparaíso, Chile
f
Laboratorio de Biotecnología Algal y Sustentabilidad, Departamento de Biotecnología, Facultad de Ciencias del Mar y Recursos Biol�ogicos, Universidad de Antofagasta,
Avenida Angamos #601, Antofagasta, Chile
g
Departamento de Química Ambiental, Facultad de Ciencias, Universidad Cat� olica de la Santísima Concepci�on, Avenida Alonso de Ribera #2850, Concepci� on, Chile
h
Núcleo Milenio en Procesos Catalíticos hacia la Química Sustentable, Universidad de Concepci� on, Avenida Víctor Lamas #1290, Concepci� on, Chile
i
Instituto del Medio Ambiente (IMA), Universidad de La Frontera, Avenida Francisco Salazar #01145, Temuco, Chile

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

Keywords: Copper contamination in watercourses is a recent issue in countries where mining operations are prevalent. In
MICP this study, the application of copper precipitation through microbe-induced carbonate precipitation (MICP) was
Copper analyzed using urea hydrolysis by bacteria to evaluate precipitated copper carbonates. This article demonstrates
Bio-precipitation
the application of a copper precipitation assay involving Sporosarcina pasteurii (in 0.5 mM Cu2þ and 333 mM
Urea
Sporosarcina pasteurii
urea) and analyzes the resultant low removal (10%). The analysis indicates that the low removal was a conse­
quence of Cu2þ complexation with the ammonia resulting from the hydrolysis of urea. However, the results
indicate that there should be a positive correlation between the initial urea concentration and the bacterial
tolerance to copper. This identifies a challenge in the industrial application of the process, wherein a minimum
consumption of urea represents an economic advantage. Therefore, it is necessary to design a sequential process
that decouples bacterial growth and copper precipitation, thereby decreasing the urea requirement.

1. Introduction Currently, the treatments options for heavy metals-enriched water

Heavy metal pollution is a relevant environmental problem world­
wide, with potentially severe impacts on both human health and wildlife
(Lesmana et al., 2009; Zhao et al., 2016). The release of heavy metals
into the environment is typically associated with the discharge of the
waste soils and wastewaters of many industries, including mining, tan­
ning, pesticide production, and electroplating (Barakat, 2011). Partic­
ularly, the production of copper via mining, which is led by Chile, Peru,
and China, produces millions of metric tons of materials containing
metals, which may pose serious environmental risks if not correctly
managed. When copper tailings are not properly contained and
managed, contamination of rivers and aquifers can become a serious
problem (Castilla, 1996; Salamanca et al., 2004; Schalscha and Ahu­
mada, 1998).
are dominated by physicochemical processes, such as chemical precip­
itation, ion exchange, adsorption, membrane filtration, coagulation and
flocculation, flotation, and electrochemical treatment (Fu and Wang,
2011; Hashim et al., 2011). However, the high energy consumption and
generation of contaminated residues associated with such processes
have motivated the search for alternatives, including the application of
biologically based processes (Nancharaiah et al., 2016). Biological
treatments can achieve high removal rates (Kiran et al., 2016;
Torres-Aravena et al., 2018). However, in most cases, the biomass
generated is rich in heavy metals. Moreover, metals may be re-mobilized
into the surrounding area, if environmental conditions change (Phillips
et al., 2013). Therefore, a sustainable process which produces stable
residues is needed. In this context, the microbe-induced carbonate pre­
cipitation (MICP) process is a potential alternative for the treatment of

* Corresponding author. Departamento de Ingeniería Química, Universidad de La Frontera, Temuco, Chile.

E-mail address: gustavo.ciudad@ufrontera.cl (G. Ciudad).

https://doi.org/10.1016/j.jenvman.2019.109938
Received 26 September 2019; Received in revised form 12 November 2019; Accepted 26 November 2019
Available online 13 December 2019
0301-4797/© 2019 Elsevier Ltd. All rights reserved.
C. Duarte-Nass et al.
wastewaters containing heavy metals.
The MICP process was first studied as a method for producing cal­
cium carbonate precipitates for different purposes, including improving
the mechanical properties of soil and sand (bio-consolidation) (DeJong
et al., 2010), bio-cementation (Ramachandran et al., 2001), CO2
sequestration (Mitchell et al., 2010), and restoration of cracks and fis­
sures in concrete structures (Achal and Mukherjee, 2015). The MICP
process is based on the use of microorganisms that hydrolyze urea (in a
reaction catalyzed by the enzyme urease), thereby generating carbonate
and ammonium ions (Phillips et al., 2013). Ammonium generation in­
creases the pH of the medium, which favors the bonding of carbonate to
calcium and finally the formation of calcium carbonate precipitates
(Krajewska, 2018). This mechanism could be applied to other metals
with a valence number of two (Dhami et al., 2013), such as copper (II),
thus representing a potential alternative bioremediation process for soils
and wastewaters. The resultant metal precipitates may remain stable
over long periods of time, since the MICP process is independent of the
redox potential of the system (Kumari et al., 2016).
Urea concentration is a key factor influencing urease activity, and is
thus a principal issue in much of the research on bio-precipitation of
calcium (Chahal et al., 2012; Cuthbert et al., 2012). Complete hydrolysis
of 1 mol of urea generates 1 mol of carbonate ions and 2 mol of
ammonium ions. However, the generation of ammonium represents an
additional source of environmental pollutant, and few studies have
directly addressed this issue (Torres-Aravena et al., 2018). Minimization
of the urea concentration during the MICP process will in turn minimize
ammonium generation, reducing the potential negative impact of the
process on watercourses. Moreover, lower urea consumption also im­
plies a reduction in substrate costs. The pH is another key factor gov­
erning the MICP process (Achal and Mukherjee, 2015). High-pH
conditions favor the formation of CO23 from HCO3 (Achal et al., 2015).
Further, pH influences the solubility and bioavailability of heavy metals,
as well as the enzymatic activity (Hashim et al., 2011). The tolerance of
microorganisms to heavy metals may represent another relevant factor.
Specifically, high metal concentrations lead to metabolic changes that
can alter cell functions or even cause cell death (Yin et al., 2018). Until
now, research dealing with the MICP process for heavy metal precipi­
tation has been limited to applications in soil bioremediation. In the case
of copper, reported removal rates vary from 6% to 95% (Li et al., 2013;
Mugwar and Harbottle, 2016). However, reasons for such a wide range
of reported removal levels are not yet evident.
The objective of this study was to investigate the potential feasibility
of MICP as an alternative process for the removal of copper from copper-
enriched waters. To achieve this, pH and urea and copper concentrations
that favor optimal copper precipitation in the MICP process were iden­
tified. Sporosarcina pasteurii, a well-known urease-positive bacteria
(Tobler et al., 2011), was used in this study. Currently, S. pasteurii is
considered to be a model bacterial strain for MICP applications (Declet
et al., 2016). Simulations using Visual MINTEQ® and experimental as­
says were carried out to study the effects of pH, urea concentration, and
bacterial copper tolerance on the successful implementation of MICP as
a tool for the removal of copper from copper-enriched waters.
2. Materials and methods
2.1. Bacterial growth conditions
S. pasteurii is a known urease-positive microorganism and has been
used in several studies on MICP (Achal et al., 2015). It was obtained
from the Chilean Culture Collection of Type Strains (CCCT 16.12) and
was kept in a solid medium, whose composition has been reported
previously (Achal et al., 2012), consisting of 1 g/L beef extract, 5 g/L
peptone, 2 g/L yeast extract, 5 g/L NaCl, 12 g/L agar, and 20 g/L urea
(333 mm). The pH was fixed at 7 using 1 m solutions of NaOH or HCl. All
components of the medium were first autoclaved, except for urea, which
was sterilized by filtration (0.22 μm pores). Petri dishes containing the
2
Journal of Environmental Management 256 (2020) 109938
bacteria were incubated at 30 � C for 48 h and then stored at 4 � C until
their utilization in the assays. The temperature selected for the bacterial
cultivation was based on the propagation methodology of the American
Type Culture Collection (ATCC® 11859™) for S. pasteurii.
For bacterial growth assays, a colony of S. pasteurii was cultivated in
a liquid growth medium as described previously (Achal et al., 2012) (but
without the addition of agar) until its optical density at 600 nm (OD600),
measured at regular intervals, reached an absorbance value between 0.6
and 0.8. Then, this culture was used to inoculate new growth media at
5% (v/v) according to the conditions of each assay. All liquid cultures
were grown in 100 mL of the corresponding medium in 250 mL flasks,
incubated at 30 � C, and shaken at 130 rpm over 3 days.
2.2. Evaluation of copper bio-precipitation by MICP
The copper bio-precipitation assays were evaluated as previously
reported (Mugwar and Harbottle, 2016). The assays were inoculated at
5% (v/v) in 100 mL of the precipitation medium. The precipitation
medium consisted of 3 g/L of beef extract, 333 mM of urea, and 0.5 mM
of Cu2þ, added as CuCl2⋅2H2O. This copper concentration should allow
at least 50% growth of S. pasteurii, according to previous assays. The pH
was adjusted to 7. Control assays without bacteria were also performed.
Assays were performed in triplicate, and a fourth flask was used only for
sampling in order to determine pH evolution. All assays were carried out
for 4 days in an orbital shaker at 30 � C and 130 rpm. At 4 days of culture,
the maximum urease activity is reached according reported data (Achal
et al., 2011, 2009). Metal removal was calculated based on the differ­
ence between the final copper content of each inoculated flask and the
copper content of the blank flasks. The precipitates generated were
recovered by centrifugation at 6000 rpm for 10 min, dried at 45 � C for
72 h, weighed, and analyzed using scanning electron
microscope-energy-dispersive X-ray spectroscopy (SEM-EDX).
2.3. Simulation of copper precipitation conditions
To evaluate the combinations of pH and urea concentration which
optimize copper carbonate precipitation in the liquid media, simulations
were carried out with the Visual MINTEQ 3.1 software, which re­
produces the most likely compounds present at equilibrium under given
conditions. The software considers only chemical effects and does not
consider the biological processes involved in MICP. Simulations were
conducted with the goal of maximizing copper precipitation as mala­
chite (Cu2CO3(OH)2) or azurite (Cu3(CO3)2(OH)2), which are both
considered to be stable compounds (Ferris et al., 2004). The conditions
applied in the simulations were: 0.5 mM Cu2þ; 1 mM Cl ; 100, 200, and
2
666 mM NHþ 4 ; 50, 100, and 333 mM of CO3 ; pH from 5 to 9; and 30 C.
�
In the experimental assays, copper would be added to the culture media
as CuCl2, and therefore Cl ions were included in simulations. The
simulations operated on an assumption that complete urea hydrolysis
occurred; hence 1 M of hydrolyzed urea would equate to 1 M of car­
bonate ions and 2 M of ammonium ions.
2.4. Urea requirement for bacterial growth
The minimal urea requirement which enabled satisfactory bacterial
growth, and by extension urease activity, were determined through
experimental assays. S. pasteurii was cultivated at different urea con­
centrations (1, 10, 100 and 333 mM) in the liquid medium described in
Section 2.1. The initial pH was selected based on the results of previous
simulation. Bacterial growth was measured at regular time intervals.
Assays were performed in triplicate, although a fourth flask was used for
sampling in order to determine pH evolution.
2.5. Copper tolerance at different urea concentrations
S. pasteurii was grown under different urea and copper
C. Duarte-Nass et al.
concentrations to evaluate the influence of these substrates on the
bacterial copper tolerance; urea and copper concentrations evaluated
were 50, 100, and 333 mM, and 0.5, 1.0 and 1.5 mM, respectively. The
initial pH and urea concentrations tested were defined according to
simulation results. The range of copper concentration was defined ac­
cording to the copper tolerance of S. pasteurii previously reported (Kang
and So, 2016; Mugwar and Harbottle, 2016). All components of the
media were sterilized by filtration (0.22 μm) and all assays were per­
formed in triplicate. A sample of 10 mL was taken from each flask at
regular intervals to measure pH, ammonium concentration, and urease
activity.
2.6. Analytical techniques
Bacterial growth was determined via optical density measured at
600 nm using a spectrophotometer (Jenway 6320D). The pH was
measured with a pH meter (Thermo Scientific Orion Star A-121). Copper
was measured by Inductively Coupled Plasma Optical Emission Spec­
trometry (ICP-OES), according to the Standard Methods for the Exami­
nation of Water and Wastewater, method SM3120B (American Public
Health Association et al., 2012). Ammonium concentration was
measured using a Hach High Range Ammonia Nitrogen by the AmVer™
Salicylate Test ‘N Tube™ Method (Hach Method 10031). Urea hydro­
lysis was calculated based on ammonia concentration and the
theoretical-maximum urea hydrolysis, considering complete stoichio­
metric hydrolysis wherein 1 mol of urea produces 2 mol of ammonium.
An analysis of variance was applied to evaluate the influences of initial
urea concentration and copper concentration on ammonium generation.
Variable pressure scanning electron microscopy (SEM) with a
transmission module STEM SU-3500 (Hitachi, Japan) and a QUANTAX
100-energy-dispersive X-Ray (EDX) detector (Bruker-Germany) was
used to semi-quantitatively analyze the compositions of the precipitates.
The urease activity was evaluated using the phenol-hypochlorite
method (Achal et al., 2009), and ammonium chloride (50–1000 μM)
was used as the standard. The culture filtrates (250 μL) were added to a
mixture containing 1 mL of 0.1 M potassium phosphate buffer (pH 8.0)
and 2.5 mL of urea (0.1 M). The mixture was incubated at 37 � C for 5
min, after which 1 mL of both phenol nitroprusside and alkaline hypo­
chlorite were added, and the mixture was incubated at 37 � C for 25 min.
Optical density was measured at 626 nm, and one unit of urease was
defined as the amount of enzyme hydrolyzing 1 μmol urea/min.
3. Results and discussion
3.1. Evaluation of copper bio-precipitation through MICP
An assay for copper precipitation through MICP was carried out. The
pH of the blank flask (without inoculum) remained around 7, indicating
that urease hydrolysis by bacteria did not occur. On the other hand, the
flask inoculated with S. pasteurii registered an initial pH of 9, even
though the medium was prepared at pH 7. This could indicate that a
certain degree of urease activity was present immediately after the
inoculation took place. Despite the initial urease activity, no significant
pH evolution was observed; pH varied only from 9.0 to 9.3 on day 4.
These pH values are similar to those reported by Mugwar and Harbottle
(2016), who evaluated pH evolution at different copper concentrations.
These authors observed more significant increases in pH at lower metal
concentrations.
The analysis of the supernatant using ICP-OES indicated that the
removal of copper was only 10%, beginning from an initial copper
concentration of 0.5 mM. This result does not match those reported by
Achal et al., (2011), Li et al., (2013), or Mugwar and Harbottle (2016),
who observed removals of 95%, 90%, and 30%, respectively. Achal
et al., (2011) used a bacterial strain isolated from a contaminated soil.
Therefore, the degree of Cu removal reported in their study may be
resultant of the adaptation of the bacteria to the high concentration of
3
Journal of Environmental Management 256 (2020) 109938
heavy metal and to the alkaline medium. Moreover, the 90% copper
removal reported by Li et al., (2013) involved an initial copper con­
centration of 2 g/L, which is more than 60 times greater than the con­
centration used in this assay. Unlike these studies, Mugwar and
Harbottle ,(2016) studied the co-precipitation of a few heavy metals,
including calcium, in MICP. In their study, copper presented the lowest
degree of removal of the heavy metals. The authors stated that precip­
itation occurred mainly due to the alkaline environment and via the
formation of copper carbonate complexes on the surfaces of calcite and
vaterite, thereby hindering further sorption as well as growth or disso­
lution of the mineral. On the other hand, Kang et al., (2016) reported a
copper removal of 5.6% when using a mixed culture isolated from soils
contaminated with heavy metals and a mix of metals. In their case,
competitive formation of metal carbonates may have occurred, and
copper was the metal least likely to form carbonates in comparison to
the other metals studied.
A small quantity of precipitate (0.037 mg/mL) was obtained and
subsequently analyzed. The elemental composition obtained by SEM-
EDX showed that no copper carbonates were present in the pre­
cipitates (data not shown). Simulations were carried out in order to find
the reasons for the low copper removal observed in the precipitation
assay and to identify the pH and urea concentration that would favor
copper carbonate precipitation.
3.2. Software simulation of copper precipitation
Copper precipitation in the liquid media was simulated using Visual
MINTEQ® software. In this simulation, precipitation of copper com­
pounds was studied under different conditions, considering different pH
values and urea concentrations. As Visual MINTEQ® software does not
consider biotic variables, an assumption of 100% efficiency was applied
for urea hydrolysis. Thus, carbonate and ammonium concentrations
were established considering complete urea hydrolysis. Fig. 1a shows
the possible copper precipitates within a pH range of 5–9 and with a urea
concentration of 333 mM. This urea concentration has typically been
used in assays for heavy metal removal through MICP in order to ensure
that there are sufficient carbonate ions for precipitation (Achal et al.,
2011; Dhami et al., 2013; Kumari et al., 2014). The simulations indi­
cated that, at a pH of 8 or 9, there is no precipitation of malachite or
azurite. This could explain why no copper was removed from the liquid
phase during the copper precipitation assay, since the pH was 9 or
higher during most of the assay. At pH values between 5 and 7, pre­
cipitation of copper carbonate would take place in the form of azurite.
Simulations also indicated that the main copper-containing com­
pounds present at pH 9 would be copper-ammonia complexes (data not
shown). Copper (II) has a high affinity for ammonia, and the formation
of coordination complexes keeps the metal soluble (Liu et al., 2019),
thus impeding formation of copper carbonate and subsequent precipi­
tation of copper. Vital et al., (2018) demonstrated that a
copper-ammonia complex formed in a copper solution containing
ammonium bicarbonate (NH4HCO3); this can be verified by the intense
blue color that develops, as was observed in our assays (data not shown).
Further, they confirmed that the copper carbonate precipitate formed in
a solution containing 160 mM of copper and 1 M of ammonium bicar­
bonate was malachite. This could indicate that a higher ratio of copper:
ammonium than that used in the copper precipitation assay (0.5:666
mM) would shift the chemical balance toward copper carbonate pre­
cipitation, as a result of the lower initial urea concentration.
New simulations were then carried out utilizing a lower urea con­
centration. Fig. 1b shows that, when the urea concentration is reduced to
100 mm, copper precipitation would occur in the form of azurite, within
the pH range of 5–6, and in the form of malachite, between pH 7 and 8.
At pH 9, the copper-ammonia complex would still be formed. When urea
concentration is reduced further to 50 mM (Fig. 1c), azurite formation
would occur between pH 5 and 6, while malachite would form between
pH 7 and 8, similarly in the case of 100 mM of urea concentration.
C. Duarte-Nass et al. Journal of Environmental Management 256 (2020) 109938

a) 100%
80%

composi on
precipitate
Simulated
60%
40%
20%
0%

100%

b) 80%
composi on
precipitate
Simulated

60%
40%
20%
0%

100%
80%
composi on
precipitate
Simulated

60%
c)
40%
20%
0%
pH 5 pH 6 pH 7 pH 8 pH 9

Precipitate Azurite Precipitate Malachite Precipitate Tenorite Soluble Cu(II)

Fig. 1. Simulation of copper compounds expected to form at different pH and hydrolyzed urea concentrations: a) 333 mm urea; b) 100 mm urea; c) 50 mm urea.

However, at pH 9, copper would precipitate in the form of tenorite

a) 3.5
(CuO). This product is undesirable since it is not stable over long periods 3.0
of time, instead tending to react readily with other compounds (Schock 2.5
and Lytle, 1995). Conversely, carbonate compounds have been shown to 2.0
OD600

be stable over time (Ferris et al., 2004). Moreover, the solubility con­ 1.5

stants at 25 � C indicate that malachite (pKsp ¼ 33.16) and azurite (pKsp 1.0
0.5
¼ 44.89) precipitate more readily than tenorite (pKsp ¼ 20.36) (Ball and
0.0
Nordstrom, 2018). Thus, both the azurite and malachite forms would be 0 10 20 30 40 50 60 70 80
desirable compounds for copper precipitation.
Hence, it can thus be inferred that copper precipitation would occur 10
when urea concentration is maintained at 50 mM or lower. Therefore, 9
b)
reducing the initial urea concentration confers a double benefit. As 8
fewer reagents would be needed, associated costs would decrease, and
pH

7
less ammonium would be released into the liquid medium, diminishing
6
the impact of this pollutant in the wastewater (Zhu and Dittrich, 2016).
5
In addition, the results of these simulations indicate that the pH 0 10 20 30 40 50 60 70 80
range should be fixed from 5 to 8 to favor the formation and precipi­ Time (h)
tation of copper carbonate. Further, this range would not exert any
Urea 1 mM Urea 10 mM Urea 100 mM Urea 333 mM
detrimental effect on the urease enzyme, which has been reported to
have hydrolytic activity within a pH range of 6–9 (Krajewska, 2016).
Fig. 2. a) S. pasteurii growth as measured via optical density at 600 nm and b)
pH evolution of the culture at different urea concentrations. The bars on Fig. 2a
indicate one standard deviation (n ¼ 3).
3.3. Urea requirements for bacterial growth

to that in a urea concentration of 333 mM. At a urea concentration of

Although the simulations indicated that a lower urea concentration
100 mM, there is no significant difference in the bacterial growth as
should increase copper precipitation, it is also necessary to consider the
compared to that at 333 mM. Under both concentrations (100 and 333
potential effect of urea concentration on bacterial growth, in order to
mM), the exponential phase of growth was reached after 3 h. This in­
ensure that the conditions are compatible with bacterial growth (Kap­
dicates that urea concentration could be reduced to one -third of the
paun et al., 2018; Krajewska, 2016).
value originally tested, without impairing bacterial growth. Fig. 2b
For this reason, an assay was carried out with the aim of determining
shows the pH evolution of the medium. Although all culture media were
the minimum urea concentration needed for bacterial growth. Fig. 2a
prepared to provide an initial pH of 6, during the assays utilizing 100
shows that no bacterial growth would be expected at a urea concen­
and 333 mM of urea, the addition of the inoculum produced an imme­
tration of 1 mM. At a urea concentration of 10 mM, there is an initial lag
diate rise in pH, to 7 and 7.5, respectively. The pH profiles display
phase of 8 h, after which time the bacterial growth becomes comparable

4
C. Duarte-Nass et al. Journal of Environmental Management 256 (2020) 109938

similar behavior to those of the bacterial growth curves (Fig. 2a), since
the pH of the medium in the assay with 1 mM urea remained under 7.5.
For urea concentration of 100 and 333 mM, the pH nearly reaches 9 as a
result of ammonium release due to the urea hydrolysis.
Li et al., (2014) studied conditions to improve the MICP processes for
calcium precipitation, reporting that urea degradation depends on the
initial urea concentration. These authors stated that maximum urea
degradation is obtained when the initial urea concentration is in the
range of 80–93 mM. These values agree with the results of this study,
which suggest a suitable range of 10–100 mM. Işik et al., (2012) also
studied the effect of initial urea concentration, reporting an optimum
concentration of 15 mM. However, De Muynck et al., (2010) reported
that the optimum urea concentration was 333 mM. Moreover, Omoregie
et al., (2017) evaluated urea concentration between 333 and 1665 mm,
and identified concentrations between 999 and 1332 mM as the optimal
conditions when utilizing S. pasteurii. Evidently, the precipitation of
copper carbonate differs from that of calcium carbonate, especially as a
result of the tendency for copper to form ammonia complexes at high
pH. This contrasts with calcium, the affinity of which for carbonate ions
increases as pH increases. Thus, it seems clear that the optimal MICP
conditions for copper precipitation would involve a lower urea con­
centration than that necessary for calcium carbonate precipitation.
3.4. Copper tolerance at different urea concentrations
Tolerance to heavy metals is an important study parameter, since it
indicates the maximum concentration which still allows bacterial
growth or another particular function of organisms such as enzyme ac­
tivity (Hu et al., 2014; Khalid et al., 2016). Therefore, the tolerance of
S. pasteurii to copper was determined at different urea concentrations.
The copper concentrations evaluated were 0.5, 1.0, and 1.5 mm, which
were chosen according to previously reported tolerance levels (Kang and
So, 2016; Mugwar and Harbottle, 2016). Fig. 3 presents the results for a
urea concentration of 50 mm. The results indicate that no bacterial
growth would occur at any of the copper concentration evaluated
(Fig. 3a). Fig. 3b and c indicate that pH and ammonium concentration,
respectively, remained constant at a copper concentration of 0.5 mM,
indicating that urea hydrolysis did not occur. Further, Fig. 3c points out
that without the addition of copper, urea hydrolysis would reach 100%,
clearly indicating the detrimental effect of copper under the tested
conditions.
Fig. 4 presents the results for a urea concentration of 100 mm. Fig. 4a
shows bacterial growth at a copper concentration of 1.0 mm. Growth
under these conditions was similar to that observed in the absence of
copper. The same result was observed with 0.5 mm of copper (data not

3.5
a) 3.0
2.5
2.0
OD600

1.5
1.0
0.5
0.0
0 10 20 30 40 50 60 70 80

10
b)
9

8
pH

5
0 10 20 30 40 50 60 70 80

120
c)
100

80
NH3-N (mM)

60

40

20

0
0 10 20 30 40 50 60 70 80
Time (h)

0 mM Cu 0.5 mM Cu 100% hydrolysis

Fig. 3. Assays with 50 mm urea and different copper concentrations: a) S. pasteurii growth; b) pH evolution; c) ammonium concentration evolution. The bars on the
figures indicate one standard deviation (n ¼ 3).

5
C. Duarte-Nass et al. Journal of Environmental Management 256 (2020) 109938

3.5
a)
3.0
2.5
2.0

OD600
1.5
1.0
0.5
0.0
0 10 20 30 40 50 60 70 80

10
b)
9

8
pH

5
0 10 20 30 40 50 60 70 80

c) 200

150
NH3-N (mM)

100

50

0
0 10 20 30 40 50 60 70 80
Time (h)

0 mM Cu 1.0 mM Cu 1.5 mM Cu 100% hydrolysis

Fig. 4. Assays with 100 mm urea and different copper concentrations: a) S. pasteurii growth; b) pH evolution; c) ammonium concentration evolution. The bars on the
figures indicate one standard deviation (n ¼ 3).

shown). Fig. 4b presents pH evolution, showing that the same behavior
is expected for both 0 and 1.0 mm copper concentrations. These results
indicate that, in the presence of 100 mM of urea, S. pasteurii would
tolerate a copper concentration of 1.0 mm. No bacterial growth was
observed in the presence of 1.5 mM of copper. The maximum degrees of
urea hydrolysis reached at copper concentrations of 0 mm, 0.5 mm and
1.0 mm were 90.1%, 75.9%, and 70.0%, respectively (Fig. 6a).
In order to compare these results with those of experiments involving
the urea concentration commonly used in MICP studies, a copper
tolerance evaluation was performed at a urea concentration of 333 mM.
Fig. 5a shows that at a copper concentration of 1.5 mm, bacterial growth
was comparable to that observed under the other tested copper con­
centrations after 72 h of culture. Comparing Figs. 3a, 4a and 5a, it can be
seen that the bacterial growth behavior would depend on the initial
concentration of both urea and copper, since under 50 mm of urea, no
growth was registered at any copper concentration. Nevertheless, under
333 mm of urea, bacterial growth was registered under copper con­
centrations up to a 1.5 mm, although the lag phase was longer under
higher concentrations.
To evaluate the effects of initial urea and copper concentrations on
ammonium generation by S. pasteurii, as an indicator of urea hydrolysis,
an analysis of variance was performed. The statistical analysis indicated
that both initial urea and copper concentration significantly influence
ammonium production, as does the interaction of the two factors. Fig. 6
shows the positive correlation between ammonium generation and
initial urea concentration, and that a higher initial urea concentration is
required as copper concentration increases in order to generate
ammonium.
In this sense, the induction of urease activity may be a strategy used
by the bacteria as a defense mechanism to either precipitate metals as
carbonate minerals or bind them in ammonia complexes. Fig. 5b shows
that pH evolution was similar under copper concentrations of 0 and 1.0
mm. At a copper concentration of 1.5 mm, the pH and ammonia profiles
were comparable to those corresponding to the copper concentrations of
0.5 and 1.0 mm, as evaluated after 72 h of culture (Fig. 5b and c).
Maximum levels of urea hydrolysis observed at copper concentrations of
0.5 mm, 1.0 mm and 1.5 mm were 64.7%,61.5% and 46.6%, respec­
tively (Fig. 7a). Without copper, the maximum urea hydrolysis was
61.7%, which is similar to the degree of hydrolysis in the presence of 1.0
mM of copper. This could indicate that although copper tolerance is
enhanced at higher urea concentrations, urea hydrolysis decreases.
Ureases have many functions in a variety of organisms, including the
modification of harmful environments through balancing of pH, and
other defense mechanisms such as anti-fungal activity, platelet exocy­
tosis, and intercellular communication, among others functions that are
currently under investigation (Kappaun et al., 2018). However, these

6
C. Duarte-Nass et al. Journal of Environmental Management 256 (2020) 109938

3.0
a)
2.5

OD600 2.0
1.5
1.0
0.5
0.0
0 10 20 30 40 50 60 70 80

10
b)
9

8
pH

5
0 10 20 30 40 50 60 70 80

c) 700
600
500
NH3-N (mM)

400
300
200
100
0
0 10 20 30 40 50 60 70 80
Time (h)

0 mM Cu 1.0 mM Cu 1.5 mM Cu 100% hydrolysis

Fig. 5. Assays with 333 mm urea and different copper concentrations: a) S. pasteurii growth; b) pH evolution; c) ammonium concentration evolution. The bars on the
figures indicate one standard deviation (n ¼ 3).

other defense mechanisms do not involve urea hydrolysis. Although
urease activity has been identified as a defense function, no studies have
reported urea hydrolysis as a mechanism in heavy metal tolerance.
Nevertheless, urease activity is a common indicator used to asses heavy
metal tolerance in soil (Yan-qing and Hong-mei, 2012), and is also a key
factor for improving the MICP process (Dhami et al., 2013). During the
assays for copper tolerance at different urea concentrations, urease ac­
tivity was determined at the beginning of the experiments and when
ammonium concentration in the medium reached a maximum. Fig. 7b
shows that initial urease activity slightly increased with increasing urea
concentration. This could explain why the pH changed immediately
from 6 to 8 (Fig. 5b) when the medium containing 333 mm of urea was
inoculated. Urease activity was also analyzed considering maximum
ammonium release into medium (Fig. 7c). The highest values were
found in the media containing 100 mm of urea, and not in those con­
taining the highest urea concentration evaluated (333 mm), under the
conditions allowing bacterial growth (at copper concentrations of 0.0,
0.5 and 1.0 mm). At a copper concentration of 1.5 mm, urease activity
was highest in the medium containing 333 mm of urea. These results
support the idea that initial urea concentration could be reduced from
333 mm to 100 mm to improve urea hydrolysis efficiency and to reduce
the degree of ammonium discharge.
As mentioned previously, the mechanism by which urea hydrolysis
acts as a defense mechanism to enhance tolerance to high copper con­
centrations and support bacterial growth has not been reported. How­
ever, the mechanism by which copper or other heavy metals inhibit
urease activity in soil is a topic of current research. Krajewska, (2008)
studied the mechanism of inhibition of urease by copper, and reported

7
C. Duarte-Nass et al. Journal of Environmental Management 256 (2020) 109938

500
450
400
350

NH4+ (mM)
300
250
200
150
100
50
0
0 50 100 150 200 250 300 350 400
Urea (mM)

0.0 mM Cu 0.5 mM Cu 1.0 mM Cu 1.5 mM Cu

Fig. 6. Relationship between initial urea and copper concentrations and ammonium concentration in S. pasteurii cultures. The bars on the figure indicate one
standard deviation (n ¼ 3).

a) 120%
100%
Urea hydrolysis
efficiency (%)

80%
60%
40%
20%
0%
0 0.5 1 1.5

1.5
ac vity (U/mL)
Ini al urease

b) 1.0

0.5

0.0
0 0.5 1 1.5

30
Urease ac vity at maximum

25
urea hydrolysis (U/mL)

20
c) 15
10
5
0
0 0.5 1 1.5
Copper concentra on (mM)

50 mM urea 100 mM urea 333 mM urea

Fig. 7. a) Urea hydrolysis efficiency, b) initial urease activity, and c) urease activity at maximum urea hydrolysis by S. pasteurii corresponding to different urea and
copper concentrations. The bars above the columns indicate one standard deviation (n ¼ 3).

that copper binds to thiol and other functional groups on the enzyme,
thereby distorting the architecture of the active site. In their study, a
urea concentration of 50 mM was tested along with copper in concen­
trations between 1 and 10 μM, but no analysis of the influence of urea
concentration was reported.
The biggest challenge involved in optimizing copper precipitation by
the MICP process has to do with the pH of the system. Visual MINTEQ®
simulations indicated that a pH of 9 favors the formation of copper-
ammonia complexes, and even more significantly so when the urea
concentration was between 100 and 333 mm. Figs. 3b, 4b and 5b show
pH values above 9, independent of the urea concentration evaluated,
which is due to urea hydrolysis. Orel et al., (2007) reported malachite
precipitation during thermal hydrolysis of urea (500 mM) when copper
concentration was 5 mm, but the pH of the mixture was between 7.7 and

8
C. Duarte-Nass et al.
8.0, which is in accordance with the pH range determined to be
adequate for copper precipitation through the simulations carried out
herein. Therefore, it is crucial to control pH throughout the MICP pro­
cess in order to optimize copper precipitation and, by extension, its
removal from copper-enriched waters.
The challenge of pH control during the MICP process may be
addressed by separating the treatment into two steps. In the first step,
bacterial growth would be allowed to proceed in the absence of any
copper. This strategy will prevent inhibition of growth by copper, and it
will thus not be necessary to add high concentrations of urea to the
process, decreasing the final ammonium concentration in the effluent.
Then, the second step would consist of mixing the biomass from the first
step with a medium containing a high concentration of copper, at a
controlled pH between 6 and 8, and would be the step wherein the metal
precipitation takes place. In this second step, it would not be necessary
for the biomass nor the enzyme be tolerant to high concentrations of
copper, since carbonate ions resulting from urea hydrolysis were already
produced in the first step. In the second step, no urea would be added, so
further urea hydrolysis would not be expected. By implementing this
two-step process, high degrees of copper removal could be achieved; pH
would be easily controlled in the second step since urea hydrolysis
would have occurred mostly in the first step. A similar system was
proposed by Torres-Aravena et al., (2018) in their application of MICP
for the precipitation of heavy metals in different industrial wastewaters.
4. Conclusions
The challenges involved in the application of MICP in the bio-
precipitation of copper from copper-enriched waters are related to pH
control and urease activity.
According simulations, the conditions of pH 9, which was recorded
in the assays, did not favor copper carbonate bonding. The equilibrium
went toward copper-ammonia complexes formation. So, less initial urea
addition would diminish ammonia production, and so, copper-ammonia
complexes.
However, initial urea concentration affects S. pasteurii copper-
tolerance. The bacteria copper-tolerance was evaluated with urea
requirement and the results indicated that there is a positive relation on
it.
With a sight on the industrial application of this technology, if the
process is carried out in a single step, the high urea requirement to the
bacteria copper-tolerance increases copper-ammonia complexes for­
mation. Therefore, it is proposed a process involving two separate steps:
the first for bacterial growth, without excess urea; and the second
serving exclusively for copper precipitation. The pH control would be
simpler in the last step since urea hydrolysis would not be occurring.
Credit author statement
Carla Duarte-Nass: Conceptualization, Methodology, Validation,
Formal analysis, Investigation, Writing - Original Draft, Visualization,
Project administration, Funding acquisition, Katherina Rebolledo:
Investigation, Tamara Valenzuela: Investigation, Matías Kopp: Investi­
gation, David Jeison: Conceptualization, Writing - Review & Editing,
Mariella Rivas: Writing - Review & Editing, Laura Azo �car: Writing -
Review & Editing, Alvaro
� Torres-Aravena: Conceptualization, Re­
sources, Writing - Review & Editing, Gustavo Ciudad: Conceptualiza­
tion, Resources, Writing - Review & Editing, Supervision, Project
administration, Funding acquisition.
Declaration of competing interest
None.
9
Journal of Environmental Management 256 (2020) 109938
Acknowledgements
This study was supported by the CONICYT PAI Doctoral Thesis in
Productive Sector 2017 (T7817110005), CONICYT FONDECYT/POST­
DOCTORADO 2018 (3180648), and by CRHIAM center (CONICYT/
FONDAP/15130015).
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